How to write
Plenary Lecture
Nature Medicine
Symposium
Y.I.A.
Presentation
Selected Oral
Presentation
Poster
Index
Aⴊ▤↢‛කቇળKPFD
Special Talk
81
Special
Lecture
The 8th Korea-Japan Joint Symposium on Vascular Biology
President
Lecture
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
抄録
Program
Osaka University Medical School, Osaka, Japan
Medicine
PhD(3/91)
Osaka University Medical School, Osaka, Japan
Medicine
Postdoctoral Fellow
Osaka University Medical School
Department of Geriatric Medicine (T. Ogihara)
Postdoctoral Fellow
Stanford University School of Medicine,
Division of Cardiovascular Medicine
(Victor J. Dzau)
Visiting Instructor
Stanford University School of Medicine,
Division of Cardiovascular Medicine
(Victor J. Dzau)
Research Fellow of the Japan Society for the Promotion of Science
Assistant Professor
Department of Geriatric Medicine (T. Ogihara)
Osaka University Medical School
Associate Professor
Division of Gene Therapy Science (Y. Kaneda)
Osaka University Medical School
Visiting Professor
The University of Hong Kong
Professor
Department of Clinical Gene Therapy
Osaka University Medical School
(Donated by Dai-ichi Pharmaceutical)
MD(3/87)
13：30〜14：20
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Professional Societies:
Board; Japan Society of Vascular Biology Organization, Society for Intellectual Property, Japan Society of Anti-Aging, Japan Society
of Gene Therapy, Japan Society of Venture
Professional activities & appointments (selected)
• Committee for Industry-Academia Relationship, Ministry of Education, Science and Culture of Japan
• Committee for Human Resource, Ministry of Education, Science and Culture of Japan
• Vice Chairman, Bio-Venture Association Originated from Universities
• Past Member of Headquarter for Intellectual Property Strategy, Cabinet Office (Koizumi & Abe Cabinet)
Editorial (selected from over 30):
Circulation (1999-), Hypertension (2006-), The Open Gene Therapy Journal (2007-; Chief-in-editor; 2008-), Cardiovascular Research
(2008-), Gene Therapy (2008-), Arteriosclerosis, Thrombosis & Vascular Biology (2009-), Immunology, Endocrine and Metabolic
Agents in Medicinal Chemistry (Associate Editor, 2009-)
Honors (selected):
1993 Upjohn Young Investigator Award in Cardiology, Stanford University
1994 Young Investigator Award of the Dr. C & F. Demuth Medical Foundation at the 15th Scientific Meeting of the International
Society of hypertension in Melbourne, Australia.
1996 Young Investigator Award (First winner) in Japanese Circulation Society
1996 Young Investigator Award in Japanese Atherosclerosis Society
1996 Harry Goldbratt Award in Council of High Blood Pressure, American Heart Association
& Endocrinology
2001 Takamine Jokichi Award in 5th annual meeting of the Society of Cardiovascular
2002 Sato Award in 27th annual meeting of the Japanese Circulation Society
2003 Japan Innovator Award in 1st meeting (Nikkei BP)
2003 Award from Minister of Education in 1st meeting
2005 Invitrogen-Nature-Biotechnology Award
01/2000
03/2003-present
10/98-03/2004
4/95-96/9
10/96-10/98
5/94-96/8
8/91-4/94
4/91-8/91
4/87-3/91
Carrier:
4/81-3/87
Birthday:
5/12/1962
Degree:
MD, PhD
Morishita Ryuichi
会長講演／President Lecture
12月2日
（木）
／Thursday, December 2
disease.
Selected Oral
Presentation
Poster
Index
83
Y.I.A.
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
will be initiated soon. In this lecture, I will introduce recent progress in molecular therapy to treat cardiovascular
Symposium
neointimal formation after balloon injury as compared to normal balloon catheter. Clinical trial of DEB catheter
developed NFkB decoy eluting balloon (DEB) catheter. In rabbit study, DEB balloon catheter inhibited
against NFkB, an essential transcription factor for inflammation and adhesion. Using decoy ODN, we have
In addition to cell and gene therapy, oligo-based therapy is also promising. We have developed decoy ODN
How to write
Plenary Lecture
Nature Medicine
regenerative drug in the world.
will be started soon, as FDA accepted fast track status. HGF (CollategeneTM) will be as a first vascular
were no major safety problems. HGF gene therapy is safe and effective for CLI. Currently, global phase III trial
improvement rate (100% [11/11]) than placebo (40% [2/5]; P=0.018). HGF plasmid also improved QOL. There
group, showing a significant difference (P=0.014). In Rutherford 5 patients, HGF achieved a significantly higher
overall improvement rate of the primary endpoint was 70.4% (19/27) in HGF group and 30.8% (4/13) in placebo
randomized, double-blind, placebo-controlled trial in PAD patients was reported (Gene Therapy 2010). The
HGF as most potent mitogen to stimulate angiogenesis. Recently, an exciting positive result from phase III
Special Talk
one of most promising strategy to treat PAD is therapeutic angiogenesis using gene therapy. Our group identified
found by Prof. Yamanaka are also highlighted as promising cell sources for regenerative medicine. Currently,
outcome, although further placebo control studies are necessary. In addition, embryonic stem cells and iPS cells
Special
Lecture
open label pilot studies to treat no option patients with PAD and IHD demonstrated the beneficial clinical
cardiovascular diseases. It is noteworthy that using bone marrow stem cells initiated by Japanese researchers,
including stem cell therapy, gene therapy, oligonuclotides therapy and peptide therapies to treat various
Recent progress in vascular biology and medicine has led to the development of novel molecular therapy
Department of Clinical Gene Therapy, Osaka University Graduate School of Medicine
Ryuichi Morishita
End of The Beginning; A New Era of Vascular Biology &
Medicine
President
Lecture
Aⴊ▤↢‛කቇળKPFD
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Program
Second Department of Surgery, Osaka University School of Medicine
Research fellow
Institute for Molecular and Cellular Biology
Osaka University
Research Associate
Howard Hughes Medical Institute
University of Utah
Research Assistant Professor
Department of Human Genetics
University of Utah
Senior Associate
Howard Hughes Medical Institute
University of Utah
Head of Biochemistry Department
Cancer Institute, Tokyo
Professor, Department of Clinical Genetics
Osaka University School of Medicine
Head, Division of Genome Analysis
Cancer Institute, Tokyo
Professor, Laboratory of Molecular Medicine
Institute of Medical Science
The University of Tokyo
Director, Human Genome Center
Institute of Medical Science
The University of Tokyo
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Awards
1991 Honarary Citizenship, The State of Maryland, USA
1992 Princess Takamatsu Cancer Research Award
1993 The Research Award of Japanese Foundation for Cancer Research
1995 The Award of the Japanese Society of Human Genetics
1996 Takeda Medical Prize
2000 Keio Medical Science Prize
2002 The Tomizo Yoshida Award of the Japanese Cancer Association
2004 The Medal with a Purple Ribbon (for contributions to education and culture)
2006 Bulgarian Academy of Medical Science, Foreign Member
2010 Chen Award for Distinguished Academic Achievement in Human Genetic and Genomic Research (HUGO)
Members in
Japan Society of Human Genetics (President, 2007-present)
Japanese Cancer Association (board member)
American Association of Cancer Research
American Society of Human Genetics
Science Council of Japan (2005-present)
Member in the Editorial (or Advisory) Board for
Annals of Human Genetics
Cancer Science (Editor-in-Cheif)
Cancer Research (Senior Editor 2003-2006, Deputy Editor 2007-2009)
Cell Cycle
Clinical Genetics
Genes Chromosomes & Cancer
Molecular Cancer Research
International Journal of Oncology
Journal of Human Genetics (Editor-in-Chief 1997-2007)
Neoplasia
1995 April - present
1994 October - present
1995 April – 2000 March
1996 April - 1999 March
1989 April - 1995 March
1989 January - 1989 August
1987 September - 1989 August
1984 October - 1988 December
Occupation:
1977 May - 1981 March
1981 April - 1984 October
Education:
Graduated from Osaka University Medical School in March 1977
1977 May Medical Doctorʼs License in Japan
1984 August Ph. D. of molecular genetics from
Osaka University for a thesis entitled: “Sequences of cDNAs for
human salivary and pancreatic alpha-amylases”
Business Address:
Human Genome Center
Institute of Medical Science
The University of Tokyo
4-6-1, Sirokanedai,
Minato-ku, Tokyo 108-8639, Japan
TEL 03-5449-5372, FAX 03-5449-5433
Nationality:
Japanese
Date of Birth:
December 8th, 1952
Position:
Director, Human Genome Center
Professor, Laboratory of Molecular Medicine
Institute of Medical Science
The University of Tokyo
Yusuke Nakamura
特別講演 1／Special Lecture 1
12月1日
（水）
／Wednesday, December 1
90
Yttrium-labeled MAb 92-13 ( 90Y-MAb 92-13) to a mouse SS-
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Index
85
Poster
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
development of novel therapeutic drugs and diagnostic tools.
analysis should be a very effective approach for identification of molecules that are potential targets for
translational research will be introduced in the meeting. These results indicated that systematic expression
than 1100 cancer patients have been enrolled by the end of July 2010. The promising clinical output of our
translational research using some of them in August 2006. We are now running 30 different protocols and more
T lymphocytes that would specifically kill cancer cells in an HLA-A restricted manner. We have so far isolated
also screened 9- or 10-amino acids peptides corresponding to a part of such oncoantigens that induce cytotoxic
growth of SS in mice, indicating that MAb 92-13 could be utilized as the novel treatment modality for SS. We
xenograft model. Expectedly, a single intravenous injection of
Y.I.A.
Presentation
nearly 60 peptides (HLA-A02 or HLA-A24 restricted) derived from about 50 oncoantigens and started
90
Symposium
Y-MAb 92-13 drastically inhibited tumor
attempted radioimmunotherapy using an
against FZD10 was effectively internalized into the SS cells after its binding to FZD10 on the cell surface, we
activity against native FZD10 product expressed in synovial sarcoma (SS) cell lines. Since the MAb 92-13
How to write
Plenary Lecture
Nature Medicine
established a murine monoclonal antibody (MAb), namely, 92-13 for FZD10 product that had specific binding
cycle arrest, apoptosis, or suppression of anchoring-dependent cell growth. Using such information, we
function as oncogenes in various cancers. The suppression of expression of such genes with siRNA induced cell
in any important vital organs. The further functional analysis identified dozens of genes that are likely to
in comparison with their corresponding normal tissues and (2) expression was not observed or hardly detectable
genes by the criteria as follows; (1) gene expressions were transactivated in a large proportion of cancer tissues
breast, and soft tissues as well as acute and chronic myeloid leukemias. We have selected hundreds of candidate
pancreas, stomach, colon, esophagus, bile duct, uterus, lung, ovary, kidney, urinary bladder, testis, prostate,
Special Talk
cancers in those tissues. So far, we have analyzed more than 1,300 clinical cancer samples of the liver,
experiments disclosed a number of genes that appeared to be involved in development and/or progression of
cancerous tissues using a cDNA microarray that consists of more than 30,000 genes or ESTs. These
Special
Lecture
comparing expression profiles of cancer cells originated from various organs with their corresponding non-
tumor marker) and for treatment of cancer (molecular-targeting drug, cancer vaccine, antibody), we have been
human cancers. To isolate novel targets for diagnosis (predictive marker for the efficacy of treatment as well as
cDNA microarray technologies have enabled us to obtain comprehensive data for gene expression profiles of
Director, National Cancer Center Research Institute
Human Genome Center, Institute of Medical Science, The University of Tokyo
Yusuke Nakamura
From cancer genomics to cancer treatment
President
Lecture
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Program
10：40〜11：30
Philosophy Doctor (PhD) at the School of Medicine, Kyoto University
Obtained a medical licence (Licence# 220365)
Graduate School of Medicine, Kyoto University
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Prize:
Fritz von Siebold Prize (Germany)
Mochida Medical Foundation Prize (Japan)
Kiyohara Makoto Prize (Japan)
Government commitments:
The Bioethical Committee of the Cabinet of Japan
The Bioethical Committee of the Ministry of Education, Science, and Technology of Japan
The Stem Cell Therapy Committee of the Ministry of Health, Labour and Welfare of Japan
International Stem Cell Initiative
Academic Associations:
International Society for Stem Cell Research
Japanese Society for Immunology
Japanese Cancer Association
Japanese Society of Inflammation and Regeneration
Japanese Society of Haematology
Member of Advisory Council
Welcome Trust Centre for Stem Cell Research (Cambridge)
Stem Cell Institute of Bangalore (Bangalore)
Institute for Frontier Medical Sciences (Kyoto University)
Korea Advanced Institute of Science and Technology (KAIST)
Editorial Board
Development
Cell Stem Cell
Education:
Jan. 1987
Nov. 1973
April 1967 → Mar. 1973
Deputy Director of RIKEN Center for Developmental Biology and Group Director of the laboratory for
Stem Cell Biology
May 1993 → March 2003: Professor, Dept Molecular Genetics, Graduate School of Medicine, Kyoto University
April 1987 → Sep. 1993
Professor, Dept. Morphogenesis, Institute of Embryology and Molecular Genetics, Kumamoto University
Medical School
Sep. 1983 → March 1987 Associate Professor, Dept. Microbiology, Chest Disease Research Institute, Kyoto University
Nov. 1980 → Jan. 1983
Postdoctoral Fellow, Institute for Genetics, Cologne University
Dec. 1973 → Oct. 1980
Intern, resident, and principal physician
Hospital of Chest Disease Research Institute, Kyoto University
Occupation:
May 2000 → present :
Research Interest
1) Molecular and cellular basis of the stem cell niche
2) Steering ES cell differentiation
3) Molecular mechanisms underlying vascular remodeling
4) Developmental biology of the secondary lymphoid organ
Correspondence address :
Lab. for Stem Cell Biology,
RIKEN Center for Developmental Biology,
2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe,
Hyogo 650-0047, Japan.
E-mail : [email protected]
Personal Information
Date of birth:03/06 1948
Gender: Male
Nationality: Japan
Shin-Ichi Nishikawa M.D., Ph.D.
特別講演 2／Special Lecture 2
12月2日
（木）
／Thursday, December 2
Selected Oral
Presentation
HSC.
Index
87
Poster
The 8th Korea-Japan Joint Symposium on Vascular Biology
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
present our current hypothesis on the integrated process of embryonic vasculogenesis and differentiation of
and which region of vascular network is derived from YS Runx1+ area. In the symposium, I would like to
project is currently underway, we are starting to understand how vascular network is formed in early embryo
etv2/er71 is extremely useful for deleting progenitors of EC and HSC in a region specific manner. While this
areas of mesodermal tissues of E7.5 embryo formed an integrated vascular system and how the hemogenic
Runx1+ region. Hence, the final issue is to dissect the process by which Flk1+ cells distributing in distinct
and may rarely differentiate to EC. Indeed, our own study suggested that only 2-3% of EC are derived from
Y.I.A.
Presentation
endothelial cells derived from Runx1+ area is integrated to this network. For this purpose, conditional KO of
Symposium
hypothesis predicts that Flk1+ cells in Gata1 or Runx1 area are driven more strongly to hematopoietic lineage
expressed give rise to primitive erythrocytes and YS cells that express Runx1 alone give rise to HSC. This
region specific manner. Namely, the cells in the most proximal region where both Gata1 and Runx1 are
to more distal region where Flk1+ cells distribute. Thus, likely scenario is that HSC fate is determined in a
How to write
Plenary Lecture
Nature Medicine
expressed in the most proximal part of YS. Likewise, Runx1 expression spans over whole YS but not extends
and Runx1 in this Flk1+ population is regulated by extrinsic factors. Indeed, in E7.5 embryo, Gata1 is
factors (TF) involved in HSC development such as Gata1 and Runx1. We are thinking that expression of Gata1
default fate is EC. However, this default fate is able to be interrupted by additional expression of transcriptional
etv2/er71 expression ? We speculate that the transient expression etv2/er71 induces a ground state whose
expression, two lineage start to diverge after its expression. How then is the fate divergence is regulated after
whose major fate is to generate EC and HSC. While EC and HSC share the common pathway until etv2/er71
phenotype is due to differentiation arrest at the process of the primitive mesoderm to the Flk1+ lateral mesoderm
Special Talk
indicating the presence of common pathway for both cell lineages. Our own observations suggested that this
Dr. Lee of KAIST reported that null mutation of etv2/er71 resulted in complete lack of both HSC and EC,
Since then, we have been investigating both HSC and EC as two lineages integrated within a system. Indeed,
Special
Lecture
derived from a set of endothelial cells (EC) that are fully integrated within the embryonic vascular network.
explained. During the course of this study, we came across a possibility that hematopoietic stem cells are
the most extensively studied stem cell, but yet its developmental pathway in mammals has not been fully
The main focus of my group is to elucidate embryonic development of hematopoietic stem cell (HSC) that is
Center for Developmental Biology, RIKEN
Shin-ichi Nishikawa
Embryonic development of vascular system including hemogenic
endothelial cells.
President
Lecture
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Program
14：20〜15：10
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EDITORIAL BOARD:
Endocrine J. ( Editorial Board, 1990-1999 )
Diabetologia ( Associate Editor, 2000-2002 )
Diabetes (Editorial Board, 2002-)
J. Clin. Invest. (Editorial Board, 2002-)
Metabolic Syndrome and Related Disorders (Editorial Board, 2002-)
The Journal of Endocrine Genetics (Editorial Board, 2003-)
Diabetes, Obesity and Metabolism (Editorial Board, 2004-)
Current Diabetes Reviews (Editorial Board, 2004-)
Arteriosclerosis Thrombosis and Vascular Biology (Editorial Board, 2004-2007)
Endocrinology (Editorial Board, 2004-2007)
Diabetes Care (Editorial Board, 2007-)
The Journal of Clinical Endocrinology & Metabolism (Editorial Board, 2007-)
Best Practice & Research Clinical Endocrinology & Metabolism, (Editorial Board, 2007-)
Journal of Diabetes (Editorial Board, 2009-)
Journal of Diabetes Investigation (Executive Editor, 2010-)
Diabetology International (Editorial Board Member, 2010-)
HONORS AND FELLOWSHIPS:
Fogarty International Fellowship (1986-1990)
Young Investigator Award of the Japanese Society of Internal Medicine (1992)
Research Award of the Japan Endocrine Society (1993)
Shionogi-Lilly Award of the Japan Diabetes Society (1993)
Research Support Award of the Japan Medical Association (1993)
Research Award of the Tokyo Medical Association (1995)
Research Award of the Association of Tokyo University Medical School (1995)
Erwin von Baelzprize (2001)
The Best Teacherʼs Award of Tokyo University Medical School (2001)
Academic Award of the Mochida Memorial Foundation (2002)
Hagedorn Award of the Japan Diabetes Society (2004)
Sankyo Takamine Memorial Award (2004)
Medical Award of The Japan Medical Association (2007)
The Uehara Prize (2007)
Award of the Japan Society of Experimental Diabetes and Obesity (2008)
Awardee of a Medal with Purple Ribbon (2010)
SOCIETIES:
The Japan Diabetes Society (Chairman of the Board of Directors)
The Japan Endocrine Society (Board Member)
Japan Society for the study of obesity (Board Member)
American Diabetes Association
The Endocrine Society
POSITIONS HOLD:
1978-1980
Resident in Internal Medicine, Tokyo University Medical School
1981-1982
Medical Staff in Endocrinology and Metabolism Department, Institute for Adult Diseases
1983-1986
Clinical Research Fellow, Diabetes Section, the Third Department of Internal Medicine,Tokyo University Medical
School (Section Chief: Dr. Masato Kasuga)
Visiting Fellow, Biochemistry and Molecular Pathophysiology Section, Diabetes Branch, National Institute of Diabetes,
1986-1990
Digestive, and Kidney, Disease, National Institutes of Health, Bethesda, MD, U.S.A. (Branch Chief: Dr. Simeon Taylor)
1990-1998
Chief, Diabetes Branch, Assistant Professor, the Third Department of Internal Medicine, Faculty of Medicine,
University of Tokyo
1998-2000
Chief, Diabetes Branch, Lecturer, the Department of Metabolic Diseases, Graduate School of Medicine, The University
of Tokyo
2001-2003
Chief, Diabetes Branch, Associate Professor, the Department of Metabolic Diseases, Graduate School of Medicine, The
University of Tokyo
2003-present Chief, Diabetes Branch, Professor, the Department of Metabolic Diseases, Graduate School of Medicine, The
University of Tokyo
Advisor to the President of the University of Tokyo
2004-2006
2005-present Vice-director of Tokyo University Hospital
2008-present Chairman of the Board of Directors, The Japan Diabetes Society
2009-present Special Advisor to the President of the University of Tokyo
EDUCATION:
Tokyo University Medical School / Tokyo M.D. 1978 Medicine
TETLE:
Professor
WORKING ADDRESS:
The Department of Metabolic Diseases, Granduate School of Medicine, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
TEL: 81-3-5800-8815
FAX: 81-3-5800-9797
E-mail: [email protected]
BIRTH DATE:
August 29, 1952
Takashi Kadowaki, M.D., Ph.D.
特別講演 3／Special Lecture 3
12月2日
（木）
／Thursday, December 2
Index
89
Poster
The 8th Korea-Japan Joint Symposium on Vascular Biology
Selected Oral
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Y.I.A.
Presentation
ameliorating skeletal muscle insulin resistance.
and insulin delivery. Improving endothelial insulin signaling may serve as a novel therapeutic strategy for
causes skeletal muscle insulin resistance as a consequence of the impaired insulin-induced capillary recruitment
We conclude that a genetically and/or environmentally induced insulin signaling defect in the endothelial cells
Symposium
result, significantly restored glucose uptake by the skeletal muscle in HF diet-fed obese and ETIrs2KO mice.
the endothelial cells completely reversed the reduction in the capillary recruitment and insulin delivery, and as a
indeed recapitulated these phenotypes. Moreover, restoration of the insulin-induced eNOS phosphorylation in
and interstitial concentrations of insulin. Mice with the Irs2 deletion in the endothelial cells (ETIrs2KO mice)
How to write
Plenary Lecture
Nature Medicine
endothelial nitric oxide synthase (eNOS), resulting in a reduction of the insulin-induced capillary recruitment
cells, with reduction of insulin receptor substrate (Irs)2 expression and insulin-stimulated phosphorylation of
mice with decreased glucose uptake in the skeletal muscle, exhibit impaired insulin signaling in the endothelial
skeletal muscle are known to be delayed and impaired. Here, we demonstrate that high-fat (HF) diet-fed obese
In subjects with type 2 diabetes and obesity, insulin delivery and insulin-dependent glucose uptake by the
2. Role of endothelial cell insulin signaling in obesity-linked muscle insulin resistance
as well as strategies to increase AdipoR1 in muscle could be exercise-mimetics.
may play causal roles in mitochondrial dysfunction and insulin resistance seen in diabetes. Agonism of AdipoR1
Special Talk
bioenergetics stimulated with adiponectin in muscle cells. Decreased levels of adiponectin/AdipoR1 in obesity
activation via extracellular Ca2+ influx and AMPK/SIRT1, respectively, and subsequent mitochondrial
sensitivity and exercise endurance, and 2) be required for adiponectin-induced PGC-1α expression and
Special
Lecture
AdipoR1 appears to 1) regulate mitochondrial function and oxidative stress in muscle as well as insulin
muscle, we generated muscle-specific AdipoR1 knockout mice. Based up on the analysis of these mice,
Adiponectin is an anti-diabetic adipokine. To clarify the physiological roles of adiponectin receptors in skeletal
1. Role of adiponectin / adiponectin receptors in obesity-linked muscle insulin resistance
The Department of Diabetes and Metabolic Diseases, Granduate School of Medicine, The University of Tokyo
Takashi Kadowaki
Molecular mechanism of obesity-linked insulin resistance and
type2 diabetes
President
Lecture
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Program
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Area of Specialization:
Cardiovascular Development, Regeneration and Ageing, Mechanisms of Cardiac Hypertrophy and Heart Failure, Signal Transduction
Editorial Board:
Journal of Clinical Investigation, Circulation Research (Associate Editor),
Arteriosclerosis, Thrombosis, and Vascular Biology, Journal of Molecular
Cellular Cardiology, Circulation Journal, Japanese Heart Journal, Heart &
Vessel, Genes to Cells
Awards and Honors:
1987
Gold Medal for Erwin von Balz Preiz
1990
American College of Cardiology/Merck Award
1993
Louis N. Katz Basic Science Research Prizes for Young Investigators(Finalist), American Heart Association
2000
Sato Award of Japanese Circulation Society
2003
Award of Japanese Society of Molecular Medicine
Outstanding Investigator Prize of the International Society of Heart Research
2010
Presidentʼs Distinguished Lecture Award of the International Society of Heart Research
Professional Experience:
1982-1984 Resident in Internal Medicine, Tokyo University Hospital
1984-1989 Clinical and Research Fellow in Cardiology, Department of Medicine III,
University of Tokyo School of Medicine
1989-1993 Research Fellow and instructor, Molecular Medicine Unit and Cardiovascular Division, Beth
Israel Hospital and Harvard Medical School
1993-1998 Instructor in Medicine, Chief of Molecular Cardiology Division, Department of Medicine III, University of Tokyo
School of Medicine
1998-2000 Assistant Professor in Medicine, Department of Cardiovascular Medicine, University of Tokyo Graduate School of
Medicine
2000-2001 Associate Professor in Medicine, Department of Medicine III, Chiba University School of Medicine
2001-2010 Professor in Medicine, Chairman, Department of Cardiovascular Science and Medicine, Chiba University Graduate
School of Medicine
2005-2007 Vice president of Chiba University Hospital
2009Professor, Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine
2010Visiting Professor in Medicine, Department of Cardiovascular Science and Medicine, Chiba University Graduate School
of Medicine
Education:
1978-1982 M.D., Faculty of Medicine, University of Tokyo
1985-1989 Ph.D., Faculty of Medicine, University of Tokyo
Issei Komuro, M.D., Ph.D.
特別講演 4／Special Lecture 4
12月3日
（金）
／Friday, December 3 10：40〜11：30
Poster
Index
91
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
dysfunction with coronary artery disease.
therapy is also effective for cardiac ischemia and are starting the clinical study for patients with cardiac
angiogenic factors, thereby promoting neovascularization in ischemic tissues. We have recently found that this
Symposium
secret angiogenic factors sufficient for neovascularization but, instead, stimulate muscle cells to produce
and its efficacy was associated with increases in the plasma levels of angiogenic factors. Implanted cells do not
Cell therapy using peripheral mononuclear cells was very effective for ~70% of patients with limb ischemia,
patients with critical limb ischemia and treated ischemic limbs by implantation of peripheral mononuclear cells.
How to write
Plenary Lecture
Nature Medicine
mechanism by which cell therapy improves tissue ischemia remains obscure. We enrolled ~80 “no-option”
endothelial progenitor cells effectively promotes neovascularization in ischemic tissues. However, the
Accumulating evidence has suggested that implantation of bone marrow mononuclear cells which contain
failure.
the anti-angiogenic property of p53 has a crucial function in the transition from cardiac hypertrophy to heart
hypertrophy further and restored cardiac dysfunction under chronic pressure overload. These results suggest that
cardiac angiogenesis by introducing angiogenic factors or by inhibiting p53 accumulation developed
inhibited Hif-1 activity and thereby impaired cardiac angiogenesis and systolic function. Conversely, promoting
Special Talk
hypertrophy and induced systolic dysfunction. Sustained pressure overload induced an accumulation of p53 that
Hif-1 and angiogenic growth factors. Inhibition of angiogenesis prevented the development of cardiac
hypertrophy but cardiac function was impaired, and the vascular density was reduced with downregulation of
Special
Lecture
(Hif-1)-dependent induction of angiogenic factors. After 14 days, however, there was no further cardiac
dysfunction until 14 days and initially promoted vascular growth in the heart by hypoxia-inducible factor-1
which is produced by constricting transverse aorta of mice, induced cardiac hypertrophy without cardiac
Prolonged cardiac hypertrophy causes heart failure, but its mechanisms are largely unknown. Pressure overload,
Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine
Issei Komuro
Angiogenic therapy for heart failure
President
Lecture
Aⴊ▤↢‛කቇળKPFD
SP04
Program
14：40〜15：10
Actress
Naomi Kawashima
女優
川島 なおみ
Aⴊ▤↢‛කቇળKPFD
92
主な出演
TV
映画
舞台・ステージ
2010年10月
小説・エッセイ
CD
4月〜 CX系東海テレビ『エゴイスト〜egoist〜』レギュラー出演
2010年1月 『サヨナライツカ』イ・ジェハン監督
2010年8月 舞台『鬼灯町鬼灯通り三丁目（再演）
』
ミュージカル『FAME』
2009年6月23日 扶桑社『熟婚のすすめ』
2009年6月24日 『Actrice』リリース
受 賞
名誉ソムリエ 就任 / パラオ共和国 親善大使 就任
フランスワイン三大産地（シャンパーニュ・ボルドー・ブルゴーニュ）より騎士号を叙任
2005年サンテミリオン・ジェラード騎士号を叙任
1998年第35回 ゴールデン･アロー賞 放送賞
コマンドール騎士号を叙任
資 格
日本ソムリエ協会認定 ワインエキスパート
チーズプロフェショナル協会認定 チーズプロフェッショナル
全日本ソムリエ連盟認定 シガーアドバイザー
スキューバーダイビングライセンス
キューバシガー協会認定 名誉シガーマスター
2008年4月〜広島国際学院大学 現代社会学部 客員教授
経 歴
昭和35年、愛知県名古屋市出身。
青山学院大学在学中にデビューし、平成9年に主演した、テレビドラマ『失楽園』や、映画『鍵』などの作品で、大きな
話題を呼ぶ。
現在も、テレビ、映画、舞台など幅広く活躍している。
また、ワインエキスパート、名誉ソムリエの称号、茶道、料理、フランス語、クラシックバレエなど多彩な趣味も、魅
力に花を添えている。
SL01
スペシャルトーク 1／Special Talk 1
12月1日
（水）
／Wednesday, December 1
12：50〜13：20
Index
93
Poster
The 8th Korea-Japan Joint Symposium on Vascular Biology
Selected Oral
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Y.I.A.
Presentation
Aⴊ▤↢‛කቇળKPFD
2002
2003
2004
2005
2006
Symposium
2001
Born
Graduated from the Department of Political Science, Faculty of Law, Seikei University
Entered Kobe Steel, Ltd.
Executive Assistant to the Minister for Foreign Affairs
Elected for the first time to the House of Representatives
Director of the Committee on Health and Welfare, House of Representatives
Director of the Social Affairs Division, Liberal Democratic Party (LDP)
Deputy Chief Cabinet Secretary, Second Mori Cabinet
Deputy Chief Cabinet Secretary, Reshuffled Second Mori Cabinet
Deputy Chief Cabinet Secretary, Reshuffled Second Mori Cabinet
Deputy Chief Cabinet Secretary, First Koizumi Cabinet
Deputy Chief Cabinet Secretary, Reshuffled First Koizumi Cabinet
Secretary General, LDP
Acting Secretary General and Chairman of Reform Promotion Headquarters, LDP
Chief Cabinet Secretary, Reshuffled Third Koizumi Cabinet
90th Prime Minister of Japan
How to write
Plenary Lecture
Nature Medicine
2000
Career
1954
1977
1979
1982
1993
1999
2002年
2003年
2004年
2005年
2006年
2007年
Special Talk
2001年
Special
Lecture
2000年
安倍晋太郎、洋子夫妻の二男として生まれる
成蹊大学法学部政治学科卒業
株式会社神戸製鋼所入社
外務大臣秘書官
衆議院議員初当選
衆議院厚生委員会理事
自由民主党社会部会長
内閣官房副長官（第二次森内閣）
内閣官房副長官（第二次森改造内閣）
内閣官房副長官（第二次森改造内閣）
内閣官房副長官（第一次小泉内閣）
内閣官房副長官（第一次小泉改造内閣）
自由民主党幹事長
自由民主党幹事長代理・党改革推進本部長
内閣官房長官（第三次小泉改造内閣）
第９０代内閣総理大臣
内閣総理大臣を辞任
The 90th Prime Minister of Japan
Shinzo Abe
第90代内閣総理大臣
安倍 晋三
President
Lecture
略 歴
1954年
1977年
1979年
1982年
1993年
1999年
SL02
スペシャルトーク 2／Special Talk 2
12月3日
（金）
／Friday, December 3
Program
15：20〜16：20
Aⴊ▤↢‛කቇળKPFD
94
Juan Carlos López was born in Oaxaca, México, in 1967. He obtained his first degree on Biomedical
Research at the Universidad Nacional Autónoma de México, majoring in neuroscience. Juan Carlos got his
Ph.D. degree from Columbia University (New York) the laboratory of Eric Kandel, studying synaptic
plasticity in neuronal cultures. He then carried out postdoctoral work at the Instituto Cajal (Madrid),
studying presynaptic mechanisms of transmitter release. During this period, Juan Carlos wrote a book on
the neurobiology of memory (“El Telar de la Memoria”, Algar Editorial), with which he won the IV
European Award of Scientific Dissemination in 1998. Two years later, Juan Carlos left experimental
research to become Editor of Nature Reviews Neuroscience in London. In January 2004, he returned to
New York to become the Chief Editor of Nature Medicine.
In addition to his work at Nature Medicine, Juan Carlos has pursued his interest in translational research through other activities
within and outside Nature Publishing Group. So, he is heavily involved in running a project known as SciCafé̶a NPG activity that
aims to facilitate translational research by introducing young scientists with a proven track record of high-quality, commercially viable
research to venture capital firms in the San Francisco, Boston and London regions.
Juan Carlos is also a member of the Scientific Advisory Board and of the Board of Directors of Noscira, a Spanish biotechnology
company interested in neurodegeneration, a member of the Board of Directors of the Eureka Institute, an international initiative that
aims to promote translational research by fostering the education of MDs and PhDs interested in bridging the gap between bench and
bedside, and a member of the Board of BioBusiness.tv, an independent internet TV network for the life sciences that provides news
and analysis for investors and executives.
Editor of Nature Medicine
Juan Carlos López
How to write Nature Medicine
12月2日
（木）
／Thursday, December 2
How to write
Plenary Lecture
Nature Medicine
Symposium
Y.I.A.
Presentation
Selected Oral
Presentation
Poster
Index
Aⴊ▤↢‛කቇળKPFD
Special Talk
95
Special
Lecture
The 8th Korea-Japan Joint Symposium on Vascular Biology
President
Lecture
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Program
13：00〜13：50
Aⴊ▤↢‛කቇળKPFD
96
PL01
the regulation of angiogenesis will be described in the symposium.
active system for the termination of angiogenesis. The detailed role of VASH-1 and its homologue, VASH-2, for
preferentially in ECs at the site of angiogenesis. Our subsequent analysis indicates that VASH-1 constitutes an
The expression of VASH-1 is induced in ECs by the stimulation with VEGF, and VASH-1 protein is shown
or negative-feedback fashion. Our group isolated one of such inhibitors and named it vasohibin-1 (VASH-1).
process. Such inhibitors intrinsic to the vasculature are thought to regulate angiogenesis by an auto-regulatory
vessels. However recently, ECs themselves have been found to produce angiogenesis inhibitors during this
inhibitors is mostly extrinsic to the vasculature, and they may act as barriers to restrict the invasion of neo-
A number of endogenous angiogenesis inhibitors have been found in the body. The origin of these angiogenesis
Angiogenesis is thought to be regulated by the local balance between stimulators and inhibitors of this process.
been unknown whether or not there is an active system for the termination of angiogenesis.
relieved, that decreases the synthesis of angiogenesis stimulators and angiogenesis subsides. However, it has
they initiate and promote angiogenesis. When new vessels are formed and blood starts to flow, hypoxia is
thought to be hypoxia. Hypoxia increases the synthesis of certain angiogenesis stimulators such as VEGF, and
including age-related macular degeneration and diabetic retinopathy. The principal trigger of angiogenesis is
development, and wound healing; whereas pathologic conditions include various cancers and ocular diseases
physiological and pathological conditions. Physiological conditions include reproduction, body and organ
in the body, but has the capacity to form neo-vessels. This process, known as angiogenesis, occurs under both
The vascular system, a hierarchical network of arteries, capillaries and veins, is one of the most quiescent organs
Professional Experiences
Research Associate, First Department of Internal Medicine, Oita Medical University, Oita, Japan
1985-1987
1987-1989
Postdoctoral Fellow, Department of Cell Biology, New York University Medical Center, New York, USA
1989-1994
Assistant Professor, First Department of Internal Medicine, Oita Medical University, Oita, Japan
1994-Present Professor, Department of Vascular Biology, Institute of Development, Aging and Cancer, Tohoku
University, Sendai, Japan
Education
1978 M.D. Kobe University School of Medicine, Kobe, Japan
1987 Ph.D. Kyushu University Postgraduate School of Medicine, Fukuoka, Japan
Current Address
Department of Vascular Biology, Institute of Development, Aging and Cancer, Tohoku University
4-1, Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
Phone 81-22-717-8528
Fax
81-22-717-8533
E-mail [email protected]
Yasufumi Sato
University
Department of Vascular Biology, Institute of Development, Aging and Cancer, Tohoku
Yasufumi Sato
A system for the termination of angiogenesis
プレナリーレクチャー 1／Plenary Lecture 1
12月1日
（水）
／Wednesday, December 1
13：00〜13：50
Poster
Index
97
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Symposium
The concept of reprogramming of somatic cells has opened a new era in regenerative medicine. Transduction of
defined factors has successfully achieved pluripotency. However, during the generation process of induced
pluripotent stem (iPS) cells, genetic manipulation of certain factors may cause tumorigenicity, which limits
further application. We report that a single transfer of embryonic stem (ES) cell–derived proteins into primarily
cultured adult mouse fibroblasts, rather than repeated transfer or prolonged exposure to materials, can achieve
full reprogramming up to the pluripotent state without the forced expression of ectopic transgenes. During the
process, gene expression and epigenetic status were converted from somatic to ES-equivalent status. We verified
that protein-based reprogramming was neither by the contamination of protein donor ES cell nor by DNA/RNA
from donor ES cell. Protein-iPS cells were biologically and functionally very similar to ES cells and
differentiated into 3 germ layers in vitro. Furthermore, protein-iPS ells possessed the potentials of in vivo
differentiation (well differentiated teratoma formation) and development (chimeric mice generation and germline transmission through a tetraploid blastocyst complementation). Our results provide an alternative and safe
strategy for the reprogramming of somatic cells that can be used to facilitate pluripotent stem cell–based cell
therapy.
How to write
Plenary Lecture
Nature Medicine
RESEARCH INTERESTS
[1] stem cell biology
[2] gene therapy ; prevention of restenosis by gene therapy using in vivo vascular injury model
[3] vascular biology ; implication of GSK3b, beta-Catenin, Integrin-linked kinase, or Forkhead transcription factor in
vascular cells
[4] pathogenesis of atherosclerosis and coronary artery disease
Special Talk
BRIEF CHRONOLOGY of EMPLOYMENT
Mar. 1984 - Feb. 1985
Internship, Seoul National University Hospital, Seoul, Korea
Mar. 1985 - Feb. 1988
Residency in Internal Medicine, Seoul National University Hospital, Seoul, Korea
Mar. 1988 - Apr. 1991
Military Medical Officer, Captain, Department of Internal Medicine, Seoul District Army
General Hospital, Seoul, Korea
May. 1991 - Aug. 1992 Clinical Fellowship in Cardiology, Seoul National University Hospital, Seoul, Korea
Sep. 1992 - Feb. 1994
Visiting Researcher in Molecular Cardiology, 3rd Department of Internal Medicine, Faculty of
Medicine, University of Tokyo, Tokyo, Japan
Mar. 1994 - Feb. 1996 Instructor, Division of Cardiology, Department of Internal Medicine, Seoul National University
Hospital
Mar. 1996 - Feb. 2000 Assistant Professor, Division of Cardiology, Department of Internal Medicine, Seoul National
University Hospital
Jan. 2000 – Jan. 2002
Visiting scholar, Division of Cardiovascular Research, St. Elizabeth Medical Center, Boston,
MA, USA
Mar. 2000 – Mar. 2006 Associate Professor, Division of Cardiology, Department of Internal Medicine / Cardiovascular
Center Seoul National University Hospital
Apr. 2006 – present
Professor, Director of Cardiac Catheterization Laboratory & Coronary Intervention,
Department of Internal Medicine / Cardiovascular Center Seoul National University Hospital
Director of National Research Laboratory for Cardiovascular Stem Cell Seoul National
University.
Premedical Course, College of Liberal Arts & Science, Seoul National University
Seoul National University College of Medicine ( M.D. )
Postgraduate School, Seoul National University ( Master of Medical Science )
Postgraduate School, Seoul National University ( Ph.D. in Medical Science )
Special
Lecture
EDUCATION
Mar. 1978 - Feb. 1980
Mar. 1980 - Feb. 1984
Mar. 1985 - Feb. 1987
Mar. 1987 - Aug. 1994
Personal
Age and Sex ; 51 / Male
Date of birth ; March 26th, 1959
Place of birth ; Busan, Korea
Citizen ship Korea (Republic of Korea)
Languages Korean, Japanese, English
Hyo-Soo Kim, MD, PhD.
Institute for Cell Therapy, Seoul National University Hospital, Seoul, Korea
National Research Laboratory for Cardiovascular Stem Cell, Innovative Research
Hyo-Soo Kim
Induction of pluripotent stem cells from adult
somatic cells by protein-based reprogramming
without genetic manipulation
President
Lecture
Aⴊ▤↢‛කቇળKPFD
PL02
プレナリーレクチャー 2／Plenary Lecture 2
12月1日
（水）
／Wednesday, December 1
Program
10：00〜11：40
（日本語）
Aⴊ▤↢‛කቇળKPFD
98
Cellular senescence was originally defined as the finite replication of human somatic cells in culture. Various
stimuli, such as telomere dysfunction and oxidative stress, can activate p53-dependent DNA damage signals and
therefore induce cellular senescence. There is evidence that senescent cells promote changes related to aging or
age-related diseases. Aging is known to increase the prevalence of metabolic disorders like diabetes. Therefore,
we hypothesized that cellular aging might influence insulin resistance and accelerate the development of
diabetes. By using various genetic models, we show that p53 expression in adipose tissue is critically involved
in insulin resistance, which underlies age-related cardiovascular and metabolic disorders. Telomerase-deficient
mice with short telomeres developed insulin resistance when fed a high-calorie diet. The adipose tissue of these
mice showed senescence-like changes, such as increases in expression levels of p53 and production of proinflammatory cytokines. We also found that excessive calorie intake led to the accumulation of oxidative stress
in the adipose tissue of type 2 diabetic mice and promoted senescence-like changes. Inhibition of p53 activity
significantly ameliorated these senescence-like changes of adipose tissue and improved insulin resistance in
type 2 diabetic mice as well as in telomerase-deficient mice. Conversely, up-regulation of p53 in adipose tissue
caused an inflammatory response that led to insulin resistance. Our results demonstrate a previously
unappreciated role of adipose tissue p53 in the regulation of insulin resistance and suggest that cellular aging
signals in adipose tissue could be a novel target for the treatment of diabetes and diabetic vasculopathy.
千葉大学大学院医学研究院 循環病態医科学、2科学技術振興機構さきがけ
1
南野 徹1,2
SY01-02 Lifestyle-related disease and cellular aging signal network
Since our ancestor employed glucose as circulating sugar, mammals including human have been destined to
suffer diabetes and its vascular complications, because glucose and its derivatives possess glycating carbonyls.
An in vitro screen conducted in this lab revealed a class of senescent macromolecules - advanced glycation
endproducts (AGE) as the major environmental account for vascular cell changes characteristic of diabetes, and
the receptor for AGE (RAGE) as the primary cellular factor that responds to AGE. We then demonstrated with
gene-manipulated animals that RAGE overexpression accelerates, but RAGE deficiency ameliorates, the
development of diabetic nephropathy. Low molecular-weight heparin was found to be a RAGE antagonist, being
capable of reversing as well as preventing diabetic glomerulosclerosis. In collaboration with the group of
Professor Kobayashi, Osaka University, we determined the three-dimensional structure of human RAGE
protein, and have conducted structure-based virtual screen for RAGE antagonists. Further, through an analysis
of polysomal RNA from human vascular cells we identified a splice variant coding for a decoy form of RAGE
and termed it endogenous secretory RAGE (esRAGE). esRAGE was able to neutralize AGE actions on
endothelial cells. More recently, agents that elevate intracellular cyclic AMP were found to convert cell surface
RAGE to soluble RAGE through ectodomain shedding.
Diabetic complications apparently have been
exploiting RAGE but may be counteracted by soluble RAGE. Means that could halt abuse of the former device
or could induce the latter should help overcome this life- and QOL-threatening disease.
金沢大学大学院医学系研究科血管分子生物学、2金沢医科大学生化学
1
山本 博1、渡邉 琢夫1、山本 靖彦1、米倉 秀人2、棟居 聖一1、大江 和代1、杉原 崇大1、齋藤 英仁1、
本吉 創1、韓 冬1、ミャット ミンチュチュ1、アボウゼッド タレク1、大原 拓郎1、牧石 祥平1、
高辻 樹理1
SY01-01 Senescent Macromolecule AGE and Vascular Derangement in Diabetes
シンポジウム 1：血管の代謝・老化
Symposium 1：Vascular Metabolism and aging
12月1日
（水）
／Wednesday, December 1
Poster
Index
Aⴊ▤↢‛කቇળKPFD
Selected Oral
Presentation
99
Y.I.A.
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Symposium
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Recent major increases in obesity and related metabolic diseases, due to sedentary lifestyle and overnutrition in
developed and developing countries, are an exploding medical and social problem. These conditions are
associated with increased risk of cardiovascular disease, the leading cause of death. Thus, it is necessary to
understand the molecular basis underlying metabolic diseases and cardiovascular disease for developping
effective preventive and therapeutic approaches against cardiovascular disease. Recently, we found that adipose
tissue-derived angiopoietin-like protein 2 (Angptl2) activates an inflammatory cascade in endothelial cells and
induces chemotaxis of monocytes/macrophages in obesity, resulting in initiation and propagation of
inflammation within adipose tissues and obesity-related metabolic diseases (Cell Metabolism 2009). More
recently, we found that endothelial cells-derived Angptl2 contributes to chronic vascular inflammation, resulting
in vascular endothelial dysfunction. Furthermore, we found that elevated circulating levels of Angptl2 were
associated with vascular inflammatory status in centenarians, and were associated with increased carotid
atherosclerosis in the multivariate regression model adjusted for cardiovascular risk factors in the communityliving seniors aged 85 years or older. In this presentation, I would like to focus my talk on the roles of Angptl2
in cardiovascular disease, and discuss the possibility that Angptl2 could function as molecular targets for
prevention and treatment of cardiovascular disease.
How to write
Plenary Lecture
Nature Medicine
熊本大学大学院生命科学研究部（医学系）分子遺伝学分野
Special Talk
尾池 雄一
Special
Lecture
SY01-04 Roles of Angiopoietin-like protein 2 in cardiovascular diseases
President
Lecture
Angiogenesis is a phenomenon in which new blood vessels emerge from an existing vascular network in
developmental and pathological contexts, including age-related and metabolic diseases. Understanding of the
phenomenon sheds light on therapeutic development against the diseases. In the process of angiogenesis,
vascular endothelial cells (ECs) and mural cells coordinately behave and then form tree-like structures. We
currently give much interest to unveil a yet-to-be-defined question how vascular ECs move individually and/or
collectively during angiogenic morphogenesis. For the purpose, we have established a time-lapse imaging
system, in which the real-time movement of ECs is visualized with nuclear marking in in vitro angiogenesis and
the following computer-assisted analyses can characterize the ECs behavior quantitatively. Furthermore, in
order to monitor the behavior of individual cells on a multicellular structure, we have recently developed an
analyzing system, in which dynamics of cell polarity or filopodia formation can be examined. Through the
analyses, we have identified unexpected but intriguing patterns of multicellular movement, some of which
appears to reflect in vivo phenomenon in retinal angiogenesis. In this presentation, I will discuss cell-based
mechanisms underlying angiogenesis on the basis of our recent findings and the merit of our methodological
strategy “from ex vivo to in vivo” for the understanding of angiogenic morphogenesis.
東京大学大学院医学系研究科代謝生理化学
西山 功一、有馬 聡、栗原 裕基
time-lapse live imaging
SY01-03 A novel approach toward an understanding of angiogenesis using in vitro
Program
Aⴊ▤↢‛කቇળKPFD
100
SY02-2
SY02-1
10：00〜11：40
（日本語）
The epidermal growth factor (EGF) family is synthesized as a type I transmembrane protein (pro-form). The
cleavage at the juxtamembrane domain of proHB-EGF, a member of the EGF family, is mediated by a
disintegrin and metalloprotease (ADAM) and matrix metalloprotease (MMP) with various stimuli such as
ultraviolet, hypoxia, oxidative and osmotic stress, and growth factors and cytokines. We previously reported that
mice expressing an uncleavable proHB-EGF mutant (uc-proHB-EGF) develop severe cardiac dilation, which is
not observed in proHB-EGF null mice. However, the molecular mechanism underlying the tissue disturbance
that depends on insufficient proHB-EGF shedding remains unclear. We investigated the role of proHB-EGF
shedding in the survival of a rat cardiomyoblast cell line, H9c2. Overexpression of uc-proHB-EGF or abrogation
of proHB-EGF shedding by a metalloproteases inhibitor enhanced cell death both under normoxic and hypoxic
conditions, but the effect was considerably more under the hypoxic conditions. Further we found that uc-proHBEGF caused the activation of Caspase-3 and c-Jun N-terminal kinase as well as the accumulation of reactive
oxygen species. Based on these findings, we would like to discuss the role of C-terminal fragment signaling of
proHB-EGF evoked by its ectodomain shedding, and present unpublished data about the regulatory mechanism
of the ectodomain shedding.
愛媛大学プロテオ医学研究センター細胞増殖・腫瘍制御部門、2愛媛大学大学院医学系研究科生化学分子遺伝学分野
1
東山 繁樹1,2、井上 博文1,2
C-terminal fragment signaling of proHB-EGF for protection of cardiac cell
death
Heart failure (HF) is a major health problem in all countries including Japan, associated with high morbidity and
mortality. Our ultimate goal of HF treatment is to improve the prognosis of patients. Despite advances in both
pharmacological and nonpharmacological treatment of HF over the last 25 years, many patients still progress to
a stage of advanced HF with high mortality. There are two approaches to solve this crucial issue; the
development of better therapeutic strategies based on a novel insight into the pathophysiology of myocardial
remodeling and the improvement of quality of the standard treatment in routine clinical practice. Our basic
approach is to develop the therapeutic strategy of myocardial remodeling by regulating mitochondrial oxidative
stress. Chronic increases in oxygen radical production in the mitochondria can lead to a catastrophic cycle of
mitochondrial DNA damage as well as functional decline, further oxygen radical generation, and cellular
injury. These cellular events play an important role in the development and progression of maladaptive
myocardial remodeling and failure. Therefore, mitochondrial oxidative stress is a good therapeutic target for HF.
Our clinical approach is to develop the effective strategies of HF management for the patients encountered in
clinical practice (“real world”). Nearly half of all patients with HF have preserved ejection fraction (HFPEF).
Compared to those with reduced EF, patients with HFPEF are older, more women, and more likely to have
hypertension and atrial fibrillation. However, the effective treatment for HFPEF has not been established.
Further research based on both basic and clinical approaches are critically needed to establish the novel and
more effective treatment strategies for patients with HF.
北海道大学大学院医学研究科循環病態内科学
筒井 裕之
Pathophysiological Insight and Effective Treatment Strategies for Heart
Failure -From both basic and clinical science-
シンポジウム 2：心不全・冠動脈
Symposium 2：Heart Failure
12月1日
（水）
／Wednesday, December 1
Index
Aⴊ▤↢‛කቇળKPFD
Poster
101
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Symposium
The angiotensin II (AngII) type 1 (AT1) receptor is a seven transmembrane spanning G protein-coupled receptor
(GPCR), and the activation of AT 1 receptor is involved in regulating the pathophysiological process of
cardiovascular system. In principle, AT1 receptor is activated upon binding to AngII. However, in practice,
recent in vitro studies demonstrated that the AT1 receptor inherently shows spontaneous constitutive activity
even in the absence of AngII. In addition, we have demonstrated that the AT1 receptor can be activated by
mechanical stress independently of AngII through conformational switch of the receptor. To elucidate the role
of AngII-independent AT 1 receptor activation in the pathogenesis of cardiac remodeling, we generated
transgenic mice overexpressing AT 1 receptor under the control of 㱍-myosin heavy chain promoter in
angiotensinogen-knockout background (AT 1 Tg-AgtKO). In AT 1 Tg-AgtKO hearts, redistribution of G㱍 q11
subunits into cytosol and phosphorylation of ERKs were significantly increased, compared with
angiotensinogen-knockout mice hearts, suggesting that AT1 receptor is constitutively activated independently of
AngII. As a consequence, AT 1 Tg-AgtKO mice showed spontaneous systolic dysfunction and chamber
dilatation, accompanied by severe interstitial fibrosis. Cardiac remodeling in AT 1Tg-AgtKO mice was prevented
by treatment with candesartan, an inverse agonist of AT1 receptor, but not by its derivative candesartan-7H,
deficient of inverse agonism due to a lack of the carboxyl group at the benzimidazole ring. Our results
demonstrate that constitutive activity of AT 1 receptor under basal conditions contributes to the cardiac
remodeling even in the absence of AngII, when AT1 receptor is up-regulated in the heart.
How to write
Plenary Lecture
Nature Medicine
大阪大学大学院医学系研究科循環器内科学
Special Talk
赤澤 宏、小室 一成
Special
Lecture
Agonist-independent activation of angiotensin II receptor in the pathogenesis
of cardiac remodeling
Signals activated by increased hemodynamic overload to the heart finally reach nuclei of cardiac myocytes,
change patterns of gene expression and cause their maladaptive hypertrophy. Nuclear acetylation controlled by
histone deacetylases and an intrinsic histone acetyltransferase, p300, is a critical event during this process.
However, a pharmacological heart failure therapy that targets this nuclear pathway has yet to be established.
Curcumin is a cell-permeable natural compound, which possesses HAT inhibitory activity with specificity for
p300/CBP. Curcumin is a polyphenol that is responsible for the yellow color of Indian curry spice turmeric, and
commonly used as a health-food diet. Here, we demonstrate that this non-toxic dietary compound provides a
novel heart failure therapy that targets nuclear signaling pathway in cardiac myocytes. In cultured neonatal rat
cardiac myocytes, curcumin inhibited hypertrophy-induced acetylation of GATA4, one of hypertrophyresponsive transcription factors, and its DNA binding in cardiac myocytes. Curcumin also disrupted p300/
GATA4 complex and repressed agonist- and p300-induced hypertrophic responses in these cells. The acetylated
form of GATA4 and p300/GATA4 complex markedly increased in hypertensive hearts of adult rats. Then, we
examined the effects of curcumin in 2 different heart failure models in vivo: one model was hypertensive heart
disease in salt-sensitive Dahl rats, and the other model was myocardial infarction in rats. In both models,
curcumin prevented deterioration of systolic function as well as increase in wall thickness and hypertrophy of
cardiac myocytes. Moreover, combined treatment of curcumin and ACE inhibitor, lisinopril, had synergistic
effects on the improvement of the post-MI LV systolic function.
静岡県立大学薬学部分子病態学、2国立病院機構京都医療センター展開医療研究部
1
森本 達也1、長谷川 浩二2
Inhibition of p300 histone acetyltransferase activity by curcumin provides a
novel heart failure therapy
President
Lecture
SY02-4
SY02-3
Program
Aⴊ▤↢‛කቇળKPFD
102
SY02-5
We identified a Toll-like receptor family member ST2, with transmembrane (ST2L) and soluble (sST2)
isoforms, as an inducible gene product due to mechanical stress in myocardium. As we observed the increased
serum ST2 level in mouse model after acute myocardial infarction (AMI), we sought the clinical importance of
ST2 and surrounding mechanisms against acute myocardial stress. Serum ST2 levels at presentation predicted
30-days outcome of patients with AMI as well as mortality or transplantation of patients with severe nonischemic heart failure (HF). Several following studies confirmed that elevated baseline serum ST2 level
independently predicted mortality after 1-year, and better in combination with NT-proBNP, in patients with AMI
or AHF.
Recently, a novel protein IL-33 was identified as a functional ligand of ST2L, allowing exploration
of pathophysiological role of ST2 in myocardium. We found that IL-33 is a biomechanically-induced protein
predominantly from cardiac fibroblasts. IL-33 significantly antagonized angiotensin-II- and phenylephrinederived NF-kappaB activation and following cardiomyocyte hypertrophy. In a pressure overload model, ST2KO mice had more left ventricular hypertrophy, more fibrosis, more chamber dilation, reduced contraction and
impaired survival compared with WT littermates. Treatment with recombinant IL-33 protein markedly reduced
these changes and restored survival only in WT mice, not in ST2-KO littermates. Furthermore, IL-33 also
inhibited apoptosis, prevented cardiac remodeling and improved survival after AMI. In conclusion, ST2 is a
good biomarker to predict mortality after acute heart events, and composes a critical mechanically-activated
signaling system with IL-33 to protect myocardium against cardiac overload or stress.
国立循環器病研究センター心臓血管内科、2大阪大学大学院医学系研究科循環器内科学
1
真田 昌爾1,2、小室 一成2、北風 政史1
A new TLR family member that predicts cardiac mortality and comprises
stress-induced cardioprotective signaling
10：00〜11：40
（日本語）
Poster
Index
Aⴊ▤↢‛කቇળKPFD
Selected Oral
Presentation
103
Y.I.A.
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Symposium
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Erectile dysfunction (ED) is defined as an unstable or shorter duration of erection which greatly affects menʼs
QOL. Nitric oxides sustainably provided by the endothelium are essential to achieve erection. The main cause
of ED is the decrease in the production of nitric oxide in endothelium by oxidative stress. ED is a risk factor for
the life-style diseases since it indicates the endothelium dysfunction. Fortunately one can be aware of ED and
appreciate ED as a sentinel for the upcoming more serious vascular diseases. In this lecture, the topics of PDE5
inhibitors and the new application of shock waves to treat ED will be presented.
How to write
Plenary Lecture
Nature Medicine
帝京大学医学部泌尿器科
Special Talk
堀江 重郎
Special
Lecture
ED is a vascular disease that you can aware of.
Recent evidence suggests that acute coronary syndrome (ACS) results from plaque rupture in most of the
cases. Vulnerable plaques are characterized by thinning of fibrous cap, increased lipid content, decreased
smooth muscle cell content, and enhanced infiltration of inflammatory cells. However, the molecular mechanism
of plaque destabilization is not fully understood. Thus, there is no established method to predict and prevent
ACS.
We have been studying the pathogenesis of plaque progression and destabilization, using animal models and
clinical specimen. ApoE-deficient mice showed exaggerated atherosclerotic lesions with aging. Accumulation of
macrophages in adventitia was first detected prior to plaque formation. Proliferation of vasa vasorum was
observed only after atherosclerotic lesion formation. Local delivery of an angiogenic growth factor promoted
lesion formation with enhanced neovascularization in the adventitia.
Periadventitial fat is distributed ubiquitously around arteries. By using fat transplantation method, we found
that periadventitial fat may protect against neointimal formation after angioplasty under physiological conditions
and that inflammatory changes in the periadventitial fat may have a direct role in the pathogenesis of vascular
disease accelerated by obesity.
Moreover, by analyzing epicardial adipose tissue obtained during elective cardiac surgery, we found that
epicardial adipose tissues in patients with coronary artery disease display inflammatory phenotype, which may
play a role in the pathogenesis of coronary artery disease.
In this symposium, I will show our findings on the molecular mechanism of atherosclerosis. Moreover, I will
present our new data how angiogenesis is regulated by androgen and a phosphodiesterase-5 inhibitor.
徳島大学大学院ヘルスバイオサイエンス研究部循環器内科学
佐田 政隆
Atherosclerosis, Angiogenesis and Sex hormones
President
Lecture
SY03-2
SY03-1
シンポジウム 3：性ホルモン
（抗加齢医学会とのジョイントシンポジウム）
Symposium 3：Anti-aging
12月1日
（水）
／Wednesday, December 1
Program
Aⴊ▤↢‛කቇળKPFD
104
SY03-4
SY03-3
Background: Dehydroepiandrposterone (DHEA), a most abundantly produced adrenal steroid, plasma
concentration usually far exceeds that of any other sex steroid hormones and its concentration decreases with
aging. DHEA may have a protective effect against age-related illnesses including cardiovascular disease.
Recently, spectral analysis of intravascular ultrasound (IVUS) radiofrequency (IVUS-Virtual Histology [VH])
data demonstrated a potential to provide detailed quantitative information on plaque composition. We assessed
the hypothesis that DHEA was associated with coronary plaque volume and composition and verified the
difference of morphological distribution along coronary vessel walls.Methods and Results: Pre-intervention
IVUS-VH using a continuous pullback (0.5 mm/s) was performed prospectively in 111 coronary vessels in 111
male patients with stable effort angina pectoris (average 67 years old). Dense calcium volume increased as
DHEA decreased (Pearson r=-0.287,p<0.03). Fibro fatty volume also was prone to decrease as DHEA decreased
(Pearson r=0.056,p=0.06). However, there was no significant change in plaque volume or necrotic core volume
regardless of DHEA. In addition, either of the free testosterone or estrogen did not have any relation with the
plaque morphological distribution data estimated by IVUS-VH. Conclusions: The dense calcium volume has
close relation with DHEA. DHEA is well known to decrease with aging. The morphological distribution of
plaque is affected by old age. These findings may suggest that the stable plaque was more in old male patients
with low DHEA. On the other hand, the vulnerable plaque may be more in relative young male patients with
coronary risk factors.
佐賀大学循環器・腎臓内科
河野 宏明
Relationship between dehydroepiandrposterone and plaque morphological
distribution in male patients with angina pectoris
In contrast to the vasoprotective action of estrogen, androgen has been considered a bad guy for cardiovascular
disease. However, recent epidemiological studies have found that androgen deficiency is associated with higher
mortality largely due to cardiovascular disease in community-dwelling elderly men. Also, the results of smallscale studies support the vasoprotective effects of androgen replacement therapy in men with androgen
deficiency, although clinical trials are required to establish the efficacy of androgen replacement therapy. In
parallel with the progress in clinical studies of menʼs health, mechanisms of androgen action are being
investigated in the field of vascular medicine. At the symposium, I will talk about our recent clinical and basic
studies on vasoprotective action of androgen. These include: 1) Low plasma testosterone levels are associated
with cardiovascular disease risk, metabolic syndrome and endothelial dysfunction in Japanese men (Akishita M,
et al. Atherosclerosis 2010; Hypertens Res 2010; Hypertens Res 2007). 2) Testosterone rapidly induces NO
production via androgen receptor (AR)-dependent non-genomic activation of eNOS in vascular endothelial cells
via direct interaction of AR with p85㱍 (Yu J, et al. Endocrinology 2010). 3) AR-dependent transactivation of
growth arrest-specific gene 6 mediates inhibitory effects of testosterone on vascular smooth muscle cell
calcification (Son BK, et al. JBC 2010).
東京大学大学院医学系研究科加齢医学
秋下 雅弘
Vasoprotective action of androgen and the role of androgen receptor
15：10〜16：50
Index
Aⴊ▤↢‛කቇળKPFD
Poster
105
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Symposium
CD34+-derived progenitor cells (CD34PCs) play a dual role in protecting against and promoting atherosclerosis.
We investigated the role of vascular niches composed of endothelial cells (ECs) and smooth muscle cells
(SMCs) in CD34PC commitment and its underlying mechanisms. CD34PCs and subpopulations CD34+CD31+
and CD34+CD31- cells were isolated from fresh human peripheral blood by using immunomagnetic beads and
placed over ECs co-cultured with SMCs on a porous membrane or in 3-dimentional collagen gels.
CD34 + CD31 + cells exhibit higher adhesiveness and potential to differentiate into endothelial and
myelomonocytic cell lineages than CD34 + CD31 - cells. Flow cytometric analysis, scratched-wound,
bromodeoxyuridine incorporation, pseudo-tube formation, and foam cell formation assays demonstrated that
CD34 + CD31 + cells adhered on and transmigrated across ECs commit to mature ECs and macrophages,
respectively. SMC-co-culture induced adhesion and transmigration of CD34 + CD31 + cells through increased
expressions of endothelial intercellular adhesion molecule-1, delta like-4, and Jagged-1 and activations of their
counterreceptors 㱎2 integrin and Notch-1 in CD34+CD31+ cells; these two pathways play differential roles in
modulating CD34+CD31+ cell commitment. Femoral artery wire-injury and cell implantation assays in severe
combined immunodeficiency and apolipoprotein E-deficient mice confirmed the differential roles of adherent
and transmigrated CD34 +CD31+ cells in preventing and promoting atherosclerosis. Our findings demonstrate
the importance of vascular niches in directing CD34+CD31+ cell fate through opposite regulation by 㱎2 integrin
and Notch.
How to write
Plenary Lecture
Nature Medicine
National Health Research Institutes
Special Talk
Yu-Tsung Shih、Tung-Lin Yang、Mei-Chun Wang、○Jeng-Jiann Chiu
Special
Lecture
Vascular Niches Direct CD34+CD31+ Progenitor Cell Fate: Roles of β2
integrin and Notch
Background - Renal dysfunction is commonly accompanied by a worsening of atherosclerosis; however, the
underlying molecular mechanism is not fully understood. We examined the role played by soluble fms-like
tyrosine kinase-1 (sFlt-1), an endogenous antagonist of the proatherogenic cytokine placental growth factor
(PlGF), in the worsening of atherosclerosis in patients with renal dysfunction and in an animal model of renal
failure. Methods and Results In this study, 329 patients who received cardiac catheterization and 76 patients
who underwent renal biopsy were studied. Both plasma sFlt-1 levels and renal sFlt-1 mRNA expression were
positively correlated with estimated glomerular filtration rate (eGFR) (p＜0.01). PlGF/sFlt-1 ratio was
negatively correlated with eGFR (p＜0.01), while plasma PlGF levels were not affected by eGFR. PlGF/sFlt-1
ratio was significantly lower in patients with multi-vessel coronary artery disease (CAD) than patients with
single or no CAD. The reduction of circulating sFlt-1 and renal sFlt-1 mRNA levels were confirmed in 5/6
nephrectomized apoE-deficient mice, which developed experimental renal dysfunction. Atherosclerotic plaque
area and macrophage infiltration into the plaque were significantly higher in 5/6 nephrectomized apoE-deficient
mice than in control, but replacement therapy with recombinant sFlt-1 significantly reduced both the plaque
formation and the macrophage infiltration. Conclusions - The present study indicates that sFlt-1 is involved in
the worsening of atherosclerosis in chronic kidney disease.
The First Department of Internal Medicine, Nara Medical University, Kashihara, Japan
Yoshihiko Saito
Soluble Flt-1 Is Involved in the Mechanism for Aggravation of
Atherosclerosis in Chronic Kidney Disease
（English）
President
Lecture
SY04-2
SY04-1
Symposium 4：Atherosclerosis
Wednesday, December 1
Program
Aⴊ▤↢‛කቇળKPFD
106
SY04-4
SY04-3
Background: Acute coronary syndrome is a major cause of death in Japan and western countries. Current
medical treatments, however, are insufficient to prevent atherosclerotic plaque rupture. Here we developed a
novel nanoparticle-based drug delivery system (nano-DDS) that attains selective delivery of therapeutic agents
into peripheral monocytes/macrophages that play a key role in atherogenesis.
Methods & Results: Nano-DDS is composed of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) and
formed by emulsion-solvent-diffusion method to incorporate therapeutic agents. In atherosclerotic ApoE
deficient mice fed with high fat diet and infused with angiotensin II, intravenously injected FITC-NP was found
in the peripheral CD11b + monocytes 1-hour after injection, and then atherosclerotic lesions in the aortas and
brachiocephalic arteries. We prepared nanoparticles that incorporate peroxisome proliferator activated receptor㱏 (PPAR㱏) agonist pioglitazone (Pio-NP) that is known to modulate macrophage phenotypic polarity less
inflammatory. After 4-week treatment of Pio-NP, we evaluated atherosclerotic plaque destabilization (fibrous
cap thinning, macrophage infiltration) and rupture (disruption of fibrous cap, buried fibrous caps) in the
brachiocephalic arteries. Pio-NP (7 mg/kg i.v. weekly) but not oral pioglitazone (1 mg/kg daily) significantly
reduced plaque destabilization and rupture. Oral pioglitazone, but Pio-NP, significantly induced ENaC-alpha
and gamma in the kidney that underlies its clinical side effects including edema and heart failure.
Conclusion: Monocyte-selective nano-DDS potentiated therapeutic efficacy of pioglitazone to inhibit
macrophage-mediated inflammation leading to plaque destabilization and rupture, while reducing possibility of
side effects.
Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences
Tetsuya Matoba、Kensuke Egashira
Nanoparticle-Mediated Monocyte-Selective Drug Delivery System for
Treatment of Atherosclerotic Plaque Rupture
Background: HDL and apolipoprotein A-I (ApoA-I) take up cholesterol from macrophages and transport it back
to the liver. ApoA-I and ABCA1 generate HDL in the liver. We identified anti-atherogenic adiponectin (APN)
from adipocytes and noted a positive correlation between plasma HDL-C and APN concentrations. Plasma APN
is reduced in patients with metabolic syndrome. We showed APN increases the hepatic expression of ApoA-I
and ABCA1 and attenuates the secretion of ApoB100 from hepatocytes. To evaluate the molecular mechanism,
we tested whether COUP-TFII and LXR alpha are involved in this mechanism. Methods and Results: HepG2
cells were incubated in medium containing recombinant APN after incubation with siRNA or control RNA for
COUP-TFII or LXR alpha and mRNA levels of ApoA-I and ABCA1 were measured. APN up-regulated mRNA
and protein levels of ApoA-I and ABCA1. Expressions of COUP-TFII and LXR alpha were significantly
increased by APN, while these were significantly lower in APN-KO mice. Inhibition of COUP-TFII by siRNA
reduced APN-induced enhancement of ApoA-I but not ABCA1 in HepG2 cells. Serum APN level is inversely
correlated with serum TG level. APN down-regulated the mRNA and protein levels of ApoB100 in HepG2 cells,
resulting in attenuation of ApoB100 secretion. In APN-KO mice, serum ApoB100 and hepatic ApoB100 mRNA
expression were increased compared with WT mice. Reduction of COUP-TF2 after incubation with siRNA
enhanced APN-induced reduction of ApoB100 mRNA in HepG2 cells. Conclusion: APN enhances hepatic HDL
synthesis through COUP-TFII- and LXR alpha-dependent pathways. APN inhibits hepatic VLDL synthesis via
COUP-TFII-dependent pathway, which may explain the molecular mechanism of hypertiglyceridemia
associated with metabolic syndrome.
Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine
Shizuya Yamashita
Adiponectin Enhances Hepatic Synthesis of HDL and Inhibits Secretion of
VLDL
How to write
Plenary Lecture
Nature Medicine
Symposium
Y.I.A.
Presentation
Selected Oral
Presentation
Poster
Index
107
Special Talk
The 8th Korea-Japan Joint Symposium on Vascular Biology
Special
Lecture
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Emerging roles of adipose tissue in metabolic disorders has been extensively studied. Adipose tissue can directly
modulate vessel wall homeostasis by influencing the endothelial cells, smooth muscle cells, and leukocytes in
the vessel wall via secretion of cytokines and chemokines. Recent studies have shown that the pattern of gene
expression and secretory factors in visceral fat would be more atherogenic compared with that in subcutaneous
fat. However, a direct effect of visceral as well as subcutaneous adipose tissues on vascular inflammation is not
fully tested in vivo. We examined the direct effect of adipose tissue on vascular inflammation by intravital
microscopic analysis of the femoral artery after adipose tissue transplantation. Methods and Results
Subcutaneous (SQ) or visceral (VIS) adipose tissue harvested from C57/BL6 mice was transplanted to the
perivascular area of the femoral artery of recipient C57/BL6 mice. A quantitative intravital microscopic analysis
was performed on the artery. The number of adherent leukocyte was increased after adipose tissue
transplantation in a time-dependent manner (1day to 7days). Visceral (VIS) adipose tissue transplantation
induced significantly more leukocyte accumulation than Subcutaneous (SQ) adipose tissue at 7 days after
transplantation. Simultaneous flow cytometry showed the activation of peripheral neutrophils and monocytes
after adipose tissue transplantation. Conclusion These data indicate that adipose tissue directly stimulates
athero-prone vascular inflammation.
Department of Life Sciences and Medical Ethics, Tokyo Medical and Dental University
Masayuki Yoshida
Intravital microscopy reveals a potential role of adipose tissue in vascular
inflammation
President
Lecture
Aⴊ▤↢‛කቇળKPFD
SY04-5
Program
Aⴊ▤↢‛කቇળKPFD
108
SY05-2
SY05-1
（English）
Patterning of new vascular structure is a coordinated multi-step process that involves sprouting and
morphogenesis of endothelial cells (ECs) and requires the formation of asymmetric EC phenotypes and their
dynamic interconversion. These processes are precisely controlled by a large number of genes and specific gene
expression within a developing vessel is indispensable for establishing functional vascular network. Recently,
we have identified several genes, which are potentially involved in regulating angiogenesis and vascular
patterning, by employing in vitro endothelial cell differentiation model and microarray-based gene expression
analysis. Interestingly, biochemical and genetic studies revealed that Wnt regulators, Dickkopf-1 and -2 (DKK1 and -2), play distinct roles in neovascularization, with their temporal and reciprocal expression during EC
proliferation and morphogenesis. We also found that LDB2, a LIM domain binding (Ldb) protein 2, is a novel
regulator of vascular sprouting and morphogenesis which controls the expression of DLL4 in ECs. Thus, our
findings provide new insight for controlling new vessel formation and offer a new opportunity for treating
angiogenesis-dependent vascular diseases.
Yonsei University
Hong-Ryul Park、Seong Sik Noh、Hyun-Jung Choi、Young-Guen Kwon
Novel Regulators of Angiogenesis and Vascular Patterning
Blood vessels in the tumor environment are structurally and functionally abnormal such as lack of mural cell
(MC) adhesion to endothelial cells (ECs), leakiness by lose cell-to-cell contact of ECs and fenestration, and so
on. These abnormal phenotypes lead to interstitial hypertension, resulted in interference with the delivery of
therapeutics to solid tumors and continuous hypoxia leading to angiogenesis. Therefore, idea of normalization
of blood vessels in the tumor environment has emerged to overcome these issues. We recently reported that
apelin, a ligand for GPCR expressed on ECs, is upregulated upon stimulation with angiopoietin-1 on ECs.
Apelin induced enlargement of blood vessels (EMBO J 2008) and stabilization of VE-cadherin (Blood 2010) for
functional recovery from tissue ischemia, suggesting that apelin is involved in maturation process of blood
vessels. Here we show that apelin induces normalization of immature blood vessels in tumor environment and
injection of activated dendritic cells with apelin effectively induced natural killer T cell infiltration into tumor,
resulted in suppression of tumor growth. This suggests that control of permeability by the normalization of
blood vessels is indeed important for cancer therapy. However, we found that cancer stem cells are located in
the perivascular region in the tumor edge (Cancer Research 2010). Those blood vessels for cancer stem cell
niche are usually maturated in the tumor environment but cancer stem cells show resistance against several anticancer therapies, suggesting that merely normalization is not enough for total tumor cell kill. Here, we propose
the sequential anti-angiogenic therapies, normalization and destabilization of blood vessels, for total tumor cell
kill including cancer stem cells.
Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University
Nobuyuki Takakura
The impact of blood vessel maturation on tumor growth
Symposium 5：Angiogenesis / Lymphangiogenesis
Wednesday, December 1 15：10〜16：50
Index
Aⴊ▤↢‛කቇળKPFD
Poster
109
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Symposium
Protein kinases have been shown to play important roles in vascular development and also implicated in various
diseases. We have been trying to understand the role and mechanism of receptor tyrosine kinases and the
downstream mediator Akt in angiogenic and lymphangiogenic vessel growth. Several genetically modified
mouse models was employed in this study, targeting VEGFR-3 or its downstream mediator Akt, including: 1) To
characterize VEGFR-3 function in angiogenesis and lymphangiogenesis, we used two genetically modified
mouse models, one targeting the coding region for the ligand-binding domain (Vegfr3LBD) or the other
targeting the tyrosine kinase domain with an inactivation point mutation (Vegfr3TKmut). We found that
VEGFR-3 ligand-binding and kinase activity are required for lymphangiogenesis but not for angiogenesis. 2) To
analyse Akt mediated signaling in vascular development, we used mice null for Akt1, Akt2 or Akt3, or
compound Akt knockout mice. We showed that Akt is required for lymphatic network formation, remodeling
and valve development, and that in spite of the compensatory roles of other Akt isoforms, Akt1 is more critically
required in the process. Specific details will be discussed in the presentation.
How to write
Plenary Lecture
Nature Medicine
Laboratory of Vascular and Cancer Biology, Model Animal Research Institute, Nanjing University
Special Talk
Yulong He
Special
Lecture
Role of VEGFR-3 and Akt mediated pathway in the regulation of angiogenesis
and lymphangiogenesis
Macrophages are white blood cells within tissues, produced by the differentiation of monocytes via the Colony
stimulating factor 1 (CSF-1) signaling. Although their roles in the immune response have been well studied,
those in angiogenesis/lymphangiogenesis are largely unclear. In this study, we used CSF-1-mutant (CSF-1 op/op)
mice to demonstrate that lack of macrophages results in developmental defects in vascular and lymphatic
remodeling. In ischemic retinopathy, one of the neovascular disease models in the retina, lack of macrophages
in CSF-1 op/op mice resulted in profound reduction in pathological neovascularization, but no alteration in the
regeneration of normal vasculature. In mouse osteosarcoma model, CSF-1 inhibition effectively suppressed
tumor angiogenesis and lymphangiogenesis by suppressing the remodeling of vascular extracellular matrices. In
contrast to VEGF blockade, interruption of M-CSF inhibition did not promote rapid vascular regrowth.
Continuous CSF-1 inhibition did not affect healthy vascular and lymphatic systems outside tumors. These
results suggest macrophage-targeted therapy is an ideal strategy for treating ocular neovascular diseases and
cancer, which may be an alternative to VEGF blockade.
2
Department of Integrated Medical Research, Keio University, Tokyo, Japan
Department of Plastic Surgery, Keio University
1
Yoshiaki Kubota1,2
Role of macrophages in angiogenesis and lymphangiogenesis
President
Lecture
SY05-4
SY05-3
Program
Aⴊ▤↢‛කቇળKPFD
110
SY05-5
Pathologic lymphangiogenesis is a multistep process that potentially requires the decrease of cell-matrix contact
in lymphatic endothelium. However, it remains unclear whether VEGF family members such as VEGF-A might
alter the cell-matrix contact, initiating lymphangiogenesis in the skin. LYVE-1, a type I transmembrane protein,
is specifically expressed by lymphatic vessels. Although LYVE-1 is known as a cell surface receptor for
hyaluronan, its potential role in lymphangiogenesis remains unclear. Therefore, to test the hypothesis whether
VEGF-A might alter LYVE-1 during lymphangiogenesis, we initially investigated the lymphatic vessels in K14promoter-driven VEGF-A transgenic mice. Targeted overexpression of VEGF-A induced dermal leakage of
colloidal carbon from lymphatic capillaries that developed among red plaques resembling psoriasis.
Immunofluorescence analysis of LYVE-1 further showed that VEGF-A-expressing plaques attenuated LYVE-1
stains in the lymphatic vessels. Therefore, next to investigate whether VEGF-A mediates the biochemical
process of LYVE-1, we subjected cultured human dermal LEC to adenoviral transduction to induce alkaline
phosphatase (AP) that is fused to LYVE-1 by the extracellular domain. VEGF-A promoted marked release of a
soluble form of AP-conjugated LYVE-1 in conditioned media, indicating that VEGF-A induces the ectodomain
shedding of LYVE-1. Furthermore, Western blot analyses showed that LYVE-1 shedding is mediated through
the activation of VEGFR-2 and ERK. Moreover, siRNA technique identified ADAM17 as a responsible MMP
for the LYVE-1 shedding in cultured LEC. Together, these results indicate, for the first time, that pathologic
lymphangiogenesis may be initiated by the enzymatic digestion of extracellular matrix receptors such as LYVE1.
2
The Department of Dermatology, Ehime Graduate School of Medicine
Department of Cell Growth and Tumor Regulation, Ehime Proteo-Medicine Research Center, Ehime University
1
Satoshi Hirakawa1,2、Shigeki Higashimaya2
Ectodomain shedding of LYVE-1: VEGF-A promotes the initial step of
pathological lymphangiogenesis
15：10〜16：50
Poster
Index
Aⴊ▤↢‛කቇળKPFD
Selected Oral
Presentation
111
Y.I.A.
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Symposium
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Cardiovascular functions, including blood pressure and vascular functions, exhibit diurnal oscillation. Circadian
variations have been clearly shown in the occurrence of cardiovascular events such as acute myocardial
infarction. Circadian rhythm strongly influences human biology and pathology. Recently, the molecular
mechanisms underlying these circadian processes have been elucidated. The biological clock is composed of
transcriptional-translational feedback loops. The clock genes are expressed almost everywhere throughout the
body in a circadian fashion. In contrast to the central clock in the suprachiasmatic nucleus (SCN), the clock in
each tissue or cell is designated as a peripheral clock. It is now accepted that peripheral clocks have their own
roles specific to each peripheral organ by regulating the expression of clock-controlled genes. However, little
was known about how the peripheral clock in the vasculature contributes to the process of cardiovascular
disorders. The biological clock allows each organ or cell to anticipate and prepare for changes in external
stimuli. Recent evidence obtained using genetically engineered mice with disrupted circadian rhythm showed a
novel function of the internal clock in the pathogenesis of endothelial dysfunction, hypertension and hemostasis.
Loss of synchronization between the central and peripheral clock also contributes to the pathogenesis of
cardiovascular diseases since restoration of clock homeostasis could prevent disease progression. Identification
of clock-controlled genes in each organ, as well as discovery of tools to manipulate the phase of each biological
clock, will be of great help in establishing a novel chronotherapeutic approach to the prevention and treatment
of cardiovascular disorders.
How to write
Plenary Lecture
Nature Medicine
長崎大学大学院循環病態制御内科学
Special Talk
前村 浩二
Special
Lecture
The Role of Biological Clock in Cardiovascular Diseases
Cardiovascular disease is a major consideration in the patients with diabetes and chronic kidney disease (CKD).
Vascular calcification is an important problem among these patients, and contributes to the increased risk of
cardiovascular events by a variety of mechanism, including an increase in arterial stiffness by medial
calcification or an increase in plaque vulnerability by a specific type of atherosclerotic calcification. Valvular
calcification is highly prevalent in patients with vascular calcification, and both type of calcification share a
mechanism in common. Increasing evidence demonstrates that vascular smooth muscle cells (SMC) undergo
osteogenic differentiation through the mechanisms involving key osteogenic transcription factors such as Msx2
and Runx2. We have long been interested in the role of Notch signaling pathway in SMC differentiation. We
have recently demonstrated that Notch signaling induces osteogenic differentiation and mineralization of SMC.
Our subsequent analysis revealed that Notch confers on the Msx2 gene the responsiveness to BMP2, a member
of TGF-㱎 superfamily that is essential in bone formation. BMP2 and Notch signaling synergistically induced
Msx2 gene expression, and hence robustly augmented Notch1-induced vascular calcification. In addition,
Receptor for Advanced Glycation End products (RAGE) provokes inflammatory response, and hence RAGEligand interaction plays a major role in the pathogenesis of vascular disease in diabetics and CKD patients. In
this symposium, the role of FGF23, a novel potential mediator of cardio-renal syndrom e, in the vascular
calcification will also be discussed.
群馬大学大学院医学系研究科臓器病態内科学
倉林 正彦
血管石灰化の分子メカニズム
（日本語）
President
Lecture
SY06-2
SY06-1
シンポジウム 6：血管細胞の転写調節
Symposium 6：Transcriptional Regulation
12月1日
（水）
／Wednesday, December 1
Program
Aⴊ▤↢‛කቇળKPFD
112
SY06-4
SY06-3
In subject with type 2 diabetes and obesity, insulin (INS) delivery and INS-dependent glucose uptake by the
skeletal muscle (SM) are known to be delay and impaired. However, the mechanism how INS delivery into the
SM interstitium is regulated remains to be elucidated. Here, we demonstrate that impaired INS signaling in the
endothelial cells reduces INS-induced glucose uptake by the SM. Endothelial-cell-specific Irs2-knockout
(ETIrs2KO) mice showed reduced INS-stimulated Akt and eNOS phosphorylation in endothelial cells.
ETIrs2KO mice also showed diminished INS-induced capillary recruitment and increase of interstitial
concentrations of INS, and consequently, impaired glucose uptake by the SM. Improvement of INS-mediated
eNOS activation ameliorated the aforementioned diminishment of capillary recruitment and increase of
interstitial concentrations of INS in the SM, resulting in improvement of the glucose uptake by the SM. High-fat
-diet-fed obese (HF) mice also showed reduced INS-stimulated phosphorylation of Akt and eNOS. These mice
also showed impaired glucose uptake by the SM, associated with diminished INS-induced capillary recruitment
and increase of interstitial concentrations of INS in the SM. Restoration of INS-mediated eNOS activation in the
endothelial cells of the HF mice also restored the INS-induced capillary recruitment and increase of interstitial
concentrations of INS, resulting in amelioration of glucose uptake by the SM. Taken together, it is postulated
that a genetically and/or environmentally induced INS signaling defect in the endothelial cells might be
associated with a decrease in INS-induced capillary recruitment and increase of interstitial concentrations of
INS in the SM, consequently resulting in SM INS resistance.
東京大学大学院医学系研究科糖尿病・代謝内科
窪田 直人、窪田 哲也、門脇 孝
Impaired insulin signaling in the endothelial cells reduces insulin-induced
glucose uptake by the skeletal muscle
Endothelial cell activation and dysfunction underlie many vascular disorders,including tumor growth and
inflammation. Endothelial cell activation, in turn, is mediated primarily at the level of gene transcription. We
previously reported that VEGF induced Down syndrome critical region 1 short variant (DSCR-1s) expression,
which negatively feeds back to attenuate endothelial cell activation. Here, to characterize the role of the
promoter that drives DSCR-1s expression in mediating inducible expression in vivo, we targeted a DNA
construct containing DSCR-1s promoter-lacZ to the Hprt locus of mice. Systemic administration of VEGF and
LPS resulted in NFAT-and GATA-dependent DSCR-1s promoter activation. The promoter was similarly induced
in the endothelium of tumor xenografts. Besides NFAT, GATA2 is also well recognized as a key transcription
factor of cell type specificity and differentiation. To characterize the GATA2-regulation in endothelium, here we
show, comparative chromatin immunoprecipitation with deep-sequencing (ChIP-seq) to determine genome wide
occupancy of GATA2 in endothelial cells and erythroid cells, and compared the occupancy to the respective
gene expression profile in each cell type. By using the ChIP-seq with epigenetic histone-mark and chromatin
conformation capture assays in endothelial cells; we elucidated the mechanistic regulation of endothelial
specific GATA2-mediated endomucin gene expression. Abrogation of GATA2 in endothelium demonstrated not
only a reduction of endothelial specific markers, but also induction of the mesenchymal transition promoting
gene expression. In this session, we provide new insights into the correlation of endothelial expressed GATA2
binding, epigenetical modification, and the determination of endothelial cell specificity.
東京大学先端科学技術研究センター
南 敬
Tissue specific gene expression, insights from the genome-wide and
epigenetical analysis in vitro and in vivo
How to write
Plenary Lecture
Nature Medicine
Symposium
Y.I.A.
Presentation
Selected Oral
Presentation
Poster
Index
113
Special Talk
The 8th Korea-Japan Joint Symposium on Vascular Biology
Special
Lecture
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
The Rho/Rho-associated coiled-coil forming kinases (ROCK1 and ROCK2) are important regulators of the actin
cytoskeleton. We have recently shown that ROCK activity is increased during the onset of ischemic stroke and
that inhibition of the actin cytoskeleton by the ROCK inhibitor, fasudil, decreased the severity of focal cerebral
ischemia, in part, through the upregulation of endothelial nitric oxide synthase (eNOS). Using RNA affinity
chromatography with 3ʼUTR of eNOS mRNA followed by MALDI-TOF-MS analysis, we have identified
eukaryotic elongation factor 1 alpha 1 (eEF1A1) as an actin-binding protein, which directly binds to eNOS
mRNA and regulates eNOS mRNA stability. Overexpression of eEF1A1 decreased, and knock-down of eEF1A1
by siRNA increased, eNOS expression. Protein kinase assays using eEF1A1 with purified ROCK1 and ROCK2
showed that eEF1A1 is strongly phosphorylated by ROCK2, but not ROCK1, and that eEF1A1 phosphorylation
by ROCK2 is required for binding to and destabilization of eNOS mRNA. Indeed, treatment of human
endothelial cells (EC) with fasudil decreased eEF1A1 phosphorylation, but not expression. This was associated
with increased eNOS mRNA stability and expression, and NO production. Furthermore, compared to that of
WT and ROCK1+/- mice, eNOS mRNA stability and expression were increased in ECs from ROCK2+/- mice
and endothelium-dependent relaxation was enhanced in both ROCK2+/- and EC-specific ROCK2 KO (ECROCK2-/-) mice. These results correlated with decreased cerebral infarct size and neurological deficits in both
ROCK2+/- and EC-ROCK2-/- mice subjected to transient focal cerebral ischemia. When ROCK2+/- mice were
place on eNOS-/- background (double mutant ROCK2+/-/eNOS-/- mice), the neuroprotective effects observed
in ROCK2+/- mice were abolished.
1
東京大学医学部循環器内科、2広島大学心臓血管生理医学講座、3ハーバード大学ブリガムアンドウィメンズ病院ヴァスキュラー
メディシンリサーチ、4三重大学循環器腎臓内科学、5ニュージャージー大学心臓血管研究所
廣井 透雄1、野間 玄督2、金 何3、李 予日斤3、岡本 隆二4、神崎 綱1、孫 何5、梁 何3
Deletion of ROCK2 Increased eNOS Expression and Neuroprotection
byInhibition of eEF1A1 Phosphorylation
President
Lecture
Aⴊ▤↢‛කቇળKPFD
SY06-5
Program
9：00〜10：40
Aⴊ▤↢‛කቇળKPFD
114
SY07-2
SY07-1
（English）
The Angiopoietin (Ang)-Tie2 pathway is almost contributory to the VEGF axis, yet its participation is
indispensable for the formation of blood vessels and maintenance of endothelial integrity. The cardinal feature
of angiopoietin-1 (Ang1)-induced vascular remodeling in vivo is the enlargement of blood vessels by promotion
of circumferential proliferation of endothelial cells in the normal tissues. Ang1 is also a strong angiogenic factor
in the pathologic conditions. However, unlike VEGF-A, Ang1 produces a non-leaky and non-inflammatory
neovascularization; so called “healthy angiogenesis.” Underlying mechanisms how Ang1 produces healthy
angiogenesis are currently being defined. Ang1-induced Tie2 mobilization to the cell-to-cell contact junctional
region (we nominated “endothelial tightening”) could be a part of the mechanism. In fact, Ang1 is a strong antiinflammatory agent mediated through reductions in productions of inflammatory cytokines and adhesion
molecules in the endothelial cells. Taking such advantages of Ang1ʼs actions, regeneration abilities of Ang1 in
ischemic diseases, delayed skin-wound healing, artherosclerotic erectile dysfunction, delayed bone healing,
retinal vasculopathies and avascular necrosis in the hip joint have been tested. Moreover, priming of endothelial
precursor cells or stromal vascular fraction with Ang1 enhances homing or sprouting for promotion of
neovascularization. I will discuss the current progresses and issues regarding potential use of Ang1 as a
regenerative therapeutic molecule.
Graduate School of Medical Science and Engineering, KAIST, Deajeon, Korea
Gou Young Koh
Angiopoietin-1 is a Regeneration Factor through Healthy Angiogenesis
Regenerative potential of stem and progenitor cells has been under intense investigation. Endothelial progenitor
cells (EPCs) have been isolated from the peripheral blood of adult individuals, cultured in-vitro and committed
into an endothelial lineage in a specific condition. Based on initial EPC biology studies, researchers then
pursued the notion of “therapeutic vasculogenesis”, whereby systemic delivery of EPCs may augment
neovascularization. Following preclinical studies, a phase I/ IIa clinical trial regarding transplantation of
autologous CD34+ cells, the EPC-enriched fraction, was performed in no-option patients with atherosclerotic
peripheral artery disease (PAD) or Buergerʼs disease representing critical limb ischemia (CLI) for 5 years in our
institutes. Our collaborators in USA performed the Phase III trial of transplantation into chronic severe
myocardial ischemia (CMI) patients for 3 years. The outcomes of both prospective clinical studies indicate
safety and feasibility of CD34+ cell therapy in patients with CLI and CMI. However, despite lots of research
development on EPC biology for 10 years following the isolation of EPC, EPC biology is still controversial
among researchers without the definitive concept of EPC identification and differentiation hierarchy. I introduce
the development of basic and translational researches regarding “What is EPC?” and “How do we use EPCs?” in
these days. Furthermore, recent basic and preclinical studies indicate optional role of EPCs for organ
reconstruction, including the tissue preparation for regeneration and trigger signals for organ differentiation.
This issue will be discussed for the future direction of regenerative medicine.
2
Regenerative Medicine, Tokai University School of Medicine, Isehara, Kanagawa
Institute of Biomedical Research and Innovation
1
Takayuki Asahara1,2
Stem Cell Biology for Vascular Medicine
Symposium 7：Regeneration
Thursday, December 2
Poster
Index
115
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
The large number of patients suffering from heart failure have only poor prognosis, who are not eligible for
heart transplantation. Cardiac cell therapy is generating a great interest as a means of restoring a loss of cardiac
function through cardiomyocyte regeneration. We generated human cardiac stem cells (hCSCs) obtained from
endomyocardial biopsy tissues by the identical technique as clonally cell isolation from “cardiosphere”. hCSCs
expressing Es-marker genes and mesenchymal properties exhibit a highly potential of cardiomyocyte
regeneration. To evaluate the effect of hCSCs transplantation, we conducted preclinical trials that chronically
myocardial ischemic pigs randomly received placebo or bFGF hydrogel-sheet implantation combined with or
without hCSCs. As a result, only transplantation of hCSCs with bFGF improved the loss of cardiac function by
contributing to cardiomyocyte regeneration. Now, we just started Phase I trial to the patients (N=6) with low
LVEF because of ischemic cardiomyopathy through this biotherapy concomitant with CABG (ALCADIA trial)
from September, 2009. A first case with reduced LVEF (22%) have succeeded in this biotherapy using
autologous hCSCs (31.5 million), and the second case was conducted at August 24, 2010. Surprisingly, cardiac
function in first patient was restored 10% of LVEF without adverse event at 4 week after transplantation. Our
findings suggest that the transplantation of hCSCs with bFGF reconstruct post-ischemic environment, and this
novel biotherapy may have a potential to lead injured myocardium to functional repair. After we confirm the
efficacy of this integrated-strategy in Phase II trial (N=40), we will move forward to spread therapeutic
indication for patients suffered from severely heart failure.
Symposium
Aⴊ▤↢‛කቇળKPFD
The Department of Cardiovasular Regeneration and Innovation, Asahikawa Medical University
Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine
3
The Department of Cardiovascular Medicine, The Cerebral and Cardiovascular Research Institute
4
Department of Biomaterials, Institute for Frontier Medical Sciences,Kyoto University
5
Department of Cardiovascular Surgery, Kyoto Prefectural University of Medicine
2
How to write
Plenary Lecture
Nature Medicine
1
Special Talk
Naofumi Takehara1、Katsuya Amano2、Yoshiaki Tsutsumi3、Tomosaburo Takahashi2、
Satoaki Matoba2、Yasuhiko Tabata4、Hitoshi Yaku5、Hiroaki Matsubara2
Special
Lecture
Surgical hybrid cell therapy using Autologous Human Cardiac Stem Cell with
control-release of bFGF ; the ALCADIA trial
The reprogramming of fibroblasts to induced pluripotent stem (iPS) cells raises the possibility that a somatic
cell could be reprogrammed to an alternative differentiated fate without first becoming a stem/progenitor cell. A
large pool of fibroblasts exists in the post-natal heart, yet no single “master regulator” of direct cardiac
reprogramming has been identified. Here, we report that a combination of three developmental transcription
factors rapidly and efficiently reprogrammed post-natal cardiac or tail-tip fibroblasts directly into differentiated
cardiomyocyte-like cells. Induced cardiomyocytes expressed cardiac-specific markers, had a global gene
expression profile similar to cardiomyocytes, and contracted spontaneously. Fibroblasts transplanted into mouse
hearts one day after transduction of the three factors also differentiated into cardiomyocyte-like cells. These
findings demonstrate that functional cardiomyocytes can be directly reprogrammed from differentiated somatic
cells by defined factors. Reprogramming of endogenous or explanted fibroblasts might provide a source of
cardiomyocytes for regenerative approaches.
Department of Clinical and Molecular Cardiovascular Research, Keio University School of Medicine
Masaki Ieda
Direct Reprogramming of Fibroblasts into Functional Cardiomyocytes by
Defined Factors
President
Lecture
SY07-4
SY07-3
Program
Aⴊ▤↢‛කቇળKPFD
116
SY07-5
Recently cell therapy was introduced to clinical setting and proved safety and feasibility, but results were
inadequate for regeneration of heart failure. We developed cell sheet technology and introduced this to the
treatment of severely damaged myocardium.We implanted myoblast sheets to the impaired heart in small and
large animal models. In a series of pre-clinical trial, we proved that myoblast sheets could regenerate the
impaired heart mainly by paracrine effect. Evidenced by these pre-clinical trials, we applied myoblast sheets to
DCM patient receiving LVAD and showed the recovery from LVAD. To improve the effectiveness of cell sheet,
the delivery of many cells to the impaired myocardium and the development of autologous beating cells are
most crucial. The development of cardiomyocyte sheets derived from iPS cells was succeeded and demonstrated
functional recovery in rat MI model. We also succeeded in making thick cardiac tissue with rich vascular
network by new cell sheet implantation technique using omentum in porcine model. Newly developed cell sheet
technology may be a promising armamentarium for regeneration of severely damaged myocardium.
Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine
Shigeru Miyagawa、Yoshiki Sawa
Cell sheet-based myocardial regeneration therapy for heart failure
9：00〜10：40
Poster
Index
Aⴊ▤↢‛කቇળKPFD
Selected Oral
Presentation
117
Y.I.A.
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Symposium
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
The increasing prevalence in metabolic syndrome (MS), a clustering of obesity and metabolic disturbances,
becomes a great challenge to health burden in the 21st century. The risk of type 2 diabetes (T2D) and
cardiovascular disease (CVD) in individuals with MS is 2 to 5 folds higher than those without MS. Therefore,
the joint impact of MS and T2D/CVD are enormous in human health management. Both genetic and
environmental factors are demonstrated to responsible for development of MS and T2D. More importantly, there
are shared underlying mechanism linking the MS and T2D, i.e. obesity and associated insulin resistance. To
begin with understanding genetic component of MS and T2D, there are several ways to identify the genes
responsible for insulin resistance and related clinical disorders. We and others have employed candidate gene
approach such as using the in vitro. cell culture system as well as obese/diabetes animal model to isolate the
genes that might involve in pathogenesis of insulin sensitivity, obesity, MS and T2D. On the other hand, we can
also employ random approaches via genome-wide linkage study and genome-wide association studies (GWAS)
to isolate some more type 2 diabetes susceptibility genes that are beyond our previous understanding in
pathogenesis of obesity and T2D. We expect the new knowledge about the gene or biological pathways involved
might benefit better understanding of pathogenesis and future development of clinical diagnostic and
interventional tools for management clinical disorders caused by insulin resistance, obesity and related
disorders.
How to write
Plenary Lecture
Nature Medicine
Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
Special Talk
Lee-Ming Chuang
Special
Lecture
Genetics of metabolic syndrome - an Asian perspective
The spontaneously hypertensive rat (SHR), first described by Okamoto and Aoki close to 5 decades ago, is the
most widely studied animal model of essential hypertension. Since the original report on the development of
the SHR by Okamoto and Aoki in 1963, this strain has proven to be very valuable for studying mechanisms and
treatments for essential hypertension. More recently, the identification of specific DNA sequence variants that
regulate blood pressure and associated metabolic phenotypes in the SHR has demonstrated that this model can
also be successfully used to identify genes involved in the primary pathogenesis of hypertension and related
metabolic disorders. This presentation will discuss the use of SHR recombinant inbred, congenic, transgenic,
and conplastic strains to identify specific variants in nuclear or mitochondrial genes including Cd36, Srebf1,
Folr1, and mt-Co1 that influence an assortment of biochemical and hemodynamic features of the metabolic
syndrome. The relevance of these genes to mechanisms and therapies for hypertension and related metabolic
disorders in humans will also be discussed.
Department of Laboratory Medicine, University of California, San Francisco
Theodore W. Kurtz
Molecular Genetics and Therapeutics of the Metabolic Syndrome: Lessons
from the Spontaneously Hypertensive Rat
（English）
President
Lecture
SY08-2
SY08-1
Symposium 8：Metabolic syndrome / Diabetes
Thursday, December 2
Program
Aⴊ▤↢‛කቇળKPFD
118
SY08-4
SY08-3
The metabolic syndrome is a state of chronic low-grade inflammation. In contrast to “acute inflammation”
which resolves by an active termination program, “chronic inflammation” is characterized by persistent crosstalk
between parenchymal and stromal cells in response to tissue stress or malfunction, thereby leading to functional
maladaptation and tissue remodeling. Obese adipose tissue is characterized by adipocyte hypertrophy, followed
by increases in angiogenesis and macrophage infiltration, which is known as “adipose tissue remodeling”. We
have provided evidence that a paracrine loop involving saturated fatty acids and tumor necrosis factor 㱍 (TNF
㱍), which are derived from adipocytes and macrophages, respectively, establishes a vicious cycle that
aggravates inflammatory changes in obese adipose tissue. During the paracrine interaction between adipocytes
and macrophages, saturated fatty acids, which are released in large quantities from hypertrophied adipocytes via
the macrophage-induced lipolysis, serve as an endogenous ligand for TLR4/MD-2 complex, a major pathogen
sensor in innate immunity, to activate macrophages for the regulation of metabolic homeostasis. Sustained
interaction between endogenous ligands derived from parenchymal cells and pathogen sensors expressed in
stromal immune cells should lead to chronic/homeostatic inflammatory responses ranging from the basal
homeostatic state to diseased tissue remodeling, which has been referred to as “homeostatic inflammation” (J.
Leukoc. Biol. 88:33-39, 2010). Understanding the molecular mechanism underlying adipose tissue remodeling
as homeostatic inflammation would lead to the identification of molecular targets that may prevent or reduce
obesity-related metabolic diseases.
Department of Molecular Medicine and Metabolism, Medical Research Institute, Tokyo Medical and Dental
University
Yoshihiro Ogawa
The metabolic syndrome and chronic inflammation
Recently, obesity become popular medical and social problems with potentially devastating consequences
because it clusters with type 2 diabetes, hypertension and hyperlipidemia in the metabolic syndrome, which is
an important risk factor for cardiovascular disease incidence and mortality. So far, caloric restriction and
increasing energy expenditure through exercise are only effective methods to solve these problems. AMPactivated protein kinase (AMPK) functions as a fuel sensor in the cell and is activated when cellular energy is
depleted. In previous report, we presented that α-lipoic acid (α-LA) decreased hypothalamic AMPK activity
and causes profound weight loss in rodents by reducing food intake and enhancing energy expenditure.
Recently, we found that pharmacologically-induced cytoplasmic NAD(P)+/NAD(P)H ratio might stimulate the
rates of glycolysis, fatty acid oxidation through the increase mitochondrial oxidative phosphorylation and
adaptive mitochondrial biogenesis. Furthermore, this might be a useful therapeutic approach for patients with
metabolic syndrome. When cytoplasmic NAD(P)H:quinone oxidoreductase 1 (NQO1) is activated by exogenous
compounds, the cytoplasmic NAD(P)+/NAD(P)H equilibrium is shifted towards oxidized NAD(P)+. Under
these conditions, the high NAD(P)+/NAD(P)H ratio stimulates mitochondrial oxidative phosphorylation and
glycolysis and activates sirtuins. Here we show that the mechanism by which NQO1-mediated oxidation of
NAD(P)H leads to enhanced mitochondrial fatty acid oxidation involves activation of AMP-activated protein
kinase (AMPK). Furthermore, NQO1-mediated oxidation of NAD(P)H ameliorates most of phenotypes of
metabolic syndrome, including obesity, glucose intolerance, dyslipidemia, and fatty liver disease in ob/ob mice
and in mice on a high-calorie diet with diet-induced obesity. Neointimal formation, the leading cause of
restenosis, is caused by proliferation of vascular smooth muscle cells (VSMCs). In this study, we found that Bl,
one of NQO1 activators which regulates NAD(P)/NAD(P)H redox potential reduces neointimal formation after
balloon injury in vivo. Bl prevents VSMCʼs proliferation caused by G1 cell cycle arrest via an AMPK dependent
mechanism. These data suggest that cellular NAD/NADH level followed by treatment of Bl in NQO1expressing cells displays beneficial effects in the treatment of metabolic syndromes including atherosclerosis at
least in part via upregulation of AMPK. Therefore these studies will provide the regulation of NAD+/NADH
redox potential may be novel therapeutic target for the treatment of metabolic syndromes.
Department of Internal Medicine, Kyungpook National University, School of Medicine, Daegu, Korea
In-kyu Lee
Importance of NAD/NADH Regulation in Metabolic Syndrome.
9：00〜10：40
循環器内科
Selected Oral
Presentation
Poster
Index
119
Y.I.A.
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Symposium
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Acute coronary syndrome (ACS), including myocardial infarction, unstable angina, and sudden cardiac death,
contributes to the high cardiovascular mortality. Plaque rupture and plaque erosion of vulnerable plaque is
considered the most important cause of ACS. However, the diagnosis of the high risk patients with vulnerable
plaques is still difficult. Therefore, it is important to understand the mechanisms of vulnerable plaque formation,
and to find the biomarkers of vulnerable plaque for precise clinical management.
Vulnerable plaques are composed of a thin fibrous cap overlying a large lipid core, an inflammatory infiltrate,
such as macrophages, T cells, dendritic cells in the shoulder, a high incidence of apoptotic cells, and neoangiogenesis as sign of inflammation. Recently, we found that activated plasmacytoid dendritic cells (pDC)
accumulate in vulnerable plaque and produce CCL19 and CCL21 to traffic activated T cell into the plaque.
Plaque-infiltrating CD4 T cells induce vascular smooth muscle cell (VSMC) and endothelial sell (EC) apoptosis
by triggering the TRAIL/TRAIL receptor 2 (DR5) pathway. We also found that activated pDC produce INF㱍
through TRL9 stimulation and amplify cytotoxic CD4 T cell functions by enhancing TRAIL expression. In
addition, DR5 was strongly expressed in the advanced atherosclerotic plaque, and TNF㱍 enhanced the
expression of DR5 on VSMC and EC in the atherosclerotic plaque. Finally, monitoring of soluble TRAIL levels
in ACS patients with both acute myocardial infarction and unstable angina was found useful in predicting
vulnerability in patients with vulnerable plaques.
東京女子医科大学
How to write
Plenary Lecture
Nature Medicine
佐藤 加代子
Special Talk
Roles of CD4 T cells in the vulnerable plaque formation
Special
Lecture
Aⴊ▤↢‛කቇળKPFD
循環器内科
It is increasingly appreciated that chronic inflammatory processes are crucially involved in various chronic
diseases and cancer. We recently demonstrated that obesity induces inflammation in visceral adipose tissue
using novel confocal microscopy-based techniques. During obesity of adipose tissue adipocytes, immune cells
and vascular cells dynamically interact with one another, which leads to adipose dysfunction and systemic
insulin resistance. We also found that similar interactions between parenchymal and stromal cells play
important role in cardiovascular, renal and metabolic diseases. For instance, we found that cardiac fibroblasts
are essential for cardiac hypertrophy. Cardiac fibroblast-specific deletion of Klf5 resulted in reduced
cardiomyocyte hypertrophy in response to pressure overload, indicating that the interaction between
cardiomyocytes and fibroblasts is required for the hypertrophic response of cardiomyocytes. More importantly,
high-intensity pressure overload caused severe heart failure and early death in fibroblast-specific Klf5 knockout
mice, suggesting that cardiac fibroblasts are essential for the myocardial adaptive response. Moreover, we
found that the parenchymal-stromal interactions are required for initiation and development of inflammatory
processes in chronic kidney disease and diabetes.
東京大学大学院医学系研究科
真鍋 一郎
Chronic inflammatory processes in cardiovascular, renal and metabolic
diseases
（日本語）
President
Lecture
SY09-2
SY09-1
シンポジウム 9：血管炎症
Symposium 9：Vascular Inflammation
12月2日
（木）
／Thursday, December 2
Program
Aⴊ▤↢‛කቇળKPFD
120
SY09-4
SY09-3
Macrophage activation participates pivotally in the pathophysiology of chronic inflammatory diseases including
atherosclerosis. Through the receptor EP4, prostaglandin E2 (PGE2) exerts an anti-inflammatory action in
macrophages, suppressing stimulus-induced expression of certain pro-inflammatory genes including
chemokines. We recently identified a novel EP4 receptor-associated protein, EPRAP/FEM1A, whose function
in PGE2-mediated anti-inflammation remains undefined. Here we demonstrate that PGE2 pretreatment
selectively inhibits lipopolysaccharide (LPS)-induced nuclear factor 㱖B1 (NF-㱖B1) p105 phosphorylation and
degradation in mouse bone marrow-derived macrophages (BMDM) through EP4-dependent mechanisms.
Similarly, directed EPRAP expression in RAW264.7 mouse macrophage-like cells suppresses LPS-induced
p105 phosphorylation and degradation, and subsequent activation of mitogen-activated protein kinase (MAPK)kinase (MEK) 1/2. Forced expression of EPRAP also inhibits NF-㱖B activation induced by various proinflammatory stimuli in a concentration-dependent manner. In co-transfected cells, EPRAP, which contains
multiple ankyrin-repeat motifs, directly interacts with NF-㱖B1 p105/p50 and forms a complex with EP4. In
EP4-overexpressing cells, PGE2 enhances the protective action of EPRAP against stimulus-induced p105
phosphorylation, whereas EPRAP silencing in RAW264.7 cells impairs the inhibitory effect of PGE2-EP4
signaling on LPS-induced p105 phosphorylation. Additionally, EPRAP knockdown as well as deficiency of NF㱖B1 in macrophages attenuate the inhibitory effect of PGE2 on LPS-induced MIP-1㱎 production. Thus,
PGE2-EP4 signaling augments NF-㱖B1 p105 protein stability through EPRAP after pro-inflammatory
stimulation, limiting macrophage activation.
京都大学医学部附属病院探索医療センター探索医療臨床部
南 学、横出 正之
EPRAP/FEM1A interacts directly with NF-κB1 p105 and attenuates
macrophage activation.
Large randomized studies in type 1 and type 2 have established that early intensive glycemic control reduces the
risk of diabetic microvascular complications, with less impact on macrovascular complications. However,
follow-up data of these trials reveal a long-term influence of early metabolic control on longer cardiovascular
outcomes, even though the influence on glycemic control has been immediately disappeared after the trials.
This phenomenon has recently been defined as “metabolic memory”. Potential mechanisms for propagating this
“memory” are the non-enzymatic glycation of cellular and tissue proteins which are conceptualized as advanced
glycation end-products (AGEs). Accumulation of AGEs leads to crucial biomedical pathway generating
intracellular oxidative stress and inflammatory mediators through receptor for AGEs (RAGE), which could
result in vicious circle through further amplification of the pathway involved in AGE generation. Several lines
of evidence suggest that AGEs/RAGE axis can profoundly be involved in vascular inflammation and
cardiovascular diseases. Inflammatory signaling is augmented by overexpression of RAGE in human
endothelial cells. On the other hand, inflammatory signaling is crucial for regulation of RAGE shedding, which
might by important for modulation of RAGE-mediated toxic signaling. In this symposium, I would like to
summarize the recent findings of RAGE axis as a crucial mediator of metabolic memory and vascular
inflammation, and to discuss their potential usefulness as therapeutic targets to overcome the effect of metabolic
memory, and also as biomarkers for the cardiovascular diseases.
大阪市立大学大学院医学研究科代謝内分泌病態内科学
小山 英則
Metabolic memory, AGE/RAGE and vascular inflammation
How to write
Plenary Lecture
Nature Medicine
Symposium
Y.I.A.
Presentation
Selected Oral
Presentation
Poster
Index
121
Special Talk
The 8th Korea-Japan Joint Symposium on Vascular Biology
Special
Lecture
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Endothelial dysfunction is the initial step in the development of atherosclerosis, leading to cardiovascular
diseases, and plays an important role in the maintenance and development of cardiovascular diseases. Several
lines of evidence have shown that cardiovascular diseases are associated with endothelial dysfunction. It is well
known that there is an association between inflammation and endothelial dysfunction. We have reported that
chronic infection with Helicobacter pylori impairs endothelial function in healthy male subjects. Patients with
periodontitis are ideal models for determining how endothelium-dependent vasodilation is affected by
inflammation. Indeed, periodontitis has been shown to be associated with endothelial dysfunction in subjects
without cardiovascular risk factors as well as patients with cardiovascular diseases through a decrease in NO
bioavailability, suggesting that systemic inflammation might be, at least in part, a cause of endothelial
dysfunction. In addition, periodontal therapy has been shown to improve endothelium-dependent vasodilation in
patients with periodontitis. Endothelial dysfunction promotes inflammation of the vascular wall, leading to a
vicious circle between endothelial dysfunction and inflammation. Under the condition of chronic inflammation,
production of proflammatory cytokines results in activation of endothelial cells, leading to the excessive
induction of adhesion molecules, cytokines, growth factors, and vasoconstrictors. Proflammatory cytokines
downregulate the expression of eNOS and decrease the half-life of eNOS mRNA in human endothelial cells.
Several pathways of proinflammatory factors may contribute to downregulation of the expression of eNOS and
decrease in enzymatic activity.
広島大学大学院医歯薬学総合研究科心臓血管生理医学
東 幸仁
Inflammation and Endothelial Function in Cardiovascular Disease
President
Lecture
Aⴊ▤↢‛කቇળKPFD
SY09-5
Program
15：20〜17：00
Aⴊ▤↢‛කቇળKPFD
122
SY10-2
SY10-1
（日本語）
Recent clinical studies demonstrated that treatment with angiotensin II receptor blocker (ARB) reduces the
onset of stroke, stroke severity, and the incidence and progression of dementia. We could expect that ARB
exerts these effects by both AT1 receptor blockade and AT2 receptor stimulation. We reported that focal brain
ischemic lesion after middle cerebral artery occlusion is smaller in AT1 receptor null mice with the increase in
cerebral blood flow in penumbra lesion and the decrease in oxidative stress, whereas brain damage is
exaggerated in AT2 receptor null mice (AT2KO). These results suggest that AT1 and AT2 receptor stimulation
has antagonistic effects on ischemic brain damage. Furthermore, we reported that treatment with ARB enhances
cognitive function before stroke and improves cognitive impairment after stroke, and attenuates the impairment
of cognitive function associated with hypertension or diabetes in mice. We observed that ARB prevents
cognitive decline after amyloid㱎 injection. Moreover, we observed that ARBs with partial PPAR㱏 activity (so
called “Metabosartan”) showed some additional effects on prevention of brain ischemic damage and cognitive
decline. It has been reported that AT2 receptor stimulation enhances neurite outgrowth and decreases neural
damage. We demonstrated that cognitive function is impaired in AT2KO, and that administration of AT2
receptor agonist (Compound 21) increased leaning ability in wild-type mice. Taken together, these results
support the notion that AT1 receptor blockade and AT2 receptor stimulation play some important roles in
reducing brain ischemic damage and increasing cognitive function.
愛媛大学大学院医学系研究科分子心血管生物・薬理学
堀内 正嗣
Effects of Stimulation of Angiotensin II Receptor Subtypes on Cerebral
Ischemia and Cognitive Function
Angiotensin receptor blockers (ARBs) have proven to be very useful for treating high blood pressure and
protecting against various forms of cardiovascular and renal damage. However, recent double blind, placebo
controlled clinical trials have raised doubts about the ability of some ARBs to provide added CV protection
beyond their ability to reduce blood pressure. For example, in the NAVIGATOR study, addition of the ARB
valsartan on top of standard therapies failed to provide added CV protection compared to placebo. In the
ROADMAP and ORIENT trials, treatment with olmesartan was associated with a 3 to 5 fold increased risk of
CV death compared to placebo. Because many mechanisms are involved in the pathogenesis of CV disease,
there is mounting interest in the identification of better antihypertensive drugs that do more than just inhibit the
renin-angiotensin system and lower blood pressure. Accordingly, next generation ARBs are being developed
that go beyond inhibition of the renin angiotensin and that function as nitric oxide donors, neprilysin inhibitors,
PPAR gamma activators, or endothelin receptor antagonists. This presentation will: 1) address the potential
importance of multi-functional ARBs that may reduce cardiovascular, renal, and metabolic risk through
multiple mechanisms that go beyond just inhibition of the renin-angiotensin system; 2) discuss functional
differences among existing ARBs; and 3) introduce four classes of next generation ARBs intended to do more
than simply inhibit the renin-angiotensin system and lower blood pressure.
Department of Laboratory Medicine, University of California, San Francisco, U.S.A.
Theodore W. Kurtz
Molecular Genetics and Therapeutics of the Metabolic Syndrome: Lessons
from the Spontaneously Hypertensive Rat
Sponsored シンポジウム 10：Renin-angiotensin system
Sponsored Symposium 10：Renin-angiotensin system
12月2日
（木）
／Thursday, December 2
Poster
Index
123
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Symposium
Obesity and associated diseases such as metabolic syndrome and cardiovascular diseases represent a steadily
growing health burden in industrialized societies. The favorable metabolic effects of telmisartan have been
attributed to its angiotensin (Ang) II receptor blockade and action as a partial agonist of peroxisome proliferators
activated receptor (PPAR)-㱏. Indeed, administration of telmisartan significantly improved endothelial
dysfunction as assessed by the vasodilator response to acetylcholine and markedly inhibited lipid accumulation
in the liver in mice with a high-fat diet. Interestingly, telmisartan, but not losartan, significantly increased the
expression of HGF (hepatocyte growth factor), but not VEGF (vascular endothelial growth factor), in the aorta.
In the further study, telmisartan showed a potential action to improve a non-alcoholic steatohepatitis (NASH)
induced by feeding Wistar rats an L-methionine- and choline-deficient (MCA) diet, possibly due to increased
HGF production through partial agonist of PPAR-㱏. As for adipose tissue, a better understanding of the
molecular mechanisms that control adipose tissue development and function should improve our understanding
of the pathogenesis and pathophysiology of metabolic syndrome. White adipose tissue is the primary site of
triglyceride and energy storage, and brown adipose tissue is important in both basal and inducible energy
expenditure in the form of thermogenesis, and of note PPAR-㱏 may contribute both differentiation. In this
session , we will also talk about the novel molecule in adipose tissue differentiation.
大阪大学連合小児発達学研究科健康発達医学
How to write
Plenary Lecture
Nature Medicine
中神 啓徳
Special Talk
The potential role of peroxisome proliferators activated receptor (PPAR)-γ
in metabolic syndrome
Special
Lecture
Aⴊ▤↢‛කቇળKPFD
腎臓・高血圧内科
Chronic kidney Disease (CKD) is defined as structural or functional abnormalities of the kidney that persist for
at least 3 months and are manifested by either kidney damage (most frequently detected as persistent
albuminuria) or a decreased glomerular filtration rate. CKD is strongly associated with cardiovascular disease
(CVD). Individuals with CKD are more likely to die of CVD than to develop kidney failure. High blood
pressure, poor glycemic control, obesity, metabolic syndrome, dyslipidemia, smoking and older age are the risk
factors for susceptibility to and initiation of CKD. In these conditions, systemic blood pressure can be
transmitted to the glomerular capillary network because of impairment of autoregularatory mechanism existing
on the afferent arteriole of glomerulus. Glomerular hypertension and endothelial dysfunction are regarded as the
common mechanisms underlying in the development of CKD. The renin-angiotensin system (RAS) is
implicated in the development of glomerular hypertension. RAS activation also underlie in endothelial
dysfunction through activation of NADPH oxidase and eNOS uncoupling. Numerous clinical trials and
experimental studies have demonstrated that angiotensin converting enzyme inhibitor (ACEI) and angiotensin
receptor blocker (ARB) ameliorate albuminuria/proteinuria and decline in renal function. We have successfully
innovated two-photon lasermicroscopy to visualize microcirculation and alteration of filtration status in the
kidney in the living animals. We will demonstrate how ARB could ameliorate imbalance of NO/ROS and
abnormal excretion of albumin in the kidney using these techniques along with the discussion of reno-protective
effects of ARB.
川崎医科大学
柏原 直樹
Renoprotective benefits of RAS inhibition : focus on prevention and treatment
of chronic kidney disease
President
Lecture
SY10-4
SY10-3
Program
15：20〜17：00
Aⴊ▤↢‛කቇળKPFD
124
SY11-2
SY11-1
（English）
RNA interference (RNAi) offers unique opportunities to become a novel therapeutic modality in combating
cancer. Although highly target specific, its use has been limited by its short duration of therapeutic gene
expression. To overcome this shortcoming, we constructed an oncolytic adenovirus (Ad)-based shRNA
expression system (Ad-deltaB7-shVEGF) against vascular endothelial growth factor (VEGF), a key mediator in
angiogenesis. To demonstrate VEGF-specific nature of this newly engineered Ad-based shRNA, replicationincompetent Ad expressing VEGF-specific shRNA (Ad-deltaE1-shVEGF) was also generated. Ad-deltaE1shVEGF was highly effective in reducing VEGF expression, and elicited anti-angiogenenic effect in vitro as
well as in vivo. Similarly, Ad-deltaB7-shVEGF exhibited potent anti-angiogenic effects in the matrigel plug
assay in vivo. Moreover, Ad-deltaB7-shVEGF also demonstrated enhanced antitumor effect and survival
advantage compared to its cognate control oncolytic Ad, Ad-deltaB7. Tumor histological analysis revealed that
Ad-deltaB7-shVEGF induced significant reduction in tumor vasculature, verifying the anti-angiogenic
mechanism. Furthermore, the duration and magnitude of the gene silencing effect following infection with AddeltaB7-shVEGF was longer and more effective than the replication-incompetent Ad, Ad-deltaE1-shVEGF. We
also constructed IL-8-specific shRNA-expressing oncolyticAd, Ad-deltaB7-shIL-8, and found that it is efficient
in killing cancer cells in vitro and in vivo. Taken together, these results suggest that the combined oncolytic
viral therapy and cancer cell-specific gene targeting is a powerful cancer therapeutical strategy.
Institute for Cancer Research, Yonsei University College of Medicine
Chae-Ok Yun
Exploiting gene-expressed RNAi for vascular-targeted cancer gene therapy
Isolation of candidate genes with therapeutic potential is indispensable for promoting molecular therapy of
acquired diseases such as vascular diseases. Hemagglutinating Virus of Japan envelope vector (HVJ-E) is a
powerful tool for this purpose. First, we attempted to find out the candidate genes for accelerating or inhibiting
endothelial cell (EC) growth. We isolated a cDNA fragment encoding a small peptide with an alpha-helix
structure consisting of 30 amino acids (angiogenic peptide-30; AG-30). AG-30 exhibited antimicrobial activity
against various bacteria and induced EC growth and tube formation in a dose-dependent manner. In the ischemic
mouse hind limb, slow-release AG-30 treatment resulted in an increase in blood flow and in capillary density.
Using the same system, we also identified a novel anti-angiogenic factor, the prime candidate gene was “fourand-a-half LIM only protein-2” (FHL-2). The mechanism of anti-angiogenic signaling of FHL-2 in EC was that
FHL-2 regulated phosphatidylinositol 3-kinase/Akt via direct suppression of the sphingosine kinase-1/
sphingosine-1-phosphate pathway in EC.Next, we tried to isolate genes responsible for suppressing neointimal
formation in artery. Several candidate genes such as ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) and
cylindromatosis (CYLD) were isolated. Both genes attenuated mainly NF-kappaB activity in vascular cells by
their deubiquitinating activities and suppressed neointimal formation in the balloon-injured artery. Those genes
will contribute to elucidation of the mechanisms of angiogenesis and atherosclerosis, and some of them will be
used for molecular therapy of vascular diseases.
Division of Gene Therapy Science, Graduate School of Medicine, Osaka University
Yasufumi Kaneda
Gene hunting for molecular therapy of vascular diseases
Sponsored Symposium 11：Gene Therapy
Thursday, December 2
Poster
Index
Aⴊ▤↢‛කቇળKPFD
Selected Oral
Presentation
125
Y.I.A.
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Symposium
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Renal disease including slight renal injuries has become to be supposed as one of risk factors for cardiovascular
events. Most conventional therapy at present is inefficient and tends to treat the symptoms rather than the
underlying causes of the disorder. Gene therapy based on oligonucleotides (ODN) offers a novel approach for
the prevention and treatment of renal diseases. Gene transfer into somatic cells to interfere with the pathogenesis
contributing to renal disease may provide such a novel approach for better prevention and treatment of renal
disease. The major development of gene transfer methods has importantly contributed to intense investigation of
the potential of gene therapy in renal diseases. Amazing advances in molecular biology have provided a
dramatic improvement of the technology that is necessary to transfer target genes into somatic cells. Gene
transfer methods, especially mediated by several viral vectors, have been surprisingly improved. Moreover,
recent progress in molecular biology has provided new techniques to inhibit target gene expression. Transfer of
cis-element double-stranded ODN (= decoy) has been reported as a novel powerful tool in a new class of antigene strategies as gene therapy. Transfer of decoy ODN corresponding to the cis sequence resulted in attenuation
of the authentic cis-trans interaction, leading to removal of trans-factors from the endogenous cis-elements with
subsequent modulation of gene expression. In this talk we would like to introduce some examples.
How to write
Plenary Lecture
Nature Medicine
Department of Internal Medicine, Kawasaki Medical School
Special Talk
Naruya Tomita
Special
Lecture
Usefulness of NF-κB Decoy Oligonucleotides Transfer to Renal Disease
Since the first clinical study on 1990, the technologies related to human gene therapy and the knowledge of
target diseases have shown steady progress. The current frustrations of the physicians and scientists, however,
are largely based on relatively lower gene transfer efficiencies of the current vectors and vector-related safety
concerns. To accelerate the clinical studies of human gene therapy, therefore, the supporting infrastructure and
improvement of vectors are keys for jump-up progress in this field.
We recently have focused on the
development of a new class and highly efficient gene transfer vector, namely recombinant Sendai virus vectors
(rSeVs). Unlikely to the currently available vectors, rSeV is a RNA virus vector expressing exogenous genes by
its own RNA polymerase in the cellular cytoplasm, without requirement of DNA phase, indicating that gene
therapy using this type of vector is theoretically free from insertional mutagenesis in cellular genomic DNA, a
great safety advantage of rSeV. In addition, rSeV has allowed us to show dramatically high gene transfer and
expression efficiencies in vivo, indicating the great potential in clinical application.
The mass production of
good manufacturing practice (GMP) of 1st generation vector, rSeV lacking F-gene (rSeV/dF), is now available
and a clinical study to treat critical limb ischemia using rSeV/dF expressing human basic fibroblast growth
factor (rSeV/dF-hFGF2: DVC1-0101) is now under evaluation in Kyushu University Hospital (SeVAT trial:
Sendai virus-mediated Angiogenic Therapy).
We here summarize the current status of SeVAT trial and its
scientific rationale for new concept as hierarchical actions of multiple angiogenic factors that are downstream
regulators of FGF-2.
R&D Laboratory for Innovative Biotherapeutics, Graduate School of Pharmaceutical Sciences, Kyushu
University
Yoshikazu Yonemitsu
Recombinant Sendai Virus Expressing Human FGF-2 (DVC1-0101) as a Novel
RNA Drug to Treat Peripheral Arterial Disease
President
Lecture
SY11-4
SY11-3
Program
Aⴊ▤↢‛කቇળKPFD
126
SY11-5
Dual-targeted therapy for anti-angiogenesis and anti-lymphangiogenesis represents a potentially effective
strategy for the treatment of various malignancies. Therefore, the goal of the present study was to identify genes
that encode inhibitors of both angiogenesis and lymphangiogenesis. The screening was performed using the
functional screening method with the HVJ-E vector. Using a cDNA library obtained from Lewis lung carcinoma
(LL/2), a candidate gene was identified by the evaluation of growth inhibition in aortic and lymphatic
endothelial cells (EC) as that coding for the mouse cold shock domain protein A (mCSDA). Over-expression of
mCSDA significantly repressed cell proliferation and c-fos promoter activity in aortic, venous, and lymphatic
ECs. CSDA is a DNA binding protein that binds to the hypoxia response element (HRE). Furthermore, of
importance, we revealed that CSDA could directly bind to the serum response element (SRE) sequence, which
may lead to growth inhibition in ECs. In an LL/2-inoculated mouse model, tumor growth was significantly
repressed in a mCSDA-injected group. Furthermore, the number of metastasis in the CSDA group was
significantly decreased. Histopathological analysis revealed that expression of blood and lymphatic endothelial
cell markers was significantly decreased in mCSDA-injected groups when CSDA plasmid was transferred to the
boundary between tumor and normal tissue by ultrasound-sonoporation. These data strongly suggested that
over-expressed CSDA repressed the growth and metastasis of LL/2 by the anti-angiogenic and antilymphangiogenic effect. In conclusion, the present study demonstrated that gene transfer of CSDA plasmid
DNA into endothelial cells repressed both angiogenesis and lymphangiogenesis in vitro and in vivo.
Department of Surgery, Asahikawa Medical University
Yukihiro Saito
Cold Shock Domain Protein A, Novel Repressor of Tumor-Angiogenesis and
Lymphangiogenesis for Cancer Gene Therapy.
9：00〜10：40
Index
Aⴊ▤↢‛කቇળKPFD
Poster
127
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Symposium
We have been investigating molecular mechanisms of vascular development and regeneration with a systematic
differentiation of mouse embryonic stem (ES) cells and induced pluripotent stem cells (Nature, 2000;
Circulation, 2008). We previously showed that cAMP together with vascular endothelial growth factor (VEGF)
potently induced endothelial cell (EC) differentiation and arterial EC formation. Recently, we demonstrated
novel mechanisms of EC differentiation and diversification through a pivotal role of cAMP pathway. We found
that Notch and GSK3㱎-mediated 㱎 -catenin signaling was simultaneously activated downstream of cAMP
through phosphatidylinositol-3 kinase, and induced arterial specification from vascular progenitors. Intracellular
domain of Notch and 㱎-catenin formed a protein complex on RBP-J binding sites of arterial genes (ephrinB2,
neuropilin1 (NRP1), Dll4, CXCR4, Hes1) in arterial, but not in venous ECs. Forced dual activation of Notch
and 㱎-catenin synergistically enhanced arterial gene expression both in vitro and in vivo (J Cell Biol, 2010).
On the other hand, Protein kinase-A (PKA) was involved in common EC differentiation. Overexpression of
constitutive active form of PKA (CA-PKA) potently induced EC differentiation and vascular formation.
Expression of VEGF 165 -specific receptors, Flk1 and Neuropilin-1 (NRP1), were increased by CA-PKAexpression, enhancing the sensitivity of the progenitors responding to VEGF 165 by more than 10 times. PKA
activation induced the formation of a protein complex with VEGF165, Flk1, and NRP1 in vascular progenitors
(Blood, 2009). These molecular machineries would offer novel understandings in vascular development and
clues for therapeutic strategies with vascular regeneration.
How to write
Plenary Lecture
Nature Medicine
Institute for Frontier Medical Sciences/Center for iPS Cell Research and Application, Kyoto Univeristy, Japan
Special Talk
Jun K. Yamashita、Kohei Yamamizu
Special
Lecture
Dual roles of cyclic AMP pathway in endothelial cell differentiation and
specification
We have recently found several genes that are upregulated in the heart where VEGF is overexpressed. One of
them is secreted frizzled related protein 2 (sFRP2), a known secreted Wnt antagonist. Through our biochemical
and cell culture experiments, we have identified a novel activity of sFRP2, as an enhancer of procollagen
C-proteinase (pCP) such as BMP-1. Furthermore, we have found that sFRP2 is upregulated specifically in
myofibroblasts during fibrosis following myocardial infarction. This specific expression pattern of sFRP2 was
also found to be correlated with its important role in fibrosis and cardiac function following myocardial
infarction, based on our studies of sFRP2-deficient mice. These studies indicate that sFRP2 is a putative VEGFinduced pCP enhancer which plays a critical role in pathogenesis following myocardial infarction, suggesting
that sFRP2 could serve a therapeutic target to treat human myocardial infarction.
Graduate School of Biological Sciences, Nara Institute of Science and Technology
Thomas N. Sato
sFRP2 is a pCP enhancer that plays a critical role in fibrosis
（English）
President
Lecture
SY12-2
SY12-1
Symposium 12：Stem cells
Friday, December 3
Program
Aⴊ▤↢‛කቇળKPFD
128
SY12-4
SY12-3
Cell transplantation has been used for myocardial ischemia as new therapeutic strategies. Since cell
transplantation has been reported to induce angiogenesis though paracrine mechanisms, tissue-like morphology
of cell sheet is suitable for engraftment, which lead to enhance angiogenesis for the long period. Recently we
have reported that cardiac Sca-1(+) cell sheet transplantation improved the cardiac function of myocardial
infarction model though in part VCAM-1/VLA-4 signaling-mediated angiogenesis. Endothelial cell co-cultured
tissue-engineered cardiomyocytes sheets improved the cardiac function of ischemic heart and blood vessels
originating from the engineered endothelial cell/cardiomyocyte tissue bridged into the infracted myocardium to
connect with capillaries of the host heart. We previously reported to construct 3-D cell-dense tissues by stacking
cell sheets, however poor vascularization remains a major obstacles in bioengineering cell-dense tissues,
limiting the viable size of constructs due to hypoxia, nutrient insufficiency and waste accumulation. Since
repeated transplantation of triple-layer grafts enabled to create about 1 mm thick tissue with well organized
microvascular network in vivo, we next challenged to fabricate microvasculature in vitro. Endothelial cell coculture in 3-D tissue introduced endothelial cell networks and some cells formed tube-like structure.
Furthermore when the 3-D tissues were transplanted in vivo, these vascular networks contributed to rapid blood
supply via blood vessel connections between host and graft. Now, the increase of thickness of 3-D tissue using
continuous media perfusion through in vitro fabricated endothelial cell networks is next challenge.
Institute of Biomedical Engineering and Science, Tokyo Womenʼs Medical University
Katsuhisa Matsuura、Tatsuya Shimizu、Teruo Okano
Vascular network formation in cell sheet transplantation and bioengineered
three-dimensional tissues
Generations of induced pluripotent stem (iPS) cells have been intensively studied by a variety of reprogramming
methods, but the molecular and functional properties of cells differentiated from iPS cells have not been well
characterized. Here, we generated iPS cells from human aortic smooth muscle cells (HASMCs) by using
lentiviral transduction of defined transcription factors. These iPS cells were differentiated back into SMCs to
enable a direct, comparative analysis with the HASMCs from which the iPS cells were originated. We observed
that iPS cell-derived SMCs were considerably similar to parental HASMCs and human embryonic stem cell
(hESC)-derived SMCs in gene expression patterns, epigenetic modifications of pluripotency-related genes, and
functional properties, despite the residual expression of some lentiviral transgenes. In addition, we found that
both hESC and iPS cells exhibited the similar differentiation tendency into endothelial cells. Endothelial cells
differentiated from iPS cells were also comparable to those derived from hESC in terms of their genetic and
phenotypic characteristics. Our study reports the generation of iPS cell from HASMCs and the parallel
characterization of human iPS cell-derived vascular cells with hESC-derived vascular cells, which may be
useful in addressing the safety and efficacy concerns related to the application of current human iPS cell
technology in regenerative medicine therapies.
Department of Biomedical Science, CHA University, Korea
Wonhee Suh
Vascular Differentiation of Human Pluripotent Stem Cells
9：00〜10：40
Poster
Index
129
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Vascular endothelial (VE)-cadherin is an endothelial cell-cell adhesion molecule involved in maintenance of
vascular integrity. Compromising vascular integrity leads to an increase in vascular permeability, which is
associated with various diseases such as chronic inflammation and tumor angiogenesis. Cyclic AMP (cAMP), a
second messenger downstream of Gs-coupled receptor, improves endothelial barrier functions. Previously, we
reported that cAMP-elevating agonists such as prostaglandins and adrenomedullin potentiate VE-cadherindependent cell adhesion through the Epac-mediated activation of Rap1 small GTPase. Furthermore, we have
recently shown that cAMP-Epac-Rap1 signal induces formation of cortical actin bundles to which VE-cadherin
is anchored through 㱍-/㱎-catenins at cell-cell adhesions. However, it remains unknown how Rap1 induces
cortical actin bundling. Here, we report that linear actin nucleators, both formin-like 2 and 3 (FMNL2/3), are
involved in Rap1-induced cortical actin bundling. FMNL2/3 are recruited to cell-cell junctions upon stimulation
with forskolin (FSK), an activator for adenylyl cyclase. FSK-induced cortical actin bundling was blunted by
depletion of FMLN2/3. Since Rho GTPases activate some formin family proteins, we further examined the role
of Rho GTPases in FMNL2/3-mediated cortical actin bundling. Active mutant of Cdc42, but not those of Rho
and Rac, bound to N-terminal region of FMNL2 and induced cortical actin bundling. In Cdc42-depleted cells,
FSK did not induce the accumulation of FMNL2/3 at cell-cell junctions and the formation of cortical actin
bundling. These results indicate that Rap1 induces cortical actin bundling through Cdc42-mediated activation of
FMNL2/3, thereby stabilizing VE-cadherin-dependent cell-cell junctions.
Symposium
Aⴊ▤↢‛කቇળKPFD
Deptartment of Cell Biology, National Cerebral and Cardiovascular Center Research Institute, Osaka, Japan
Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku University
2
How to write
Plenary Lecture
Nature Medicine
1
Special Talk
Shigetomo Fukuhara1、Koji Ando1,2、Kazuomi Noda1、Jianghui Zhang1、
Norimichi Nakahata2、Naoki Mochizuki1
Special
Lecture
Signaling pathways regulating endothelial barrier functions
Vascular endothelial cells (ECs) lining the inner surface of blood vessels are constantly exposed to shear stress,
a fluid mechanical force generated by flowing blood. In recent years, a biomechanical approach in which
cultured ECs are subjected to controlled levels of shear stress in a fluid-dynamically designed apparatus has
been widely used. It has become apparent that ECs respond to shear stress by altering their morphology and
functions, including the production of a potent vasodilator nitric oxide (NO) and anti-thrombotic protein
thrombomodulin. When cultured ECs are exposed to flow, the intracellular Ca2+ concentration ([Ca2+]i) increases
in a shear-stress-dependent manner. The flow-induced Ca2+ response is due to an influx of extracellular Ca2+ via
an ATP-gated ion channel P2X4, meaning that ECs can accurately convert information regarding shear stress
intensity into changes in [Ca 2+]i and that P2X4 have a shear transducer property by which shear stress signals
are transmitted into the cell interior via the Ca2+ influx. Shear-induced activation of P2X4 requires ATP, which is
supplied in the form of endogenous ATP released by ECs. Shear-stress-induced ATP release is initiated at
caveolae. To gain insight into the physiological role of the P2X4-meidated shear-stress mechanotransduction,
we generated P2X4-deficient (KO) mice. KO mice do not exhibit normal EC responses to shear stress, such as
Ca 2+ influx and subsequent production of NO. The vasodilation induced by acute increases in blood flow is
markedly suppressed in KO mice. KO mice have higher blood pressure values than wild-type mice. Thus,
P2X4-mediated shear-stress-mechanotransduction plays an important role in the vascular homeostasis, including
the control of blood pressure and flow-induced vasodilation.
Laboratory of System Physiology, Department of Biomedical Engineering, Graduate School of Medicine, The
University of Tokyo
2
Laboratory of Biomedical Engineering, School of Medicine, Dokkyo Medical University
1
Kimiko Yamamoto1、Joji Ando2
ATP-gated P2X4 ion channel serve as transducers for shear stress
mechanotransduction in vascular endothelial cells
（English）
President
Lecture
SY13-2
SY13-1
Symposium 13：Vascular Signal Transduction
Friday, December 3
Program
Aⴊ▤↢‛කቇળKPFD
130
SY13-4
SY13-3
Orchestration of differentiation, migration and re-assembly of cells is one of fundamental aspects of pattern
formation of tissues and organs. In physics, pattern formation, such as the Benard convection and the Taylor
instability, is extensively studied in physics and chemistry. In these cases, homogenous molecules can form
distinct patterns. In another case, oxidative and reductive states repeat in an oscillatory way, known as the B-Z
reaction. These indicate that similar autonomous mechanisms could exist in developing embryos, some of which
were already studied extensively by Turing and Meinhard. We have been exploring molecular mechanisms of
pattern formation of vertebrate embryos with several key transcription factors. Nonetheless, we have noticed
that extensive analyses on the genetic programs are not sufficient for understanding the dynamic pattern
formation of developing embryos. Recently, we have found that several proteins respond to physical forces
generated by cells, hereby such strains trigger next biochemical responses. These mechano-sensitive factors are
now emerging as key regulators of morphogenesis, circulatory homeostasis, remodeling of bones and muscles,
and even metabolism/obesity. We are now studying novel mechanisms to understand functional roles of physical
forces generated by cells and sensed by cells, especially in the vascular system.
Institute of Development, Aging and Cancer, Tohoku University
Toshihiko Ogura
Machanical control of gene expression in vascular system.
Although oral phosphodiesterase (PDE)-5 inhibitors have revolutionized the way we treat patients with erectile
dysfunction (ED), they are not cures for ED and have important limitations: PDE5 inhibitors must be used on
demand; and men with severe endothelial dysfunction from vascular risk factors, such as diabetes, respond
poorly to these drugs. Recently, a link between ED and systemic vascular disease was unveiled and both
diseases were known to share the same vascular risk factors with endothelial cell dysfunction being the common
denominator between these two conditions. Therefore, endothelial cell regeneration is a promising therapeutic
strategy for curing ED and is anticipated to be effective for patients who do not respond to PDE5 inhibitors and
to restore physiologic erections, i.e., spontaneity of sexual act. We examined the efficacy of potent
angiopoietin-1 (Ang1) variant, cartilage oligomeric matrix protein (COMP)-Ang1, on erectile function in animal
models for diabetic ED, including type I (streptozotocin-induced) and type II (db/db mice) diabetic models.
Local delivery of COMP-Ang1 significantly increased cavernous endothelial proliferation, eNOS
phosphorylation, and cGMP expression compared with that in the untreated control or PBS-treated diabetic
group. These changes in group receiving COMP-Ang1 restored erectile function up to 4 weeks after treatment.
Endothelial protective effects, such as marked decreases in the expression of p47phox and iNOS, in the generation
of superoxide anion and nitrotyrosine, and in the number of apoptotic cells in the corpus cavernosum tissue,
were noted in COMP-Ang1-treated diabetic mice. Intracavernous injection of COMP-Ang1 completely restored
endothelial cell-to-cell junction proteins (VE-cadherin, ZO-1, occludin, and claudin-5) and decreased cavernous
endothelial permeability. COMP-Ang1-induced promotion of cavernous angiogenesis and erectile function was
abolished in the presence of the NOS inhibitor, L-NAME, but not in the presence of NADPH oxidase inhibitor,
apocynin. These findings constitute a new paradigm toward curative treatment of both cavernous angiopathy
and ED.
National Research Laboratory of Regenerative Sexual Medicine and Department of Urology, Inha University
School of Medicine, Incheon, Korea
2
Department of Biological Sciences and Laboratory for Vascular Biology, Korea Advanced Institute of Science
and Technology (KAIST), Daejeon, Korea
1
Ji-Kan Ryu1、Hai-Rong Jin1、Gou Young Koh2、Jun-Kyu Suh1
Intracavernous Delivery of a Designed Angiopoietin-1 Variant Rescues
Erectile Function by Enhancing Endothelial Regeneration in the Diabetic
Mouse
9：00〜10：40
Poster
Index
131
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Aneurysm and dissection are the major aortic diseases in adult, of which molecular mechanism for the failure of
homeostatic balance is largely unknown. We previously discovered several key molecules that regulate the
balance between tissue destruction and repair in aortic aneurysm. To further identify the key molecules for the
homeostasis of aorta, we systematically analyzed the molecules that are expressed during the progression of
aortic aneurysm. We found that tenascin-C (TNC), an extracellular matrix protein, was expressed in the human
aneurysm tissue with active inflammation. In normal wild type (WT) mice, TNC was constitutively expressed in
the distal, but not in the proximal aorta. Biomechanical analyses revealed the loss of flexibility in distal aorta
and concomitant increase in hemodynamic stress in proximal aorta in TNC knockout (KO) mice. Periaortic
application of calcium chloride in the distal aorta and systemic administration of angiotensin II (C+A) in WT
caused the loss of flexibility of the distal aorta due to chronic inflammation, which was associated with the
compensatory increase in the hemodynamic stress, medial hypertrophy, and TNC induction in the proximal
aorta. In contrast, C+A caused a large medial dissection in the proximal aorta in KO. Transcriptome analysis
revealed the low expression of various chemokines at the baseline, but exaggerated induction in response to
C+A in proximal aorta of KO. The hypersensitive chemokine induction was recapitulated in KO-derived aortic
smooth muscle cell culture. Therefore, TNC works as a biomechanical and biochemical “shock-absorber” to
alleviate the onset of catastrophic inflammatory response to the stress, thus maintaining the homeostasis of
aortic tissue.
Symposium
Aⴊ▤↢‛කቇળKPFD
久留米大学循環器病研究所、2筑波大学大学院人間総合科学研究科循環器内科、3山口大学大学院医学系研究科器官病態外科学、
三重大学大学院医学系研究科修復再生病理学分野、5国立国際医療研究センター、6山口大学大学院医学系研究科器官病態内科
学
4
How to write
Plenary Lecture
Nature Medicine
1
Special Talk
青木 浩樹1、木村 泰三2、吉村 耕一3、今中 恭子4、吉田 利通4、青沼 和隆2、廣江 道昭5、今泉 勉1、
松崎 益徳6
Special
Lecture
Molecular network for the homeostasis of aortic tissue
All of our tissues contain the same 30,000 genes; however, in a given tissue and at a given stage, owing to an
“epigenetic code,” only a few of these genes are expressed, giving rise to the “phenotype.” Disruption of the
balance of epigenetic networks may cause several major diseases, including cancer, syndromes involving
chromosomal instabilities, and mental retardation. However, the relevance of epigenetics to other
physiopathological mechanisms in common diseases, such as metabolic syndrome, was less clear. Through
genome wide identification of PPAR㱏 targets by chromatin immunoprecipitation on Chip (ChIP-Chip)
analysis, we identified several histone modification enzymes (HKMTs) and candidatesʼ genes as new PPAR㱏
targets. We show that these HKMTs function either anti-, or pro- adipogenic factor and coordinately regulated
their gene expressions by PPAR㱏 to promote adipogenesis. We therefore propose the novel action of PPAR
gamma: controlling epigenomic status in fat cell differentiation. In addition, we demonstrate that JHDM2a, a
demehtylase of H3K9me2, regulates metabolic genes related to energy homeostasis. Mice deficient in JHDM2a
(JHDM2a-/-) develop adult onset obesity, hypertriglyceridemia, hypercholesterolemia, hyperinsulinemia, and
hyperleptinemia, which are hallmarks of metabolic syndrome. Thus, H3K9 demethylase JHDM2a is a crucial
regulator of genes involved in energy expenditure and fat storage, which suggests it is a previously unrecognized
key regulator of obesity and metabolic syndrome.
東京大学先端科学技術研究センター 代謝医学分野
酒井 寿郎
Metabolic syndrome and epigenomic regulations
（日本語）
President
Lecture
SY14-2
SY14-1
シンポジウム 14：新規疾患関連分子
Symposium 14：New therapeutic target
12月3日
（金）
／Friday, December 3
Program
Aⴊ▤↢‛කቇળKPFD
132
SY14-4
SY14-3
MicroRNAs (miRNAs) are endogenously expressed small non-coding RNAs that function as negative regulators
of gene expression. Posttranscriptional regulation by miRNAs is important for many aspects of development,
homeostasis, and disease. Sveral miRNAs are highly expressed in endothelial cells and are involved in
angiogenesis. Whereas the specific functions of miRNAs for tumor angiogenesis are less clear.
Here, we identified several miRNAs that were enriched in endothelial cells derived from LLC tumor. Among
these miRNAs, we found that mir-125 regulated the expression of endothelial cell adherent protein VE-cadherin.
Additionally, overexpression of mir-125 in endothelial cells resulted in inhibition of proliferation, tube
formation and migration activity. Furthermore, in vivo transfection of mir-125 in subcutaneous implanted LLC
tumor, inhibited functional vascular formation and suppressed tumor growth.
These findings illustrate that mir-125 act as negative regulator of tumor angiogenesis and providing a new target
for modulating vascular formation and function.
大阪大学微生物病研究所情報伝達分野
木戸屋 浩康、村松 史隆、高倉 伸幸
Role of microRNAs for tumor angiogenesis and endothelial function
Grb2-associated binder (Gab) docking proteins play critical roles for receptor tyrosine kinases-mediated
activation of MAP kinases (ERKs) and PI3-kinase-AKT. We previously reported the crucial role of Gab proteins
for maintenance of cardiac function through the analysis of cardiomyocyte-specific Gab1/Gab2 double knockout
mice (J Clin, Invest. 117, 1771, 2007). Recently, we created endothelium-specific Gab1 knockout mice
(Gab1flox/flox Tie2Cre(+); Gab1ECKO). Since Gab1ECKO were viable and showed no obvious developmental
defects, we created a murine model of operatively induced hindlimb ischemia. Angiogenesis in the ischemic
limb was significantly impaired in Gab1ECKO versus control evaluated by either doppler flow intensity or
capillary density measurement. Intriguingly, necrosis in the operated limb was observed in all of Gab1ECKO,
but not in control. In addition, we have found that Gab1 deletion in the endothelium also leads to enhanced
formation of atherosclerosis after chronic administration of angiotensin II in ApoE-null genetic background,
suggesting that Gab1 is a critical player for prevention of cardiovascular diseases. Using human endothelial
cells (ECs), we found that hepatocyte growth factor (HGF) induces the most prominent tyrosinephosphorylation of Gab1 among several proangiogenic growth factors. Phosphorylated Gab1 associates with
protein tyrosine phosphatase SHP2 and PI3-kinase p85 subunit. Furthermore, Gab1 is essential for sustained
activation of ERK1/2, ERK5, and AKT in responsen to HGF in the ECs. These data suggest that Gab1 might
have a key role for HGF-dependent vascular protection. Thus, elucidating the molecular mechanism of Gab1dependent vascular protection will enable us to discover novel therapeutic targets for cardiovascular diseases.
大阪大学大学院医学系研究科循環器内科学、2国立循環器病研究センター研究所
1
中岡 良和1、望月 直樹2、小室 一成1
Docking protein Gab1 plays critical roles for prevention of cardiovascular
diseases
How to write
Plenary Lecture
Nature Medicine
Symposium
Y.I.A.
Presentation
Selected Oral
Presentation
Poster
Index
133
Special Talk
The 8th Korea-Japan Joint Symposium on Vascular Biology
Special
Lecture
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Members of the bone morphogenetic protein (BMP) family have been implicated in the development and
maintenance of vascular systems. While members of the BMP-2/4 and osteogenic protein-1 groups signal via
activin receptor-like kinases (ALK)-2, 3, and 6, BMP-9 and 10 have been reported to bind to ALK-1 in
endothelial cells. However, the roles of BMP-9/ALK-1 signaling in the regulation of endothelial cells have not
yet been fully elucidated. Here, we examined the effects of BMP-9 on the proliferation of endothelial cells using
various systems. Vascular tube formation from ex vivo allantoic explants of mouse embryos was promoted by
BMP-9. BMP-9, as well as BMP-4 and 6, also induced the proliferation of in vitro-cultured mouse embryonic
stem cell-derived endothelial cells (MESEC) by inducing the expression of vascular endothelial growth factor
receptor 2 and Tie2, a receptor for Angiopoietin-1. Decrease in ALK-1 expression and expression of
constitutively active ALK-1 in MESEC abrogated and mimicked the effects of BMP-9 on the proliferation of
MESEC, respectively, suggesting that BMP-9 promotes their proliferation via ALK-1. Furthermore, in vivo
angiogenesis was promoted by BMP-9 in a Matrigel plug assay and a BxPC3 human pancreatic cancer xenograft
model. In consistent with these in vivo findings, BMP-9 enhanced the proliferation of in vitro cultured normal
endothelial cells from dermal tissues of adult mice and tumor-associated endothelial cells isolated from tumor
xenografts in host mice. These findings suggest that BMP-9 signaling activates the endothelium tested in the
present study via ALK-1.
東京大学大学院医学系研究科分子病理学、2科学技術振興機構さきがけ、3北海道大学大学院歯学研究科口腔病態学講座血管生
物学教室
1
渡部 徹郎1,2、鈴木 夕佳1、吉松 康裕1、大賀 則孝3、森下 保幸1、樋田 京子3、宮園 浩平1
Physiological and pathological roles of BMP-9/ALK-1 signals in the
proliferation of endothelial cells
President
Lecture
Aⴊ▤↢‛කቇળKPFD
SY14-5
Program
Aⴊ▤↢‛කቇળKPFD
134
SY15-2
SY15-1
（日本語）
Molecular medicine, the mechanistic understanding of pathophysiology in molecular terms to prevent, diagnose
and treat human disease, has made great progress at a basic level, but has failed to fully deliver on its promise of
new therapies. In this talk, I identify some of the roadblocks that have kept molecular medicine from fulfilling
our expectations, focusing specifically on three aspects: the limitations of animal models, organizational and
financial issues that impede the development of new drugs, and educational aspects related to the lack of
training that most researchers have when trying to explore the translational/commercial potential of their work.
Chief Editor, Nature Medicine
Juan Carlos Lopez
Key obstacles to translational research: an editorial view
Japanese academic community, government, and industries have made efforts to deliver the scientific findings
of biomedical research. Japanese industries, especially conventional pharmaceutical companies, were not
straightforward to initiate collaborative research and development (R&D) with universities, and academic
researchers established start-up venture companies for the R&D of their products mostly during 2000-2005,
however, most of them gave up because there were not many professionals in regulation and/or development
with these companies, and also venture capitals shrank investment to these companies. Also, Japanese clinical
trial system is too complicated since there are two separate pathways to perform clinical trials: Pharmaceutical
affairs law (PAL)-regulating clinical trial (“Chiken”) and clinical research not regulated by PAL, which has
made confusion to both researchers and industries. Later, conventional pharmaceutical companies started to
acquire biomedical seeds from the USA or European companies, not from Japanese university-spin out
companies.In this talk, current status and future directions of translational research in Japan in views of
regulation, government programs, and bioindustries will be discussed.
京都大学大学院医学研究科薬剤疫学
川上 浩司
臨床応用研究の道筋と制度的課題
シンポジウム 15：トランスレーショナル リサーチ
Symposium 15：Translational Research
12月3日
（金）
／Friday, December 3 13：20〜15：00
Index
Aⴊ▤↢‛කቇળKPFD
Poster
135
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Symposium
Direct reprogramming of somatic cells to induced pluripotent stem (iPS) cells is a recent most prominent
progress in stem cell biology. Next important issues are how to obtain the human somatic cells with less
invasiveness, how to shorten the period and increase the efficiency of iPS cell production, and how to avoid the
genome integration of extrinsic genes. Recent studies demonstrated that use of somatic stem cells, and
application of plasmid, adenovirus, protein, transpozon or protein could increase the efficiency or safety to some
extent. However, these procedures cannot overcome these 4 problems at the same time. Here we show that the
combination of activated T cell cultivation and the temperature sensitive mutated Sendai virus (SeV) encoding
human OCT3/4, SOX2, KLF4 and c-MYC can overcome these issues simultaneously, and generate the iPS cells
very efficiently and safely within one month. Terminally differentiated T cells were obtained from 1 ml of
peripheral blood, and can expand themselves by activating with plate-bound anti-CD3 monoclonal antibody and
rIL-2. SeV is negative-sense single stranded RNA virus, do not integrate into host genome, and is very
efficiently transfected into the activated T cells. Now, T cell-derived iPS (TiPS) cells can be readily used in
clinics and research centers. We generated cardiomyocytes from TiPS cells, purified them using TMRM dye and
FACS, and transplaneted them into the immunodeficient mice heart. The transplanted human cardiomyocytes do
not make teratoma, and can survive in the heart for extended period. In the future, we are planning to transplant
them in the patients with severe congestive heart failure.
How to write
Plenary Lecture
Nature Medicine
慶應義塾大学医学部循環器内科
Special Talk
福田 恵一、関 倫久、湯浅 慎介
Special
Lecture
Generation of iPS cells from a single droplet of peripheral blood and its
application to human heart failure
Human cardiac regeneration therapy has been performed by using various sources of stem cell. Myoblasts and
bone marrow cells have been injected for patients with ischemic cardiomyopathy in our clinical trial and
improved cardiac performance. We had examined the efficacy of stem cell therapy using tissue engineered sheet
technique compared to needle injection. This technique has advantages such as the ability for treatment to large
area, and less invasive for host heart such as lethal arrhythmia. In vivo, implantation of autologous myoblast
sheet had improved cardiac function of ischemic or dilated cardiomyopathy models using rat, hamster, canine
and porcine models. We also showed that myoblast sheets provided various factors inducing angiogenesis,
hematopoietic cell recruitment and anti-apoptosis, following anti-remodeling. Thus, after approved by IRB of
our institution, we have started the clinical trial of myoblast sheet implantation for DCM patients, and assessed
the feasibility and efficacy for the first patient. In this patient, any sequelae including arrhythmia have not
occurred after implantation, and the cardiac function showed recovery.
Furthermore, we have investigated several improvement of this technology for the layered implantation for
ischemic heart, for right heart failure, and another cell source to construct the sheet.
Thus, stem cell sheet implantation could be safe and eligible as cardiac regeneration therapy.
大阪大学大学院医学系研究科機能制御外科学
澤 芳樹
Stem Cell Therapy for Severe Heart Failure.
President
Lecture
SY15-4
SY15-3
Program
13：20〜14：10
Aⴊ▤↢‛කቇળKPFD
136
YF-2
YF-1
（English）
Akt mediated signaling plays an important role in blood vascular development. Here we have investigated the
role of Akt in lymphatic growth using Akt deficient mice. Firstly, we found that lymphangiogenesis occurred in
Akt1-/-, Akt2-/- or Akt3-/- mice. However, the diameter and endothelial cell number of lymphatic capillaries were
significantly less in Akt1-/- mice than that of wildtype control, while there was only slight change in Akt2-/- or
Akt3-/- mice. Secondly, valves present in the small collecting lymphatics in the superficial dermal layer of ear
skin were rarely observed in Akt1 -/- mice, although they could be detected in the large collecting lymphatics in
the deep layer of skin tissues. Fluorescence microlymphangiography assay showed that skin lymphatic network
in Akt1 -/- mice was functional but with defects as shown by FITC-dextran draining. There was abnormal
enlargement of collecting lymphatic vessels, and further analysis showed that smooth muscle cell coverage with
collecting lymphatics became much sparser in Akt deficient mice than that in wildtype control. Finally we
showed that lymphatic vessels were detected in compound Akt null mice, and that lymphangiogenesis could be
induced by VEGF-C delivered via adenoviral vectors in adult mice lacking Akt1. These results indicate that in
spite of the compensatory roles of other Akt isoforms, Akt1 is more critically required in the lymphatic
development.
Laboratory of Vascular and Cancer Biology, Model Animal Research Institute, Nanjing University, Nanjing,
China
2
Laboratory of Heart and Disease Model, Model Animal Research Institute, Nanjing University, Nanjing, China
3
Departments of Surgery and Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, Keck
School of Medicine, University of Southern California, Los Angeles
4
Department of Medical Imaging, Nanjing Jinling Hospital, Clinical School of Medical College, Nanjing
University, Nanjing, China
5
Molecular/Cancer Biology Laboratory, Department of Pathology, Haartman Institute and Ludwig Institute for
Cancer Research, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland
6
Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
1
Fei Zhou1、Zai Chang2、Luqing Zhang1、Young-Kwon Hong3、Bin Shen1、Bo Wang1、
Fan Zhang4、Guangming Lu4、Denis Tvorogov5、Kari Alitalo5、Brian̲A. Hemmings6、
Zhongzhou Yang2、Yulong He1
Akt / Protein Kinase B is required for lymphatic network formation,
remodeling and valve development
Although VEGFR-3 deficiency disrupts blood vascular development during early embryogenesis, the underlying
mechanism was not clear. To characterize its function in angiogenesis and lymphangiogenesis, we employed
two genetically modified mouse models in this study, targeting the coding region for the ligand-binding domain
(Vegfr3 deltaLBD ) or the tyrosine kinase domain with an inactivation point mutation (Vegfr3 TKmut ). We show that
lymphatic growth was disrupted in Vegfr3 deltaLBD/deltaLBD and Vegfr3 TKmut/TKmut mice, but blood vessels developed
normally in both embryo and yolk sac. Interestingly, in Vegfr3deltaLBD/deltaLBD but not Vegfr3TKmut/TKmut mice, lymph
sac was present but there was lack of lymphangiogenic sprouting. We further demonstrate that both the wildtype and mutant forms of VEGFR-3 could form heterodimers with VEGFR-2, and decreased the level of
phospho-VEGFR-2 and the downstream phospho-Erk1/2 in endothelial cells when they were treated with
VEGF-A. These findings indicate that signaling mediated via VEGFR-3 activation by its cognate ligands
(VEGF-C/-D) is not required for angiogenesis, and that VEGFR-3 may play a role in this process by modulating
VEGFR-2-mediated signals.
Laboratory of Vascular and Cancer Biology, MOE Key Laboratory for Model Animal and Disease Study, Model
Animal Research Institute, Nanjing University
2
Laboratory of Vascular Biology, Institute of Molecular Medicine, Peking University
3
Department of Molecular Oncology, Tokyo Medical and Dental University
1
Luqing Zhang1、○Fei Zhou1、Wencan Han1、Bin Shen1、Jincai Luo2、
Shibuya Masabumi3、Yulong He1
VEGFR-3 ligand-binding and kinase activity are required for
lymphangiogenesis but not for angiogenesis
Y.I.A. Presentation (for overseas nominees)
Friday, December 3
Index
Aⴊ▤↢‛කቇળKPFD
Poster
137
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Symposium
Heme oxygenase-1 (HO-1) is a stress-inducible enzyme catalyzing the oxidative degradation of heme to free
iron, carbon monoxide (CO) and biliverdin. Our previous study has demonstrated that HO-1 overexpression in
ischemic heart promoted vascular endothelial growth factor (VEGF) expression and angiogenesis, which
contributed to the attenuation of left ventricular fibrosis and cardiac dysfunctions. However, the mechanism
underlying HO-1-induced VEGF expression remained elusive. Here we show that HO-1 gene transduction in
rat primary cardiomyocytes and H9C2 myocytes resulted in significant induction of VEGF expression. A
similar effect was also observed when cells were directly exposed to low doses of CO gas (100 & 250 ppm)or a
CO-releasing compound, tricarbonyldichlororuthenium (II) dimmer (10-50 㱘M). HO-1/CO-induced VEGF
expression was significantly suppressed by pharmacological inhibition of p38 kinase, but not of AKT, activation.
VEGF promoter-luciferase reporter assays, electrophoretic mobility shift assays, supershift assay, and chromatin
immunoprecipitation revealed that CO-induced VEGF promoter activation requires the binding of the Sp1
transcriptional factor to a cis-regulatory sequence located at the VEGF promoter. Western blot analysis and
immunostaining experiments demonstrated that HO-1/CO induced p38-dependent phosphorylation of Sp1 at
Thr453 and Thr739 both in vitro and in vivo. Overexpression of Sp1 protein with an alanine mutation at Thr453
or Thr739 suppressed CO-induced Sp1 binding to the VEGF promoter and its transcriptional activation.
Collectively, these data suggest that p38-dependent phosphorylation of Sp1 at Thr543/Thr739 is crucial for
HO-1/CO-induced VEGF expression in cardiomyocytes.
How to write
Plenary Lecture
Nature Medicine
The Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
Special Talk
Heng-Huei Lin、Shao-Chuan Lai、Lee-Young Chau
Special
Lecture
P38 kinase-dependent activation of Sp1 transcriptional factor mediates heme
oxygenase-1/carbon monoxide-induced vascular endothelial growth factor
gene expression in cardiomyocytes
Wnt pathway has been implicated in angiogenesis. Although Wnt related proteins are known to be important
during development, functions of their complex interaction are still unclear in vascular development. We found
a novel angiogenic activator, DKK2 which interact with lrp6, a known receptor of Wnt. In the mouse cornea
assay, DKK2 protein recruited new vessels. Unlike VEGF, DKK2 induced more stable form of vessels, which
are coated well with pericytes. Consistently, more vessels were found in matrigel plug mixted with DKK2
compared to control plug. In vivo, Tie2 specific DKK2 expression in mice caused more sprouting at the front of
retinal vessels than in those of littermate wild mice. Retina vessel density and filopodia number of DKK2 Tg
mice was also increased compared to wild retina. Treatment of DKK2 protein improved tissue repair by
enhancing angiogenesis in hindlimb ischemia and cardiac infarction mouse model. We propose that DKK2
promotes angiogenesis by enhancing endothelial tip cell dynamics, and this may provide therapeutic potential of
DKK2 protein in angiogenesis-related disease.
2
Department of Biochemistry, Yonsei University, Seoul, Korea
Vascular System Research Center, Kangwon National University, Kangwon-Do 200-701, Korea
1
Hongryeol Park1、Jeong-Ki Min1、Hyun-Jung Choi1、Seung-sik Rho1、
Young-Myoung Kim2、Young-Guen Kwon1
DKK2, the Wnt antagonist, activates angiogenesis
President
Lecture
YF-4
YF-3
Program
Aⴊ▤↢‛කቇળKPFD
138
YF-5
Malignant melanoma is highly metastatic and notoriously resistant to conventional therapies, posing a
challenging therapeutic obstacle attributed to tumor population heterogeneity. Highly metastatic and
chemotherapy-resistant properties of malignant melanomas stand as challenging barriers to successful treatment,
yet the mechanisms responsible for their aggressive characteristics are not fully defined. We show that a distinct
population expressing CD133 (Prominin-1), which is highly enriched after administration of a chemotherapeutic
drug, dacarbazine, has enhanced metastatic potential in vivo. CD133+ tumor cells are located close to tumorassociated lymphatic vessels in metastatic organs, such as the regional lymph nodes and lung. Lymphatic
endothelial cells promote the migratory activity of a CD133+ subset to target organs and regulation of lymphatic
growth efficiently modulates the metastasis of CD133+ tumor cells. We found that lymphatic vessels in
metastatic tissues stimulate CXCR4+/CD133+ cell metastasis to target organs by secretion of SDF-1. The
CXCR4+/CD133+ cells exhibited higher metastatic activity compared to CXCR4-/CD133+ cells and,
importantly, blockade of CXCR4 coupled with dacarbazine efficiently inhibited metastasis; dacarbazine alone
could not attenuate tumor metastasis. The current study demonstrates a previously unidentified role of the
lymphatic microenvironment in facilitating metastasis of chemo-resistant melanoma cells via a specific
chemotactic axis - SDF-1/CXCR4. Our findings suggest that targeting the SDF-1/CXCR4 axis in addition to
dacarbazine treatment could therapeutically block chemo-resistant CD133+ cell metastasis toward a lymphatic
metastatic niche.
Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea
Minah Kim、Jun Yeop Lee、Kyung Min Choi、Gou Young Koh
Regulation of Chemo-resistant Melanoma Cell Metastasis by a Lymphatic
Metastatic Niche: a Key Role of CXCR4 Signaling
14：10〜15：00
Index
Aⴊ▤↢‛කቇળKPFD
Poster
139
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Symposium
糖尿病網膜症などの虚血性網膜症の進行には, 網膜血管新生が深く関与していることが知られてい
る。最近, 本疾患に対して従来のレーザー治療に加えて, VEGF中和抗体の硝子体内投与が行われ始め
た。このVEGF中和抗体は, 虚血性網膜症に対して効果的な治療効果を示す一方で, その効果が一過性
であることや, 全身性副作用がみられること, VEGF中和抗体に反応しない新生血管があることなどの
問題が指摘されている。そのため, より効果的な治療法の開発にはこれら以外の新しい標的分子の同
定が必要である。そこで今回,著者らが網膜血管形成に関与することを見出したアペリンが虚血性網
膜症モデルにおける病的な網膜血管新生に関与するか否かを検討した。マウス酸素誘発虚血性（OIR）
モデルは, 生後7日齢（P7）から5日間75％酸素条件下で飼育することで網膜血管形成を抑制し, その後,
通常大気条件下で5日間飼育することで網膜虚血領域に向かう病的な網膜血管新生を誘導するモデル
である。病的血管新生が生じる時期であるP15およびP17の網膜において, VEGFやエリスロポエチン
（Epo）の発現上昇がみられたが, これらの既存の血管新生因子よりもアペリンの発現が最も劇的に上
昇していた。受容体APJはP15において血管新生を伴っている血管網に発現していること, さらに網膜
から硝子体に侵入したAPJ陽性細胞の多くに BrdUが取り込まれていた。また, アペリン遺伝子欠損
（KO）マウスを用いたOIRモデルでは, 野生型マウスと比較して有意に網膜血管新生が抑制され, さら
に, 硝子体内に侵入する異常細胞数およびBrdU陽性細胞数も有意に低下していた。また, アペリンKO
マウスを用いたOIRモデルの網膜における遺伝子発現解析を行った結果, P15およびP17においてVEGF
やEpoの発現は野生型マウスよりも有意に高いこと, およびそれらの受容体（VEGFR1,VEGFR2,
EpoR）の発現は, 両遺伝子型間で大きな差はみられないことを見出した。以上の結果から, アペリン/
APJシステムは虚血性網膜症モデルにおいて血管内皮細胞の増殖を介して網膜血管新生を誘導してい
ることが示唆され, 虚血性網膜疾患の効果的な治療標的となる可能性が示唆された。
摂南大学薬学部薬物治療学、2大阪大学大学院医学系研究科眼科学、3大阪大学大学院薬学研究科神経薬理学
How to write
Plenary Lecture
Nature Medicine
1
Special Talk
笠井 淳司1、石丸 侑希1、吉岡 靖啓1、山室 晶子1、五味 文2、新谷 紀人3、馬場 明道3、
前田 定秋1
Special
Lecture
Apelin is a crucial factor for hypoxia-induced retinal angiogenesis
血管網と神経網は、秩序ある分岐パターンを形成し、多くの部位において並走し生命活動を担ってい
る。近年、血管発生及び神経発生の両者に関与する共通分子が報告され、分子レベルにおいて密接に
連関している可能性が示唆されている。我々は、これまでにES細胞を用いて中胚葉マーカーである
VEGF 2型受容体（Flk1）陽性血管前駆細胞より血管内皮細胞を分化誘導するシステムを構築した
(Yamashita, Nature, 2000)。さらに、VEGF及びcAMPシグナルの活性化により内皮細胞分化の促進及び
動脈内皮細胞の誘導が起こる分子機構を明らかにした (Yamamizu, Blood, 2009; J Cell Biol, 2010)。最近、
我々はcAMPシグナルを抑制的に制御すると考えられるKappaオピオイド受容体 (KOR) が血管細胞に
高発現していることを見出した。本研究では神経機能に重要な役割を果たすオピオイドの血管発生・
新生における新しい役割を検討した。ES細胞由来血管前駆細胞へのKORアゴニストであるU50,488H
及びTRK-820の添加は血管内皮細胞分化及び三次元的血管形成を抑制した。KORの活性化は、
VEGF-A発現には影響を及ぼさなかったがVEGF-A受容体であるFlk1及びNeuropilin1の発現を抑制し
た。8-bromo-cAMP及びPKA選択的アゴニストである6-Bnz-cAMPの添加はKORの活性化による内皮細
胞分化の抑制効果を消失させた。我々はさらにKOR及び内因性KORアゴニストであるダイノルフィ
ンの2種類のノックアウト（KO）マウスを用いた検討を行った。胎生10.5日の両KOマウス胎仔にお
いて血管発生・新生が有意に亢進していることをFACS及び免疫染色にて明らかにした。また、両KO
マウス胎仔の体節において異所性の血管の侵入が観察された。KOマウス胎仔から純化した血管内皮
細胞においてFlk1及びNeuropilin1の発現亢進が認められ、一方、血管走行を調節するPlexinD1の発現
が減少していることを見出した。これらの結果より血管発生及び血管走行の両者を負に制御するオピ
オイドは新規血管新生抑制因子であると考えられた。本研究は、血管発生学に新たな知見を与えると
共にオピオイド薬を用いた腫瘍血管新生抑制による抗がん治療など新たな治療戦略の開発に寄与す
ることが期待される。
京都大学再生医科学研究所幹細胞分化制御研究領域、2京都大学iPS細胞研究所 増殖分化機構研究部門、3星薬科大学薬品毒性
学教室、4北里大学生命薬化学研究室
1
山水 康平1,2、古田 貞由3、片山 志織1,2、成田 道子3、葛巻 直子3、今井 哲司3、長瀬 博4、鈴木 勉3、
成田 年3、山下 潤1,2
血管内皮細胞分化および血管走行制御におけるオピオイドの役割
（日本語）
President
Lecture
YJ-2
YJ-1
YIA審査発表
（日本人対象）
Y.I.A. Presentation (for Japanese nominees)
12月3日
（金）
／Friday, December 3
Program
Aⴊ▤↢‛කቇળKPFD
140
YJ-4
YJ-3
【背景】近年の疫学研究から糖尿病がアルツハイマー病（AD）の強力な危険因子であることが報告さ
れ注目を集めている。しかしその背景にあるメカニズムは依然不明である。本研究ではこの背景病態
を解明することを目的とし、両疾患の病態を反映する新規病態合併モデルマウスを確立しその解析を
行った。【方法】ADモデルTgマウス（APP23）と2種類の糖尿病マウス（ob/ob、NSYマウス）との掛
け合わせをそれぞれ行い、APP-ob/obマウスとAPP-NSYマウスという2系統の合併モデルマウスを確
立した。これらのマウスの代謝、及び神経機能を経時的に評価した。【結果】APP-ob/obマウスでは、
糖尿病の発症とともにADマウスの認知機能障害が顕著に重篤化し、8週齢という従来のADマウスと
比較して非常に早い時期に既に有意な認知機能障害が見られた。この時、神経毒性物質である脳内A
β量を評価したところ合併マウスと通常のAPP23マウスの間で差は見られなかったが、興味深いこと
に合併マウスでは脳血管におけるAβ蛋白の沈着が有意に亢進していた。また脳血管におけるRAGE
の発現や血管壁の炎症反応の亢進が観察され、認知機能障害増悪の原因の一つになっていることが示
唆された。またAPP-ob/obマウスでは神経細胞におけるインスリン抵抗性や脳内インスリン量の低下
が見られ、脳内インスリンシグナルに障害をきたしている可能性が示唆された。APP-NSYマウスで
も同様に記憶力障害の重症化が観察されたが、この時脳内Aβ量にはやはり変化が見られず、脳血管
における炎症反応の亢進や脳内神経細胞におけるインスリンシグナルの障害が観察された。一方で、
APP-ob/ob合併マウスはob/obマウスと比較してより高度の糖尿病症状を呈していた。糖負荷試験でも
より高度の耐糖能異常が観察され、これは主に高度のインスリン抵抗性に起因していた。APP-NSY
マウスも同様に、NSYマウスと比較してより高度の耐糖能異常を呈していた。【結論】糖尿病がADの
発症リスクを上昇させる機序として脳血管病態や脳内インスリンシグナルの障害が関与しているこ
と、又合併マウスに見られた糖尿病病体の重篤化から、両疾患が互いの病態を増悪しあう関係にある
可能性が示唆された。
大阪大学大学院医学系研究科臨床遺伝子治療学、2大阪大学大学院医学系研究科老年腎臓内科、3医薬基盤研究所生物資源研究
部実験動物開発研究室
1
武田 朱公1,2、里 直行1,2、内尾-山田 こずえ3、楽木 宏実2、森下 竜一1
Diabetes accelerates memory dysfunction via cerebrovascular dysfunction in
an Alzheimer mouse model with diabetes
Proangiogenic growth factors and receptor tyrosine kinases (RTKs) have crucial roles for angiogenesis. Grb2associated binder (Gab) docking proteins have been reported to coordinate RTKs-dependent activation of
ERK1/2 and AKT. However, the roles of Gab proteins in angiogenesis remain elusive. Therefore, we aim to
elucidate the roles of Gab1 and Gab2 expressed in the endothelium for postnatal angiogenesis. We analyzed a
hindlimb ischemia (HLI) model in control, endothelium-specific Gab1 knockout (Gab1ECKO), or Gab2
conventional knockout (Gab2KO) mice. Intriguingly, impaired blood flow recovery and necrosis was observed
in all of Gab1ECKO mice, but not in control or Gab2KO mice. Among various proangiogenic factors,
hepatocyte growth factor (HGF) induced the most prominent tyrosine phosphorylation of Gab1 and subsequent
complex formation of Gab1 with tyrosine phosphatase SHP2 and PI3K subunit p85 in HUVECs. Gab1 was
required for HGF-induced activation of ERK1/2, ERK5, and AKT and subsequent migration of endothelial
cells. Using DNA microarray analysis, we found that the stimulation with HGF upregulated the expression of
Kruppel-like factor 2 (KLF2), a crucial transcriptional factor for endothelial maintenance through interaction of
Gab1 with SHP2 in HUVECs. HGF-induced upregulation of KLF2 was mediated by MEK5-ERK5 pathway.
Furthermore, the expression of KLF2 in the endothelium was significantly decreased in the Gab1ECKO,
compared with control mice after HLI. Consistent with these findings, intramuscular injection of HGF
expression plasmid improved either doppler flow intensity or capillary density score in control, but not in
Gab1ECKO mice. Collectively, these data indicate that Gab1 is essential for HGF-dependent endothelial
signaling and angiogenesis after ischemia.
大阪大学大学院医学系研究科循環器内科、2東京大学先端科学技術研究センターシステム生物医学ラボラトリー、3大阪大学大
学院医学系研究科臨床遺伝子治療学、4大阪大学微生物病研究所情報伝達分野、5国立循環器病研究センター研究所細胞生物学部
1
塩山 渉1、中岡 良和1、樋口 香織1、南 敬2、谷山 義明3、木戸屋 裕康4、内藤 尚道4、高倉 伸幸4、
森下 竜一3、児玉 龍彦2、望月 直樹5、小室 一成1
Docking protein Gab1 is essential for postnatal angiogenesis after ischemia
via HGF/c-Met signaling
How to write
Plenary Lecture
Nature Medicine
Symposium
Y.I.A.
Presentation
Selected Oral
Presentation
Poster
Index
141
Special Talk
The 8th Korea-Japan Joint Symposium on Vascular Biology
Special
Lecture
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
目的；アンドロゲン低下は心血管病リスクとなる可能性が示唆されているが、アンドロゲンの心血管
保護効果の作用機序はいまだ十分に解明されていない。そこで我々は、アンドロゲンの標的臓器への
作用の大部分がアンドロゲン受容体（AR）を介していることに着目し、AR欠損マウスを用い、心血
管系におけるアンドロゲンーARシステムの意義について検討を行った。現在までに、ARはアンジオ
テンシンII負荷による心血管リモデリングに保護的に作用することを見出した。本研究では、心血管
病の重要な要素である虚血に対する組織保護や血管新生に、アンドロゲンがどのように関与するのか
を検討した。方法；25週齢のAR欠損雄マウス（ARKO）と野生型雄マウス（WT）に下肢虚血モデル
を作成し、虚血回復過程の表現型、虚血筋の組織学的検討、虚血筋での血管新生制御因子およびアポ
トーシス制御因子の変化を検討した。また骨髄由来血管内皮前駆細胞（EPC）の下肢虚血後の表現型
への関与について骨髄移植モデルで検討し、ARKOの血管新生能をex vivoで評価した。さらに血管内
皮細胞(HUVEC)を用い、ARシグナルの虚血における役割を検討した。結果；下肢虚血術後、WTと
比較してARKOは血流回復が著しく障害され、高頻度に下肢脱落を生じ、虚血大腿筋における毛細血
管密度の低下を認めた。また、ARKOではWTに比較して、虚血直後の骨格筋でアポトーシスを示す
細 胞 が 増 加 し、Bcl2/Bax比 が 低 下、 さ ら にcell survivalやangiogenesisに 重 要 な 因 子 で あ る Erk1/2、
Akt、eNOSのリン酸化の低下、VEGF受容体であるKdrの発現低下、血管新生抑制因子Bai-1の発現増
加を認めた。Aortic ringやマトリゲルを用いたex vivoの検討ではARKOマウスの血管新生能は減弱し
ていた。さらにARKOマウスの骨髄を野生型で置換した上で下肢虚血術を施行したところ、ARKOマ
ウスの表現型には改善を認めず、アンドロゲンによる血管新生促進作用はEPCに依存しないことが示
唆された。in vitroでは、HUVECのAR knockdownにより、VEGF刺激によるAkt-eNOS経路の活性化が
障害された。結論；アンドロゲン-AR系は、虚血に対するcell survivalやangiogenesisに重要な役割を担っ
ていることが示された。
徳島大学大学院ヘルスバイオサイエンス研究部生体情報内科学、2徳島大学大学院ヘルスバイオサイエンス研究部薬理学、3徳
島大学大学院ヘルスバイオサイエンス研究部循環器内科学
1
吉田 守美子1、粟飯原 賢一1、上田 由佳1、池田 康将2、岩瀬 俊3、赤池 雅史3、佐田 政隆3、
松本 俊夫1
アンドロゲン受容体は虚血後骨格筋細胞のアポトーシスと血管新生を制御する
President
Lecture
Aⴊ▤↢‛කቇળKPFD
YJ-5
Program
Aⴊ▤↢‛කቇળKPFD
142
AF-03
AF-02
16：50〜18：30
Red blood cell transfusion therapy administered on a long-term, recurrent basis is a mainstay in the treatment of
several chronic congenital and acquired anemias. However, patients receiving such transfusions may
consequently develop transfusional hemosiderosis, a major complication of iron overload that sometimes results
in heart failure. To determine the iron amount in the tissue is essential for the physicians to care these patients.
In this work, a high Tc superconucting quantumtinterference devices (SQUID) biosusceptometry with a
scanning coil probe was developed. The measurement principal was based on the AC susceptibility of the
magnetic ferrous particles, and the low noise of 8 pT/Hz 1/2 at 400 Hz was characterized in an unshielded
environment. To demonstrate the in-vivo and quick examining feasibility of this instrument, dextran-coated
magnetic iron nano-particles of 50 nm in diameter were administrated intravenously into the Wistar male rats.
The iron level in the heart and liver regions could be chronologically measured to demonstrate the biological
behavior of the injected magnetic nano-particles in the animals. In conclusion, this novel method could quickly
measure the tissue iron amount in an in-vivo manner.
Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan、2Department of Internal
Medicine, E-Da Hospital, Kaohsiung, Taiwan、3Institute of Electro-optical Science and Technology, National
Taiwan Normal University, Taipei, Taiwan、4Department of Physics, National Taiwan University, Taipei, Taiwan
1
Chau-Chung Wu1、○Wei-Kong Tseng2、Jen-Jei Chieh3、Shei-Yeh Yang3、
Hen-Er Horng3、Hong-Chang Yang4
A novel method with scanning SQUID biosusceptometry to in-vivo determine
the iron amount in the tissue
Background Coronary vasospasm (CVsp) has been reported to be an inflammatory disease, reflected by
elevated high-sensitivity C-reactive protein (hs-CRP). We investigated the interactions among gender, age,
hypertension, and hs-CRP in patients with CVsp.Materials and methods We retrospectively examined 722
Taiwanese patients with or without CVsp during an 8-year period. None of the patients had obstructive coronary
artery disease. Serum hs-CRP levels were examined in a subset of 375 patients to evaluate the interactions of hsCRP with gender, age, smoking, and hypertension in the development of CVsp.Results In women, only the
highest hs-CRP tertile (larger than 3 mg L-1) was independently associated with CVsp. In men, age older than
58 years and the highest hs-CRP tertile were independently associated with CVsp. In women, elevated risk was
only demonstrated in patients younger than or at 58 years with hs-CRP levels in the highest tertile. In men, a
positively monotonic trend was demonstrated between hs-CRP levels and CVsp in those older than 58 years.
The odds ratios of CVsp in both women and men with hs-CRP in the highest tertile reduced from 6.01 to 1.48
and 6.35 to 2.69, respectively, if they had hypertension.Conclusion The relationship between hs-CRP and CVsp
differed between men and women. Our findings that there is a non-threshold model in men and a threshold
model in women provide evidence that more smokers in men (life-style) and age (induction time) contribute to
the natural history of CVsp development. The negative effect of hypertension on CVsp suggests that the
pathogenesis of CVsp differs from that of coronary atherosclerosis.
2
Section of Cardiology, Department of Medicine, Taipei Medical University-Shuang Ho Hospital, Taipei, Taiwan、
Chang Gung University, Taoyuan, Taiwan、3Section of Cardiology, Department of Medicine, Chang Gung
Memorial Hospital at Keelung, Chang Gung University College of Medicine, Keelung, Taiwan
1
MingYow Hung1,2、Kuang-Hung Hsu2、Ming-Jui Hung2,3
Interactions among gender, age, hypertension, and C-reactive protein in
coronary vasospasm
優秀演題口述発表
Selected Oral Presentation
12月2日
（木）
／Thursday, December 2
Index
Aⴊ▤↢‛කቇળKPFD
Poster
143
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Symposium
Cyr61 (CYstein-Riched angiogenic inducer 61) is known to a secreted, extracellular matrix-associated, and
angiogenic regulator. Cyr61 regulates endothelial cell adhesion, migration, proliferation, survival and death by
binding to some types of integrins and heparan sulfate proteoglycans in a cell type-dependent manner. However,
specific roles of cyr61 in vascular development of zebrafish have remained largely unknown. Cyr61 was
expressed at the hypochord and notochord in zebrafish trunk region during embryo development. Using of Tg
(flk1:EGFP) zebrafish and in situ hybridization with flk-1, we assessed the effect of cyr61 knockdown on
vascular development with its morpholino antisense oligomer. Silencing of cyr61 induced ectopic expression of
flk-1 and defects of sprouting in intersegmental vessels (ISVs). Multi-directional migration and incorporate
network between microvessels were observed in cyr61-knockdown embryos. Silencing of cyr61 by siRNA
reduced vascular endothelial growth factor (VEGF)-induced tube formation in human umbilical vein endothelial
cells. Treatment of recombinant human cyr61 increased the tube formation which was blocked by the treatment
of VEGF neutralizing antibody, suggesting a VEGF involvement in cyr61 function. Cyr61 also stimulated
endothealial cell sprouting form cultured aortic rings through the activation of VEGF. Taken together, cyr61
mediates vascular development of zebrafish and activates endothelial cell sprouting through VEGF signaling.
How to write
Plenary Lecture
Nature Medicine
School of Life Sciences and Biochnology, college of Natural sciences, Kyungpook National University, Korea
Special Talk
Ae Kyoung Kim、Sun Hye Shin、○You Mie Lee
Special
Lecture
Cyr61 promotes endothelial cell sprouting through the regulation of VEGF
signaling in zebrafish vascular development
Vascular calcification (VC) is prevalent in patients with chronic kidney disease (CKD) as well as
atherosclerosis, aging and diabetes. Aortic calcification significantly increases the risk of cardiovascular
morbidity and mortality by promoting arterial wall stiffness and a progressive decline of compliance of the
blood vessels. Dimethyl fumarate (DMF) has been used to treat psoriasis and induces the antioxidant
transcription factor Nrf-2 pathway and subsequent antioxidative and phase II detoxifying enzyme. This study
was undertaken to examine the effect of DMF, an anti-psoriatic agent on VC. VSMC calcification was induced
by inorganic phosphate (Pi) and DMF significantly diminished the Pi-induced calcification in a time and dose
dependent manner, as demonstrated by decreased calcium deposition visualized by von Kossaʼs staining and
determined by measurement of calcium contents. Transient transfection assay showed that DMF repressed
several osteogenic gene promoters including Runx2, osteocalcin, alkaline phosphatase (ALP), and bone
morphogenic protein-2 (BMP-2). DMF increased protein levels and nuclear localization of Nrf-2, whereas it did
not change mRNA level of Nrf-2, suggesting that DMF stabilized Nrf-2 at the posttranslational levels but not
transcriptional levels. Moreover, Nrf-2 repressed both BMP-2 gene promoter and BMP-2-stimulated BRE
activity, suggesting that Nrf-2 directly inhibits downstream signaling pathway of BMP-2. finally, using an
animal model of calcification induced by excessive dose of vitamin D3, we demonstrated that DMF significantly
reduced vitamin D3-induced calcification of whole aorta. Taken together, these results suggest that DMF inhibits
VC through Nrf-2-dependent repression of BMP-2 expression and its downstream signaling pathway, leading to
suppression of several osteogenic genes. This study also provides a molecular basis of potential use of DMF as
a therapeutic agent for the prevention and treatment of VC.
Department of Endocrinology, Kyungpook National University Hospital, Daegu, South Korea
Ji-Hyun Kim、Young-Keun Choi、Han-Jong Kim、Joon-Young Kim、In-Kyu Lee
Protective Effect of Dimethyl Fumarate against Vascular Calcification
President
Lecture
AF-05
AF-04
Program
Aⴊ▤↢‛කቇળKPFD
144
AF-07
AF-06
Correspondence and requests for materials should be addressed to Goo Taeg Oh. e-mail:[email protected]
Atherosclerosis is a chronic inflammatory disease that both innate and adaptive immunity are involved. The
tumor necrosis factor receptor superfamily (TNFRSF) as an important T cell costimulatory molecule, such as
CD40, LIGHT, and OX40, play important roles in atherosclerosis. CD137 (4-1BB), a member of the TNFRSF,
has been reported to be expressed in human atherosclerotic lesions. However, the precise role of CD137 in
atherosclerosis and the effects of blocking CD137/CD137 ligand (CD137L) signaling on lesion formation are
not known yet. To investigate the role of CD137 in the pathogenesis of atherosclerosis, we generated CD137deficient Apolipoprotein E knockout mice (ApoE -/-CD137-/-) and LDL receptor knockout mice (Ldlr-/-CD137-/-).
The deficiency of CD137 induced a reduction in atherosclerotic plaque lesions in both atherosclerosis mouse
models, attributed to the downregulation of cytokines such as interferon-gamma; (IFN-㱏), monocyte
chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-㱍). CD137 signaling promoted the
production of inflammatory molecules including MCP-1, interleukin-6 (IL-6), vascular cell adhesion molecule-1
(VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in endothelial cells. Moreover, stimulation of
CD137L signaling activated monocytes/macrophages and increased the production of proinflammatory
cytokines in atherosclerotic vessels. Therefore, CD137/CD137L signaling plays multiple roles in the progression
of atherosclerosis, and blocking of this pathway can be a valuable therapeutic target for the disease.
Division of Life and Pharmaceutical Science, Ewha Womans University, Seoul, Korea
、2Department of
Veterinary Pathology, College of Veterinary Medicine, Seoul National University, Seoul, Korea 、3Laboratory of
Cellular Physiology and Immunology, The Rockefeller University, New York, USA 、4R & D Center for Cancer
Therapeutics, National Cancer Center, Ilsan, Korea
1
In-Hyuk Jung1,2、Jae-Hoon Choi1,3、Hyung Jun Jeon1、Dae-Yong Kim2、
Byoung S. Kwon4、Goo Taeg Oh1
CD137/CD137L (4-1BB/4-1BBL) Signaling Facilitates Atherosclerosis through
Combined Actions on Immune and Endothelial Cells in Hyperlipidemic Mice
Background: Increased Rho kinase activity is associated with cardiovascular risk factors and some of the
beneficial, non-cholesterol effects of statins may be due to their ability to decrease Rho kinase (ROCK) activity.
Previous studies suggest that statin can improve endothelial function and increase the number of circulating
EPCs. However, the role of ROCK inhibition in mediating the effect of statins on EPC function is still unknown.
Methods and Results: 24 patients with dyslipidemia were randomized to 12 weeks of treatment with
simvastatin (5-10 mg/day) or a cholesterol absorption inhibitor, ezetimibe (10 mg/day). Flow-mediated dilation
(FMD) and ROCK activity from peripheral leukocytes were determined. EPCs were analyzed by flow
cytometry. Although simvastatin and ezetimibe reduced LDL cholesterol to a similar extent (-15.8 ± 6.8 vs.
-16.1 ± 7.9%; P=0.32), there was substantial difference in mevalonate levels between the groups (simvastatin,
-1.04 ± 0.62 vs. ezetimibe, 1.79 ± 0.94 ng/mL; P<0.05). The change in mevalonate levels corresponded
inversely to changes in FMD (simvastatin, 3.5 ± 1.6 vs. ezetimibe, 1.1 ± 1.7%; P<0.01), suggesting that
decreased isoprenoids rather than cholesterol mediates FMD. Indeed, leukocyte ROCK activity, which is
dependent upon isoprenoids, was reduced after treatment with simvastatin but not ezetimibe (-30.2 ± 16.7 vs.
-6.2 ± 4.5%; P<0.01). Furthermore, decreased ROCK activity correlated with improvement in FMD and
increased number of EPCs (R2 = 0.36, P<0.05).
Conclusions: Despite equal reduction in LDL cholesterol, simvastatin is superior to ezetimibe in terms of
improvement in endothelial function and increased number of EPCs. The improvement in FMD and EPC
functions correlated with decreased ROCK activity by simvastatin but not ezetimibe, suggesting a noncholesterol effect of statin.
2
Division of Cardiology, Internal Medicine, National Cheng Kung University Hospital, Taiwan, Tainan、
Department of Biochemistry and Molecular Biology, National Cheng Kung University, Taiwan, Tainan、
3
Vascular Medicine Research, Division of Cardiology, Brigham & Womens Hospital, Harvard Medical School,
Boston, MA, USA
1
Ping-Yen Liu1,3、Ting-Hsing Chao1、Wei-Ting Lee1、Yun-Ling Tai1、Liang-Miin Tsai1、
Jyh-Hong Chen1、Hua-Lin Wu2、JK Liao3
Improvement in endothelial function and endothelial progenitor cell number
by statins in dyslipidemic subjects correlate with decreased ROCK activity
Index
Aⴊ▤↢‛කቇળKPFD
Poster
145
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Symposium
To compare the change in lipoprotein metabolism with aging, we analyzed the lipid and protein compositions of
individual lipoprotein fractions. Healthy and non-obese elderly subjects (elderly group, n=26) had a serum lipid
profile within the normal range, although slightly higher than in young subjects (control group, n=18). However,
the elderly group had a 2-fold higher serum uric acid level and triglyceride (TG)/high-density lipoprotein
(HDL)-cholesterol ratio. The elderly group had less antioxidant ability and elevated TG content in HDL with
enhanced cholesteryl ester transfer activity. An elevated level of advanced glycated end products in lipoproteins
and fragmentation of apoA-I were present in the elderly group, with detected lower apoA-I level and more
multimerized apoA-I in HDL. The protein levels of apoA-I, apoC-III, and serum amyloid A in lipoproteindeficient serum were increased in the elderly group. HDL from the elderly (E-HDL) had increased glycation
with modification of apolipoprotein (apo) A-I. Glycated apoA-I (gA-I) by fructosylation showed covalently
multimerized band without crosslinker and impaired phospholipid binding ability. Treatment of human dermal
fibroblast (HDF) cells and macrophages with E-HDL and glycated apoA-I (gA-I) caused severe premature
cellular senescence and foam cell formation, respectively; however, treatment with HDL from a young group
(Y-HDL) and native apoA-I (nA-I) suppressed senescence and atherosclerosis.
How to write
Plenary Lecture
Nature Medicine
School of Biotechnol, Yeungnam University, Gyeongsan, Korea
Special Talk
Kyung-Hyun Cho
Special
Lecture
Age-associated modification of high-density lipoprotein with an elevated level
of advanced glycated end products and glycated apolipoproteinA-I
exacerbated cellular senescence
Background: We have previously demonstrated the feasibility of biofunctionalized magnetic nanoparticles with
anti-vascular cell adhesion molecule-1 (VCAM-1) to magnetically label and locate in vivo the vulnerable aortic
lesions of hypercholesterolemic rabbits with the aid of magnetic resonance imaging (MRI). Purpose of the
study: The purpose of this study was to find the adequate timing for MRI acquisition after the intravenous
injection of these anti-VCAM-1 nanoparticles.Methods: For an in vivo study on the magnetic labeling of
VCAM-1 in animal models, control (n=9) and hypercholesterolemic rabbits (n=9) were intravenously injected
with the anti-VCAM-1 magnetic nanoparticles (MagQu Co., Taipei). The in vivo magnetic labeling on the aortic
walls was investigated with the aid of a 3T MRI system before, 4 hours, 12 hours, 24 hours, 72 hours, and 7
days after the rabbits received the injection. Immediately after the second MRI examination, the animals were
sacrificed for hematoxylin/eosin and Prussian iron blue staining. Summary of results: Before the injection of
these nanoparticles, no difference in the signal intensity can be observed in the aortic wall between control and
hypercholesterolemic rabbits. Four hours after the injection, we observed darkening of the aortic wall in both
control and hypercholesterolemic rabbits. However, this darkening persisted only in hypercholesterolemic
rabbits at 12 hours and peaked at 24 hours after the injection.Conclusions: These results demonstrated the
potential applications of anti-VCAM-1-functionalzied magnetic nanoparticles for imaging and localizing in vivo
the lesions prone to vulnerable plaque formation, and the best timing for image acquisition was 24 hours after
the injection.
Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan、2Center for
Optoelectronic Biomedicine, College of Medicine, National Taiwan University, Taipei, Taiwan、3Institute of
Electro-optical Science and Technology, National Taiwan Normal University, Taipei, Taiwan、4Department of
Physics, National Taiwan University, Taipei, Taiwan
1
Chau-Chung Wu1、Wen-I-Issac Tseng2、Shei-Yeh Yang3、Hen-Er Horng3、
Hong-Chang Yang4
Biofunctionalized magnetic nanoparticles for imaging the vulnerable aortic
lesions
President
Lecture
AF-09
AF-08
Program
Aⴊ▤↢‛කቇળKPFD
146
AF-10
Mesenchymal stem cells promote tumorigenesis by providing a tumor-supportive microenvironment in vivo.
However, the molecular mechanisms by which mesenchymal stem cells stimulate tumorigenesis are largely
unknown. In the present study, we demonstrate that conditioned medium from A549 human lung
adenocarcinoma cells stimulates expression of angiogenic cytokines including vascular endothelial growth
factor and stromal cell-derived factor-1 in human adipose tissue-derived mesenchymal stem cells (hASCs). In
addition, A549 conditioned medium induces secretion of 㱎ig-h3, an extracellular matrix protein, which is
implicated in tumorigenesis and metastasis, from hASCs. A549 conditioned medium-induced expression of
these protein factors was completely abrogated by treatment of hASCs with Ki16425, a lysophosphatidic acid
(LPA) receptor antagonist, or knockdown of lysophosphatidic acid receptor 1 (LPA1) expression in hASCs with
small interfering RNA or lentiviral short hairpin RNA, suggesting a pivotal role of LPA-LPA1 signaling
pathway in the paracrine activation of mesenchymal stem cells by tumor cells. Using a murine xenograft
transplantation model of A549 cells, we showed that co-transplantation of hASCs with A549 cells stimulated
growth of A549 xenograft tumor and angiogenesis in vivo. Knockdown of LPA1 expression in hASCs abrogated
hASCs-stimulated growth of A549 xenograft tumor and angiogenesis. Moreover, LPA-conditioned medium
from hASCs stimulated adhesion and proliferation of A549 human lung adenocarcinoma cells in vitro and
immunodepletion of 㱎ig-h3 suppressed the LPA conditioned medium-stimulated adhesion and proliferation of
A549 cells. From these results, we suggest a crosstalk mechanism that A549 cells activate mesenchymal stem
cells through an LPA-LPA1-mediated pathway and activated mesenchymal stem cells promote tumor growth
and angiogenesis by secretion of VEGF and 㱎ig-h3.
Medical Research Center for Ischemic Tissue Regeneration, School of Medicine, Pusan National University;
Yangsan, Republic of Korea
Jae-Ho Kim、Il-Hwan Lee、Sang-Hun Shin、Soon-Chul Heo
Paracrine Crosstalk between Mesenchymal Stem Cells and Tumor Cells
Stimulates Tumor Angiogenesis in vivo
Index
Aⴊ▤↢‛කቇળKPFD
Poster
147
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Symposium
アドレノメデュリン(AM)は、強力な血管拡張による降圧作用、血管保護作用、抗酸化ストレス作用、
臓器保護作用をはじめ、多彩な生理作用を有する内因性ペプチド因子として知られている。我々は、
AMホモノックアウトマウス(AM-/-)が血管の発生異常により胎生致死となる事を報告した。AMとそ
のファミリー因子は、同一の受容体CRLRを共用しており、CRLRには受容体活性調節タンパク
（RAMP） が 重 合 す る。RAMPサ ブ ア イ ソ フ ォ ー ム の 中 で も、RAMP2ホ モ ノ ッ ク ア ウ ト マ ウ ス
(RAMP2-/-)では、AM-/-と同様の血管の異常が再現され、胎生致死となる事から、AM-RAMP2系が胎
生期の血管新生に必須であることが明らかとなった。 血管におけるRAMP2の意義を解明するため
に、血管内皮細胞特異的RAMP2ノックアウトマウス(E-RAMP2-/-)と、成体おいてオンデマンドに血
管内皮細胞特異的にRAMP2を欠損させる薬剤誘導性血管内皮細胞特異的RAMP2ノックアウトマウス
(DI-E-RAMP2-/-)を樹立した。E-RAMP2-/-のほとんどは出生直前に致死であり、全身性の浮腫を認め
た。一方で、血管のRAMP2発現が2割程度残存する一部のE-RAMP2-/-では、成体が得られた。成体
のE-RAMP2-/-では、血管壁構造の異常に加え、肝硬変様の形態変化や腎臓の臓器障害が認められた。
さらに、肺、肝臓、腎臓などの主要臓器の血管周囲に、著明な炎症細胞浸潤や、血管において老化相
関β-ガラクトシダーゼ染色陽性所見が認められた。E-RAMP2-/-では得られる成体が限られるため、
次に成体でRAMP2欠損を可能とするDI-E-RAMP2-/-を用いた解析を行った。RAMP2欠損後早期から
著しい体重の増加や血管内皮細胞において接着性因子の発現の亢進、下肢虚血モデルでの血流回復の
遅延を示した。 以上の結果は、AM-RAMP2系が血管新生に必須であり、成体の血管恒常性維持に
も重要な役割を示しており、RAMP2は新たな治療標的分子として期待される。
How to write
Plenary Lecture
Nature Medicine
信州大学大学院医学系研究科臓器発生制御医学講座
Special Talk
小山 晃英、桜井 敬之、神吉 昭子、荒居 琢磨、新藤 優佳、吉沢 隆弘、楊 磊、植竹 龍一、沖村 綾乃、
山内 啓弘、田中 愛、新藤 隆行
Special
Lecture
血管内皮細胞におけるアドレノメデュリン−ＲＡＭＰ２システムの役割
Background: Lipopolysaccharide (LPS) triggers systemic inflammatory response syndrome (SIRS). LPSinduced vascular injury in these conditions results in sepsis and multi organ failure. On the other hand, recently
we reported the anti-oxidative stress effect of hepatocyte growth factor (HGF) on vessels (Hypertens 2009)
through epithelial growth factor receptor (EGFR) degradation by ubiquitin proteasome system (Cir Res 2009).
In this experiment, we tested whether HGF can protect SIRS induced by LPS and investigated its mechanism.
Methods and Results: In vivo, we intravenously injected LPS (1mg) into HGF TG mice with increased HGF
serum concentration or its litter mates. After 24 hours, we evaluated the inflammation on vessel wall. HGF TG
reduced LPS-induced VCAM-1 evaluated by western blotting, and ROS evaluated by DHE staining as
compared to control (P＜0.05) accompanied by significant EGFR degradation (P＜0.05). Moreover, HGF TG
significantly improved survival in LPS injection model, and EGFR inhibitor (AG-1478) also significantly
improved it (P＜0.05). In vitro, HGF significantly inhibited the increased expression of VCAM-1 and EGFR by
LPS in VSMCs (P＜0.05), while the EGFR inhibition by siRNA significantly inhibited them (P＜0.05). HGF
significantly promote SHIP2 translocation from EGFR to c-Met against LPS in VSMCs (P＜0.05). Moreover,
EGFR was combined with ubiquitin E3 ligase, c-Cbl, though SHIP2 translocation from c-Met to EGFR, and
was broken down by proteasome system. In addition, HGF inhibited ROS production by LPS stimulation in
HAECs (P＜0.05).
Conclusion: HGF regulated SHIP2 recruitment to EGFR and inhibited LPS induced inflammation via epithelial
growth factor receptor degradation. This protective effect of HGF may be useful for inflammatory disease.
済生会山口総合病院薬剤部、2大阪大学大学院医学系研究科臨床遺伝子治療学
1
清水 一賢1,2、谷山 義明2、眞田 文博2、家串 和真2、久徳 真梨子2、東 純哉2、楠 博2、岡山 慶太2、
岩林 正明2、アマルナット チャッタッジー2、森下 竜一2
Protective Effects of HGF on LPS Induced Inflammation via EGF Receptor
Degradation by ubiquitin proteasome system
16：50〜18：30
President
Lecture
AJ-02
AJ-01
優秀演題口述発表
Selected Oral Presentation
12月2日
（木）
／Thursday, December 2
Program
Aⴊ▤↢‛කቇળKPFD
148
AJ-04
AJ-03
(目的)vasohibin-1 (VASH1) はVEGFなどの血管新生刺激に反応して、血管内皮細胞が自ら産生・分泌
して血管新生を抑制するネガティブフィードバック調節因子である。 一般に、血管新生抑制因子は
内皮細胞の細胞死を促進して血管を退縮させるが、VASH1の遺伝子を安定導入したマウス内皮細胞
では、血管新生は抑制されるものの細胞死は増加せず、逆にH2O2などの種々の細胞ストレスに対し
て耐性を示す。一方、VASH1 の発現をノックダウンすると内皮細胞の老化がひきおこされ、細胞ス
トレスに対して細胞死が増加した。これらより我々は、VASH1は抗細胞死・抗老化作用など内皮細
胞の恒常性維持にも重要であることを考えている。ヒトVASH1遺伝子は365 アミノ酸からなる通常の
VASH1の他に、エクソン1から4の後に85bpのイントロンが付加される204アミノ酸のsplice variant
vasohibin-1 (v-VASH1)が存在する。しかしその機能には不明な点が多い。本研究ではv-VASH1の内皮
細胞での機能を抗細胞死・抗老化作用を中心に解析したので報告する。 (方法と結果) v-VASH1は
VASH1と同様に内皮細胞においてVEGFやFGF-2で発現が誘導され、初代培養細胞において血管内皮
細胞にほぼ特異的に発現していた。しかし、内皮細胞でVASH1を過剰発現するとVEGFレセプター-1
の発現が低下するが、v-VASH1の過剰発現によってその現象は認めなかった。次に、v-VASH1の抗細
胞死・抗老化作用などを明らかにするためにv-VASH1特異的siRNAとv-VASH1の発現ベクターを準備
した。HUVECにおいてv-VASH1に対するsiRNAでv-VASH1の発現をノックダウンすると、VASH1の
発現が増加し細胞は老化せず細胞ストレスによる細胞死に変化はなかった。(考察) 内皮細胞において
v-VASH1はVEGFで誘導されるなどVASH1と共通する部分も多いが、v-VASH1の発現をノックダウン
しても細胞老化がみられないなど、VASH1とは抗細胞死・抗老化作用において異なる機能が示唆さ
れた。現在、v-VASH1の発現ベクターをマウス内皮細胞株MS1に安定導入し3個のv-VASH1過剰発現
クローンを得ており、学会ではv-VASH1 siRNAとこれらのクローンを用いた解析結果についても言及
したい。
東北大学加齢医学研究所腫瘍循環研究分野
宮下 浩輝、佐藤 靖史
内皮ストレス耐性におけるsplicing variant vasohibin-1の意義
背景：小径の腹部大動脈瘤は外科的治療では生命予後が改善しないとする報告がある。そのため現在
の治療方針は経過観察となっており、早期に発見されたメリットは生かされていない。初期の動脈瘤
壁には薬物や分子治療に反応する多くの細胞を含有しており、これらの細胞をターゲットにした低侵
襲治療法の確立が期待されている。目的：動脈瘤の進展に重要なmatrix metalloproteinases（MMP）な
どのプロテアーゼ群の発現には転写因子NF㱖BとEtsが関与している。この転写因子の活性を競合的
に抑制する核酸医薬にデコイがある。これまでの検討でNF㱖BとEtsに対するキメラデコイは動脈瘤
の縮小効果を示したが、従来型デコイは二本鎖配列の開放端からヌクレアーゼで分解される。そこで
断端を環状に結合した低分解性のリボン型デコイを開発し低侵襲治療法の可能性を検討した。方法：
マクロファージに分化するTHP-1細胞を使い、各種デコイのMMP分泌抑制効果を評価した。またデ
コイの腹腔内投与によるラット動脈瘤モデルの拡大抑制効果を検討した。結果：THP-1細胞への導入
実験では従来型に比べリボン型デコイは有意にMMP-9の分泌を抑制した。動物実験では、腹腔内投
与されたFITC-デコイは動脈瘤壁に浸潤したマクロファージに認めれれている。超音波検査で行った
治療効果の評価ではコントロール群・従来型デコイ群と比較してリボン型デコイ群は有意に動脈瘤の
拡大を抑制した。組織学的検討では、リボン型デコイ群は血管中膜（弾性繊維）の破壊が抑えられて
いた。そのメカニズムとして、マクロファージの浸潤はコントロール群と同程度で影響を与えていな
いが、マクロファージが分泌するプロテアーゼであるMMP-9、MMP-12、カゼプシンBそしてカゼプ
シンKの分泌抑制を認め、マクロファージの機能面での抑制効果と考えられた。結語：NF㱖BとEtsに
対する低分解性のリボン型デコイは腹腔内投与で浸潤するマクロファージに導入することが可能で
瘤壁内でのプロテアーゼ分泌を抑制して有効な動脈瘤進展抑制効果を示した。
大阪大学連合小児発達学研究科健康発達医学、2大阪大学医学系研究科臨床遺伝子治療学
1
三宅 隆1、島村 宗尚1、中神 啓徳1、森下 竜一2
核酸医薬の全身投与による転写因子NF-kappaBとEtsの抑制が腹部大動脈瘤に及ぼ
す影響
Index
Aⴊ▤↢‛කቇળKPFD
Poster
149
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Symposium
Introduction: Mental stressors contribute to the occurrence of cardiovascular disease. Meanwhile, adipose
inflammation evokes exacerbation of prothrombotic state. Hypothesis: We hypothesized that chronic restraint
stress (RS) worsens the inflammatory reaction in adipose tissue, resulting in a procoagulant state.Methods:
Twelve male mice were subjected to daily RS for 2 weeks. Inguinal white adipose tissue (WAT) and skeletal
muscle were collected from control (n=12) and stressed mice. The expression of macrophage markers,
proinflammatory cytokines, adiponectin, and coagulation factors in WAT were analyzed with real time RT-PCR
method. Macrophage/monocyte accumulation in WAT was assessed with CD11b staining. Results: The RS
reduced body weight gain (stressed vs. control (g); 22.6 ± 1.8 vs. 25.0 ± 2.4, p＜0.05) and WAT weight (mg)
(223.9 ± 91.3 vs. 137.0 ± 74.7, p＜0.05). The accumulation of CD11b-positive cells was significantly
increased in WAT from the stressed mice (%positive cells in total cells; stressed vs. control; 25.2 ± 2.3% vs.
14.0 ± 2.9%, p＜0.01). The expression of F4/80 and CD68 was also increased in WAT significantly (% of
control F4/80 and CD68; 213 ± 8.5%, and 319 ± 32%, respectively, p＜0.01). Furthermore the RS strikingly
increased the expression of proinflammatory cytokines (TNF-α, MCP-1, and IL-6; % of control; 732 ± 112%,
440 ± 39.1%, and 493 ± 30.7%, respectively, p＜0.01), coagulation factors (PAI-1 and TF; % of control; 998
± 67.3% and 494 ± 23.1%, respectively, p＜0.01) in WAT, but did not alter that of adiponectin. Conclusions:
RS augmented adipose inflammation to promote a procoagulant state. Thus, mental stress would play critical
roles in the occurrence of cardiovasucular diseases via adipose-derived inflammatory cytokines.
How to write
Plenary Lecture
Nature Medicine
名古屋大学大学院医学系研究科循環器内科学
Special Talk
内田 恭寛、竹下 享典、菊地 良介、室原 豊明、山本 晃士
Special
Lecture
Chronic restraint stress evokes adipose tissue inflammation to promote
procoagulant state in a murine model.
Background Oxidative stress, generated by excessive reactive oxygen species (ROS), promotes cardiovascular
disease. However, specific therapeutic target still remains to be elucidated. A particularly important mechanism
for ROS-mediated cardiovascular disease appears to be stimulation of pro-inflammatory events. It has become
clear that increases in ROS represent a common pathogenic mechanism for abdominal aortic aneurysm (AAA)
and atherosclerosis. Methods and Results Cyclophilin A (CyPA) is a proinflammatory cytokine that is secreted
from vascular smooth muscle cells (VSMC) in response to ROS in mice and humans. Based on the role of CyPA
in ROS signaling we hypothesized that CyPA would promote cardiovascular diseases. First, we confirmed that
CyPA plays a crucial role in VSMC proliferation and inflammatory cell migration, resulting in the flowmediated vascular remodeling and intima formation in mice (Circulation 2008). Second, we demonstrated that
CyPA plays a crucial role for AAA formation and rupture in mice and humans (Satoh K, et al. Nat Med. 2009).
Third, our recent study provided strong mechanistic evidence of synergy between CyPA and AngII to increase
ROS generation. Since ROS stimulate myocardial hypertrophy, matrix remodeling, and cellular dysfunction,
CyPA contributed to the AngII-induced cardiac hypertrophy. Fourth, we observed strong expression of CyPA
just beneath the thin fibrous cap in atherosclerotic coronary arteries from patients with myocardial infarction.
CyPA is a novel cytokine which is a newly discovered class of ROS mediators that we term SOXF for Secreted
Oxidative stress induced Factors. Conclusion CyPA is a novel mediator of cardiovascular diseases and plays as
an important SOXF in vessels that contributes to cardiovascular diseases.
東北大学大学院・医学系研究科・循環器病態学分野、2ロチェスター大学心血管研究所
1
佐藤 公雄1、福本 義弘1、清水 亨1、鈴木 秀明1、杉村 宏一郎1、三浦 裕1、建部 俊介1、宮道 沙織1、
高橋 潤1、武田 守彦1、安田 聡1、Bradford C. Berk2、下川 宏明1
Cyclophilin A Is a Novel Biomarker for Cardiovascular Diseases
President
Lecture
AJ-06
AJ-05
Program
Aⴊ▤↢‛කቇળKPFD
150
AJ-08
AJ-07
【背景・目的】我々は種々の腫瘍モデルにおいて、組換センダイウイルスベクターによって活性化さ
せた樹状細胞(rSeV/DC)を用いることで、旧来法と比較して著しく高い抗腫瘍免疫効果を誘導可能で
あることを証明してきた。悪性腹水（MA: malignant ascities）を伴った進行消化器癌や卵巣癌は、免
疫療法を含む既存の治療に対して高い抵抗性を示す。血管内皮細胞増殖因子VEGFは当初腹水より単
離され、強力な血管透過性を示すことから、血管透過性因子（VPF）とも呼ばれるが、抗腫瘍免疫に
おけるVEGF/VPFの機能は明らかになっていない。本研究では、腹水の抗腫瘍免疫誘導に対する抵抗
性の原因を明らかにするため、マウス大腸癌腹膜播種MAモデルにおけるVEGFとそのデコイ受容体
である可溶型Flt-1（sFlt-1）の動態と機能について検討した。【方法】腹膜播種モデルとしてBalb/c、
Balb/c nu/nuマウスに大腸癌細胞株CT26を腹腔内投与したものを用い、rSeV/DCを腹腔内へ投与した。
活性化に用いたrSeV/DCは、導入遺伝子を持たないrSeV/DC(null)群と、ヒトsFlt-1を導入発現する
rSeV/DC(sFlt-1)群について検討した。
【結果・考察】既知のごとく腹水内には大量のVEGFが存在した。
皮下腫瘍モデルでのrSeV/DC(null)治療効果と比較し、MAモデルにおいては、rSeV/DC(null)治療に対
して強い抵抗性を示し、一方rSeV/DC(sFlt-1)群では、rSeV/DC(null)群と比較して明らかにMAが抑制
され、生存期間が延長された。この治療効果はBalb/c nu/nuマウスではキャンセルされたことから、
腹水内VEGFは主として抗腫瘍免疫抑制に働くことが明らかとなった。従って、sFlt-1や中和抗体によ
るVEGFの免疫抑制能の解除は、MAの治療成績向上に寄与する可能性があると考えられた。
九州大学薬学研究院革新的バイオ医薬創成学、2九州大学医学研究院消化器・総合外科、3九州大学医学研究院病理病態学、4ディ
ナベック株式会社
1
杉山 雅彦2、吉田 久美1、掛地 吉弘2、原田 結1、鬼丸 満穂3、諸富 洋介2、長谷川 護4、前原 喜彦2、
米満 吉和1,2
活性化樹状細胞の抗腫瘍効果誘導における腹水内VEGFの機能
背景:血管新生は個体発生や創傷治癒、動脈硬化、癌の発育、転移など生理的にも病的にも重要な役
割を果たしている。Rho、Rac、Cdc42といったRhoファミリーの低分子量G蛋白質は血管内皮細胞に
おいて細胞内のシグナル伝達を制御して、細胞の極性形成、遊走、接着、増殖、アクチン細胞骨格形
成などを調節しており、血管内皮細胞の機能制御に重要な役割を担っている。低分子量G蛋白質の活
性化はguanine nucleotide exchange factor (GEF)とGTPase-activating protein(GAP)によって調節されてい
るが、血管内皮細胞におけるRho ファミリー低分子量G蛋白質の制御機構についてはまだ充分には解
明されていない。我々は最近Cdc42のGEFであるFGDファミリーの1つであるFGD5が血管内皮細胞に
選択的に発現していることを見出した。そこで今回は血管内皮細胞におけるFGD5の機能について検
討した。方法・結果:RT-PCR法によりマウスにおけるFGD5の組織発現分布を検討したところ、FGD5
は肺、腎臓、脾臓、卵巣に主に発現していた。同様に培養細胞における発現を検討したところ、内皮
細胞に選択的に発現を認めた。免疫細胞染色法によりヒト臍帯静脈内皮細胞(HUVEC)におけるFGD5
の細胞内局在について検討したところ、細胞辺縁に形成されるラッフル膜にFGD5の集積を認めた。
FGD5の血管内皮細胞における役割について検討するため、HUVECにおいてsiRNAを用いてFGD5の
ノックダウンを行った。FGD5をノックダウンすると、血管内皮増殖因子(VEGF)刺激による内皮細胞
のマトリゲル上でのネットワーク形成や遊走、増殖は有意に抑制された。また、FGD5のノックダウ
ンにより細胞遊走時の極性形成は有意に抑制された。結語:以上より、FGD5はVEGFによる管腔形成、
遊走、増殖、細胞遊走時の極性形成を制御していることが明らかになった。
神戸大学大学院医学研究科循環器内科学
銕 佑介、力武 良行、小林 成美、衣笠 允雄、平田 健一
FGD5によるVEGF誘導性催血管新生能の制御
Index
Aⴊ▤↢‛කቇળKPFD
Poster
151
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Symposium
背景 血管石灰化は糖尿病や慢性腎不全などで発症頻度が高く、冠動脈石灰化は虚血性心疾患の危険
因子の一つである。血管石灰化は血管平滑筋細胞の骨芽細胞様分化転換により生じると考えられてい
るが、その発症の分子機序は不明である。新しくヒト冠動脈平滑筋細胞（HCASMC）での発現を見
出したBMP-binding endothelial regulator (BMPER)の冠動脈における発現様式と平滑筋細胞の分化転換
に及ぼす作用について検討した。方法・結果免疫組織化学法にてBMPERは、内膜肥厚や動脈硬化を伴っ
たヒト冠動脈において内皮とともに新生内膜や中膜の平滑筋に発現しており、特に、微小石灰化を
伴ったプラークの周囲に強く発現していた。免疫細胞化学法にてBMPERはHCASMCのゴルジ体に局
在 し て お り、 ウ エ ス タ ン ブ ロ ッ ト 法 に てBMPERは 培 養 液 中 へ 分 泌 さ れ て い た。BMPERに よ り
HCASMCにおける骨芽細胞マーカー分子であるアルカリホスファターゼ（ALP）やオステオポンチン、
転写因子Runx2のmRNA発現は増加したのに対し、平滑筋細胞マーカー分子であるαSMAやカルポニ
ン1の発現は減少し、骨芽細胞様分化転換の指標であるALP活性は上昇した。反対に、BMPERを
siRNAによりノックダウンすると、骨芽細胞様分化転換はほぼ完全に抑制された。こうしたBMPER
の骨芽細胞様分化転換作用はBMPに非依存性であった。BMPERの発現は、HCASMCにおいて高グル
コース負荷により、また、ストレプトゾトシン誘発糖尿病モデルマウスにおいて有意に増加しており、
高血糖状態における血管石灰化との関連が示唆された。結語以上より、BMPERは血管平滑筋細胞の
骨芽細胞様分化転換を誘導して血管石灰化を制御する分子であると考えられた。
神戸大学大学院医学研究科内科学講座循環器内科学分野、2神戸大学医学研究科生化学・分子細胞学講座シグナル伝達学分野
How to write
Plenary Lecture
Nature Medicine
1
Special Talk
小林 成美1、力武 良行2、石田 達郎1、江本 憲明1、平田 健一1
Special
Lecture
BMP-binding endothelial regulator (BMPER)は培養ヒト冠動脈平滑筋細胞の骨
芽細胞様分化転換を促進する
The hedgehog (Hh) pathway has been implicated in the pathogenesis of cancer. We have reported that the
oncogenic function of Hh in pancreatic ductal adenocarcinoma (PDAC) involves signaling in the stromal cells
rather than cell autonomous effects on the tumor cells, and this was mediated through a paracrine effect on bone
marrow-derived pro-angiogenic cells (PLoS One 2010). In the current study we sought to define at what stage
of pancreatic tumorigenesis this aberrant activation of the Hh pathway. To this end we utilized genetically
engineered mice Pdx1-Cre;LSL-Kras G12D strain as a model for precursor lesion PanIN and Pdx1-Cre;LSLKrasG12D;p53lox/+ strain for PDAC with a dense fibrous stroma. Although aberrant pancreatic epithelium highly
expresses Shh ligand in both PanIN and PDAC strains, the expression of Gli1/Gli2 in the stromal compartment
associated with the tumor vasculature was demonstrated only in PDAC strains with abundant desmoplasia.
Induction of VE-cadherin and Id1 mRNA in lineage - ; and c-Kit + BMMNC (LK cells) was observed during
progression from PanIN to PDAC, suggesting that BM cells are pro-angiogenic during tumorigenesis. However,
no increased induction of Gli1 mRNA by recombinant Shh was observed in LK cells from PDAC as compared
to PanIN strains. Significant downregulation of one of the negative regulators of Hh signaling via methylation
was observed in tumor cells at later stages of PDAC development, potentially accounting for the paracrine
switch in the PDAC stroma. Taken together, these studies suggest that primitive precursors from BM that
contribute to neovascularization are fully activated in the microenvironment of PDAC at late tumor stages.
旭川医科大学医学部内科学講座消化器・血液腫瘍制御内科学分野、2大阪医科大学医学部薬理学講座
1
河本 徹1、水上 裕輔1、杉山 祥晃1、笹島 順平1、佐藤 一也1、伊井 正明2、高後 裕1
The paracrine effect of hedgehog on the tumor vasculature emerges at later
stages during pancreatic tumorigenesis
President
Lecture
AJ-10
AJ-09
Program
18：00〜
Aⴊ▤↢‛කቇળKPFD
152
高齢化する現代社会において、認知症問題は解決が待たれている。認知症
の約半数以上を占めるアルツハイマー病は進行性の神経変性疾患である。
その原因遺伝子はアミロイド前駆体蛋白やプレセニリンが報告されており、
それらに基づき、アルツハイマー病の病因としてアミロイド・カスケード
仮説が提唱されている。現在、そのカスケードの各段階をターゲットとし
た根本的予防薬・治療薬の開発が現在進行中である。一方で、疫学的調査
により、非ステロイド性抗炎症薬や脂質異常症治療薬であるスタチンにア
ルツハイマー病発症抑制効果があることが示唆されている。今回、我々は
後者のスタチンに注目し、そのアルツハイマー病発症抑制効果の機序を解
明することを本研究の目的とした。その結果、我々はマウス生体内で、早
期エンドソームからライソゾームへ至る、ベータ・アミロイドの前駆体の
輸送が促進され、そのライソゾームでの分解が促進していることを見出し
た。このことからベータ・アミロイドの産生の材料になる前駆体が減るこ
とで、産生が低下することを示した。さらにベータ・アミロイドの産生の
みならず、クリアランスが増加していることをも見出した。これら２つの
機序が働き、スタチンはアルツハイマー病発症抑制効果を発揮することが
示唆された。
大阪大学医学部臨床遺伝子治療学、2大阪大学医学部老年腎臓内科学
1
里 直行1,2、篠原 充1,2、島村 宗尚1、武田 朱公1、楽木 宏実2、森下 竜一1
スタチンによるアルツハイマー病予防効果の機序の解明
3
【目的】慢性腎臓病（CKD）の病態において、HGFは腎組織での線維化を抑
制することが知られている。腎間質の線維化に重要な役割を果たす筋線維
芽細胞へのHGFの効果について解明するため本研究を行った。【方法】心臓
特異的にHGFを過剰発現させたトランスジェニックマウス（HGF-Tg）に対
し、アンジオテンシン IIを持続注入し，コントロールマウスと比較し、腎
臓の線維化に差が表れるかを検討した。また、HGF-Tgでは筋線維芽細胞の
アポトーシスが誘発されているかを調べた。及びヒトメサンギウム細胞に
TGF-β1を負荷した培養系では，HGFの投与による変化につき検討し、また、
FAK-MMP経路を阻害剤を用いて抑制しHGFによる筋線維芽細胞のアポトー
シス誘発に対してどのような影響が出るか調べた．【成績】アンジオテンシ
ン IIを持続注入したHGF-Tgでは，コントロールマウスと比較し、腎臓の線
維化は明らかに抑制され、間質の筋線維芽細胞も減少していた。また、
HGF-TgではTGF-β1、I型コラーゲン、IV型コラーゲン、MMP-2、MMP-9の
遺伝子発現が抑制されていた。さらに、
HGF-Tgでは筋線維芽細胞のアポトー
シスを認めた。ヒトメサンギウム細胞にTGF-β1を負荷した系においては、
HGFの投与により筋線維芽細胞のアノイキス（足場依存的なアポトーシス）
が誘発されていた。また、FAK-MMP経路を抑制したところ、HGFによる筋
線維芽細胞のアポトーシス誘発は抑えられた。【結論】HGFはメサンギウム
細胞由来の筋線維芽細胞のアノイキスを誘発することで、腎臓における抗
線維化作用を発揮すると考えられた。
大阪大学大学院医学系研究科臨床遺伝子治療学、2大阪大学大学院医学系研究
科 老年・腎臓内科
Transplantation of mesenchymal stem cells (MSC) derived from bone marrow (BM)
or adipose tissue has been expected to become hopeful cell therapy for stroke.
Here, we compared the therapeutic potential of adipose-derived stem cells (ASC)
with that of BM-derived stem cells (BMSC) in a murine model of stroke. ASC and
BMSC isolated from C57BL/6J mice were analyzed for their growth kinetics,
capacity to secrete trophic factors and capacity to differentiate into neural and/or
vascular cell lineage in vitro. In vivo study, ASC or BMSC were intravenously
administrated into recipient mice (1x105 cells/mouse) soon after reperfusion
following 90 minutes left middle cerebral artery occlusion. Neurological deficits,
the volume of infarction and swelling, the expression of trophic factors in brain,
and the fate of injected cells after 24 h reperfusion were observed. In vitro study,
ASC showed higher proliferative activity with greater production of VEGF and
HGF than BMSC. In addition, cell culture conditions allowed ASC to differentiate
into neural, glial and vascular endothelial cells. Notably, in vivo study revealed that
ASC administration showed remarkable attenuation of ischemic damages.
Furthermore, the expression of HGF and Angiopoietin-1 in ischemic brain tissues
were significantly increased in ASC-treated mice compared to the BMSC group.
Together with the general agreement about easier and safer access to adipose tissue,
our findings suggest that ASC would be a more preferable source for cell therapy
for brain ischemia than BMSC.
1
岐阜大学大学院医学系研究科細胞情報学分野、2岐阜大学大学院医学系研究科脳神
経外科学
林 真一郎1、池亀 由香1,2、山下 健太郎1,2、吉村 紳一2、岩間 亨2、
中島 茂1
Potential of adipose tissue-derived stem cells and bone
marrow-derived stem cells for ischemic stroke
therapy
4
循環器疾患の進行には性差があると考えられており、循環器疾患の発症は
閉経前女性においては男性より低いことが報告されており、閉経後の女性
では循環器障害が男性のレベルまで増加するとされる。我々はマウスの血
管傷害モデルを用いて、これまでに血管リモデリングの性差にAT2受容体
が関与することを報告してきた。今回はマウスの加齢に伴うリモデリング
の性差の変化とAT2受容体の変化を検討した。マウスの大腿動脈周囲にポ
リエチレンチューブをカフ状に留置し、炎症性血管傷害を作製した。野生
型（C57BL/6J）マウスでは、10週齢では傷害血管における細胞増殖、新生
内膜形成が雄性マウスに比べ雌性マウスにおいて低下しており、血管リモ
デリングが雌性マウスのほうが軽減されていることがわかった。傷害血管
における炎症性サイトカインの発現やスーパーオキシド産生も雌性 マウス
のほうが軽度であった。これに対し、50−55週齢の加齢マウスでは雌性マ
ウスの血中エストロゲン濃度が低下しているが、このマウスを用いて同様
に血管傷害を引き起こすと、細胞増殖、新生内膜形成が特に雌性マウスに
おいて増加するため、性差の減少が認められた。加齢マウスにエストロゲ
ンを前投与しておくと、リモデリングの性差が再び認められることから、
エストロゲンが性差の重要な因子の一つだと考えられる。一方、傷害血管
ではAT2受容体の発現が増加するが、これも10週齢では雌性マウスにおい
て発現が高値であったが、加齢マウスではその発現量が低下していた。そ
こで、AT2受容体欠損マウスの加齢マウスを用いて血管リモデリングを引
き起こすと、このマウスではエストロゲンに対する反応が低下していた。
上記実験結果から、血管リモデリングの性差は加齢により減少し、それに
はエストロゲンに加えてAT2受容体刺激の変化が関与していることが示唆
された。
1
愛媛大学大学院医学系研究科分子心血管生物・薬理学、2愛媛大学大学院医学系研
究科生殖病態外科学分野
岩井 將1、奥村 みどり1,2、中岡 裕智1、菅野 晴美1、伊藤 昌春2、
堀内 正嗣1
大津 礼1、谷山 義明1,2、家串 和真1,2、東 純哉1,2、楠 博1,2、眞田 文博1、
久徳 真梨子1、岩林 正明1、アマルナス チャタジー1、岡山 慶太1、
樂木 宏美1,2、森下 竜一1,2
1
加齢マウスにおける血管リモデリングの性差とAT2受容
体の役割
2
肝細胞増殖因子(HGF)は腎組織の筋線維芽細胞のアノイ
キスを誘導し、
腎線維化を抑制する
1
ポスターセッション：血管代謝・老化
（1）
Poster Session：Vascular Metabolism and aging (1)
12月1日
（水）
／Wednesday, December 1
How to write
Plenary Lecture
Nature Medicine
Symposium
Y.I.A.
Presentation
Selected Oral
Presentation
Poster
Index
Aⴊ▤↢‛කቇળKPFD
153
Special Talk
The 8th Korea-Japan Joint Symposium on Vascular Biology
Special
Lecture
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
President
Lecture
The vast majority of cases of Alzheimer disease (AD) are known to be due to the
sporadic (non-genetic) form of the disease, the mechanism underlying its cause and
progression still remains unclear. Here, we found that vascular beta-amyloid (A㱎),
A㱎40, inhibited the proliferative activity of human brain vascular endothelial cells
(HBEC) without toxic effects on them. This peptide also inhibited tube formation
and migration of HBEC. Moreover, A㱎40 inhibited ex vivo hippocampal
revascularization, re-endothelialization and the differentiation of adult endothelial
progenitor cells. Notably, A㱎40 suppressed the proliferative activity of HBEC
through the induction of “self-eating” autophagy. This induction involved the
intracellular regulation of class 3 phosphatidylinositol 3-kinase (PI3K) as well as
Akt signaling in HBEC. In addition, tissue culture of murine brain sections from
GFP-LC3 transgenic mice revealed that A㱎40 not only reduced the vessel density
in hippocampal lesions, but also induced autophagy in neurovascular EC.
Furthermore, in vivo neurovascular endothelial autophagy was detected in mutant
mice of AD. Our present findings indicate that the initial progression of AD might
be in part driven by A㱎40-induced endothelial autophagy and impairment of
neurovascular regeneration, suggesting important implications for therapeutic
approaches to AD.
1
岐阜大学大学院医学系研究科細胞情報学分野、2大阪大学大学院 医学系研究
科 臨床遺伝子治療学
林 真一郎1,2、里 直行2、武田 朱公2、篠原 充2、中島 茂1、森下 竜一2
Alzheimer disease associated peptide inhibits vascular
regeneration with induction of endothelial autophagy
5
Program
18：00〜
大阪大学大学院医学系研究科医科学専攻
愛媛大学大学院医学系研究科分子心血管生物・薬理学
Aⴊ▤↢‛කቇળKPFD
154
Both Angiotensin II (Ang II) and transforming growth factor-β1(TGF-β1) are
thought to be involved in the progression of chronic kidney disease (CKD). Clinical
and experimental CKD studies revealed Ang II type 1 receptor (AT1R) blocker
subsequently attenuated TGF-β1 levels, and reduced the progression of CKD.
Previous studies showed that hepatocyte growth factor (HGF) improves kidney
fibrosis in some animal models. So, we investigate whether HGF break out of the
positive feedback loop between Ang II and TGF-β1 to result in the amplification
of their profibrotic effects. In cultured human mesangial cells (HMC), TGF-β1
increased AT1R mRNA and HGF attenuated AT1R mRNA levels following TGF-β
1 stimulation. HGF also decreased AT1R mRNA stability. LY294002 (PI3K
inhibitor) and LY294002 significantly attenuated TGF-β1-induced AT1R gene
expression. These data suggest a TGF-β1-mediated up-regulation of the AT1R
gene involved in PI3K-Akt pathways. Besides, TGF-β1 also decreased PTEN
expression, a negative regulator of the PI3K/Akt pathways, and its phosphatase
activity, increasing Akt activity. However, the increased Akt activity induced by
TGF-β1, was blocked by co-treatment with HGF. This effect amplified the
phosphatase activity of PTEN. In vivo, the cardiac specific HGF transgenic mice
(HGF-Tg) reduced renal AT1R expression in the Ang II infusion. In vitro, it
increased PTEN expression and decreased Akt activity in HGF-Tg mice with Ang
II infusion. It is concluded that HGF reduces the expression of AT1R, inhibiting the
positive feedback loop between Ang II & TGF-β1. This serves to increase PTEN
expression, lowering Akt activity. HGF attenuated to decrease PTEN expression by
TGF-b1 stimulation.
大阪大学大学院医学系研究科臨床遺伝子治療学、2大阪大学大学院医学系研究科老
年腎臓内科学
1
アマルナス チャタジー1、谷山 義明1,2、家串 和真1,2、楠 博1,2、真田 文博1、
東 純哉1,2、岡山 慶太1、岩林 正明1、久徳 真梨子1、楽木 宏実2、
森下 竜一1
Hepatocyte Growth Factor attenuates TGFβand
AngII Crosstalk by Down Regulation of AT1-R
through PTEN/Akt Pathway
8
【背景】レニン・アンジオテンシン系（RAS）は血管老化に影響を与え、動
脈硬化の発症に深く関与すると考えられている。我々はRASのコンポーネ
ントと血管老化について検討してきた。今回、その概要について報告する。
【方法】 ラットおよびマウスの胸部大動脈より調整した血管平滑筋細胞を
用い、細胞の老化は、老化関連βガラクトシダーゼ (SA-㱎-gal) 染色と
p53、p21などのサイクリン依存性キナーゼ（CDKI）の発現の増大を指標と
した。また細胞内シグナルの発現をウエスタンブロットで検討し、より詳
しい検討にはsiRNAによる遺伝子ノックダウン法も用いた。酸化ストレス
についてはジヒドロエチジウムによるスーパーオキシドアニオンの産生量
で評価した。
【結果】VSMCは連日のアンジオテンシン（Ang）II (10 -7 M) 刺激により刺
激後5日目頃よりSA-㱎-gal染色陽性細胞数やCDKIの発現が増加した。これ
らの老化の指標は選択的Ang II 1型（AT1）受容体ブロッカー（ARB）の投
与でほぼ完全に抑制され、ミネラルコルチコイド拮抗薬のスピロノラクト
ンでも部分的に抑制された。また、Ang II 2型（AT2）受容体欠損マウスか
ら単離したVSMCでは老化が促進され、アンジオテンシン受容体関連タン
パク、ATRAPおよびATIPを過剰発現マウスから単離したVSMCでは老化が
抑制された。Ang II刺激によりVSMCからのアルドステロン分泌が時間依存
的に増加し、Aldoも相互に作用することで老化を伴に促進していることが
分かった。また、Ang II 刺激によりAT 1 受容体刺激により酸化ストレスや
NADPHオキシダ−ゼ活性が増加すること、その作用をAT2 受容体シグナル
が抑制することが見出された。一方、ATRAPを介した血管老化の抑制作用
には、calcineurin/NFAT pathwayも関与していることが示唆された。
【結論】以上よりVSMCの老化促進のメカニズムに、Ang IIが重要な役割を
果たしており、加齢に伴った動脈硬化性病変にRASの制御が効果的である
と考えられた。
竹内 希予、山本 浩一
茂木 正樹、閔 莉娟、堀内 正嗣
【目的】長期的なレニン・アンジオテンシン系（RAS）の活性化はアンジオ
テンシン（Ang）II 1型受容体（AT1）シグナルを介して認知機能を低下さ
せると考えられ、血圧を超えたAng IIの影響が示唆される。一方でAng II 2
型受容体（AT2）シグナルは、神経分化の促進や神経保護作用を持つことが
明らかとなり、認知機能低下への抑制効果が期待されている。最近開発さ
れたAT2 受容体の直接刺激薬であるCompound 21（C21）は心筋梗塞後の心
筋細胞保護作用を持つことが報告され、注目されているが、本実験ではC21
の認知機能に対する作用を検討した。
【方法】野生型及びAT2 受容体欠損マウスにC21を1日1回腹腔内注射により2
週間連日投与後に、モリスの水迷路にて空間認知能を評価した。脳表の血
流をレーザードップラー法により評価し、興奮性シナプス後電位（EPSP）
を生体信号測定装置で検討した。また、胎児マウスより海馬神経細胞を単
離し、C21を添加することによる神経伸長の程度を検討した。
【結果】C21の投与により、野生型マウスにおいて血圧及び脳内のAT1、
AT2 のmRNAの発現レベルには変化を認めなかった。空間認知機能は無投薬
群に比べて顕著に向上した。この改善効果はブラジキニンのB2 受容体アン
タゴニストであるicatibantの同時投与により減弱した。一方、AT2 受容体欠
損マウスにおいてはC21投与による認知機能の改善効果は認められなかっ
た。脳表血流はC21投与により顕著な増加を認めたが、AT2 受容体欠損マウ
スではその効果は認められなかった。EPSPはC21投与で濃度依存性に増加
を認めたが、この効果もAT2受容体欠損マウスでは減弱していた。最後に、
C21の添加により胎児海馬神経細胞の伸長が認められ、AT2 受容体のアンタ
ゴニストであるPD123319の同時添加でその効果は抑制された。
【結論】AT2 受容体を直接刺激するC21は認知機能を向上させる作用を有す
る。このメカニズムには、血流の増加とシナプス数の増加・神経伸長など
の多方面の作用が影響していることが示唆された。
愛媛大学大学院医学系研究科分子心血管生物・薬理学
茂木 正樹、景 斐、堀内 正嗣
アンジオテンシンII 2型受容体直接刺激薬による血流増加
を介した認知機能改善作用
9
（目的）ミトコンドリアはエネルギー産生や活性酸素種の調節に重要な役割
を果たしている。平滑筋細胞におけるミトコンドリア機能の障害は、活性
酸素を産生し、細胞障害によりアポトーシスが促進し、動脈硬化を引き起
こすことが報告されている。ＰＰＡＲγはミトコンドリア機能を調節する
重要な因子であることは既に知られているが、本研究では、ＰＰＡＲγの
部分的アゴニストとしても知られるアンジオテンシン受容体ブロッカー（Ａ
ＲＢ）、テルミサルタンのミトコンドリア機能（エネルギー産生、活性酸素
種の産生、）への影響を調べた。
（方法、結果）ヒト血管平滑筋細胞において、
テルミサルタンは用量依存的にＡＴＰ産生を亢進させたが、ＰＰＡＲγア
ゴニスト作用をもたないイプロサルタンはＡＴＰ産生には影響を及ぼさな
かった。またヒト血管平滑筋細胞におけるＨ２Ｏ２産生も、テルミサルタ
ンはコントロールに比べて減少させたが、イプロサルタンでは変化を認め
なかった。これらのテルミサルタンの作用のＰＰＡＲγへの関与を検討す
るために、ＰＰＡRγを欠損させた血管平滑筋細胞と、コントロール血管
平滑筋細胞を用いて実験を行った。結果、テルミサルタンはコントロール
及びＰＰＡRγ欠損血管平滑筋細胞において共にＡＴＰ産生を亢進させた。
またテルミサルタンはＰＰＡＲγ欠損血管平滑筋細胞においてＨ２Ｏ２産
生を増加させたが、コントロール血管平滑筋細胞においては増加させず電
子伝達系からの活性酸素の放出が、テルミサルタンによるＰＰＡＲγの活
性化による抗酸化作用で抑制されることが示唆された。さらにテルミサル
タンはＰＰＡＲγ欠損血管平滑筋細胞において、Ｈ２Ｏ２刺激によるアポ
トーシス反応を抑えたが、ＰＰＡＲγをもつコントロール細胞では抗アポ
トーシス作用は見られなかった。（結論）ＰＰＡＲγアゴニスト活性をもた
ないイプロサルタンとは異なり、テルミサルタンは平 滑筋細胞におけるＡ
ＴＰ産生を亢進させ、Ｈ２Ｏ２産生を抑制した。これらの作用はＡＴ１と
は独立した経路が関与しており、ＰＰＡＲγ依存的・非依存的な経路が組
み合わされて引き起こされることが示唆された。
老年・腎臓内科学研究室
ＰＰＡＲγ依存的・非依存的な、
テルミサルタンのミトコン
ドリア機能への影響の検討
7
血管平滑筋細胞の老化におけるレニン・アンジオテンシン
系のコンポーネントの働き
6
ポスターセッション：血管代謝・老化（2）
Poster Session：Vascular Metabolism and aging (2)
12月1日
（水）
／Wednesday, December 1
How to write
Plenary Lecture
Nature Medicine
Symposium
Y.I.A.
Presentation
Selected Oral
Presentation
Poster
Index
Aⴊ▤↢‛කቇળKPFD
155
Special Talk
The 8th Korea-Japan Joint Symposium on Vascular Biology
Special
Lecture
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
President
Lecture
老化は虚血性心血管疾患の重大な危険因子である。老齢動物およびヒト高
齢者では血管内皮機能、血管新生能が低下していることが数多く報告され
ており、高齢者における虚血性心疾患の高い罹患率および罹患後の予後不
良の直接的原因と考えられる。しかしながら老化に伴う血管内皮機能低下、
血管新生能低下の分子機構は未だ十分には解明されてない。そこで私達は
継代を重ねて老化誘導したHUVECと老齢マウスの大動脈から単離した血管
内皮細胞(AEC)を用いて、老化に伴う血管内皮機能低下とその分子メカニ
ズムの解明を試みた。老化HUVEC、老化AECの内皮機能は若い対照細胞と
比較して著明に低下していた。MAPKやAktシグナル、VEGF受容体、TIE2
の発現量は老化細胞と若い細胞の間に差を認めなかった。一方、私達は老
化血管内皮細胞ではBcl-2の発現が著明に低下していることを見出した。
Bcl-2ノックダウンおよびBcl-2阻害剤は若い血管内皮細胞中のアポトーシス
を憎悪させるのみならず遊走能や管腔形成能も有意に抑制した。Bcl-2は抗
アポトーシス作用に加えてグルタチオンとの結合を介したミトコンドリア
酸化ストレス軽減作用を有することが報告されている。老化血管内皮細胞
ではミトコンドリア酸化ストレスが有意に増大しており、抗酸化剤により
内皮機能は改善した。さらに老化血管内皮細胞にBcl-2を再発現させるとミ
トコンドリア酸化ストレスが減少し、血管内皮機能が改善した。以上の結
果からBcl-2は血管内皮細胞においてミトコンドリアを酸化ストレスから保
護する重要な作用を有しており、老化に伴うBcl-2発現低下はミトコンドリ
ア酸化ストレス増大から内皮機能低下を引き起こすと考えられた。さらに
老齢マウスの皮下に注入したBcl-2アデノを加えたMatrigelではLacZアデノ
を加えた対照群に比べて新生血管が著明に増加していた。以上より細胞老
化に伴いBcl-2発現が減少し、血管新生能低下を引き起こすことが明らかと
なった。
京都府立医科大学循環器内科
池田 宏二、浦岡 真季、小出 正洋、赤壁 佳樹、北村 洋平、中川 裕介、
栗本 律子、松原 弘明
Bcl-2発現低下はミトコンドリア酸化ストレス増大を介し
て老化血管内皮細胞の血管新生能を障害する。
10
Program
18：00〜
老年腎臓内科
Aⴊ▤↢‛කቇળKPFD
156
Klotho蛋白の血管内皮細胞における抗炎症作用背景：Klotho（KL)は、その
欠損により老化を来し、またその蛋白は、抗酸化作用、抗アポトーシス作
用を有する。我々は、血管の老化において重要な要素であり、アポトーシス、
酸化ストレスと関連が深い、炎症に対するKLの影響に着目した。目的：血
管内皮細胞において、TNF-α刺激によって誘発される炎症に対するKL蛋白
の作用を明らかにする。方法：血管内皮細胞として、ヒト臍帯静脈由来内
皮細胞(HUVEC)を用いた。炎症はTNF-α刺激により誘発し、KL蛋白の有
無による、炎症マーカー（ICAM、VCAM）発現の変化、NFκB活性の関与、
HUVECに対する単球細胞（THP-１）接着への影響等について検討した。
結果：炎症によるICAM、VCAM発現上昇は、KL添加群で減弱した（ICAM：
72%、VCAM：67%）。 同 様 にNFκB活 性 は、KL添 加 群 で71%減 少 し た。
THP-１接着は、KL添加群で73%抑制された。結語： KL蛋白は、血管内皮
細胞においてTNF-αによる炎症反応を抑制しうることを明らかにした。本
研究により、KLが炎症反応の抑制を介して血管老化制御に働く可能性が示
された。
大阪大学大学院医学系研究科
前川 佳敬、大石 充、小黒 亮輔、山本 博子、楽木 宏実
Klotho蛋白の血管内皮細胞における抗炎症作用
13
【目的】筋萎縮性側索硬化症(ALS)は上位および下位の運動ニューロン(MN)
が選択的に障害される神経変性疾患である。近年、血管構成因子の機能、
構造異常がALS病態に関与することが示唆されている。そこで、本研究で
はALSモ デ ル マ ウ ス を 用 い て 脊 髄 血 管 のneurovascular unit を 構 成 す る
endothelium, tight junction basement membraneの変化について検討した。
【方
法】実験にはALSのモデル動物であるG93Aトランスジェニック(Tg)マウス
を用いた。このマウスは14週頃に運動障害を発症し、19週で死亡するため、
10週をpre-symptomaticモデル、15週をearly-symptomaticモデル、18週をendstageモデルとした。各個体から主要な病巣部である脊髄を採取し、免疫染
色およびウェスタンブロット解析を行い、neurovascular unitを構成するタン
パク質の発現量と詳細な分布の変化を経時的に調べた。
【結果】Tgマウスで
は病気の進行に伴いendotheliumのマーカーであるPCAM-1陽性の微小血管
の径、密度が減少した。また、tight junction構成蛋白のOccludinは運動障害
が現れる前の10週から血管での発現が減少しはじめ、18週では発現が見ら
れなくなった。 basement membrane構成蛋白のCollagen IVの発現量も10週か
ら減少しはじめ、18週では血管構造が消失した。これらの異常は運動障害
を発症する前から見られ、脊髄前角で顕著であった。さらにneurovascular
unit の外側に位置する構成成分ほど損傷が激しかった。【結論】血管径、密
度の減少が見られたことから、ALSモデルマウスの脊髄では微小循環血流
量が減少している可能性が示唆された。neurovascular unit構成成分の異常は
運動障害が現れる前から見られたため、ALS病態メカニズムの比較的上流
に位置しており、今後治療ターゲットとなり得る可能性が示唆された。
恒常性制御学講座
Purpose: Optimal design of non-invasive therapy and evaluations for heart failure
(HF) remains limited due to the lack of simple clinical criteria to characterize the
patients. Compared to respiratory or gastroenterololgical fields, Kampo-medicine is
used less frequently cardiological field. Numerical scoring systems to estimate
patientʼs physical conditions have been induced in Kampo using physical
examination and medical questionnaire. Suitai-score (a scoring system for water
stagnation) is one of so-called Kampo-scores. This study was designed to
investigate the correlation between Kampo-scores and cardiological scores with
Doppler echocardiography in hemodialysis outpatients with chronic HF. Patients
and Methods: We studied 38 patients who were outpatients at the hemodyalysis unit
with simultaneous Suitai-scores and cardiological scores with doppler
echocardiography. We estimated the Suitai-scores with physical examination and
medical questionnaire. We also measured and compared them by linear regression
with E/Ea ratio (transmitral early diastolic velocity/mitral annular early diastolic
velocity ratio) and other indices, which are suggested to have strong relation to left
ventricular diastolic function or pulmonary capillary wedge pressure (PCWP).
Results: Suitai-scores had strong relation to some of the indices with Doppler
echocardiography. Conclusion: Our data and some reports suggested that simple
and noninvasive clinical assessments, Kampo-scores especially Suitai-score, can be
used to define clinical assessment of congestion, which has been shown by PCWP
with Swan-Ganz catheterization, in hemodialysis outpatients with chronic HF.
These Kampo-scores estimate outcomes and may be used to guide therapy for
future study.
1
大阪大学大学院医学系研究科臨床遺伝子治療学、2大阪大学大学院医学系研究科老
年腎臓内科学、3大阪大学医学部附属病院総合診療部
尾崎 和成1,2、牧野 寛史1,3、森下 竜一1
Noninvasive new clinical assessment in Heart
Failure:The relation between Kampo-scores and
indices with echocardiography
14
背景：細胞には分裂限界があり、培養細胞において継代を繰り返すと細胞
分裂を停止する細胞老化とよばれる現象が知られている。細胞老化が加齢
性疾患の発症に関与することが近年明らかにされた。さらにアンギオテン
シンIIによって誘導される血管細胞老化が動脈硬化進展に関与することが
示された。以上のことから血管細胞老化は動脈硬化進展に重要な役割を果
たしていると考えられる。アルドステロンは電解質バランスや血圧を調整
する重要なホルモンであるが、一方で内皮機能障害を引き起こし動脈硬化
進展に関与することが知られている。今回我々はアルドステロンによる内
皮細胞の細胞老化とその機序について検討した。方法と結果：ヒト臍静脈
内皮細胞（HUVEC）をアルドステロンで刺激し、細胞老化を老化関連βガラクトシダーゼ染色を用いて調べたところ、濃度依存的に染色陽性細胞
数の増加を認め、アルドステロン10-6 M、72時間の刺激にて有意な増加を
認めた。ウエスタンブロット法を用いて、細胞老化の重要なメディエーター
であるp21の発現レベルを検討したところ、アルドステロン刺激で、p21の
蛋白レベルが有意に増加した。In vitroの検討でアルドステロンによるプロ
テアソーム活性の抑制が確認され、p21蛋白の増加の機序として、p21蛋白
の分解抑制が関与している可能性が考えられた。続いてアルドステロンに
よる細胞老化におけるp21の役割を明らかにするためにsiRNAを用いて検討
を行った。siRNAはHUVECにおいてp21の発現レベルを20％に抑制した。
このp21蛋白発現をノックダウンしたHUVECでは、アルドステロンにより
誘導される細胞老化が抑制された。またアルドステロン刺激により炎症性
サイトカインIL-6、MCP-1のｍRNAの発現の亢進が認められたが、p21発現
レベルの抑制により、アルドステロンにより誘導される炎症性サイトカイ
ン発現レベルの増強効果は抑制された。結論：アルドステロンはp21を介し
た経路で内皮細胞の細胞老化および炎症性サイトカイン発現を誘導し、動
脈硬化性病変形成に関与する可能性が示唆された。
金沢大学附属病院内科
油谷 伊佐央、薄井 荘一郎、高島 伸一郎、古荘 浩司、高村 雅之、
金子 周一
阿部 康二、宮崎 一徳、森本 展年、倉田 智子、太田 康之、池田 佳生、松浦 徹
岡山大学 医歯薬学総合研究科 脳神経内科学
アルドステロンはp21を介した経路で内皮細胞に細胞老化
と炎症を誘導する
12
ALS モデルマウス脊髄血管におけるneurovascular unit
の異常
11
ポスターセッション：血管代謝・心不全
Poster Session：Vascular Metabolism / Heart Failure
12月1日
（水）
／Wednesday, December 1
1
静岡県立大学薬学部分子病態学講座、2国立病院機構京都医療センター臨床研究セ
ンター 展開医療研究部、3京都大学大学院医学研究科 人間健康科学系専攻
1
Symposium
Y.I.A.
Presentation
Selected Oral
Presentation
Poster
Index
Aⴊ▤↢‛කቇળKPFD
How to write
Plenary Lecture
Nature Medicine
157
Special Talk
The 8th Korea-Japan Joint Symposium on Vascular Biology
Special
Lecture
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Endothelin-1 (ET1) /Endothelin A receptor (ETAR) axis plays an important roles in
regulating cardiovascular homeostasis and organ development. We have previously
reported that ET1 and ETAR knockout mice display craniofacial defects and aortic
arch malformations. However, their phenotype in coronary vessel formation
remains largely unknown. Here we show that ET1/ETAR axis insufficiency disturbs
coronary artery development. Coronary angiograms of the mouse fetus using an
ink-injection method revealed that some septal branches were abnormally enlarged
and some had focal aneurysm in both ET-1 and ETAR KO mice at embryonic day
17.5 (E17.5). Additionally, origins of the septal branch were completely switched
from right to left coronary artery. To further clarify cell-based mechanisms, we
examined the coronary vessel structure at various developmental periods. We also
analyzed expression patterns of ETAR using ETAR-lacZ knock-in mice. Coronary
angiogarms detected no anatomical differences between normal and KO mice until
E14.5. At this stage, CD31-positive endothelial cells configured vascular network,
but neither smooth muscle actin (SMA)-positive mural cells nor ETAR expressing
cells covered these vessels in wild-type mice. At E17.5, both ETAR and SMApositive mural cells are uniformly aligned as a single cell layer covering the
endothelium in wild-type mice. However, in knockout mice, some parts of enlarged
septal branches were free of SMA-positive cells, whereas the others were covered
with multi-layered SMA-positive mural cells, collectively indicating that mural
cell-mediated coronary artery maturation was impaired. These findings suggest that
ET-1/ETAR signaling may be involved in coronary artery development through a
mural cell-mediated mechanism.
3
President
Lecture
【目的】我々はこれまでに、心筋特異的転写因子GATA4が内因性ヒストンア
セチルトランスフェラーゼ活性を持つp300と協調的に作用し、肥大反応遺
伝子の転写を調整して、心不全の発症・増悪に関与することを見いだした。
そこでp300/GATA4経路のさらなる作用メカニズムを解明するために、プロ
テオミクス解析によりGATA4の結合因子を73個同定した。この中には転写
コリプレッサーであるNucleosome Remodeling and histone Deacetylase(NuRD)
複合体の構成因子、RbAp48(以下p48)及びRbAp46(以下p46)が含まれていた。
本研究では、p48、p46がp300/GATA4と機能的コンプレックスを形成するか、
心筋細胞肥大反応遺伝子制御にどのように関与するかを検討した。
【方法と
結果】大腸菌を用いてp48、p46とp300、GATA4のリコンビナントタンパク
を 作 成 し、GST Pull-Down assay後、Western Blottingを 行 い、p48、p46は
GATA4だけでなくp300と直接結合することを見出した。次に、HEK293細胞
に心筋肥大反応遺伝子ANFプロモーターコンストラクトをトランスフェク
ションし、転写活性を測定したところ、GATA4とp300の共発現によって相
乗的に亢進した転写活性は、p48またはp46を共発現させることで抑制され
た。さらに、HEK293細胞にGATA4、p300、p48を発現させ、核タンパク質
を抽出し、免疫沈降-Western Blottingを行ったところ、p300の発現により亢
進したGATA4のアセチル化は、p48により抑制された。次に、培養心筋細胞
において、p48またはp46を過剰発現させ、肥大反応刺激であるフェニレフ
リン刺激を行い、ANF及びET-1の心筋肥大遺伝子プロモーター活性を測定
したところ、フェニレフリン刺激により上昇した転写活性は、p48、p46の
過剰発現で抑制された。さらに、フェニレフリン刺激により誘導される心
筋細胞肥大は、p48またはp46過剰発現で抑制された。【結論】以上より、
p46、p48はp300/GATA4経路に作用して心筋細胞の肥大を抑制した。p48お
よびp46が心不全治療のターゲットになる可能性が示唆された。
寺田 太士1、砂川 陽一1,2,3、渡辺 雄一1、高谷 智英2,3、和田 啓道2、
刀坂 泰史1、島津 章2、木村 剛3、藤田 正俊3、長谷川 浩二2、森本 達也1
有馬 勇一郎1、西山 功一1、宮川−富田 幸子2、有馬 聡1、淺井 理恵子1、
金 基成1、内島 泰信1、栗原 由紀子1、小川 久雄3、栗原 裕基1
東京大学大学院医学系研究科代謝生理化学教室、2東京女子医科大学循環器小児科、
熊本大学大学院医学薬学研究部循環器病態学
新規GATA4結合タンパク質RbAp46、
RbAp48の心筋細胞
肥大への影響
16
Coronary artery anomalies in Endothelin-1 and
Endothelin A receptor knockout mice.
15
Program
歯周病科、3東京医科歯科大
歯周病科、3東京医科歯科大
Aⴊ▤↢‛කቇળKPFD
158
【目的】フルバスタチンを長期投与している高脂血症患者を一次・二次予防
別に層別し、心・脳イベントの発症と血清脂質値との関連を検討する。
【調査及び対象】高脂血症患者を対象に実施されたフルバスタチン大規模市
販後調査における、19,084例を一次（冠動脈疾患、脳血管障害ともに既往・
合併なし）及び二次予防（冠動脈疾患の既往・合併あり（＋）
、脳血管障害
の既往・合併あり（＋）の2群）別に3層別し、心及び脳イベント発症と血
清脂質値（期間平均値）との関係を検討した。
【結果】
１）一次・二次予防における心・脳イベントの発現率と相対危険度（RR）
・心イベントの発症：一次予防群（発現率は0.81 %）のRRを１として推定
した二次予防群の冠動脈疾患（＋）及び脳血管障害（＋）群のRRはそれぞ
れ8.3（発現率5.86 %）及び2.2（発現率2.13 %）であった。
・脳イベントの発症：一次予防群（発現率は0.45%）のRRを１として推定
した二次予防群の冠動脈疾患（＋）及び脳血管障害（＋）群患者のRRはそ
れぞれ2.9（発現率1.11 %）及び5.3（発現率2.77 %）であった。
２）心・脳ベントの発症と血清脂質値
・LDL-C（180 mg/dL以上）は唯一心イベントの一次予防で危険因子であっ
たが、その他の層別群において危険因子とはなっていなかった。
・TGは、脳イベントの一次予防（TG 250 mg/dL以上でRR 2.3）および心イ
ベントの冠動脈疾患（＋）患者の二次予防（TG 150 mg/ dL以上でRR 1.8〜2.0）
でリスク因子であった。
・HDL-Cは脳イベントの一次予防及び心イベントの冠動脈疾患（＋）患者
の二次予防においていずれも40 mg/dL未満でリスクを高める傾向（RR 1.5
〜2.3）を、60〜70 mg/dL以上で冠動脈疾患（＋）患者の二次予防における
心イベントリスクを有意に減弱させた（RR 0.3〜0.4）
。
【結論】フルバスタチン投与患者においては、ほとんどの患者でLDL-Cは十
分に管理されており、さらに低下させる必要はないが、心・脳イベントの
発症及び再発リスクをさらに低下させるには、TGの厳格な管理とHDL-Cを
高める生活習慣等の改善が望まれる。
【目的】フルバスタチンを長期投与している高脂血症患者を一次・二次予防
別に層別し、心・脳イベントの発症と危険因子について検討する。
【調査及び対象】高脂血症患者を対象に実施されたフルバスタチン大規模市
販後調査における、19,084例を一次（冠動脈疾患、脳血管障害ともに既往・
合併なし）及び二次予防（冠動脈疾患の既往・合併あり（＋）
、脳血管障害
の既往・合併あり（＋）の2群）別に3層別し、心及び脳イベント発症と危
険因子との関係を検討した。
【結果】
１）一次・二次予防における心・脳イベントの発現率と相対危険度
・一次予防における心及び脳イベント発現率は各々0.81% および 0.45 %で
あった。
・一次予防群に対する二次予防群の相対危険度（RR）は、冠動脈疾患（＋）
患者の心および脳イベントで各々 8.3と2.9、脳血管障害（＋）患者の心お
よび脳イベントでは各々2.2と5.3であり、一次予防に比べていずれも有意に
高かった。
２）一次・二次予防における心・脳イベントの発症と危険因子
・LDL-C（180 mg/dL以上）は唯一心イベントの一次予防で危険因子（RR 2.1）
であったが、脳イベントの一次予防および心・脳イベントの二次予防では
いずれも危険因子とはならなかった。
・糖尿病は心及び脳イベントの一次・二次予防において、もっとも強力な
危険因子（RR 1.6〜3.8）であった。
・高血圧は心及び脳イベントの一次予防においていずれも危険因子（RR 1.6
〜1.8）であったが、
二次予防においては、
冠動脈疾患または脳血管障害（＋）
を問わず、危険因子ではなかった。
・喫煙は、脳イベントの一次予防において危険因子であった（RR 2.1）
。ま
た、心イベントの一次予防および冠動脈疾患（＋）患者の二次予防におけ
る心イベントリスクを高める傾向を示した。
【結論】フルバスタチン投与患者においてはLDL-Cはリスクを高める閾値
（180 mg/dL）以下にほぼ管理されており、コレステロールをさらに低下さ
せる必要はないが、心・脳イベントの発症及び再発リスクをさらに低減さ
せるには、糖尿病および高血圧治療等の厳格な管理と禁煙が推奨される。
1
1
大阪大学大学院医学系研究科 遺伝子治療学、2東京医科大学 内科学第三講座、3
日本医科大学内科学講座 循環器・肝臓・老年・総合病態部門、4東京医科歯科大
学 先進倫理医科学開発学、5應義塾大学 抗加齢内分泌学講座、6日本薬剤学会、7
茨城キリスト教大学 生活科学部食物健康科学科、8中谷内科クリニック、9LEM研
究グループ
森下 竜一1、小田原 雅人2、水野 杏一3、吉田 雅幸4、市原 淳弘5、楠 正6、
板倉 弘重7、中谷 矩章8、LEM研究 グループ9
森下 竜一1、小田原 雅人2、水野 杏一3、吉田 雅幸4、市原 淳弘5、楠 正6、
板倉 弘重7、中谷 矩章8、LEM研究 グル−プ9
大阪大学大学院医学系研究科 遺伝子治療学、2東京医科大学 内科学第三講座、3
日本医科大学内科学講座 循環器・肝臓・老年・総合病態部門、4東京医科歯科大
学 先進倫理医科学開発学、5慶應義塾大学 抗加齢内分泌学講座、6日本薬剤学会、
7
茨城キリスト教大学 生活科学部食物健康科学科、8中谷内科クリニック、9LEM
研究グループ
スタチン管理下における高脂血症患者のイベント予防は
TG及びHDL-C管理が重要である
（LEM研究：心・脳血管障
害サブ解析２）
20
【目的】炎症性応答は血管傷害後の新生内膜増殖に重要な役割を担う。歯周
炎は慢性炎症の状態である。しかし，血管傷害後の歯周病原細菌の影響に
ついては知られていない。そこで，本研究では血管傷害後の歯周病原細菌
の影響を調査した。【方法】マウスの背部皮下にチャンバーを埋入し，その
2週後，マウスの大腿動脈を0.38mmのコイルスプリングワイヤーで傷害し
た。チャンバーにPorphyromonas gingivalis (Pg) を投与した。またPBSを用い
た群をcontrolとした。傷害後，14日で大腿動脈を採取し，病理組織学的分
析をEvG染色にて行った。また，ELISA法にて血漿中の抗Pg抗体価，MCP1，IL-6値を測定した。
【結果および考察】病理所見から，Pg感染群は新生
内膜形成の有意な促進が観察された(I/M ratio Pg 4.54±0.94, control 2.65±
0.34, p＜0.05) 。また、抗体価はコントロール群と比較し、Pg投与群で有
意に上昇していた。免疫組織分析では、Pg投与群の動脈壁および周囲組織
で増殖細胞核抗原（PCNA）の発現が増強していた。血漿MCP-1値はコント
ロール群と比較し、Pg投与群で有意差は見られなかった。血漿IL-6値につ
いても同様にPg投与群とコントロール群では有意差はなかった。【結論】Pg
感染が血管傷害後の新生内膜形成を促進させることが示唆された。
東京大学先端臨床医学開発講座、2東京医科歯科大学
学 循環器内科、4東京大学 循環器内科
1
鈴木 淳一1、小林 奈穂2、青山 典生2、小川 真仁1、平田 恭信1、和泉 雄一2、
磯部 光章3、永井 良三4
歯周病菌感染はマウス血管傷害モデルにおいて新生内膜
形成を促進する
スタチン管理下における高脂血症患者のイベント発症は
糖尿病、
高血圧及び喫煙で上昇する（LEM 研究：心・脳血管
障害サブ解析１）
19
背景：腹部大動脈瘤の形成と促進には炎症が関与していることが知られて
いる。腹部大動脈瘤組織でのmatrix metalloproteinase（MMP）の上昇が多く
報告されており、腹部大動脈瘤形成においてMMPが重要な役割を担ってい
ることが知られている。また、臨床検体において腹部大動脈瘤組織から複
数の歯周病原細菌が検出されており、歯周病と大動脈瘤との関連が強く示
唆されている。本研究の目的は、腹部大動脈瘤形成に際して歯周病原細菌
感染がどのように影響しているかを明らかにすることである。方法：マウ
スの腹部大動脈壁に塩化カルシウム溶液を塗布することにより、実験的に
腹 部 大 動 脈 瘤 を 誘 導 し た。 主 要 な 歯 周 病 原 細 菌 と し てPorphyromonas
gingivalis (Pg)を用いた。マウス背部にコイルチャンバーを植え込み、Pgを
７日毎にコイル内に注入して持続感染状態を作成した。Pgを注入した群（Pg
群）と注入しない群（対照群）の２群を用いて比較検討した。大動脈瘤誘
導前と誘導後28日で肉眼的に動脈径を計測し、動脈拡張の状態を評価した。
大動脈瘤誘導後の動脈組織を採取し、組織学的、免疫組織学的解析を行った。
またELISA法により血漿中のMMP濃度を計測した。結果：Pg群では、対照
群と比べて術後の動脈拡張が有意に大きかった。組織学的に、実験群でよ
り重度のエラスチン破壊が認められ、また免疫染色により実験群で動脈組
織におけるMMPの高度な発現が観察された。対照群と比較して、実験群で
はMMPの血漿中濃度の上昇が認められた。結論：本研究結果から、歯周病
原細菌感染はMMPレベルの上昇により、腹部大動脈瘤形成を促進する可能
性があることが示唆された。
東京大学先端臨床医学開発講座、2東京医科歯科大学
学 循環器内科、4東京大学 循環器内科
1
鈴木 淳一1、青山 典生2、小川 真仁1、平田 恭信1、和泉 雄一2、磯部 光章3、
永井 良三4
歯周病菌による腹部大動脈瘤の形成促進
17
18
18：00〜
ポスターセッション：動脈硬化（1）
Poster Session：Atherosclerosis(1)
12月1日
（水）
／Wednesday, December 1
臨床遺伝子治療学、3東京医科歯科大
Symposium
Y.I.A.
Presentation
Selected Oral
Presentation
Poster
Index
159
How to write
Plenary Lecture
Nature Medicine
The 8th Korea-Japan Joint Symposium on Vascular Biology
Special Talk
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Special
Lecture
Aⴊ▤↢‛කቇળKPFD
循環器内科、3東京医科歯科
背景：歯周病菌のPorphyromonas gingivalis（P.g.菌）は口腔の慢性感染によ
り歯周病を進行させる原因菌としてよく知られているが、歯周病菌感染が
冠動脈疾患リスクとなるか否かについては議論がある。方法：冠動脈CTを
施行した連続50例（平均年齢65.6±1.2歳）で患者より唾液を採取しPCR法
によりP.g.菌を同定した。病歴より冠危険因子（肥満、高血圧、糖尿病、高
脂血症、喫煙歴、家族歴、CKD）とCRP値を評価した。患者は発熱やCRP
値の上昇はなく、かつデータが得られた45例を解析に用いた。CT所見でプ
ラークもしくは石灰化所見を有するものを冠動脈硬化患者（IHD）とした。
またP.g.菌感染群、各冠危険因子群で比較を行った。結果： P.g.菌感染群で
は 非 感 染 群 と 比 べIHDの 割 合 が 有 意 に 多 か っ た（80.7±7.2% vs. 47.4±
11.8%, P＝0.022）
。またP.g.菌感染群ではCRPが有意に高く（CRP=0.23±0.06
vs. 0.08±0.03mg/dl, p＝0.02）、IHDとP.g.菌感染の有無によるサブグループ
でのCRP値はIHD（＋）P.g.（＋）
：0.26±0.07mg/dl、IHD（＋）P.g.（−）
：0.14
：
±0.05mg/dl、IHD（−）P.g（＋）
：0.078±0.025mg/dl、IHD（−）P.g（−）
0.029±0.011mg/dlであった。冠危険因子の解析では、肥満と非肥満者では
P.g.菌の感染率に有意差は認められなかった。同様に高血圧、糖尿病、脂質
異常症、喫煙歴、家族歴、CKDの有無に関し、P.g．菌の感染率に有意差が
なかった。結論：P.g.菌感染者にCRP上昇を伴ったIHDが多くみられた。P.g.菌
感染が冠動脈硬化の危険因子となる可能性が示唆された。
東京大学先端臨床医学開発講座、2東京医科歯科大学
大学 歯周病科
1
President
Lecture
＜背景＞血管病変に対する新しい治療法を開発する際に、炎症の側面を無
視することはできない。我々は、血管形成術後再狭窄などにおける炎症反
応を活性化する接着分子であるvascular cell adhesion molecule (VCAM)-1に注
目 し た。 超 音 波 マ イ ク ロ バ ブ ル 法 を 用 い て 生 体 内 にsiRNAを 導 入 し て
VCAM-1を制御する新しい治療法の開発を試みた。＜方法と結果＞最初に、
超音波マイクロバブル法による生体内へのsiRNA導入効率を確認するため
に、FITCラベルしたスクランブルsiRNAを用いた。その結果、FITCラベル
siRNAとマイクロバブルを超音波ゲルに混入して血管外膜側に塗布し、同
部に超音波を照射した群で著しくFITC陽性細胞の増加が確認された。次に、
血管形成術後再狭窄の病態と治療効果を確認するために、ラット大腿動脈
弾性ワイヤー傷害モデルを作成した。その病態を解析したところ、血管傷
害後早期にVCAM-1が発現し、それを契機としてサイトカインなどの炎症
性物質が活性化して内膜肥厚が進行していた。この傷害血管部位に、超音
波マイクロバブル法を用いてVCAM-1に対するsiRNAを導入したところ、無
治療群に比してVCAM-1発現が制御された。その結果、炎症性細胞の浸潤
やサイトカインの産生が抑制され、血管のリモデリングが軽減した。＜考
察と結論＞これらの結果より、VCAM-1は傷害血管の早期炎症過程におい
て重要な役を演じていることが示唆された。VCAM-1に対するsiRNAを超音
波マイクロバブル法により生体内に導入することにより、傷害局所に限局
したVCAM-1の制御が可能となり、結果として傷害血管のリモデリングが
抑制できる。超音波マイクロバブル法によるsiRNA遺伝子導入は対象遺伝
子と対象臓器を限局して実施されうるため、非特異的な反応や全身投与に
よる身体への副作用が無く、近い将来臨床に応用されることが期待される。
東京大学先端臨床医学開発講座、2大阪大学
学 循環器内科、4東京大学 循環器内科
鈴木 淳一1、手塚 大介2、和泉 雄一3、磯部 光章2
鈴木 淳一1、小川 真仁1、谷山 義明2、平田 恭信1、磯部 光章3、永井 良三4、
森下 竜一2
1
冠動脈CTを施行した連続50例における歯周病菌
Porphyromonas gingivalis感染と冠危険因子との比較
22
血管リモデリングにおける接着分子の役割と生体内
siRNA導入による制御
21
Program
Aⴊ▤↢‛කቇળKPFD
160
Recently, we identified thrombomodulin (TM) is induced by inorganic phosphate
(Pi) at 24 hour in vascular smooth muscle cells (VSMC), using DNA microarray. In
the present study, we examined the role of TM in vascular calcification, a critical
event in the development of cardiovascular disease. [Results] In VSMC, both
mRNA and protein expression of TM were markedly increased by Pi in a timedependent manner. Conversely, knockdown of TM by siRNA significantly
decreased Pi-induced calcification. TM secretion into the medium was increased by
Pi, and VSMC calcification was significantly augmented by the recombinant
extracellular six-EGF-repeat domain of TM. Furthermore, cholera toxin inhibited
calcification, whereas pertussis toxin had no effect on Pi-induced calcification,
suggesting that secreted TM and Gs-coupled receptor play an important role. Piinduced ERK1/2 phosphorylation, a downstream signal of TM and EGF, was
abrogated by TM siRNA. Pi also increased the expression of krüppel-like factor 5
(KLF5), which is a key regulator of VSMC phenotype and is known to be
transactivated by ERK1/2. In fact, Pi-induced expression of KLF5 was markedly
inhibited by ERK inhibitors, PD98059 and U0126, and by TM siRNA. Finally, Piinduced apoptosis and osteoblastic phenotype transition were significantly inhibited
by TM siRNA, ERK1/2 inhibitors and KLF5 siRNA. [Conclusion] TM is a novel
molecule that mediates vascular calcification via ERK1/2 and KLF5 signaling.
東京大学大学院医学系研究科加齢医学講座、2長崎大学大学院医歯薬学総合研究科
循環病態制御内科
1
孫 輔卿1、秋下 雅弘1、飯島 勝矢1、小川 純人1、前村 浩二2、江頭 正人1、
大内 尉義1
Thrombomodulin, a novel molecule promoting
vascular calcification via ERK/KLF5 signaling
25
Sphingosine-1-phosphate (S1P), a multifunctional lipid mediator in plasma,
regulates vascular and immune cell functions by activating S1P receptors. Previous
results suggest divergent effects of S1P on atherosclerosis. However, it remains
unknown whether and how each S1P receptor subtype in endothelial cells (ECs),
smooth muscle cells (SMCs), or monocytes/macrophages stimulates or inhibits
atherosclerosis. We explored the role of G12/13-Rho-coupled S1P2 in atherosclerosis
by analyzing S1P 2 -/- mice with apoE -/- background. S1P 2 was expressed in
macrophages, ECs and SMCs in atherosclerotic lesions of aortas. Compared to
S1P2 +/+ mice, the plaque area of S1P2 -/- mice was strikingly suppressed with the
decreased macrophage density, increased SMC density, elevated eNOS
phosphorylation, and down-regulation of proinflammatory cytokines. Bone marrow
chimera experiments revealed the major role of macrophage S1P2 in atherogenesis.
S1P2 -/- macrophages showed reduced Rho-Rho kinase (ROCK)-NF-㱖B activity,
consequently reduced oxidized LDL uptake and stimulated cholesterol efflux with
concomitant decreases in scavenger receptor expression and increases in cholesterol
efflux transporter expression, and reduced cytokine expression. S1P 2 in
macrophages stimulated macrophage transmigration toward the compartment with
a lower S1P concentration both in vitro and in vivo with Rac inhibition. S1P2 -/- ECs
also showed diminished activities of ROCK and NF-㱖B with decreased MCP-1
expression and increased phosphorylation of Akt and eNOS. Pharmacological S1P2
inhibition in S1P2 +/+ mice reduced the plaque area in aortas and modified LDL
accumulation in macrophages. These observations demonstrate that S1P2 plays a
critical role in atherogenesis and is a novel therapeutic target for atherosclerosis.
1
金沢大学医薬保健研究域医学系血管分子生理学、2金沢大学医薬保健研究域医学系
内科学、3石川県立看護大学健康科学講座
王 飛1、岡本 安雄1、居軒 功2、吉岡 和晃1、多久和 典子1,3、多久和 陽1
Sphingosine-1-phosphate receptor-2 deletion inhibits
macrophage proinflammatory activities and
atherosclerosis in mice
23
[BACKGROUND] The angiotensin II (Ang II) type 2 (AT2) receptor is crucially
involved in atherogenesis; however, bone marrow (BM) AT2-mediated antiatherogenic action remains undefined.
[METHOD AND RESULT] We generated BM chimera apoE-deficient (apoE -/- )
mice whose BM cells were repopulated with AT2-deficient (Agtr2 -/-) or wild type
(Agtr2+/+) cells. Eight weeks after BM transplantation, all mice were fed a western
diet for further two months. Mean blood pressure, heart rate, and lipid profile did
not show any difference between the two groups. The numbers of BM granulocyte/
macrophage progenitor cells (GMP) and peripheral blood monocytes did not differ
between the two groups of mice. Atherosclerotic lesion area was significantly
increased in apoE -/- /BM-Agtr2 -/- mice compared with apoE -/- /BM-Agtr2 +/+ mice
(51%, P＜0.05). The accumulation of MOMA-2 positive cells in aortic root was
markedly enhanced in apoE -/- /BM-Agtr2 -/- mice (65%, P＜0.05). To further
investigate the BM-AT2-mediated action on macrophage, Thioglycollate-elicited
Peritoneal macrophages (TGPM) were isolated from Agtr2-/- or Agtr2+/+ mice. Realtime PCR analysis showed that AT2 mRNA expression level in Agtr2+/+ TGPM was
much higher than Agtr2+/+ BM cells, whereas AT1 mRNA expression did not differ
between Agtr2 -/- and Agtr2 +/+ TGPM. Treatment with lipopolysaccharide (LPS)
increased TNF-㱍 and IL-1㱎 secretion from Agtr2 +/+ TGPM in a dose-dependent
manner, which were significantly augmented in Agtr2 -/- TGPM (40%, 50%,
respectively, P＜0.01). The number of apoptotic cells evaluated by TUNEL staining
did not differ between the two groups.
[CONCLUSION] Our findings demonstrate that BM-AT2 attenuates macrophage
pro-inflammatory responses to induce AT2-mediated anti-atherogenic activities.
1
京都府立医科大学大学院医学研究科循環器内科学、2愛媛大学大学院医学系研究科
分子心血管生物・薬理学
加藤 拓1、山田 浩之1、川人 浩之1、岸田 聡1、入江 大介1、池田 宏二1、
高橋 知三郎1、沖垣 光彦1、岩井 將2、堀内 正嗣2、松原 弘明1
Bone marrow AT2 receptor deficiency aggravates
atherosclerosis by augmenting macrophage proinflammatory responses
26
【目的】慢性腎臓病（CKD）は心血管病発症の独立した危険因子であるが、
CKDにおける動脈硬化進展機序の詳細は十分明らかではない。一方、血管
周囲脂肪組織は動脈硬化や血管傷害後のリモデリングに深く関与している
ことが最近報告されているが、慢性腎臓病における血管周囲脂肪組織の役
割については不明な点が多い。本研究の目的は、軽度腎機能障害が血管周
囲脂肪組織の表現型やレニン・アンジオテンシン系（RAS）に与える影響
を検討し、動脈硬化進展機構におけるその役割を明らかにすることである。
【方法と結果】8週齢雄アポE欠損マウスの片腎を摘出後、12週齢時より高コ
レステロール食を開始した。(片腎摘出群: n=12、対照群: n=12) 20週齢時
の胸部大動脈における動脈硬化形成を検討したところ片腎摘出群において
有意に進展していた(enface法 42％、P＜0.05)。平均血圧、心拍数、脂質プ
ロファイルは両群で同等であった。高コレステロール食負荷8週後の胸部大
動脈におけるVCAM-1, ICAM-1のmRNA発現レベルは負荷前と比べて著明
に亢進していたが、両群間では有意差を認めなかった。興味深いことに血
管周囲の脂肪組織重量はUNX群で有意に増加していた(31%、P＜0.05)。一
方精巣周囲脂肪重量は両群間で同等であった。血管周囲脂肪組織における
免疫組織学的解析では、脂肪組織面積および細胞数が片腎摘出群で有意に
増加していた(58%, 37%, P＜0.01 )。しかしCD45陽性細胞の集積は両群で同
等であった。更に血管周囲脂肪組織を採取しmRNA発現レベルをreal-time
PCR法を用いて解析したところ、片腎摘出群において褐色脂肪のマーカー
あるUCP1の低下とともに白色脂肪マーカーであるIgfbp3の発現亢進を認め
(44%, 130%, P＜0.05 ), RAS系のコンポーネントであるrenin, Angiotensinogen
の遺伝子発現が有意に亢進していた(430%, 33%, P＜0.05 )。これらの変化は
精巣周囲脂肪組織では認められなかった。【結語】片腎摘出マウスでは、血
管周囲脂肪組織における脂肪細胞の増殖およびRASの亢進が認められ、
CKDにおける動脈硬化進展機序に関与している可能性が示唆された。
1
京都府立医科大学大学院医学研究科循環器内科学、2京都府立医科大学大学院医学
研究科腎臓内科学
川人 浩之1、山田 浩之1、入江 大介1、岸田 聡1、加藤 拓1、沖垣 光彦2、
森 泰清2、松原 弘明1
血管周囲脂肪表現型とレニン・アンジオテンシ系-慢性腎臓
病はＲＡ系賦活化を介して動脈硬化を進展させるー
24
18：00〜
ポスターセッション：動脈硬化（2）
Poster Session：Atherosclerosis(2)
12月1日
（水）
／Wednesday, December 1
医学系研究科 遺伝子治療
How to write
Plenary Lecture
Nature Medicine
Symposium
Y.I.A.
Presentation
Selected Oral
Presentation
Poster
Index
Aⴊ▤↢‛කቇળKPFD
Special Talk
The 8th Korea-Japan Joint Symposium on Vascular Biology
Special
Lecture
161
［背景］マクロファージのアポトーシスはプラーク内の壊死コア形成に関与
しプラーク破綻のリスクを増加させることが知られている。しかしながら
アンジオテンシII受容体拮抗薬（ARB）のマクロファージアポトーシスに
対する作用と進展プラーク内における役割については不明な点が多い。
［方法・結果］8週齢から高コレステロール食を開始した雄apo E 欠損マウス
に15週齢からARB(オルメサルタン 3.0mg/kg/day）またはヒドララジン(50mg/
kg/day）を投与し、17、19週齢時の腕頭動脈における組織学的解析を行った。
コントロール群と比較しARB群とヒドララジン群は平均血圧の低下を認め
たが(p＜0.05）、2群間において差を認めなかった。脂質プロファイルは3群
間で同等であった。腕頭動脈プラーク面積はコントロール群とヒドララジ
ン群で同等であったが、オルメサルタン群で著明に低下していた（37％、p
＜0.01）
。またプラーク内の壊死コア領域はコントロール群とヒドララジン
群では差を認めなかったが、オルメサルタン群で著明に減少していた(52%,
p＜0.05)。17週齢時の腕頭動脈プラーク内におけるTUNEL陽性マクロファー
ジ数はARB投与群で著明に減少していた(23%、p＜0.05)。
［結論］ARBは進展プラーク内のマクロファージアポトーシスを減少させ、
壊死コア形成を抑制した。
京都府立医科大学大学院医学研究科循環器内科学
岸田 聡、山田 浩之、川人 浩之、加藤 拓、入江 大介、池田 宏二、高橋 知三郎、
沖垣 光彦、松原 弘明
アンジオテンシンII受容体拮抗薬はマクロファージのア
ポトーシス減少により進展プラーク内の壊死コア形成を
抑制する
28
President
Lecture
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
背景：Klotho遺伝子欠損マウスは早期老化症状によく似た表現形を示し、
動脈硬化や血管内皮機能障害を有する。近年、ヒトKlothoの遺伝素因と動
脈 硬 化 性 疾 患 と の 関 連 が 検 討 さ れ て い る。 我 々 はKlothoのSNPで あ る
rs650439(A/T)が高血圧患者において頚動脈内中膜肥厚(IMT)と関連しTアレ
ル保因者で動脈硬化進展が著しいと報告した。
目的：Klotho遺伝子の遺伝素因を有することが生活習慣病による心血管イ
ベント発症に繋がるか否かを明らかにするために、前回の検討とは別の研
究対象を用いてKlotho rs650439と高血圧臓器障害の関連性を検討する。
方法：大阪大学附属病院老年高血圧内科において遺伝子解析研究に参加し
た高血圧患者のうちで予後調査を行い得た555人(男性315人、240人)を対象
とした。Taqman PCR法にてrs650439の遺伝子型を同定し心血管イベント発
症との関連を検討した。また、高血圧性臓器障害の指標(動脈硬化：総頚動
脈IMT・脈波伝播速度(PWV)、心肥大：左室重量係数(LVMI)、腎障害：推
算糸球体濾過率(eGFR))とKlotho rs650439との関連を解析した。
結果：カプランマイヤー法で解析したところrs650439(AA+AT vs. TT)は脳卒
中発症と有意差な関連を認めTTアレル保持者で脳卒中発症率が高いという
結果であった (P＜0.05)。交絡因子を考慮した共分散分析を用いて頚動脈
IMT、LVMI、PWV、eGFRとの関連を検討したところrs650439(AA+AT vs.
TT)はLVMI(P=0.039)およびcf PWV(P=0.046)と有意な関連を認めた。IMTお
よびeGFRとrs650439の間に有意な関連性はみとめられなかった。
考察：Klotho のSNP(rs650439)は脳卒中発症ならびに動脈硬化・心肥大との
有意な関連性が認められたことより、Klotho 遺伝子多型が高血圧患者にお
いて臓器障害促進に関与する可能性が示された。
1
大阪大学医学部老年・腎臓内科学、2大阪大学大学院
学
小黒 亮輔1、神出 計1、加藤 のぞみ1、島岡 泉1、Congrains Ada1、
勝谷 友宏2、大石 充1、楽木 宏実1
Klotho遺伝子一塩基多型と動脈硬化性疾患との関連
27
Program
名古屋大学大学院医学系研究科循環器内科講座、2浜松医科大学医学部解剖学講座、
名古屋大学大学院医学系研究科老年科講座
Aⴊ▤↢‛කቇળKPFD
162
Objective: An interaction between the renin-angiotensin system and
neovascularization in atherosclerotic plaque development is unclear. We
investigated the actions of an angiotensin II type I receptor blocker in the
pathogenesis of atherosclerosis in ApoE-/- mice with special focus into plaque
neovascularization. Methods and Results: Ten-week-old male ApoE-/- mice fed a
high-fat diet were randomly assigned to two groups given vehicle (0.5%
carboxymethyl cellulose) or olmesartan (1 mg/kg daily) for 12 weeks.
Quantification of plaque areas at the aortic sinus and in the thoracic and abdominal
aorta revealed that, in all three regions, olmesartan reduced the diet-induced intimal
area and intimal neovessel density. Olmesartan reduced the expressions of toll-like
receptors (TLR-2 and TLR-4), stromal-derived factor-1, and a CXC chemokine
receptor mRNAs in these regions of aortas. Olmesartan reduced the numbers of
macrophages and the productions of monocyte chemoattractant protein-1 and
osteopontin proteins in the aortic sinus. Olmesartan reduced the increases in the
levels of matrix metalloproteinase (MMP)-2 and MMP-9 proteins and their
gelatinolytic activities in the aortic sinus or/and thoracic aorta. Double
immunostaining of the aortic sinus showed that these MMPs were localized in the
vascular smooth muscle cells and macrophages. Plasma levels of tumor necrosis
factor-α and interleukin-1β were reduced by olmesartan. MMP-2 deficiency
impaired not only macrophage accumulation and TLR4 expression but also intimal
neossel formation and plaque growth in the aortic sinus. Conclusions: Olmesartan
appears to inhibit intimal neovascularization in the ApoE-/- mouse model, partly by
reducing inflammation and MMP activation, thus decreasing atherogenic plaque
growth and stability.
3
1
成 憲武1、佐々木 健2、井上 愛子3、胡 莉那3、宋 海珍3、坂東 泰子1、
葛谷 雅文3、奥村 健二1、室原 豊明1
Angiotensin Type 1 Receptor Blocker Reduces Intimal
Neovascularization and Plaque Growth in Apo
E-Deficient Mice
31
〔目的〕高齢化と糖尿病罹患者の急激な増加に伴い、閉塞性動脈硬化症
(Peripheral Arterial Disease(PAD))による社会的損失が急増している。近年、
ゲノムワイド関連解析による大規模スタディにより、脳梗塞や心筋梗塞な
どのcommonな多因子遺伝性疾患の疾患感受性遺伝子が同定されているが、
閉塞性動脈硬化症の成因や治療反応性を規定する疾患関連遺伝子は未だ不
明である。疾患感受性遺伝子の機能を明らかにすることができれば、末梢
血管疾患特有の病態の解明ひいてはより効果的な治療法の開発に資するこ
ととなると考えられる。そこで我々は日本人集団に対してSNPsを用いたゲ
ノムワイド関連解析を行うことにより閉塞性動脈硬化症の疾患感受性遺伝
子を同定することを目的とした。
〔方法ならびに成績〕全国より集めた日本
人サンプルより、一次スクリーニングとしてPAD群195例を抽出し、対照群
1,358例とともに、222,285 SNPsについて網羅的にタイピングを行った。両
群の比較により得られた有意差上位2,696 SNPsについて、二次スクリーニ
ングとしてPAD群699例と、対照群1,540例との比較解析を行った。両スク
リーニングの結果を総じて、最も強い関連がOSBPL10遺伝子のintronic SNP
。また
rs1902341に認められた（P = 4.7×10-7, OR = 1.31, 95% CI 1.18 - 1.46）
OSBPL10遺伝子には他にも関連を示すSNPsが複数存在した(rs2045298, P =
-5
-5
-6
2.7×10 , rs2168422, P = 2.1×10 , rs6779621, P = 2.7×10 )。他にもCSMD1
遺伝子のintronic SNP rs2554503 (P = 5.7×10-5, OR = 1.32, 95% CI 1.15 - 1.51)
などいくつかのPADとの関連を示すSNPsが同定された。〔総括〕以上の結果
から、日本人集団におけるゲノムワイド関連解析により、OSBPL10遺伝子
が閉塞性動脈硬化症の疾患関連遺伝子であることが示唆された。
1
大阪大学大学院医学系研究科臨床遺伝子治療学、2大阪大学大学院大阪大学・金沢
大学・浜松医科大学連合小児発達学研究科、3大阪大学大学院医学系研究科老年・
腎臓内科学、4大阪府立急性期・総合医療センター、5大阪大学大学院医学系研究科
遺伝子治療学
郡山 弘1、中神 啓徳2、勝谷 友宏1、杉本 研3、荻原 俊男4、楽木 宏実3、
金田 安史5、森下 竜一1
ゲノムワイド関連解析による閉塞性動脈硬化症疾患関連
遺伝子OSBPL10の同定
29
老年・腎臓内科、2国立循環器病研究センター
高
【目的】高齢者に多い両側性腎動脈狭窄(RAS)の早期発見に有用な情報を得
ることを目的として、両側性RASの患者背景を片側性と比較検討。
【方法】
対象は動脈硬化性RAS 55例。腎動脈造影で50%以上の狭窄を認める場合を
狭窄ありと判断し、両側性(B群)・片側性(U群)に群分けした。2群で動脈硬
化のリスクファクター、腎機能、血圧などを比較検討した。
【結果】対象の
内訳は、B群 20例・U群35例であった。両群の平均年齢は両側RASで高い傾
向にあった (p=0.195)。男女比も両側RASで男性がやや多かった(p=0.167)。
動脈硬化危険因子の比較では、B群で糖尿病を有する割合が多い傾向を示
した(p=0.087)。喫煙率もB群で高い傾向(p=0.156)を認め、飲酒者はB群：
86% vs. U群 58%と有意に両側で多かった(p=0.026)。血圧や腎機能には両群
で差を認めなかった。PRAは両群ともに正常より高く、群間に有意な差を
認めなかった。
【結論】両側性RASは従来から言われているように、動脈硬
化リスクの高い高齢の男性で喫煙、飲酒者、糖尿病患者に多い傾向を認め
ることが確認された。
1
大阪大学大学院医学系研究科
血圧・腎科
神出 計1、河野 雄平2、大石 充1、楽木 宏実1
両側腎動脈狭窄症の患者背景因子に関する検討
32
〔目的〕我々はこれまでに日本人集団に対してSNPsを用いたゲノムワイド
関連解析を行うことによりPADの疾患関連遺伝子としてOSBPL10を同定し
た。OSBPL10の機能に関してはこれまであまり報告がなされていない。そ
こでPADの病態のメカニズムを明らかにするため、OSBPL10の機能解析を
行うことを目的とした。〔方法ならびに成績〕ヒト各組織のNorthern blotting
により発現パターンを解析した。その結果OSBPL10は心臓、腎臓、肺、胎
盤など、血管が比較的豊富な臓器で強い発現を認めた。心臓の分画では、
大動脈に最も強い発現を認めた。培養細胞に対しwestern blottingを行ったと
ころ、ヒト血管内皮細胞（HAEC）で強い発現を認めた。抗OSBPL10抗体
を用いて免疫染色を行ったところOSBPL10はHAECの小胞体に局在するこ
とが確認された。OSBPL10転写産物をクローニングし、
HAECに発現させた。
その結果、過剰発現したOSBPL10は微小管および小胞体に局在し、両者の
形態変化を引き起こした。血管内皮細胞はin vitroにおいて酸化コレステロー
ルであるオキシステロールの添加などにより小胞体ストレスが誘導される
こと、また動脈硬化性病変で小胞体ストレスが生じていることなどが明ら
かになってきている。そこで次に小胞体ストレスにおけるOSBPL10の関与
について検討した。まずHAECの培養液に7-ketosterolやツニカマイシンを添
加、あるいは低酸素刺激を行ったところ、OSBPL10の発現上昇が認められ
た。次にOSBPL10を未刺激のHAECに過剰発現させたところ、小胞体スト
レスが誘導され、下流の現象であるオートファジーも誘導された。siRNA
を用いてOSBPL10をノックダウンしたHAECに、7-ketosterolやツニカマイシ
ン処理を行ったところ小胞体ストレスやオートファジーの有意な抑制は認
めなかった。これらの結果からOSBPL10は小胞体ストレスにさらされた血
管内皮細胞において、微小管依存的なストレス応答に関与している可能性
が示唆されたが、
小胞体ストレス経路自体に必須ではないと考えられた。
〔総
括〕OSBPL10はHAECで高い発現を認め、小胞体および微小管上に局在し、
血管内皮細胞における小胞体ストレスに関与している可能性が示唆された。
1
大阪大学大学院医学系研究科臨床遺伝子治療学、2大阪大学大学院大阪大学・金沢
大学・浜松医科大学連合小児発達学研究科、3大阪大学大学院医学系研究科老年・
腎臓内科学、4大阪大学大学院医学系研究科遺伝子治療学
郡山 弘1、中神 啓徳2、楽木 宏実3、金田 安史4、森下 竜一1
閉塞性動脈硬化症疾患関連遺伝子OSBPL10の機能解析
30
18：00〜
ポスターセッション：動脈硬化（3）
Poster Session：Atherosclerosis(3)
12月1日
（水）
／Wednesday, December 1
1
1
大阪大学大学院医学系研究科循環器内科学、2大阪大学大学院医学系研究科臨床遺
伝子治療学、3国立循環器病研究センター細胞生物部
Symposium
Y.I.A.
Presentation
Selected Oral
Presentation
Poster
Index
Aⴊ▤↢‛කቇળKPFD
How to write
Plenary Lecture
Nature Medicine
163
Special Talk
The 8th Korea-Japan Joint Symposium on Vascular Biology
Special
Lecture
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Atherosclerosis is commonly observed in obesity. Obese atherosclerosis-prone
animal models may be a promising tool for understanding the pathophysiology of
obesity-associated atherosclerosis and for developing effective therapeutic strategies
for such diseases. However, most rat strains are resistant to atherosclerosis. This
study aimed to assess the susceptibility of 2 obese hypertensive rat models, SHRSP.
Z-Lepr fa/IzmDmcr rats (SHRSP-fatty) and SHR.Cg-Leprcp/NDmcr rats (SHR-cp),
to arterial lipid deposition, an initial stage of atherosclerosis, by comparing these
strains with nonobese stroke-prone spontaneously hypertensive rats (SHRSP).
Eight-week-old male SHRSP, SHRSP-fatty, and SHR-cp were fed a high fat and
cholesterol diet containing 20% palm oil, 5% cholesterol, and 2% cholic acid for 5
weeks. Body weight, blood pressure, and fasting serum levels of total cholesterol
and triglycerides were measured in 12-week-old rats. Oil red O staining was
conducted to visualise lipid deposition in the mesenteric artery. The body weight of
12-week-old SHRSP-fatty and SHR-cp was higher than that of SHRSP (p ＜
0.005). The systolic blood pressure of SHRSP and SHRSP-fatty was higher than
that of SHR-cp (p ＜ 0.005). Serum total cholesterol and triglyceride levels were
elevated in SHRSP-fatty (p ＜ 0.005) and SHR-cp (p ＜ 0.05) compared with those
in SHRSP. Lipid deposition in the mesenteric artery was more extensive in SHRSPfatty (37.7 ± 4.9%; p ＜ 0.005) than in SHRSP (13.1 ± 2.8%), but was markedly
less in SHR-cp (1.8 ± 0.4%; p ＜ 0.05). These results indicate that SHRSP-fatty is
highly susceptible to arterial lipid deposition, whereas SHR-cp is resistant to such
deposition. SHRSP-fatty may be a useful obese rat model for studying
atherosclerotic processes.
President
Lecture
（背景と目的）動脈硬化は血管炎症に基づく内皮機能障害をベースに病態形
成がなされる。ドッキング蛋白質であるGab1は、hepatocyte growth factor
(HGF)/c-Met依存性シグナルで重要な役割を果たす事がこれまで報告されて
いる。内皮細胞特異的Gab1欠損 (Gab1ECKO)マウスを我々はこれまで作成
して、大腿動脈結紮による下肢虚血で、全例が下腿壊死を呈して、虚血耐
性の低下を呈する事を見出した。さらに、我々はGab1ECKOマウス の下腿
壊死の表現型は、内皮細胞におけるHGF/c-Met依存性シグナルによる血管
新生の異常によることも見出している。一方、Gab1の動脈硬化性疾患にお
ける役割はこれまで明らかでない。本研究は内皮細胞のGab1が動脈硬化過
程で担う役割を明らかにすることを目的とする。
（方法、結果）マイクロ
DNAアレイを用いて、ヒト臍帯静脈内皮細胞（HUVEC）をHGFで刺激して
Gab1依 存 性 に 発 現 誘 導 さ れ る 遺 伝 子 群 を 探 索 し た と こ ろ、Kruppel-like
factor (KLF2)を見出した。また、Gab1ECKOマウスではコントロールマウス
に比して内皮細胞でのKLF2の発現が有意に減少していた。KLF2は内皮細
胞の機能維持や抗動脈硬化作用に必須の遺伝子と報告されている。そこで、
我々は内皮細胞のGab1が動脈硬化に対して抑制的な働きを有するものと考
えて、Gab1ECKOマウスをapolipoprotein E欠損（apoEKO）マウスと交配し
てGab1ECKO/apoEKOマウスを作成した。正常食を給餌された24週齢のオ
ス のGab1ECKO/apoEKO 及 びapoE KOマ ウ ス（ コ ン ト ロ ー ル ） に
angiotensinII(500ng/kg/min)を浸透圧ポンプで4週間投与した。胸腹部大動脈
を摘出してOil Red O染色したところ、Gab1ECKO/apoEKOマウスでは全例
においてコントロールに比して有意に広範囲の強い動脈硬化形成が観察さ
れ た。 さ ら に マ ク ロ フ ァ ー ジ の 浸 潤 と 動 脈 壁 で のVCAM-1発 現 も
Gab1ECKO/apoEKOマウスではコントロールに比して有意に促進されてい
た。
（結論）以上より、内皮細胞のGab1はapoEKOマウスでのangiotensin II
依存性の動脈硬化を負に制御する機能を担うことが示唆された。
国政 和宏1、吉富 久恵2、三浦 ちとせ2、森 英樹1、土倉 覚3、池田 克己2、
高 明2、森 真理1、家森 幸男1
樋口 香織1、中岡 良和1、塩山 渉1、谷山 義明2、森下 竜一2、望月 直樹3、
小室 一成1
武庫川女子大学国際健康開発研究所、2武庫川女子大学薬学部、3SHR等疾患モデ
ル研究会
High susceptibility of obese hypertensive SHRSP.
fa
/IzmDmcr rats to lipid deposition in the
Zmesenteric artery
34
内皮細胞のドッキング蛋白質Gab1はapolipoprotein E欠
損マウスでのangiotensin II依存性動脈硬化を負に制御す
る
33
Program
18：00〜
Aⴊ▤↢‛කቇળKPFD
164
2Cl-C.OXT-A is a synthesized nucleoside analogue with the molecular weight of
284. It is stable, easy to synthesize and small; these properties of this agent may
thereby allow various systemic and topical delivery methods.
In vitro, 2Cl-C.OXT-A significantly stimulated tube formation of human umbilical
endothelial cells (HUVEC). Its maximum potency at 100 㱘M was stronger than
that of vascular endothelial growth factor (VEGF), a positive control. At this
concentration, 2Cl-C.OXT-A moderately stimulated proliferation as well as
migration of HUVEC. Immunoblot analyses suggest that 2Cl-C.OXT-A induces
angiogenic responses in HUVEC mediated by a MAP kinase cascade comprising
MEK and ERK1/2, but independently of VEGF receptor tyrosine kinase.
In vivo assay using chicken chorioallantoic membrane (CAM) and rabbit cornea
also suggested the angiogenic potency of 2Cl-C.OXT-A.
2Cl-C.OXT-Aは分子量284の合成核酸誘導体である。我々はこれまでin vitro
において2Cl-C.OXT-A が強力なHUVECの管腔形成促進作用を有することを
見いだし、報告してきた。2Cl-C.OXT-AはHUVECの管腔形成の他、増殖や
遊走も同じ濃度（10-100㱘M）で促進する。同時にMEKやERK1/2の活性化
を促進し、MEK阻害剤PD98059は2Cl-C.OXT-AによるERK1/2の活性化だけ
でなく管腔形成促進をも阻害した。VEGFR inhibitor SU5416 は2Cl-C.OXT-A
によるERK1/2の活性化に影響せず、従って作用点はVEGFRの下流でMEK
よりも上流と考えられた。今回、この2Cl-C.OXT-Aのin vivoでの効果を鶏漿
尿膜とウサギ角膜を用いて検証したので報告する。
香川大学医学部薬物生体情報学講座、2香川大学医学部自律機能生理学講座、3香川
大学医学部細胞情報生理学講座、4香川大学医学部皮膚化学講座、5徳島文理大学香
川薬学部、6東京都臨床医学総合研究所、7帝國製薬株式会社
1
Angio- and lymphangiogenesis are inherently related processes, in part driven by
the same growth factors (GFs). However, how blood and lymphatic vessels regulate
each otherʼs growth is unknown. This work introduces a novel mechanism
explaining the temporal and spatial relation of blood and lymphatic vessels. Using
the corneal micropocket assay, PBS or VEGF-A (100, 200, 400 or 1600ng) was
implanted in mice corneas to quantify angio- and lymphangiogenesis using
immunostaining of LYVE-1 and CD31, respectively. 100ng VEGF-A induced
significant angiogenesis but not lymphangiogenesis, while 400 and 1600ng
VEGF-A induced both lymph- and angiogenesis, suggesting the dose threshold for
VEGF-A-induced corneal lymphangiogenesis to be higher than angiogenesis.
200ng VEGF-A-induced lymphangiogenesis could be occurred on day 10 but not
day 6 when angiogenesis could be observed, suggesting that VEGF-A induced
angiogenesis peaks earlier than lymphangiogenesis. VEGF-A surprisingly reduced
VEGF-C in the supernatant of blood vessel endothelial cells, suggesting GF
clearance by the growing endothelium. The orientation of lymphatic sprouting
toward angiogenic vessels and away from exogenous GFs was VEGF-C dependent.
In vivo molecular imaging revealed higher VEGFR-2 in angiogenic tips when
compared to normal vessels. Consistently, lymphatic growth was impeded in the
angiogenic front. VEGF-C/R-2 complex in the cytoplasm of VEGF-A-treated
endothelium indicated that receptor-mediated internalization causes GF clearance
from the extra cellular matrix. VEGF-A-induced lymphangiogenesis is delayed by
angiogenic vessels where upregulated VEGFR-2 can trap VEGF-C. GF clearance
by receptor-mediated internalization is a new paradigm explaining how growing
vasculature regulates its environment.
マサチューセッツ眼科耳鼻
中尾 新太郎1,2
塚本 郁子1、榊原 紀和5、丸山 徳見5、五十嵐 淳介2、小坂 博昭2、窪田 泰夫4、
徳田 雅明3、芦野 洋美6、川田 光裕7、服部 健一7、田中 真司7、
小西 良士1
1
九州大学大学院医学研究院眼科学、2ハーバード大学
科病院 眼科
Blood vessel endothelial VEGFR-2 delays
lymphangiogenesis
38
Background: Deferoxamine (DFO), an iron chelator, is clinically used for excess
iron disorders. DFO has been reported to upregulate expression of angiogenic
factors such as vascular endothelial growth factor (VEGF), therefore DFO has
potential action for contributing to angiogenesis. Herein we clarify the effect and
mechanism of DFO on angiogenesis in vitro and in vivo. Methods and Results: In
vitro study, DFO induced 2-fold increase in phosphorylation of Akt and endothelial
nitric oxide synthesis (eNOS) in human aortic endothelial cells (HAEC). DFO
action on eNOS phosphorylation was inhibited by phosphatidylinositol 3-kinase
inhibitor LY294002. Tube formation, cell proliferation and migration were
promoted by DFO stimulation, which was significantly reduced by LY294002 in
HAEC. In vivo study, C57BL/6J mice at 8 weeks old age were subjected to
unilateral hindlimb surgery with or without DFO administration daily. Laser
Doppler analysis demonstrated that DFO treatment accelerated blood flow recovery
in response to ischemic hindlimb (LDBF of ischemia to non-ischemia ratio on
postoperative day 28 days, 84+/-4% vs 72+/-4%, p＜0.01). Capillary density was
significantly higher in ischemic muscle of DFO-treated mice compared to vehicletreated mice (capillaries to muscle fiber ratio; 1.30+/-0.11 vs 0.90+/-0,01, p＜0.05).
Phosphorylation of Akt and eNOS in ischemic muscles were markedly augmented
1.5-times in DFO-treated mice compared to vehicle-treated mice. Urinary NOx
excretion was significantly increased in DFO-treated mice. This DFO effect on
angiogenesis was abolished in ischemic hindlimb model of eNOS-deficient mice.
Conclusion: DFO promotes vascular endothelial cell function and enhances
angiogenesis through Akt-eNOS pathway.
1
徳島大学大学院ヘルスバイオサイエンス研究部薬理学、2徳島大学大学院ヘルスバ
イオサイエンス研究部生体情報内科学
池田 康将1、田島 壮一郎1、吉田 守美子2、山野 範子1、木平 孝高1、
石澤 啓介1、粟飯原 賢一2、冨田 修平1、土屋 浩一郎1、玉置 俊晃1
Deferoxamine, an iron chelator, enhances
angiogenesis through Akt-eNOS-dependenat pathway
in endothelial cells
36
2Cl-C.OXT-A stimulates angiogenesis both in vitro and
in vivo.
37
【背景及び目的】PI3キナーゼ(PI3K)は、クラスI, II及びIIIに分類され、8種
のアイソフォームが存在する。これまで血管内皮細胞において、クラスI型
に属するp110㱍は血管内皮増殖因子(VEGF)やアンジオポエチン-1(Ang-1)な
どの刺激により細胞膜上でPtdIns (3,4,5)P3 を産生し、その下流のAktやRacを
介して内皮細胞の増殖、分化、生存、遊走等を制御し、脈管形成及び血管
新生に関与することが分かっていた。一方、3種のクラスII型のうち、C2㱍
は心臓、血管平滑筋及び内皮細胞に豊富に発現し、ある種の細胞では細胞
内小胞輸送に関わる可能性が示唆されていたが、動物個体レベルでの生理
的役割は全く不明であった。本研究では、C2㱍の動物個体における生理機
能を明らかにするために、全身型および組織特異的C2㱍遺伝子ノックアウ
ト(KO)マウスを作製し、その表現型を解析した。
【結果及び考察】C2㱍全身性KOマウスは、
顕著な血管新生・成熟異常を伴い、
胎生致死
（E9.5-E11.5）であった。内皮細胞特異的コンディショナルKO (CKO)
マウスも血管新生・成熟異常によりE18.5までに致死となった。また、平滑
筋及び心筋細胞CKOマウスは正常に発育し、心血管系には異常が見られな
いことから、内皮細胞に発現するC2㱍が胎生期血管形成に必須であること
が明らかとなった。一方、C2㱍ヘテロKOマウスは正常に発生・発育するも
の、生後の生理的網膜血管新生・成熟の異常、血管透過性亢進、アンジオ
テンシンII慢性投与による解離性動脈瘤を呈した。更に、RNA干渉法を用
い たC2㱍 ノ ッ ク ダ ウ ンHUVECに お い て、 １） 初 期 エ ン ド ソ ー ム で の
PtdIns(3)P産生、２）細胞遊走及び管腔形成、
３）低分子量Gタンパク質（RhoA,
Rac1, Rap1）活性化、
４）アクチン骨格、
トランス-ゴルジネットワーク（TGN）
形態、５）VE-カドヘリンの細胞膜局在及び透過性制御、の著しい異常が
示された。以上の結果から、C2㱍はVEGF-Akt/ERKシグナル伝達系制御に
は関与せず、クラスI型とは全く異なるメカニズムで機能する新たな細胞内
シグナル分子であることが明らかとなった。
金沢大学大学院医学系研究科血管分子生理学、2石川県立看護大学看護学部健康科
学講座
1
吉岡 和晃1、多久和 典子1,2、岡本 安雄1、多久和 陽1
クラスIIα型PI3キナーゼC2αは血管新生及びバリアー機
能に必須である
35
ポスターセッション：血管・リンパ管新生
（1）
Poster Session：Angiogenesis / Lymphangiogenesis(1)
12月1日
（水）
／Wednesday, December 1
Special Talk
How to write
Plenary Lecture
Nature Medicine
Symposium
Y.I.A.
Presentation
Selected Oral
Presentation
Poster
Index
Aⴊ▤↢‛කቇળKPFD
165
Special
Lecture
The 8th Korea-Japan Joint Symposium on Vascular Biology
President
Lecture
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
近年、VEGFシグナル遮断による血管新生阻害薬が臨床導入されたが、特
に癌に対する治療に際し、最初から効かないことや初めは効いてもやがて
効かなくなること（薬剤不応性・耐性）
、正常な血管の内皮細胞を障害する
ことが問題となっている。前者はVEGF以外の血管新生促進因子の関与、後
者はVEGFが内皮生存因子として機能していることによる。我々は、血管内
皮細胞が産生し、自らに作用して血管新生を制御するVasohibin-1 (VASH１)
と、そのホモログのVasohibin-2 (VASH２) を単離・同定し、臨床応用に向け
た研究を進めている。VASH１は、広いスペクトルムで血管新生を抑制する
が、このことはVEGF以外の血管新生促進因子が関与する場合でも有用と考
えられる。VASH１の他の血管新生抑制因子と異なる特徴として、種々のス
トレスに対する血管内皮細胞の抵抗性を増し、血管内皮細胞の生存因子と
して機能することを見出している。今回は、VEGFR阻害剤によって生じる
血管内皮細胞障害をVASH1が制御出来るか否か検討した。
ヒト臍帯静脈血管内皮細胞(HUVEC)にVEGFR1/2阻害剤SU5416(1μM)を
作用させ、72時間後の細胞死を定量的に解析した。HUVEC をVASH１遺伝
子搭載アデノウイルスベクター(AdVASH1)で感染させておくと、SU5416に
よ る 細 胞 死 はAdLacZの コ ン ト ロ ー ル と 比 較 し て 約40%抑 制 さ れ た(p＜
0.001)。アデノウイルスベクターの替わりに、レコンビナントVASH１蛋白
（100 nM）を投与して評価したところ、SU5416による細胞死は約30%抑制
された(p＜0.001)。
以上より、VEGFシグナル阻害薬とVASH-1を併用することで、内皮細胞
障害を回避し、より効果的な血管新生抑制効果が期待される。学会では、
マウスを用いたVEGFシグナル遮断により起こる正常血管退縮に対する
VASH１の効果についても報告したい。
東北大学加齢医学研究所腫瘍循環研究分野
林 英樹、宮下 浩輝、鈴木 康弘、小林 美穂、佐藤 靖史
VasohibinはVEGFシグナル遮断による内皮細胞障害を防
止する
39
Program
18：00〜
Aⴊ▤↢‛කቇળKPFD
166
Endothelial cell activation and dysfunction are associated with many vascular
disorders, including atherosclerosis, tumor growth, and sepsis. Here, we showed
that vascular endothelial growth factor (VEGF), tumor necrosis factor-㱍, and
thrombin rapidly and dramatically induced early growth response (Egr) families,
Egr-1 and Egr-3. Egr-3 expression in response to VEGF was more sustained
compared with Egr-1, which was mediated by NFATc1, NFATc2, CREB, and SRF
binding to the Egr-3 promoter. RNAi-mediated knockdown of Egr-3 specifically
inhibited transcription of the target genes, including TF, Ets-1, E-selectin and
VCAM-1. Genome-wide analysis of Egr-3 binding sites using chromatin
immunoprecipitation followed by sequencing (ChIP-seq) revealed that Egr-3 bound
to the transcription initiation sites of target genes where histone H3 lysine 4 monoand tri-methylation were highly enriched. Egr-3 knockdown compromised VEGFmediated proliferation, migration, and tube formation of endothelial cells and
blocked monocyte adhesion. Knocking down Egr-3 using an adenoviral miRNA
approach abrogated VEGF-mediated vascular outgrowth from ex vivo aortic rings
and attenuated vascularization and melanoma tumor growth in vivo. Together, these
findings suggest that Egr-3 is a critical determinant of VEGF signaling in activated
endothelial cells. Thus, Egr-3 represents a potential therapeutic target in VEGFmediated vasculopathic diseases.
Semaphorin3E (Sema3E)は胎生期における神経 ‐ 血管ガイダンス分子であ
ることが知られているが、生後の血管新生における役割はよく知られてい
ない。その役割を調べるため、ヒト臍帯静脈血管内皮細胞 (HUVEC)を用い
て検討したところ、Sema3EはHUVECにおいて血管内皮増殖因子（VEGF）
の受容体であるVEGFR2のリン酸化を抑制し、濃度依存性にVEGFの血管新
生促進作用を抑制することがわかった。またマウス虚血肢においてSema3E
の発現は著明に上昇しており、Sema3Eの阻害剤として働くPlexinD1-Fc融合
蛋白（PlexinD1-Fc）によりSema3Eを抑制すると、有意な血流の改善を認め
た。さらにSema3Eノックアウトマウスでは下肢虚血後の血管新生が野生型
に比して改善していた。老化分子であるp53も血管新生に対して抑制的に働
くことが知られているが、HUVECにおいては、低酸素刺激によってp53が
活性化しSema3Eの発現が誘導された。マウス虚血下肢においてもp53依存
性にSema3Eの発現の増加が認められたが、これらの上昇は糖尿病マウスの
虚血下肢においてさらに増強されていた。そして糖尿病マウスの虚血肢に
VEGFとPlexinD1-Fcの発現ベクターを同時に投与すると、VEGF単独投与群
と比較して著明な血流改善が得られた。骨格筋や脂肪組織といったエネル
ギー代謝の標的臓器では、その機能に関して血管の形成が重要な役割を担っ
ている。我々はこれらの臓器におけるSema3Eの発現が糖尿病モデルにおい
て上昇していること、さらにSema3Eのヘテロノックアウトマウスにおいて
糖負荷試験を行うと、野生型に比して血糖値の上昇が抑制されていること
を確認した。さらに高脂肪高ショ糖負荷をした野生型マウスにPlexinD1-Fc
の発現ベクターを筋注すると、コントロールに比べて空腹時血糖および糖
負荷試験による血糖値の上昇が抑制されていた。これらの結果から、
Sema3Eを抑制することは、特にVEGF等の治療のみでは効果のない症例に
対する新たな治療的血管新生の標的となり得ると考えられた。さらに
Sema3Eは、血管新生の抑制作用のみならず、糖尿病をはじめとする代謝性
疾患の病態生理に関わっていることが示唆された。
千葉大学大学院医学研究院循環病態医科学
東京大学先端科学技術研究センターシステム生物医学部門、2Department of
Molecular and Vascular Medicine, Beth Israsel Deaconess Medical
Center/Harvard Medical School, Boston, MA, USA
1
森谷 純治、南野 徹
末弘 淳一1、浜窪 隆雄1、児玉 龍彦2、William C Aird2、南 敬1
血管新生抑制因子を標的とした次世代の血管再生治療お
よび代謝性疾患治療の開発
43
Vasohibin-1(VASH1)は、VEGFやFGF-2をはじめとする血管新生刺激に応答
し、血管内皮細胞特異的に発現誘導され、血管新生を負に制御する分泌性
調節因子として機能する。VEGFやFGF-2によるVASH1遺伝子の発現誘導は
PKC-㱐のシグナル伝達経路を経ていること、その誘導はタンパク合成阻害
により消失すること、低酸素はVEGFやFGF-2によるVASH1遺伝子の発現誘
導を抑制することなどが判明しているが、その分子メカニズムついては不
明である。最近、ChIP-on-chip法を用いて転写因子GATA-2の結合をゲノム
ワイドにスクリーニングする過程でVASH1遺伝子の5ʼ上流域及びエクソン
領域にGATA-2の結合サイトが幾つか存在することを見出した。CHIPアッ
セイによってさらに結合領域を絞り込んだところ、VASH1遺伝子の5ʼ上流
域-1282~1138bpの間にGATA-2結合サイトが存在し、その結合はVEGFの刺
激に応答して増強することが確認された。siRNAによってGATA-2をノック
ダウンすることによって、VEGF刺激によるVASH1遺伝子の発現亢進が顕
著に抑制された。一方、プロテアソーム阻害剤MG-132を用いた解析から、
内皮細胞内においてGATA-2蛋白はプロテアソーム経路によって迅速に分解
を受けており、VEGF刺激によりGATA-2蛋白が安定化することが示唆され
た。GATA-2に対する特異抗体を用いた免疫染色の結果、血管内皮細胞内で
のGATA-2は、VEGF、FGF-2、血清刺激によって核へと集積することが確
認された。また、低酸素状態ではGATA-2のmRNA発現レベルや血管新生刺
激による核への集積に対してほとんど影響はなかった。以上の結果、血管
新生刺激によりGATA-2は内皮細胞の核へと集積し、VASH1遺伝子の５ʼ上
流域に結合して転写促進に寄与すること、低酸素状態においてはGATA-2以
外の抑制メカニズムが関与することが示唆された。
1
東北大学加齢医学研究所腫瘍循環研究分野、2東京大学先端科学技術研究センター
システム生物医学
鈴木 康弘1、神吉 康晴2、南 敬2、佐藤 靖史1
転写因子GATA-2によるVasohibin-1遺伝子の発現調節
41
Vascular endothelial growth factor activation of
endothelial cells is mediated by early growth
response-3
42
目的：成体において種々の臓器に組織幹細胞が存在し、組織傷害に対し修
復を行うことが示唆されている。血管においても最近、血管外膜に血管に
分化し得る血管幹細胞、血管内皮前駆細胞の存在が報告されている。今回
我々は、ラット胸大動脈における血管に局在する血管内皮前駆細胞の確認
および血管構成細胞との相互作用を検討した。方法：7週齢雄性SDラット
より胸大動脈を摘出しリング状に切断し、マトリゲルに包埋して7日間3次
元培養を行い、組織より発芽した細胞（sprout細胞）を内皮マーカーである
von willbrand factor (vWF)、 平 滑 筋 細 胞 マ ー カ ー で あ る 㱍-Smooth Muscle
Actin (㱍-SMA)による免疫染色を行った。さらに3次元培養を施行中の大動
脈リングを経時的にマトリゲルより取り出し組織切片を作成し、CD31、
CD34および細胞増殖マーカーであるKi-67による免疫染色を行った。a)内膜、
中膜、外膜を有する大動脈、b)外膜を取り除いた大動脈、c)大動脈外膜のみ、
の3種の組織をそれぞれ3次元培養しsprout細胞を観察した。また、大動脈外
膜より細胞を採取し、外膜に存在する細胞の特性を確認した。結果：3次元
培養により発芽したsprout細胞はvWF陽性、α-SMA陰性であった。3次元培
養中の大動脈はsprout細胞発芽時において外膜にCD31陰性かつCD34陽性、
Ki-67陽性細胞の存在が確認された。b)外膜を取り除いた大動脈、c)大動脈
外膜のみを用いた3次元培養ではsprout細胞の発芽を認めなかった。外膜よ
り採取した細胞からCD34陽性かつCD31陰性の細胞を確認した。結論：ラッ
ト大動脈外膜には血管内皮前駆細胞の存在が示唆され、sprout細胞の発芽に
寄与している可能性が示唆された。またsprout細胞は、内膜、中膜、外膜を
有する大動脈でのみ発芽し、外膜を取り除いた動脈、外膜のみの組織では
発芽しないことより血管外膜に局在する血管内皮前駆細胞は中膜平滑筋と
の相互作用により血管内皮に分化し、血管新生に寄与する可能性が示唆さ
れた。
日本大学大学院総合科学研究科生命科学、2日本大学医学部先端医学講座細胞再生・
移植医学分野
1
山元 智衣1、松本 太郎2、福田 昇1
ラット胸大動脈における血管内皮前駆細胞の局在および
血管構成細胞との相互作用についての検討
40
ポスターセッション：血管・リンパ管新生
（2）
Poster Session：Angiogenesis / Lymphangiogenesis(2)
12月1日
（水）
／Wednesday, December 1
1
1
東京大学先端科学技術研究センター システム生物医学部門、2興和株式会社
創薬研究所
How to write
Plenary Lecture
Nature Medicine
Symposium
Y.I.A.
Presentation
Selected Oral
Presentation
Poster
Index
Aⴊ▤↢‛කቇળKPFD
Special Talk
The 8th Korea-Japan Joint Symposium on Vascular Biology
Special
Lecture
167
Flk1/VEGFR2 is a receptor tyrosine kinase for vascular endothelial growth factor
(VEGF-A). It has been demonstrated that VEGF-A/Flk1 signals play important
roles in proliferation, survival, migration, and cell-cell adhesion of nascent
endothelial cells and are essential for embryonic blood vessel development in mice.
However, it remains to be elucidated whether Flk1 is also required for the
development of lymphatic vessels originated from blood vessels at later stages. In
this study, we analyzed the expression patterns and roles of Flk1 in lymphatic
endothelial cells (LECs). The expression analysis of flowcytometry,
immunohistochemistry, and a lacZ reporter knocked into the Flk1 locus confirmed
that Flk1 is expressed in CD31+Prox1+Podoplanin+ LECs throughout lymphatic
vessel development in mice. To address if Flk1 plays a role in lymphatic vessel
development, we conditionally inactivated the Flk1 gene in LECs by the tamoxifeninduced Cre-loxP system using Prox1-CreERT2 and Flk1-flox mice. Tamoxifen or
4-hydroxytamoxifen was administrated into pregnant and neonatal mice to induce
loss of Flk1 specifically in embryonic and neonatal LECs, respectively. We have
detected to date abnormal distribution of LECs in Flk1 conditional knockout
embryos. In addition, a lymphatic flow along the ischiatic vein was missing with fat
deposition, indicating the regression of a collecting lymphatic vessel during
postnatal periods after Flk1 was inactivated in neonates. These results suggest that
Flk1 plays a crucial role in migration and cell survival during physiological
lymphatic vessel formation in mice.
President
Lecture
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
血管内皮細胞においてKLF2およびKLF4はThrombomodulinやeNOSなど血管
内 皮 機 能 の 改 善 作 用 を 有 す る 遺 伝 子 の 発 現 を 増 強 さ せ、VCAM-1や
E-selectinといった接着因子や炎症性因子の発現を抑制させることが明らか
となっている。HMG-CoA reductase阻害剤であるピタバスタチンは血中LDL
コレステロールを低下させるだけでなく血管内皮細胞におけるKLF2および
KLF4の発現を増強させることが明らかとなった。KLF2ならびにKLF4はマ
ウス大動脈においてもピタバスタチンにより誘導されることが確認された。
しかし、ピタバスタチンによるKLF2ならびにKLF4の発現増強機序は明ら
かにはなっていない。そこで、今回、血管内皮細胞であるHUVECを用いて
ピタバスタチンによるKLF2およびKLF4発現増強機序の解析を行った。そ
の結果、ピタバスタチンによりHUVECにおいてERK5のリン酸化が2時間よ
り誘導されることが明らかとなった。ERK5は血管内皮細胞においてMEK5/
ERK5/MEF2経路の一端を担っていることが報告されている。そこで、ピタ
バスタチンによるKLF2およびKLF4発現増強機序にMEK5/ERK5/MEF2経路
が 関 与 し て い る かsiRNA実 験 を 行 い、 解 析 し た。 そ の 結 果、MEK5、
ERK5、MEF2のいずれのタンパクもノックダウンさせるとピタバスタチン
によるKLF2およびKLF4発現増強作用は減弱した。さらに、抗MEF2Aおよ
び抗MEF2C抗体を用いたChIP-SequenceならびにChIP-PCR法により、KLF2
promoter領域ならびにKLF4 promoter領域のMEF2結合領域が明らかとなっ
た。以上の結果から、血管内皮細胞においてピタバスタチンによりMEK5/
ERK5/MEF2経路を介してKLF2ならびにKLF4の発現が増強されることが明
らかとなった。ピタバスタチンによるKLF2、KLF4発現増強作用はピタバ
スタチンのpleiotropic effectの一端を担っている可能性が示唆された。
2
神戸大学大学院医学研究科生理学・細胞生物学講座血管生物学分野、
Department of Genetics and Tumor Cell Biology, St. Jude Childrenʼs
Hospital、3筑波大学大学院人間総合科学研究科解剖学発生学講座、4Program of
Developmental Biology, The Hospital for Sick Children、5慶應義塾大学医学
部坂口光洋記念講座発生・分化生物学
佐野 圭吾1、R. Sathish Srinivasan2、依馬 正次3、Janet Rossant4、
Guillermo Oliver2、須田 年生5、平島 正則1
前島 崇司1,2、大田 佳宏1、南 敬1、和田 洋一郎1、井原 茂男1、酒井 寿郎1、
浜窪 隆雄1、児玉 龍彦1
東京
Flk1/VEGFR2 is required for proper distribution and
survival of lymphatic endothelial cells in mice
45
ピタバスタチンによる血管内皮細胞におけるKLF2および
KLF4発現増強機序の解析
44
Program
18：00〜
Aⴊ▤↢‛කቇળKPFD
168
Sprouting step during angiogenesis is the growth of new capillary vessels from
preexisting ones. Inangiogenic sprouting, vascular endothelial growth factor
(VEGF)-A is essential for the induction ofsprouting (Gerhardt et al. 2003). In
contrast, Notch signaling inhibits angiogenic sprouting (Hellstrom et al. 2007).
Notch ligand, Dll4 is expressed in endothelial cells, in particular to arteries and
capillaries (Villa et al. 2001, Shutter et al. 2000, Claxton & Fruttinger 2004,
Benedito & Duarte 2005). Notch1 receptor was expressed in endothelial cells both
in vitro and in vivo (Taylor et al. 2002). Sprouting angiogenesis and induction of tip
cells are inhibited by Notch signaling. However, Notch signaling is detected in
vessels and tip cell using a transgenic Notch-reporter mouse (Hellstrom et al. 2007).
We assumed that an initial step by an angiogenic stimulation such as VEGF
transiently needs to suppress Notch signaling to abrogate anti-angiogenic effects on
endothelial cells. In order to identify the molecules that link VEGF signaling to
Notch signaling, we performedDNA microarray analysis and identified a zinc finger
protein of 50 kDa (ZF50). The knockdownof ZF50 by its specific siRNA disrupted
completely VEGF-induced network formation of HUVEC,which was recovered by
an inhibitor of Notch signaling. Conversely, overexpression of ZF50induced
vascular sprouting from aorta in ex vivo culture without VEGF stimulation. These
resultssuggest that ZF50 would be a downstream component in VEGF signaling
that interferes withNotch signaling pathway. And then, we also found that ZF50
interacts with CBF1, a nuclear mediator of Notch signaling, and decreased its
stability. Here we identify ZF50 as an OFF-switch for Notch signaling in the
downstream of VEGFsignaling.
愛媛大学大学院医学系研究科生化学・分子遺伝学分野、2愛媛大学プロテオ医学研
究センター
1
井上 博文1,2、東山 繁樹1,2
ZF50, a novel component of CBF1-degradation
complex, mediates VEGFR and Notch cross-signaling
in angiogenesis.
48
【背景】創傷治癒過程における血管の新生は、創傷部位への栄養や酸素の供
給に不可欠であるうえに、炎症性細胞の動員にも深く関与していると考え
られる。また、血管新生には、macrophage系細胞が分泌する血管内皮増殖
因子(Vascular endothelial growth factor;VEGF-A)をはじめとした成長因子等の
関与が指摘されている。そこで、本研究では創傷部位の血管新生メカニズ
ムを解明するため、マウス創傷治癒モデルを作製し、創傷部位における血
管新生に関与する遺伝子群および細胞群の詳細な解析を行った。
【方法】野
生型マウスを用い、創傷治癒モデルの作製を行った。本モデルにおいて、
創傷治癒部位の遺伝子発現を経時的にmicro arrayで解析し、血管新生に関連
する遺伝子群の変化を評価した。さらに、創傷部位の血管新生様式および
血管新生に関与する細胞群を免疫組織化学的手法によって経時的に観察し
た。創傷部位にはmacrophageマーカー陽性細胞を含む多くの炎症性細胞が
浸潤してくることが観察された。そこで、macrophage系細胞形成不全マウ
スを用いて創傷治癒過程における血管新生を免疫組織化学的手法により可
視化し、野生型マウスと比較検討した。【結果】創傷治癒過程における遺伝
子発現のmicro array解析の結果、創傷10日目で血管新生を正に調整する遺伝
子群が高発現していることが認められた。また、血管新生部位の免疫組織
学的解析により、野生型マウスの創傷治癒部位では、創傷3〜10日目にかけ
て既存血管からのsproutingが観察され、活発な血管新生が認められた。一方、
macrophage系細胞形成不全マウスの創傷治癒部位では、血管新生が極度に
抑制され、さらに血管の形状に明らかな異常が認められた。
【結論】野生型
マウスおよびmacrophage系細胞形成不全マウスを用いた創傷治癒モデルの
解析結果から、創傷治癒過程における正常な血管新生には、macrophage系
細胞の直接的または間接的な関与が強く示唆された。
COUP-TFII orphan nuclear receptor is composed of amino-terminal DNA binding
domain and carboxy-terminal trans-activating domain, and exerts a pleiotropic
effect on development and homeostasis. During embryonic vascular development,
COUP-TFII is expressed in venous blood vascular endothelial cells (BECs) and
plays critical roles in venous specification, and induces the expression of Prox1
transcription factor, which plays central roles in the differentiation of lymphatic
endothelial cells (LECs). Furthermore, we previously reported that COUP-TFII
physically and functionally interacts with Prox1 during differentiation and
maintenance of lymphatic vessels. However, the molecular mechanisms that
regulate the functions of COUP-TFII have not yet been fully elucidated. Here, we
isolated a novel COUP-TFII isoform lacking a DNA binding domain (hereafter
referred to as COUP-TFII variant 2) by PCR cloning. We found that COUP-TFII
variant 2 protein is expressed in the nuclei of human umbilical vein endothelial
cells (HUVEC) and human dermal lymphatic endothelial cells (HDLEC) as well as
blood vascular and lymphatic vessels of mouse embryos. While COUP-TFII has
been shown to form a homodimer, COUP-TFII variant 2 also forms a homodimer
and a heterodimer with full-length COUP-TFII. In order to examine the possibility
that the DNA binding abilities of COUP-TFII may be modulated by COUP-TFII
variant 2 lacking DNA binding domain, we carried out gel shift assay. We found
that COUP-TFII variant 2 is not capable of binding to COUP-TFII binding
consensus sequence, and interferes with the DNA binding ability of full-length
COUP-TFII. These results suggest that this novel isoform may modulate COUPTFII function as an endogenous antagonist during vascular development.
1
東京大学医学系研究科病因病理学専攻分子病理学講座、2東京大学先端科学技術研
究センターシステム生物医学、3PRESTO, 科学技術振興機構
山崎 智子1、末弘 淳一2、南 敬2、児玉 龍彦2、渡部 徹郎1,3、宮園 浩平1
Identification of novel COUP-TFII isoform in blood
vascular and lymphatic endothelial cells
49
To understand how vascular endothelial cells (ECs) move individually and
collectively during angiogenesis, we have recently established a microscopy-based
system in which real-time behavior of ECs is visualized during in vitro
angiogenesis. Branch elongation was driven mostly via EC movement, which was
unexpectedly complex and heterogeneous. In elongating branches, ECs moved
forward and backward, overtaking each other, and at a point they stopped. As a
result, relative positional relationship of ECs was dramatically and dynamically
changed (“Cell-mixing”). Contrary to previous understanding, even “tip cell” was
frequently replaced by a newly coming cell. Next to verify whether “cell-mixing”
also occurs in vivo, we labeled ECs by an intravascular injection of lectin and
tracked them during retinal angiogenesis. At short interval, labeled proximal(lumen forming) and unlabeled distal portions (including the tip) could be
reproducibly distinguished by lectin. However, mixed mosaic pattern of lectinlabeled (even at the tip) and unlabeled ECs was observed 6 hours after the injection,
strongly suggesting “cell-mixing” phenomenon during in vivo angiogenesis. To
analyze the behavior of individual ECs in more detail, we marked cell cytoplasm in
the vessel branch sporadically by adenovirus-mediated overexpression of EGFP and
monitored the real-time behavior at high resolution. We found that high motile ECs
display forward-rear cell polarity whereas low motile ECs were spindle-shaped
without cell polarity, and individual ECs switch the two motile modes dynamically.
Taken together, we propose a possible cell-based mechanism: ECs provide other
ECs with a “rail” to move along by dynamically changing the modes, which enables
efficient elongation of vessel branches.
東京大学大学院医学系研究科代謝生理化学
西山 功一、有馬 聡、候 俊之、有馬 勇一郎、小関 宏明、内島 泰信、
栗原 由紀子、栗原 裕基
富山大学大学院分子医科薬理学講座、2金沢工業大学 ゲノム生物工学研究所、3国
立長寿医療センター研究所遺伝子蛋白質解析室
東 英梨月1,2、山本 誠士1、新飯田 俊平3、堂本 光子2、服部 裕一1
1
A newly identified phenomenon “Cell-mixing” during
angiogenesis
47
マウス創傷治癒モデルにおける創傷面血管新生への
macrophage系細胞の関与
46
ポスターセッション：血管・リンパ管新生
（3）
Poster Session：Angiogenesis / Lymphangiogenesis(3)
12月1日
（水）
／Wednesday, December 1
Symposium
Y.I.A.
Presentation
Selected Oral
Presentation
Poster
Index
Aⴊ▤↢‛කቇળKPFD
How to write
Plenary Lecture
Nature Medicine
169
Special Talk
The 8th Korea-Japan Joint Symposium on Vascular Biology
Special
Lecture
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
VEGF and its receptors including Flt-1 and Flk-1 are involved in angiogenesis
under physiological and pathological conditions. Recently, Flt-1-expressing cells
were reported to contribute to the intracranial growth of glioma cells. However, the
role of Flt-1 signaling in solid tumor growth in subcutaneous tissue has not been
elucidated. To investigate how Flt-1 signaling is involved in the proliferation of
solid tumors, we implanted tumor cells into wild-type (Wt) and Flt-1 tyrosine
kinase (TK)-deficient (Flt-1 TK-/-) mice. Growth of HSML and B16 but not LLC
in subcutaneous tissue was significantly decreased in Flt-1TK-/- mice. Angiogenesis
in HSML and B16 tumors was remarkably reduced in Flt-1TK-/- mice. Moreover,
the infiltration of macrophage-lineage cells into HSML and B16 tumors was clearly
suppressed in Flt-1 TK-/- mice. Pericyte-marker+ cells were also reduced in Flt-1
TK-/- mice. However, in the border area of tumor, angiogenesis and theinfiltration
of macrophage-lineage cell were basically similar between Wt and Flt-1 TK-/-mice.
In bone marrow transplantation (BMT) experiments, tumor angiogenesis,
infiltrationof macrophage-lineage cells and tumor growth were significantly
suppressed in Wt/Flt-1TK-/- mice implanted with Flt-1 TK-/- bone marrow (BM)
cells compared with thoseimplanted with Wt BM cells. We conclude that Flt-1
signaling is involved in the function of BM-derived cell such as the migration of
macrophages into cancerous tissues, and significantly contributes to angiogenesis
and tumor progression.
President
Lecture
GATA2 is well recognized as a key transcription factor regulated with cell type
specificity and the differentiation. Currently in vascular endothelial cells, GATA2
has been reported in the regulation of some endothelial specific genesʼ expression.
Although investigating the molecular mechanisms behind endothelial cell specific
gene regulation might uncover the vascular homeostasis and onset of the vascular
diseases, these remain to be uncovered. In this report, we performed chromatin
immunoprecipitation with deep sequencing (ChIP-seq) to determine the binding
regions of GATA2 on a genome-wide scale. Comparison of GATA2 occupancies
between endothelial cells and non-endothelial cells, we identified that GATA2
binding regions were different and correlated with specific expressed genes within
each type of cells. Our microarray analysis with si-RNA against GATA2 revealed
that GATA2 regulates a new endothelial marker gene, endomucin. In the endomucin
locus, GATA2 was recruited not only to the promoter region but to the distal
enhancer region with enriched H3K4me1, p300, and active PolII only in the
endothelial cells. Besides, by using chromatin conformation capture (3C) assays,
endothelial specific chromatin loop was mediated by GATA2, which is a new
molecular mechanism GATA2 epigenetically regulates the expression of endothelial
specific gene. Moreover, abrogation of GATA2 function in endothelium
demonstrated not only reduction of the endothelial specific markers, but also
induction of the EndMT (endothelial-mesenchymal transition) promoting gene
expression. These results provide new insights into the cooperation between
endothelial expressed GATA2 binding and epigenetic modification, resulting in the
determination of endothelial cell specificity.
1
独立法人国立長寿医療研究センター研究所 遺伝子蛋白質解析、2東京医科歯科大
学 大学院医歯学総合研究科 分子腫瘍医学、3富山大学 医学薬学研究部 分子医科
薬理学講座、4上武大学副学長・東京医科歯科大学客員教授
村松 昌1,2、山本 誠士3、大澤 毅2、澁谷 正史4
神吉 康晴、堤 修一、大田 佳宏、三村 維真理、和田 洋一郎、浜窪 隆雄、
油谷 浩幸、児玉 龍彦、南 敬
東京大学先端科学技術研究センターシステム生物医学
VEGFR-1 signaling promotes mobilization of
macrophage-lineage cells from bone marrow and
stimulated tumor growth
51
Epigenetically coordinate GATA2 occupancy is
necessary for the determination of the endothelial cell
specificity
50
Program
18：00〜
Aⴊ▤↢‛කቇળKPFD
170
真核生物において、遺伝子が正常に機能するためには、転写された未熟な
mRNAのイントロンを取り除き、エクソンを正確につなぎ合わせるスプラ
イシング反応が必須である。抗癌活性物質として発見された天然化合物
FR901464の安定型誘導体であるスプライソスタチンA（SSA）は、スプラ
イシング因子であるSF3bを標的とする世界初のスプライシング特異的阻害
剤である。今回我々はSSAの血管内皮細胞増殖因子（VEGF）の発現に対す
る影響を調べ、SSAの抗癌活性発揮の一因としての血管新生阻害活性につ
いて解析したので報告する。SSA処理をしたヒト癌細胞（HeLa）では、ス
プライシング阻害の結果VEGFの成熟mRNAの生成がGAPDHと比較して著
しく抑制され、細胞外に分泌されるVEGFタンパク量は30%程度にまで抑制
された。鶏胚漿尿膜（CAM）上に移植した癌細胞周囲に形成される腫瘍血
管の新生は、SSAによって50％に阻害され、その阻害はVEGF添加により解
除された。さらにHeLa細胞を用いたマイクロアレイによる解析で、SSA処
理によって全遺伝子の、VEGFを含む約38%の遺伝子の発現が抑制されるこ
とがわかった。また、VEGFがオートクライン作用で血管内皮細胞の維持、
生存に深く関わることが報告されていることから、我々はさらにSSAの血
管内皮細胞に与える影響について調べたところ、SSAは血管内皮細胞の成
熟mRNAの生成を抑制し、また増殖及び管腔形成を阻害した。SSAは鶏胚
漿尿膜の血管新生を顕著に阻害し、その阻害はVEGF添加により解除され
た。以上のことから、スプライシング阻害剤SSAは、癌細胞におけるVEGF
の発現抑制を介する腫瘍血管新生抑制と、血管内皮細胞のオートクライン
VEGF生成の抑制による血管新生抑制の両方の効果を有することがわかっ
た。腫瘍血管新生により起こるがんの成長や転移、さらには、異常な血管
新生亢進によって起こる様々な疾病などの治療にSSAが有用である可能性
が示唆された。
Transglutaminase 2 (TG2) has been implicated in formation of new blood vessels
(angiogenesis) in wound healing process and progression of atherosclerosis. Little
is known about a role of TG2 in cancer angiogenesis. (Sane DC, 2007, Front
Biosci., 12:2530-45.) Although expression of TG2 is known to increase in vascular
endothelial cells during vessel remodeling and in wound healing response of vessel
wall, whether TG2 is involved in tumor-angiogenesis is not clear. Conversely, it is
reported that TG2 crosslinks ECM components, resulting in blockade of malignant
cell invasion and angiogenesis. (Kotsakis P, Griffin M, 2007, Amino Acid, 33;37384.) Using the dorsal air sac (DAS) animal model, we looked if TG2 might be
involved in tumor-angiogenesis. Lewis lung carcinoma cells were loaded into a
chamber ring, which was then implanted into the dorsal inter-space made in mice.
After 7 days, we compared formation of neovessels developed from back skin of
control B6 mice, TG2 hetero mice and TG2 knockout mice. Formation of
neovessels was seen in B6 mice, but not in TG2 knockout mice. Furthermore, the
result of matrigel plug assay, in which angiogenesis formed within VEGFcontaining matrigels implanted into the back of mice were observed, showed no
angiogenesis when matrigels were implanted into TG2 knockout mice and less
angiogenesis was observed in the matrigels containing a TG2 inhibitor, cystamine,
suggesting that TG2 activity was important for migration of endothelial cells (ECs).
These results suggest that TG2 plays an important role in tumor-induced
angiogenesis, and that TG2 activity might regulate EC migration and new vessel
formation. To know the underlying molecular mechanism, the gene chip analysis is
under way.
1
Tokyo Medical and Dental University、2Mol. Ligand Biol. Res. Team,
RIKEN ASI、3Victor Chang Cardiac Institute
李 殷瑞1,2、Kazuyo UCHIDA2、Siiri E. IISMAA3、
Robert M. GRAHAM3、Soichi KOJIMA1,2
独立行政法人理化学研究所基幹研究所分子リガンド生物研究チーム、2独立行政法
人理化学研究所基幹研究所吉田化学遺伝学研究室、3独立行政法人理化学研究所基
幹研究所分子リガンド探索研究チーム
内田 和代1、古米 亮平2、吉田 稔2,3、小嶋 聡一1
1
Promising Role of Transglutaminase 2 in TumorAngiogenesis
55
ARIA is novel gene preferentially expressed in endothelial cells. We generated
theARIA -/- mice. ARIA -/- in Aortic endothelial cells reduced apoptosis and
enhancedtube-formation on matrigel and migration capacity as compared with WT.
In vivo ARIA-/- remarkably enhanced blood flow recovery and ischemia-induced
neovasculalizationin hind-limb ischemia model than WT. Because ARIA also highy
expressed inendothelial cells as well as endothelial progenitor cells (EPCs), we
expected theimpotant roles of ARIA in EPCs. Knockdown of ARIA by using siRNA
in humancord-derived endothelial progenitor cells (HCEPCs) also dramatically
reduced apoptosisand enhanced migration capacity. In vivo ARIA -/- in bone
marrow-derived cellsenhanced ischemia-induced neovasculalization and promoted
blood flow recovery inischemia-limb, and increased EPCs homed to ischemia limb
as compared with WT.Furthermore ARIA -/- in lung endothelial cells and
Knockdown of ARIA in HCEPCsactivated the phosphoinositide 3-kinase (PI3K)/
Akt/ eNOS signaling pathway in ligandnon-specificity mannar. Inhibition of PI3K
completely blocked enhancement ofmigration capacity by ARIA-knockdown in
HCEPCs. In vivo L-NAME, NOS inhibitorcompletely canceled enhancement of
blood flow recovery and ischemia-inducedneovasculalization of ARIA -/- mice.
Taken together ARIA regulates cell survival andmigration in endothelial cells as
well as endothelial progenitor cells, contributed topromote ischemia-induced
angiogeneisis and vasculogenesis through the PI3K/ Akt/eNOS signaling pathway.
ARIA is attractively a new therapeutic target for ischemiccardiovascular diseases.
スプライシング阻害剤スプライソスタチンAによるVEGF
発現抑制を介した血管新生阻害
54
【目的】Vascular endothelial growth factor (VEGF) は、血管形成や造血に関与
することが知られているが、その作用機構や関与する下流因子はほとんど
分かっていない。我々はこれまでに、正常ヒト臍帯静脈血管内皮細胞のマ
イクロアレイ解析により、VEGFがArl4A、LPP3、pmch、TSPAN13などの
遺伝子発現を制御することを明らかにしてきた。そこで、本研究ではこれ
らの遺伝子が血管形成や造血に及ぼす影響をin vivoで解析し、VEGFの役割
を明らかにすることを目的とした。【方法】発生初期における遺伝子の機能
解析を行うため、アフリカツメガエル（Xenopus laevis）をモデル動物とし
て用い、Arl4A、LPP3、pmch、TSPAN13遺伝子のアフリカツメガエルホモ
ログをクローニングした。次に、各遺伝子のcDNAから合成したmRNAをア
フリカツメガエル受精卵に注入して過剰発現させた。最後に幼生期まで発
生した個体を用いて、血管形成や造血への影響を解析した。【結果】pmch
やTSPAN13を過剰発現させた個体ではaortic archから分岐する血管の退縮が
起こらず、血管形成への影響がみられた。LPP3を過剰発現させた個体では
血液細胞数が著しく減少し、造血への影響がみられた。また、Arl4Aを過剰
発現させた個体では発生が原腸胚で停止したため、血管形成と造血のいず
れ も 解 析 不 可 能 で あ っ た。【 結 論 】 こ れ ら の こ と か ら、VEGFはLPP3、
pmch、TSPAN13の発現を制御することで、血管形成や造血を調節している
ことが推測された。
日本医科大学老人病研究所病理部門、2日本医科大学生物学
京都府立医科大学循環器内科
小出 正洋、池田 宏二、北村 洋平、赤壁 佳樹、中川 祐介、松原 弘明
1
藤原 正和1、長谷部 孝2、岡 敦子2、ガジザデ モハマド1
ARIA regulates hypoxia-induced angiogenesis and
vasculogenesis through the PI3K/AKT/eNOS
signaling pathway
53
Vascular endothelial growth factorによって発現制御さ
れる遺伝子とその役割
52
ポスターセッション：血管・リンパ管新生
（4）
Poster Session：Angiogenesis / Lymphangiogenesis(4)
12月1日
（水）
／Wednesday, December 1
Symposium
Y.I.A.
Presentation
Selected Oral
Presentation
Poster
Index
Aⴊ▤↢‛කቇળKPFD
How to write
Plenary Lecture
Nature Medicine
171
Special Talk
The 8th Korea-Japan Joint Symposium on Vascular Biology
Special
Lecture
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
ets1遺伝子は心血管系に強く発現している転写因子であり、内皮細胞の増
殖・遊走を促す作用をもつ血管新生因子であることが知られている。しか
しながら、現在のところ、発生過程におけるets1の血管内皮細胞に関する役
割はあまり分かっていない。そこで我々は、発生過程における内皮細胞の
分化形成に対するets1の役割を検討した。アフリカツメガエルは興味のある
遺伝子のmRNAを受精卵に注入するだけで容易にその遺伝子の強発現胚を
作成できるというメリットを有している。さらに他の脊椎動物と同様に発
生期においては、血管の出現場所および時期は厳密に決まっているので、
わずかな血管形成の異常も検出可能である。血管の形成に関する研究をす
る際、アフリカツメガエルの実験系は極めて有用である。そこで我々はア
フリカツメガエルを用いてets1の強発現胚を作成し、内皮細胞マーカーであ
るtie2、PECAM遺伝子の発現をrealtime PCR法で観察した。その結果、ets1
の強発現胚では内皮細胞マーカーの発現が上昇している事が分かった。こ
のことは、ets1は発生過程においても血管の形成を促進する作用があること
を示している。さらにin situ hybridization法で、どの領域で内皮マーカーの
発現が上昇しているかを検討した。その結果、ets1の強発現胚では尾部に異
所性の血管内皮細胞の出現が観察された。このことは、本来は血管になら
ない尾部の中胚葉細胞がets1遺伝子によって血管へと分化誘導されたこと
を示している。また、
静脈マーカー（EphB4）および動脈マーカー（Ephrin-B2）
の発現を同様に検討した。その結果、静脈マーカーの発現には変化が見ら
れなかったが、
動脈マーカーでは発現の上昇が観察された。これらのことは、
ets1は中胚葉細胞を血管へと分化誘導をし、さらにその血管を動脈に誘導し
ている可能性を示唆している。
1
東京大学医学系研究科先端臨床医学開発講座、2大阪大学医学系研究科臨床遺伝子
治療学
鯉渕 信孝1、谷山 義明2、鈴木 淳一1、森下 竜一2
血管内皮細胞の分化過程におけるets1の役割の解析
57
President
Lecture
Lymphatic endothelial cells (LECs) are derived from venous endothelial cells
(VECs) during embryogenesis, but lymphatic vessels are subsequently established
as a vascular system separated from blood vessels. It has been shown that lymphatic
vessels fail to separate from blood vessels in mice lacking the hematopoietic
signaling molecules Syk ,Slp-76, or Phospholipase C gamma 2 (Plcg2), all of which
are important for platelet activation. Recent studies actually indicated that a critical
role for platelets in separation between blood vessels and lymphatic vessels during
an early phase of lymphatic vessel development, but detailed mechanisms of how
platelets are involved in this process remain to be elucidated. In this study, we
analyzed vascular phenotypes in mice lacking Plcg2 or CLEC-2, a platelet receptor
activated by its ligand-podoplanin expressed on the surface of LECs. Blood-filled
and enlarged lymphatic vessels were detected in both Plcg2-/- and CLEC-2-/embryos, which is consistent with previous reports on a role for platelet activation.
We also detected abnormal connections between blood vessels and lymphatic
vessels in peripheral regions such as the back skin of CLEC-2-/- embryos or hind
limb of Plcg2-/- adult mice. Previous studies reported that platelets form aggregates
around the orifice of the lymph sacs, leading to separate the lymphatic sac from the
jugular vein, whereas no report suggests to date that LECs are derived from VECs
in the peripheral regions. Thus we would like to discuss another mechanism
underlying the role for platelets in keeping apart blood vascular and lymphatic
vascular compartment after these two vascular systems are separately formed.
1
神戸大学大学院医学研究科生理学細胞生物学講座血管生物学分野、2山梨大学大学
院医学工学総合研究部・臨床検査医学、3京都大学医学研究科次世代免疫制御を目
指す創薬医学融合拠点、4独立行政法人理化学研究所 免疫・アレルギー化学統合研
究センター・分化制御研究グループ
丁 国1、井上 克枝2、井上 修2、疋田 正喜3、黒崎 知博4、尾崎 由基男2、
平島 正則1
Mechanisms of keeping apart blood and lymphatic
vascular compartment
56
Program
60
Aⴊ▤↢‛කቇળKPFD
172
Recently heterogeneity of ECs in angiogenesis such as tip and stalk cells, and
Phalanx cells are suggested. Here we show there are heterogeneity in pre-existing
peripheral ECs in the adulthood that act as EC supplying cells in angiogenesis. By
using Hoechst efflux method, we identified endothelial side population cells (ECSPs) from peripheral vasculature which is not in the peripheral blood or bone
marrow cells and distinct from EPCs. We isolated ECs from many organs and found
that among CD31+CD45- ECs, about 1% of cells are Hoechst low EC-SPs. These
EC-SPs were VE-Cadherin+, Sca-1+, Flk-1+ and such phenotype was identical to
main population (MP) of CD31+CD45- endothelial fraction. However, compared to
MP, CD133 expression of EC-SPs was higher and cell cycle study by Pyronin Y
revealed that 95% of EC-SPs were quiescent. In vitro, EC-SPs showed colony high
forming capacity and formed fine endothelial networks compared to MP. Limiting
dilution analysis revealed that frequency of colony forming cells in SP was 1/6.62,
and that was 10 times higher than that of MP. Cell cycle analysis after induction of
ischemia revealed EC-SPs escaped G0 and drove its cell cycle, and after 2 to 3 days
the number and proportion of EC-SPs increased temporarily and peaked out within
2 weeks. To assure that EC-SPs contribute to angiogenesis in vivo, EC-SPs were
transplanted into ischemia model mice. After 2weeks, EC-SPs gave rise vascular
network and these were connected to systemic circulation. This study revealed that
EC-SPs are functionally and phenotypically different ECs and that look like
undifferentiated endothelial progenitor/stem cells systemically distributing in the
body. This suggests that endothelial cells have their original hierarchical stem cell
system in the peripheral level.
大阪大学微生物病研究所 情報伝達分野
内藤 尚道、木戸屋 浩康、高倉 信幸
Vascular Endothelial Side Population Cells and its
Role in Angiogenesis
１、背景・目的
血管内皮前駆細胞（EPC）は、虚血性心疾患や慢性閉塞性動脈硬化症、脳
梗塞などの組織虚血部における血管新生を促進させる治療法として研究、
臨床応用されている。EPCは骨髄から末梢血に動員され、既存の血管の内
皮細胞に接着後、増殖・分化して新たな血管を形成する。この過程において、
EPCは血流に起因する機械的刺激である流れずり応力を受けると考えられ
る。そこで我々は、流れずり応力が循環している(浮遊している)EPCの分化
誘導に及ぼす効果に関して検討した。
２、方法
ヒト臍帯血から比重分離法により単核球を分離後、磁気ビーズでCD133陽
性細胞を単離して1週間培養し、EPCを精製した。この細胞に回転円盤型流
れずり応力負荷装置を用いて流れずり応力(2.5ダイン)を24〜72時間負荷し
た。その後細胞を回収して蛋白の発現レベルをフローサイトメトリーで、
遺伝子の発現変化をPCR法で検討した。また、VEGFあるいはSCFに対する
増殖能をWST8アッセイで、細胞死をフローサイトメトリーで、過酸化水素
水に対する抗アポトーシス能をELISAで、脈管形成能を３Dマトリゲルで検
討した。さらには内皮コロニー形成能をメチルセルロースで検討した。
３、結果毛細血管レベルの流れずり応力(2.5ダイン)をEPCに負荷すること
により、血管内皮のマーカーであるKDR、PECAM、Tie2の蛋白発現レベル
はいずれも有意に増大したが、VE-cadherinの発現は変化しなかった。また、
遺伝子発現レベルは流れずり応力によりKDRの増大を認めた。次に細胞の
機能を検討したところ、流れずり応力により増殖能、抗アポトーシス能、
接着能、内皮コロニー形成能、脈管形成能はいずれも増大した。一方、流
れずり応力により細胞死は減少した。
４、考察
我々は、流れずり応力が接着したEPCを血管内皮に分化誘導することを以
前報告してきた。今回、流れずり応力が循環している（浮遊している）
EPCも血管内皮に分化誘導することが明らかになった。このことはEPCが
流れずり応力を感知し、その情報を細胞内部に伝達して細胞応答をおこす
ことを意味する。今後流れずり応力を用いたEPC治療を血管新生の臨床に
応用することにより、一層の治療効果が得られると期待される。
1
東京大学医学系大学院生体物理医学専攻医用生体工学講座、2東海大学医学部基盤
診療学系再生医療科学、3独協医科大学医学部生体医工学
小尾 正太郎1,2、増田 治史2、山本 希美子1、安藤 譲二3、阿部 裕輔1、
浅原 孝之2
流れ刺激が循環血管内皮前駆細胞に及ぼす効果
61
Vascular endothelial growth factor receptor 2 (VEGFR2) plays critical roles in
vasculogenesis, a process involving cell proliferation, migration, and differentiation.
However, the molecular mechanism by which VEGFR2 signaling directs vascular
endothelial differentiation of VEGFR2 + vascular progenitors is not well understood.
In this study, we examined the signal transduction pathway downstream of
VEGFR2 for endothelial differentiation using an in vitro differentiation system of
mouse embryonic stem cell-derived VEGFR2 + cells. We found that signaling
through tyrosine-1175 (Y1175, corresponding to mouse Y1173) of VEGFR2 is
crucial for endothelial differentiation by means of expressing chimeric receptors,
composed of intracellular domain of VEGFR2 and extracellular and transmembrane
domain of VEGFR3, in VEGFR2 + cells. Endothelial differentiation involves two
processes that are dependent on VEGFR2 signaling; endothelial specification of
VEGFR2 + cells, and subsequent survival of endothelial cells. We confirmed that
signaling through VEGFR2 Y1175 is involved in both processes. Furthermore, we
revealed that PLC㱏1, which interacts with VEGFR2 through phophorylated
Y1175, is an inducer of endothelial specification, though PLC㱏1 is not involved in
endothelial survival process. Endothelial specification of VEGFR2 + cells induced
by PLC㱏1 signaling abrogated by treatment with FTI-277, a farnesyltransferase
inhibitor of H-Ras, suggesting that H-Ras is downstream of PLC㱏1. Moreover,
U0126, MEK inhibitor, also blocked endothelial differentiation. These findings
indicate that VEGFR2-PLC㱏1-Ras/ERK pathway plays essential roles in
endothelial specification of VEGFR2+ cells.
東京大学大学院医学系研究科病因・病理学専攻分子病理学
大阪大学大学院薬学研究科応用医療薬科学専攻臨床薬効解析学分野
【目的】近年、心筋組織内に存在する幹細胞が、心筋細胞や血管内皮細胞な
どへの分化能を有することが報告されている。これまでに我々はinterleukin
(IL)-6ファミリーサイトカインがgp130/STAT3シグナルを介し、心筋組織幹
細胞の血管内皮細胞分化を誘導することを明らかにしてきた。本研究は、
内皮細胞分化機構を解明することを目的とした。
【方法】雄性C57Bl/6マウスの心臓よりMACS法を用いてSca-1陽性細胞を単
離した。DNAアレイにより、心筋組織幹細胞においてIL-6ファミリーサイ
トカインであるLeukemia inhibitory factor (LIF)により誘導される遺伝子を解
析した。また、心筋組織幹細胞の内皮細胞分化を、RT-PCR法により検討し
た。さらに、LacZを発現するアデノウイルスベクターによりマーキングし
たSca-1陽性細胞を心筋梗塞モデルマウスに移植し、生体内において分化機
構の解明を行った。
【結果・考察】DNAアレイにより、Sca-1陽性細胞においてセリン・スレオ
ニンキナーゼであるPim-1がLIF刺激後、発現誘導されることを見出した。
培養心筋組織幹細胞において、抑制型STAT3遺伝子導入による、あるいは、
STAT3遺伝子のノックアウトによるSTAT3の機能阻害は、LIFによるPim-1の
発現を有意に抑制した。さらに抑制型Pim-1遺伝子を導入することにより、
LIFによる培養Sca-1陽性細胞の血管内皮細胞への分化が有意に抑制された。
次に、マウス心筋梗塞モデルを用いた検討において、梗塞巣に移植された
Sca-1陽性細胞の血管内皮細胞への分化効率は、野生型STAT3遺伝子を導入
することにより有意に上昇したが、STAT3遺伝子のノックアウトまたは抑
制型Pim-1遺伝子の導入により有意に減少した。以上のことから、心筋組織
の修復・再生過程では、IL-6ファミリーサイトカインによるSTAT3/Pim-1シ
グナルの活性化が心筋組織幹細胞を血管内皮細胞へと分化誘導し、新規血
管形成を促進する可能性が示唆された。
佐瀬 仁志、渡部 徹郎、川崎 京子、宮園 浩平、宮澤 恵二
岩倉 智彦、毛利 友美、濱谷 辰斗、尾花 理徳、山下 朋美、中山 博之、藤尾 慈
59
18：00〜
Analysis of VEGFR2 signaling for endothelial
differentiation of embryonic stem cell-derived
VEGFR2+ cells
STAT3/Pim-1シグナルは心筋組織幹細胞を血管内皮細胞
に分化誘導する
58
ポスターセッション：再生
Poster Session：Regeneration
12月1日
（水）
／Wednesday, December 1
How to write
Plenary Lecture
Nature Medicine
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Y.I.A.
Presentation
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Poster
Index
Aⴊ▤↢‛කቇળKPFD
173
Special Talk
The 8th Korea-Japan Joint Symposium on Vascular Biology
Special
Lecture
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
President
Lecture
Background-Exercise stimulates the vascular response in pathological conditions,
including ischemia. However, the molecular mechanisms by which exercise
improves the impaired hypoxia-induced factor (HIF)-1㱍-mediated response to
hypoxia associated with aging are poorly understood. Here, we report that
swimming training (ST) modulates the vascular response to ischemia in aged
(24-month-old) mice.Methods and Results- Aged wild-type mice (MMP-2+/+) that
maintained ST (swimming 1 hour/day) from day 1 after surgery were randomly
assigned to four groups that were treated with either vehicle, LY294002, or
deferoxamine for 14 days. Mice maintained sedentary served as controls. ST
increased blood flow, capillary density, and the levels of p-Akt, HIF-1㱍, vascular
endothelial growth factor, Fit-1, and matrix metalloproteinase-2 (MMP-2) in MMP2+/+ mice and reduced and prolyl hydroxylases and factor inhibiting HIF-1. ST
also increased the numbers of circulating endothelial progenitor cells (EPCs) and
their function associated with activation of HIF-1㱍. All of these effects were
diminished by LY294002, an inhibitor of phosphatidylinositol-3-kinase (PI3K);
enhanced by deferoxamine, a HIF-1㱍 stabilizer; and impaired by knockout of
MMP-2. Finally, bone marrow transplantation confirmed that ST enhanced EPC
homing to ischemic sites in aged mice. In vitro, siHIF-1 impaired old human
umbilical vein endothelial cell adhesion, invasion and tubulogenesis.Circulation-ST
can thus improve neovascularization in response to hypoxia via a PI3K-dependent
mechanism that is mediated by the HIF-1㱍/VEGF/MMP-2 pathway in advanced
age.
1
名古屋大学大学院医学系研究科循環器内科学講座、2名古屋大学大学院医学系研究
科講座、3浜松医科大学医学部解剖学講座
成 憲武1、葛谷 雅文2、井上 愛子2、佐々木 健3、宋 海珍2、胡 莉那2、
奥村 健二1、室原 豊明1
Exercise Stimulates Ischemia-induced
Neovascularization via PI3K/Akt-dependent HIF-1α
Reactivation in Aged Mice
62
Program
18：00〜
1
大阪大学医学部臨床遺伝子治療学、2大阪大学大学院健康発達医学
骨 粗 鬆 症 の 進 展 に お い て は、 骨 芽 細 胞 上 に 発 現 す るRANKL (Receptor
activator of NFkB ligand) が破骨細胞上に存在するRANK (Receptor activator of
NFkB)と結合することにより破骨細胞が活性化することが重要であり、
RANKLの デ コ イ 型 レ セ プ タ ー で あ るOPG (Osteoprotegerin) はRANKLと
RANKの結合を阻害することで、破骨細胞の活性化を抑制する。一方で、
肥満状態のマウスの脂肪組織においては慢性炎症が存在するとされている
が、我々は高脂肪食下のマウスの脂肪組織においてTNF-α、MCP-1と同様
にRANKLの発現が上昇することを確認し、培養マクロファージ細胞におい
てはRANKL刺激によりTNF-α、MCP-1の発現が上昇することを確認した。
OPG欠損マウスは、RANKLの持続刺激により骨粗鬆症を発症するモデルマ
ウスであるが、このマウスは高脂肪食下において、Glucose Tolerance Testで
高インスリン血症を認め、Insulin Tolerance Testでインスリン反応性の低下
を認めた。また、脂肪組織ではマクロファージの浸潤を多く認め、TNF-α、
MCP-1の著明な発現上昇が認められた。これらのことから、骨粗鬆症で重
要とされるOPG-RANK-RANKL systemは、脂肪組織における炎症を惹起し、
インスリン抵抗性に関与する可能性が示唆された。
東京大学大学院医学系研究科循環器内科
Recent studies are clarifying that inflammation is crucially involved in metabolic
abnormalities. However, it is still unclear whether inflammation is involved in
pancreatic 㱎 cell dysfunction seen in type 2 diabetes (T2D). Serum free fatty acid
(FFA) levels are independent predictor of cardiovascular and metabolic diseases.
The deleterious effects of FFAs on 㱎 cells in vitro are well known. To see the
effect of palmitate in vivo, we established a novel method to selectively increase
the circulating free palmitate level and found that palmitate induces rapid 㱎 cells
dysfunction in mice via TLR4/MyD88 pathway. However, the TLR4/MyD88
pathway was dispensable for palmitate-induced 㱎 cell dysfunction in vitro,
indicating mechanisms essentially different from the previously known one are
important in vivo. Interestingly, palmitate infusion induced expression of CCL2 and
CXCL1 in 㱎 cells. Indeed, flow cytometric analysis showed that palmitate induced
accumulation of CD11b+Ly-6C+ M1 inflammatory macrophages (M㱢) within
islets, which was not observed in Tlr4-/- or Myd88-/- mice. Moreover, depletion of
M1 M㱢s protected mice from palmitate-induced 㱎 cell dysfunction. Coculture
experiments demonstrated that reciprocal interactions between 㱎 cells and M㱢s
further augment M㱢 recruitment and 㱎 cell dysfunction. Finally, we demonstrated
that same mechanisms are playing causative roles in 㱎 cell dysfunction in db/db
mice, a T2D model. We also found that palmitate activates inflammatory processes
and exacerbates neointima formation. Our results thus highlight the causative
involvement of inflammation in 㱎 cell dysfunction in T2D and suggest that
saturated FFAs and chronic inflammation are the unified pathogenic factors and
mechanisms for both cardiovascular and metabolic diseases.
Aⴊ▤↢‛කቇળKPFD
174
Adipose tissue plays a central role in metabolic syndrome, by producing various
adipokines and controlling systemic energy metabolism. Recent studies indicated
that inflammation takes place in obese adipose tissue, which affects not only
adipose function but also systemic insulin sensitivity. We developed a highthroughput, functional imaging-based screening strategy to identify new regulators
involved in adipocyte differentiation and function. Using this strategy, we found
that Rad51, a key molecule of homologous-recombination and DNA-repair, was
required for adipocyte differentiation. Rad51 knockdown suppressed adipocyte
differentiation in 3T3-L1 preadipocytes by inhibiting cell cycle progression at the
mitotic clonal expansion, which is an essential step for adipocyte differentiation. In
obese mice, Rad51 expression was increased in white adipose tissues (WAT). Using
FCM analysis, Rad51+/- suppressed CD34(+)/Sca1(+)/Pref1(+) pre-adipocyte and
CD34(+)/Sca1(+)/CD24(+/-) adipocyte-precursor cells as well. Interestingly,
differentiation function of adipocyte precursor cells was suppressed in Rad51+/-,
which means that high fat diet-induced adipocyte hyperplasia was reduced in
Rad51+/- compared with wild type. Additionally, we clearly demonstrated that the
adiposity related-adipocyte hyperplasia would induce macrophages infiltration into
WAT and regulate adipose tissue inflammation. Subsequently, Rad51+/- reduced
macrophage infiltration and protected from high fat diet-induced systemic insulin
resistance. These results elucidate that Rad51 is a novel key molecule that controls
diet-induced adipose tissue proliferation and inflammation, which leads to systemic
insulin resistance.
背景・目的：イルベサルタンはPPARg活性化作用を有するアンジオテンシ
ン 受 容 体 拮 抗 薬 で あ る。 今 回、 我 々 は 肥 満 糖 尿 病 モ デ ル で あ るZucker
diabetic fatty（ZDF）ラットを用いて、イルベサルタンの薬効評価を行った。
方法：Lean群（Leprfa/: hetero）
、ZDF群(Leprfa/Leprfa: homo)、ZDF+Irebe群（イ
ルベサルタン30 mg/kg/day強制経口投与）の３群（各群６匹）に対して１２
週齢から１２週間観察した。体重・随時血糖は毎週測定、摂餌量・血圧・
生化学検査（総コレステロール、中性脂肪、BUN、Cre）・尿たんぱくは４
週間に１度測定、OGTTおよびITT試験は投与後１０週後に一度行った。
１２週後に血管を摘出し内皮機能測定（アセチルコリンに対する反応性試
験）を行い、各臓器重量を測定した。結果：体重・血糖は試験開始の１２
週齢ですでにLean群に比してZDF群では著明に増加していたが、その後の
経過でもZDF群とZDF+Irebe群では有意な差はなかった。また、OGTTおよ
びITT試験においてもZDF群とZDF+Irebe群には有意な差はなく、イルベサ
ルタンによる耐糖能改善効果は認められなかった。脂質値は、総コレステ
ロ ー ル と 中 性 脂 肪 の 値 がlean群 と 比 べZDF群 で 著 明 に 増 加 し た が、
ZDF+Irebe群ではその増加が軽減していた。腎機能評価においては、BUN,
Creの 値 は ３ 群 で 有 意 な 差 が な か っ た が、 尿 蛋 白 がZDF群 に 比 し て
ZDF+Irebe群で有意に減少していた。肝機能評価では、GOT/GPTがlean群に
比してZDF群で有意に増加していたが、ZDF+Irebe群で有意に改善していた。
血管内皮機能評価では、ZDF群で著明に悪化していたが、ZDF+Irebe群でほ
ぼlean群レベルまで改善を認めた。以上の結果により、イルベサルタンは
肥満糖尿病ラットの治療モデルにおいて耐糖能改善は認めなかったが、内
皮機能・腎機能・肝機能・脂質異常などの臓器障害の改善を認めた。
大阪大学健康発達医学講座、2大阪大学臨床遺伝子治療学講座、3日精バイリス株式
会社 滋賀研究所 研究部
1
島村 宗尚1、中神 啓徳1、中神 太1、三宅 隆1、志水 秀郎1、
マリアナ オオサコ1、下里 貴3、森下 竜一2
松本 佐保姫、真鍋 一郎、永井 良三
東京大学医学部循環器内科
イルベサルタンはZucker diabetic fatty
（ZDF）ラットの
臓器障害を改善する
Rad51 is a novel key regulator of adipocyte
proliferation and adiposity inflammation
66
中神 太志1、中神 啓徳2、大迫 今日美1、森下 竜一1
江口 航生、真鍋 一郎、永井 良三
65
脂肪組織においてRANKLシステムはインスリン抵抗性を
惹起する
64
Islet inflammation induced by free fatty acid-TLR4
pathway leads to β cell dysfunction in type 2 diabetes
63
ポスターセッション：メタボ・糖尿病
（1）
Poster Session：Metabolic syndrome / Diabetes(1)
12月1日
（水）
／Wednesday, December 1
喫煙は心血管リスクであるが、禁煙後、短期的には肥満と２型糖尿病発症
の割合が増える。肥満に伴う脂肪組織増大には血管新生が不可欠である。
血管内皮増殖因子(VEGF)のホモログである胎盤由来増殖因子(PlGF)は、生
理的な血管新生には影響を及ぼさないが病的血管新生においては重要な役
割を果たす。PlGFノックアウトマウスでは、高脂肪食負荷による脂肪組織
の増大が抑制されている。しかしながら、肥満及び禁煙後の内臓脂肪増加
と血清PlGFの関連は不明である。連続176名の健常男性から血清を採取し、
VEGF、可溶性VEGF受容体1(sVEGFR-1)および２（sVEGFR-2）
、PlGFレベ
ルを測定した。VEGF、sVEGFR-1、sVEGFR-2はBMI 23未満(n=71)とBMI23
以上(n=105)で有意差がなかった。しかしながら、PlGFは後者で有意に上昇
していた。ステップワイズ回帰分析の結果、PlGFの規定因子はsVEGFR-1、
non-HDL-C、腹囲、年齢であった。次に3ヶ月の禁煙プログラムで成功した
男性43名の禁煙前後の血清レベルを比較した。プログラム前後でsVEGFR-1
レベルは変化なかった。しかしながら、BMI、VEGF (P = 0.01)、sVEGFR-2 (P
= 0.01)、PlGF (P ＜ 0.0001) は有意に上昇した。面白いことに、禁煙前後の
PlGFの変化率はadiponectinの変化率と有意な負の相関を示した。以上から、
PlGFは、禁煙後の内臓脂肪蓄積、インスリン抵抗性増悪の指標である可能
性が示唆された。
血管内皮増殖因子C (VEGF-C) はVEGF受容体3(VEGFR-3)に結合してリンパ
管 新 生 に お け る 中 心 的 役 割 を 果 た す。 最 近、 可 溶 性VEGF受 容 体 ２
(sVEGFR-2)がVEGF-Cの内因性拮抗因子として働くことが明らかとなった。
我々はsVEGFR-2がメタボリックシンドローム (MetS) で上昇していること
を報告した。しかしながら、MetSにおけるVEGF-Cと可溶性VEGF受容体３
(sVEGFR-3) の血中レベルならびにsVEGFR-2との関連は不明である。いか
なる薬物治療も受けていない連続する健常成人239名から血清を採取し、
VEGF-C, sVEGFR-2, sVEGFR-3の血中レベルをELISA法で測定した。その結
果、VEGFとsVEGFR-1はMetS群 とnon-MetS群 で 有 意 差 が な か っ た が
VEGF-C, sVEGFR-2, sVEGFR-3はMetS群で有意に上昇していた。年齢、性
別で調整後、VEGF-CはBMI、収縮期 (SBP) および拡張期 (DBP) 血圧、中性
脂 肪 (TG)、LDL-C、non-HDL-C、sVEGFR-2と、sVEGFR-3はSBP、DBP、
sVEGFR-2と各々有意な相関を認めた。ステップワイズ回帰分析の結果、
TG、sVEGFR-2、DBPがVEGF-Cの、sVEGFR-2がsVEGFR-3の各々独立した
規定因子であった。面白いことにBMI、DBP、TGを含む多変量解析の結果、
VEGF-Cの規定因子はTGとDBPであったが、対照的にVEGFの規定因子は
BMIであった。以上から、VEGF-C/VEGFR-3シグナリングがsVEGFR-2と関
連してMetSの病態に関与している可能性が示唆された。
How to write
Plenary Lecture
Nature Medicine
Symposium
Y.I.A.
Presentation
Selected Oral
Presentation
Poster
The 8th Korea-Japan Joint Symposium on Vascular Biology
Special Talk
Index
Aⴊ▤↢‛කቇળKPFD
1
国立病院機構京都医療センター 展開医療研究部、2国立病院機構京都医療セン
ター 健診センター、3国立病院機構京都医療センター 糖尿病研究部、4国立病院
機構京都医療センター 循環器科、5静岡大学薬学部 分子医学、6京都大学大学院
医学研究科 人間健康科学、7奈良女子大学 健康管理センター、8国立病院機構京
都医療センター 臨床研究センター
1
国立病院機構京都医療センター 展開医療研究部、2国立病院機構京都医療セン
ター 健診センター、3国立病院機構京都医療センター 糖尿病研究部、4国立病院
機構京都医療センター 循環器科、5静岡大学薬学部 分子医学、6京都大学医学
部 人間健康科学、7国立病院機構京都医療センター 臨床研究センター、8京都大
学 循環器内科
Special
Lecture
175
和田 啓道1、○山田 明1、北岡 修二2、浦 修一1、浅原 哲子3、赤尾 昌治1,4、
阿部 充1,4、森本 達也1,5、藤田 正俊6、高橋 裕子7、島津 章8、
長谷川 浩二1
和田 啓道1、○浦 修一1、北岡 修二2、浅原 哲子3、高谷 智英1,8、森 利依子1、
赤尾 昌治1,4、阿部 充1,4、森本 達也1,5、藤田 正俊6、島津 章7、
長谷川 浩二1
President
Lecture
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
胎盤由来増殖因子(PlGF)は禁煙後の内臓脂肪蓄積、
インス
リン抵抗性増悪の鋭敏な指標である
68
血管内皮増殖因子C (VEGF-C) はメタボリックシンドロー
ムで上昇している
67
Program
18：00〜
Aⴊ▤↢‛කቇળKPFD
176
Recent epidemiological studies suggest that vascular risk factors play a significant
role in the development of Alzheimer disease (AD). Furthermore, the use of
antihypertensive drugs has been suggested to reduce the incidence of dementia
including AD. In this study, we examined the effects of an angiotensin receptor
blocker (ARB), olmesartan, on Aβ-induced cerebrovascular dysfunction and
memory impairment. Oral administration of a low dose of olmesartan attenuated
cerebrovascular dysfunction in young AD model transgenic mice (APP23), without
a reduction of brain Aβ level. Moreover, treatment of APP23 mice with olmesartan
decreased oxidative stress in brain microvessels. Using an acute mouse model
induced by intracerebroventricular administration of Aβ, we assessed the effect of
oral administration of olmesartan on spatial learning evaluated with the Morris
water maze. Olmesartan significantly improved cognitive function independent of
its blood pressure-lowering effect, whereas there was no improvement by other
types of antihypertensive drugs (hydralazine and nifedipine). We found that pretreatment with a low dose of olmesartan completely prevented Aβ-induced
vascular dysregulation, and partially attenuated the impairment of hippocampal
synaptic plasticity. These findings suggest the possibility that amelioration of
cerebrovascular dysfunction with an ARB could be a novel therapeutic strategy for
the early stage of AD.
1
大阪大学大学院医学系研究科臨床遺伝子治療学、2大阪大学大学院医学系研究科老
年腎臓内科、3大阪府立急性期・総合医療センター、4東京大学大学院医学系研究科
神経生理学教室
武田 朱公1,2、里 直行1,2、荻原 俊男3、狩野 方伸4、楽木 宏実2、
森下 竜一1
アンジオテンシンII受容体拮抗薬はAβによる脳血管反応
性障害を抑制しアルツハイマー病モデルマウスの認知機
能障害を改善する
71
Background: Abdominal aortic aneurysm (AAA) is a common disease and lethal
when it progresses to rupture, but current medical treatments are insufficient to
suppress AAA progression. Recent studies suggest a key role of monocyte-mediated
inflammation in the pathogenesis of AAA.
Methods and Results: We prepared bioabsorbable PLGA nanoparticles (NP),
which were incorporated into CD11b + monocytes and then delivered into the
abdominal aorta after intravenous injection. In cultured monocytes, pitavastatincontaining NP (Pitava-NP), but not control FITC-NP or Pitava alone, inhibited
lipopolysaccaride-induced NF-㱖B activation by ELISA and MCP-1 mRNA
expression by real-time PCR. To examine therapeutic effects of Pitava-NP, we used
a murine model of AAA in ApoE-deficient mice fed with high-fat diet and infused
with angiotensin II. Treatment with Pitava-NP (weekly intravenous injection of NP
containing 0.4 mg/kg Pitava for 4 weeks), but not that with FITC-NP or Pitava
alone, prevented AAA formation (5/9 vs 0/11 for FITC-NP and Pitava-NP), reduced
monocyte recruitment and MCP-1 expression in both AAA and non-AAA sites, and
reduced the composite endpoint of AAA and sudden death. In contrast, no such
beneficial effects were seen in the mice treated with oral administration of Pitava at
1 and 10 mg/kg/day for 4 weeks, proving the advantage of NP-mediated drug
delivery system. Importantly, the AAA formation was blunted in ApoE/CCR2double deficient mice and the mice treated with NP containing dominant-negative
inhibitor of MCP-1, suggesting the importance of MCP-1/CCR2 signaling in AAA
formation.
Conclusion: NP-mediated monocyte-selective delivery of Pitava is effective to
prevent AAA progression. Pitava-NP inhibits monocyte-mediated inflammation
through MCP-1 signaling.
九州大学大学院医学研究院循環器内科学、2松山赤十字病院
レニン−アンジオテンシン系は心血管リモデリングにおいて重要な役割を
果たしている。近年、アンジオテンシン変換酵素２（ACE2）とその働きに
よりアンジオテンシンIIから産生されるアンジオテンシン(1-7)が生理活性
因子として循環器系の調節に関わっていることが報告されている。今回我々
はマウス大腿動脈周囲にポリエチレンチューブをカフ状に留置し血管傷害
モデルを作製し、これに伴うリモデリングにおけるACE2−アンジオテンシ
ン（1-7）系の役割を検討した。カフ留置により大腿動脈には炎症性血管傷
害と新生内膜形成が引き起こされた。組織におけるmRNA発現をリアルタ
イムRT-PCRにて測定すると、ACE2の発現は傷害血管においては著明に低
下していた。最近の報告により、アンジオテンシン（1-7）の受容体タンパ
クがMasであることが明らかとなったが、傷害血管組織では、MasのｍRNA
発現も非傷害血管に比べて著明に低下していた。これらの結果は、ACE2−
アンジオテンシン(1-7)−Mas経路の機能低下が血管リモデリングに影響す
る可能性を示唆している。そこで、Masアンタゴニストである[D-Ala(7)]
Ang(1-7)（0.5 mg/kg/day）をカフ留置と同時にミニポンプで投与すると、新
生内膜形成が増強した。一方、ARBであるオルメサルタンは新生内膜形成
を抑制するが、このとき傷害血管でのACE2およびMasの発現低下を同時に
抑制していた。これらの結果から、血管リモデリングにおいてACE2−アン
ジオテンシン(1-7)−Mas経路が抑制的な作用を有する可能性が示唆され、
またオルメサルタンによるリモデリング抑制にも、この経路の活性化が関
与していると考えられた。
愛媛大学大学院医学系研究科分子心血管生物・薬理学
岩井 將、中岡 裕智、仙波 泉、菅野 晴美、堀内 正嗣
マウス炎症性血管傷害におけるACE2−アンジオテンシン
（1-7）
系の役割
72
【目的】脳内における炎症反応の亢進はアルツハイマー病（AD）の重要な
病態の一つとして認識されつつある。一方で、肺炎や尿路感染症などの末
梢臓器の感染症に伴う全身性の炎症はしばしば脳内に波及して急性の認知
機能障害を惹起することが知られており、特に認知症高齢者ではその頻度
が高い。本研究ではAD脳病態の全身性炎症反応に対する脆弱性について、
ADモデルマウスを用いて検討を行った。
【方法】各種脳内ペプチドを生体
マウスで経時的に回収可能な新規マイクロダイアリシス回収系を確立し、
これを用いてマウス脳内における脳内Aβ、炎症性サイトカイン（IL-6、
TNF-α ） の 変 動 をin vivoで 評 価 し た。11〜12週 齢 のADモ デ ルTgマ ウ ス
（APP-Tｇ）及び野生型マウスに対しLPS（10mg/kg）腹腔内投与を行い、そ
の前後の脳内及び血中Aβ、炎症性サイトカインの量を測定した。またLPS
投 与 に よ る 行 動 変 化（ 活 動 量、Social Behavior、 摂 食 量 ） を 評 価 し た。
Blood-brain-Barrier（BBB） の 透 過 性 の 評 価 と し てFITC-Albumin leakage
assayを用いた。【結果】LPS腹腔内投与により血中炎症性サイトカインは
ADモデルマウスと野生型マウスで同程度に上昇したが、脳内IL-6の上昇の
程度は野生型マウスに比しADモデルマウスで有意に大きかった。またこれ
に伴い、LPS投与後に生じる行動変化（Sickness behavior）もADモデルマウ
スで有意に重篤化していた。LPS投与により脳内Aβ量自体は変化しなかっ
た。全身性の炎症亢進によるBBB破綻の程度をFITC-Albumin Leakage Assay
で評価したところ、ADモデルマウスでは野生型マウスに対してBBB透過性
がより亢進していることが示された。
【考察】以上の結果から、ADマウス
では野生型マウスに比して全身性炎症に伴う脳炎がより重篤化すること、
その原因の一つとしてADマウスでは炎症に対するBBBの脆弱性がより亢進
している可能性が示唆された。このことは認知症高齢者の感染症に伴うせ
ん妄や急性脳障害の発症の病態を考える上で重要な示唆を与えると考えら
れる。
1
大阪大学大学院医学系研究科臨床遺伝子治療学、2大阪大学大学院医学系研究科老
年腎臓内科
武田 朱公1,2、里 直行1,2、楽木 宏実2、森下 竜一1
香月 俊輔1、的場 哲哉1、古賀 純一郎2、中野 覚1、佐藤 敬1、砂川 賢二1、
江頭 健輔1
1
Increased blood-brain barrier vulnerability to
systemic inflammation in Alzheimer disease mouse
model
70
Nanoparticle-Mediated Monocyte-Selective Delivery of
Pitavastatin Prevents Abdominal Aortic Aneurysm
Formation in Mice
69
ポスターセッション：血管炎症（1）
Poster Session：Vascular Inflammation(1)
12月1日
（水）
／Wednesday, December 1
18：00〜
Index
Aⴊ▤↢‛කቇળKPFD
Poster
177
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
【目的】Apoptosis signal-regulating kinase(ASK) 1は、酸化ストレス応答に重
要な役割を果たしているMAPKKKであることが知られている。食塩負荷は、
心血管病のリスクを増加させることはよく知られているが、その分子機序
については不明 である。今回我々は、
ASK1ノックアウトマウス（ASK1KO）
を用いて、
食塩負荷による臓器障害におけるASK1の役割を検討した。
【方法】
18週齢の ASK1KO、野生型マウス（WT）をそれぞれ、正食塩食群（0.5%
NaCl）と高食塩食群（8% NaCl）に分け、10週間の高食塩負荷に対する反
応を比較検討した。【成績】高食塩食を投与すると、WT, ASK1KOともに、
尿量、飲水量が顕著に増加したが、その反応には両群間で有意差はなかった。
また、尿中のNa排泄にも両群間で有意差を認めなかった。血圧の推移も
WTとASK1KO間に大きな差は認めなかった。血漿レニン活性、血中アルド
ステロン濃度は食塩負荷により、WT, ASK1KOともに低下し、両群間で差
はなかった。心臓組織中のASK1とその下流分子であるp38のリン酸化をウ
エスタンブロットにて調べたところ、食塩負荷により、WTではASK1、p38
のリン酸化が増加したが、ASK1KOではp38のリン酸化が増加しなかった。
10週間の食塩負荷後、心臓の線維化を評価したところ、WTでは心臓の著明
な線維化がみられたが、ASK1KOでは線維化は有意に少なかった。WTでは、
食塩負荷により心臓組織中のgp91の発現、スーパーオキシド産生および炎
症細胞浸潤が増加していたが、ASK1KOでは食塩負荷により有意な増加を
認めなかった。また、WTでは食塩負荷によりTGF-βの発現が亢進してい
たが、ASK1KOでは、有意な増加を認めなかった。胸部大動脈のアセチル
コリンによる内皮依存性弛緩反応を調べたところ、WTでは食塩負荷により
有意な低下を認めたが、ASK1KOでは変化しなかった。血管組織における、
スーパーオキシド産生および炎症細胞浸潤も、心臓と同様、WTでは食塩負
荷により増加したが、ASK1KOでは、変化をしなかった。
【結論】食塩負荷
による心臓線維化、血管内皮障害には、ASK1, p38を介した酸化ストレスと
炎症の亢進が関与している。
熊本大学生体機能薬理学、2熊本大学循環器病態学
1
片岡 恵一郎1、外山 研介1、末田 大輔1、董 一飛1、小川 久雄2、
光山 勝慶1
Symposium
＜背景＞ 高齢化とイメージング診断の発達に伴い大動脈瘤罹患数は増加
傾向にあり、それら治療法として、侵襲の大きい人工血管置換術に加え、
高齢者などではステントグラフトなどの低侵襲治療が用いられるように
なっている。一方で、手術適応に満たない大動脈瘤では降圧療法などで経
過観察されていることが多い。しかし、外科的視点に立った動脈瘤進行を
抑えるような効果的な管理方法は、モデル作製の困難さ・不確かさから明
らかにされてこなかった。
＜目的＞ マウスで作為的に動脈瘤モデルを作製し、その病態解明を目指
すとともに薬剤介在による効果について検討を行う
＜方法＞ C57BL/6系マウスを使用し、誘発薬剤としてAngiotensinII (AngII)
と㱎-aminopropionitrile (BAPN)を使用し、両者を皮下注射しモデル作製を
図った。検討項目として、開始前と開始後4週間定期的に尾動脈による血圧
測定を行い、血圧変化を測定した。薬剤投与開始4週間後にSacrficeを行い、
組織評価を行った
＜結果＞ 血圧変化は、投与前収縮期圧90 mmHg前後であるが、投与開始4
週間後には150mmHg前後まで上昇した。約25%の対象マウスが、急性大動
脈乖離＋大動脈破裂で開始後1週間以内突然死した。4週間後のSacrificeにて、
AngII＋BAPN投与群では全例で胸部大動脈瘤または腹部大動脈瘤が形成さ
れていた。一方で、AngIIのみの群では血圧上昇を認めるものの、20%程度
しか動脈瘤が形成されていなかった。組織解析では、形成群では動脈瘤部
に一致して、細胞浸潤を認めていた。免疫染色・組織中炎症性サイトカイン、
MMP変化等については現在追加実験中である。
＜まとめ＞ 作為的に動脈瘤を形成させるモデルはこれまでにもいろいろ
と報告されてきたが、形成する確率が安定せず、薬物治療効果判定などに
用いることができなかった。今回、我々は、安定的にかつ効率的に動脈瘤
を形成するモデル系の確立に成功した。今後これらモデルを使用して、さ
らにヒト動脈瘤形成のメカニズムを検討するとともに、トランスレーショ
ナルリサーチとして薬剤介入が及ぼす影響について検討する予定である。
徳島大学大学院ヘルスバイオサイエンス研究部心臓血管外科、2徳島大学大学院ヘ
ルスバイオサイエンス研究部循環器内科学分野、3徳島大学大学院ヘルスバイオサ
イエンス研究部脳神経外科学分野
1
松岡 裕貴1、黒部 裕嗣1、平田 陽一郎2、兼松 康久3、佐田 政隆2、
北川 哲也1
76
How to write
Plenary Lecture
Nature Medicine
食塩負荷による心血管障害の分子機序 ーASK1を介した
炎症と酸化ストレスの役割ー
Special Talk
75
Special
Lecture
大動脈瘤モデルの作製とその評価系の確立
背景：腹部大動脈瘤の発症・進展には複数の因子が関与すると考えられて
いる。疫学調査では加齢、性別（男性）そして喫煙が動脈瘤の進展因子と
して明らかにされているが、腎機能障害の明らかな関与は証明されていな
い。しかしApoE欠損マウスにAngiotensis II (Ang II) を持続投与すると動脈
瘤が形成される事が報告され、レニン・アンジオテンシン系の役割が注目
されるようになった。目的：動脈瘤の動物モデルで投与されるAng IIは非生
理的な高用量であり、実際の病態を反映していない。そこで、慢性腎機能
障害の動脈瘤への関与を動物実験で検討した。方法：ApoE欠損マウスに5/6
腎臓摘出術を行い、慢性腎障害モデルを作成した。その後2週間ごとの経時
的な超音波検査で腎動脈上の大動脈の拡張および循環動態を６週間評価し
た。結果： 5/6腎臓摘出群は非摘出群（Sham群）と比較して、脈拍・血圧に
有意な変化は見られなかったが、血清レニン活性とAng II濃度は上昇してい
た。動脈瘤径の検討では、5/6腎臓摘出群では約150％に拡大し、Sham群に
比べ有意な変化を示した。さらに、この動脈拡大は、循環動態に影響を与
えない投与量のAT1受容体阻害薬で抑制された。組織検査では5/6腎臓摘出
群は弾性線維の伸長・分断化が認められ、そのため瘤化が進んでいると考
えられた。メカニズムの検討では、接着因子であるICAM-1の発現が上昇し
ており、その結果として血管壁へのマクロファージの浸潤が認められた。
またマクロファージから各種のプロテアーゼ群が分泌されるが、主要な細
胞外器質の分解酵素であるmatrix metalloproteinases (MMP)-2, MMP-9そして
MMP-12の分泌が亢進していた。結語：臨床では透析などの治療により明
らかな関係は証明されていないが、慢性腎障害によるレニン・アンジオテ
ンシン系の亢進は動脈瘤の進展因子になる可能性が示唆された。
1
大阪大学連合小児発達学研究科健康発達医学、2大阪大学医学系研究科臨床遺伝子
治療学
President
Lecture
【背景】High mobility group box-1(HMGB1)は核内非ヒストン蛋白として当初
同定されたが、近年、種々のストレスによって細胞外に放出され、炎症性
サイトカインの発現に寄与することが報告されている。一方で、動脈硬化
は血管壁の慢性炎症であることが明らかとなっており、動脈硬化などの血
管のリモデリングの過程でHMGB1の果たす役割を明らかにすることを目的
として以下の研究を行った。
【方法】野生型マウスの大腿動脈にWire Injuryを行い、術後2時間から2週間
までの各時間で血清HMGB1濃度を測定した。次に、Wire Injuryを行った傷
害血管周囲に、コラーゲンジェルを用いてPBS(PBS群)、抗HMGB1中和抗
体（局所抗体群）を持続投与した。また中和抗体を術後2、12、24時間後に
腹腔内に投与した（全身投与群）
。以上の3群（各群n=5）で、4週間後の血
清ＨＭＧB1濃度と新生内膜増殖面積を測定した。さらに、マウスマクロ
ファージ（RAW264.7）、ヒト臍帯静脈内皮細胞、ラット平滑筋細胞をLPS
（100ng/ml）で刺激し、1から24時間後の培養上清のHMGB1濃度を測定した。
【結果】Wire Injury後の血清HMGB1濃度は傷害直後には上昇せず、4日目に
高値となりその後徐々に低下した。傷害後4週間での新生内膜/中膜比は、
局所投与群において有意に抑制された(p＜0.05)が、全身投与群はPBS群と
差がなかった。血清HMGB1濃度も同様に、PBS群と腹腔内投与群には差が
なく局所抗体群で有意に低下した(p＜0.05)。RAWでは刺激6時間後をピー
クにHMGB1濃度が上昇したが、内皮細胞や平滑筋細胞では上昇しなかった。
【結論】中和抗体を局所投与することで新生内膜増殖が抑制されたことから、
傷害後の血管リモデリングにHMGB1が関与することが示された。また血清
濃度の変化やvitroの結果から、傷害を受けた細胞から放出されるHMGB1だ
け で は な く、 マ ク ロ フ ァ ー ジ な ど の 免 疫 細 胞 か ら2次 的 に 分 泌 さ れ る
HMGB1によって、炎症が増幅される負の連鎖が起きていることが示唆され
た。
徳島大学大学院ヘルスバイオサイエンス研究部代謝栄養学分野、2徳島大学医学部
医学科、3徳島大学大学院ヘルスバイオサイエンス研究部循環器内科学分野、4徳島
大学大学院ヘルスバイオサイエンス研究部心臓血管外科学分野
三宅 隆1、浜田 日月1、島村 宗尚1、中神 啓徳1、森下 竜一2
1
動脈瘤の進展における慢性腎障害の影響
東田 真由子1、西雄 千佳1、松岡 裕貴2、平田 陽一郎3、黒部 裕嗣4、中屋 豊1、
北川 哲也4、佐田 政隆3
74
血管リモデリングにおけるHMGB1の果たす役割
73
ポスターセッション：血管シグナル（1）
Poster Session：Vascular Signal Transduction(1)
12月1日
（水）
／Wednesday, December 1
Program
Aⴊ▤↢‛කቇળKPFD
178
【目的】ASK1, ASK2は酸化ストレスにより活性化されるMAPKKKである。
ASK2はASK1の結合タンパクとして同定されたが、ASK2の循環器系におけ
る役割については不明である。今回、ASK2の循環器系における生理的な役
割とAngiotensin IIによる心血管障害における役割をASK1と比較検討すると
ともに、ASK1, ASK2の相互作用を検討した。【方法・結果】
（実験1）野生
型マウス（WT）, ASK1KO, ASK2欠損マウス（ASK2KO）、ASK1/ASK2両欠
損マウス(ASK1/2DKO)の生理的な循環器系の表現型を比較した。12週令で
のASK2KO, ASK1/2DKOの血圧は、WT, ASK1KOよりも有意に高かった。
17週 令 で の 心 臓 の 線 維 化 を 組 織 学 的 に 評 価 し た と こ ろ、ASK2KOと
ASK1/2DKOの線維化はWT, ASK1よりも有意に増加していた。すなわち、
ASK2をノックアウトすると高血圧と心臓の線維化を呈し、これらの表現型
はASK1を同時にノックアウトしても有意な変化がない事から、ASK1非依
存性のものと考えられた。 (実験2) これらのマウスに4週間AII（600ng/kg/
min）投与を行った。AIIにより全てのマウスの血圧は同程度に上昇した。4
週間のAII投与により、WTとASK2KOの心重量は有意に増加していたが、
ASK1KOとASK1/2DKOではAIIによる有意な変化は認めなかった。心臓の
組織評価を行ったところ、WT, ASK2KOではAII投与により線維化の亢進が
起こっていたが、ASK1KOとASK1/2DKOではそれが抑制されていた。組織
中のCD68の増加も線維化と同様の傾向を示していた。すなわち、AII投与
時の炎症と心臓線維化はASK1依存性である事が示された。アセチルコリン
による血管内皮依存性の血管弛緩反応を調べたところ、WTでみられたAII
による血管内皮機能の障害は、ASK1KO, ASK2KO, ASK1/2DKOでは見られ
なかった。
【結論】1) ASK2を欠損させると高血圧と心臓の線維化がおこり、
ASK1KOとは異なった表現型を呈する。 2) ASK2欠損時の高血圧と心肥大
はASK1非依存性である。3) AII投与による心肥大と炎症はASK1依存性であ
る。 4) AIIによる血管内皮機能障害にはASK1, ASK2の両者が関与している。
5)ASK2はASK1の結合タンパクとして同定されたが、血圧調節と心臓の線
維化に関しては、ASK1に依存しない機能がある。
熊本大学生体機能薬理学、2熊本大学循環器病態学
1
片岡 恵一郎1、外山 研介1、末田 大輔1、董 一飛1、小川 久雄2、
光山 勝慶1
ASK2欠損マウスはASK1非依存性に高血圧、
心肥大を呈
する〜ASK1/2ダブルノックアウトマウスを用いた検討
77
Poster
The 8th Korea-Japan Joint Symposium on Vascular Biology
Selected Oral
Presentation
Index
Aⴊ▤↢‛කቇળKPFD
Additional blood pressure (BP) control provided by the combination of the
angiotensin II receptor blocker valsartan and the dihydropyridine calcium channel
blocker amlodipine had been recognized as the predominant benefits for
cardiovascular and cerebral protection in hypertension. However, whether there is
a BP-independent component that may contribute to the additional beneficial effect
of this combination strategy is needed to be confirmed. Salt-loaded stroke-prone
spontaneously hypertensive (SHRSP) rats were treated with (1) vehicle, (2)
valsartan (2mg/kg/day), (3) amlodipine (2mg/kg/day) and (4) combined valsartan
and amlodipine for 4 weeks. The cerebral and cardiovascular protective effects
were compared among these groups. Subantihypertensive dose of valsartan and
hypotensive dose of amlodipine similarly and significantly improved the results of
stroke incidence, left ventricle mass, brain edema, cardiac and cerebral cortical
ED-1-positive cell infiltration, cerebral penetrating arteriolar thickening, pericoronary arteriolar fibrosis, cardiac interstitial fibrosis, and endothelium-dependent
vascular relaxation. Increased cerebral vascular endothelial growth factor (VEGF)
in salt-loaded SHRSP rats was significantly inhibited by either monotherapy.
Addition of valsartan to amlodipine did not significantly change the blood pressure
of rats compared with those treated with amlodipine alone in this study. However
such a combination therapy, compared with either monotherapy, significantly
improved the above cerebral and cardiovascular damages. Combination of valsartan
and amlodipine provided BP-independent additional protective effects on cerebral
and cardiovascular injuries in SHRSP rats. This therapeutic strategy is useful for
hypertensive patients.
Objective: We administrated angiotensin converting enzyme (ACE) inhibitor
perindopril to a normotensive amyloid 㱎 (A㱎)(1-40)-injected Alzheimerʼs disease
(AD) mouse model and investigated the effect of perindopril on memory
impairment and hippocampal damage. Methods: ICR mice were pretreated with (1)
vehicle, (2) perindopril (1 mg/kg/day) and (3) perindopril (3 mg/kg/day) for 2 days
before icv injection of A㱎(1-40). Memory impairment was evaluated with Y-maze
test. Results: Accompanying with a significant decline of spontaneous alternation,
A㱎-injected mice presented significant increase of oxidative stress, activated
microglia and astrocytes in the hippocampus. A㱎 injection induced significant
overexpressions of hippocampal iNOS and nitrotyrosine-positive protein while
significantly decreased hippocampal Ec-SOD level . Moreover, iNOS was found to
be expressed in the activated astrocytes and endothelial cells in the hippocampus.
A㱎-induced hippocampul damage and memory impairment were significanlty
ameliorated by prindopril treatments as compared with vehicle mice. Perindopril
dose-dependently decreased the cerebral ACE activity. Likewise, perindopril dosedependently decreased the expression of iNOS, prevented the nitration of proteins,
and increased the expression of Ec-SOD in the hippocampus. Conclusions:
Perindopril attenuated hippocampal oxidative stress and inflammation via inhibition
of overexpressed iNOS and nitrotyrosine-positive proteins, and via suppression of
the activation of microglia and astrocytes in the hippocampus. Our data suggest
that priondopril may be a potential theraputic agent for hypertensive AD patients.
Y.I.A.
Presentation
179
熊本大学大学院生命科学研究部生体機能薬理学分野
熊本大学大学院生命科学研究部生体機能薬理学分野
Symposium
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
董 一飛、片岡 恵一郎、中村 太志、名幸 久仁、外山 研介、光山 勝慶
董 一飛、片岡 恵一郎、中村 太志、名幸 久仁、外山 研介、光山 勝慶
How to write
Plenary Lecture
Nature Medicine
Valsartan and amlodipine combination provided
additional effects on cerebral and cardiovascular
injuries in SHRSP rats
Special Talk
Perindopril protects amyloid β-evoked memory
dysfunction in mice via inhibition of oxidative stress
and inflammation
Special
Lecture
81
Background: The pathogenesis of inflammatory bowel disease (IBD) involves with
local expression of inflammatory cytokines. Nuclear factor-kB (NFkB), a
transcriptional factor, plays an important role in the coordinated transactivation of
the inflammatory cytokines. It is reported that decoy oligonucleotide (ODN)
targeting NF-kB offers a therapeutic approach for prevention and treatment for
inflammatory diseases. We developed a novel Hybrid Staple-Modified Partial
Phosphorothioated NFkB decoy ODN (HSODN) that increase the stability. HSODN
is resistant to digestion by endonucleases, whereas double-stranded decoy ODN
(dsODN) is easily digested. Aim: To investigate the therapeutic potential of an
intravenously administered HSODN in dextran sulphate sodium (DSS)-induced
chronic colitis, experimental model of IBD. Methods: We administered NFkB
decoys (HSODN, dsODN) in mice with DSS-induced IBD. Scrambled decoy was
used as a control. Decoy ODNs was administrated intravenously on day 7,11,14.
The therapeutic effects were evaluated on day 0,4,7,11,14,16 with the survival rate,
length of large intestine, and disease activity index (DAI). Furthermore, expression
of inflammatory cytokines was assessed using real-time RT PCR.Results: The
survival rate, the length of large intestine and DAI significantly improved in the
mouse treated with HSODN than in the control group and dsODN group. Although
expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the IBD
group significantly increased, treatment with HSODN significantly reduced
expression of these cytokines. Conclusions: These data suggest that intravenous
injection of HSODN is effective in DSS-induced colitis, experimental model of
IBD, and support the potential use of HSODN as a future therapeutics in human
IBD.
1
大阪大学医学部附属病院総合診療部、2大阪大学大学院医学系研究科臨床遺伝子治
療学、3大阪大学大学院医学系研究科老年腎臓内科学
牧野 寛史1,2、尾崎 和成2,3、樂木 宏実3、森下 竜一2
President
Lecture
80
Background and Purpose: Periostin, a novel secreted and soluble extracellular
matrix, has been reported to play pivotal roles in cell survival, migration, and
regeneration in various tissues. However, its function was still unknown in
mammalian brain. In the present study, we explored the role of periostin in normal
and ischemic brain.Methods and Results: RT-PCR and in situ hybridization showed
expression of periostin mRNA in normal brain, especially in cerebral cortex and
hippocampus. Immunohistochemistry revealed that periostin was expressed mainly
in matured neurons. When periostin was added in medium in adult primary
neuronal culture, the length of neurite at 3 days after plating was longer in periostintreated neurons than that of control neurons. Also, pre-treatment of periostin
increased cell survival under hypoxic condition induced by CoCl 2, indicating that
periostin had neurotrophic effects. Next, the role of periostin was examined in
transient middle cerebral artery occlusion (tMCAo) model. The expression of
periostin mRNA was decreased in ischemic core and increased in peri-infarct region
at 3 hrs after tMCAo, followed by increased expression in both regions at 24 hrs
after tMCAo. Immunohistochemistry showed that neuron and activated astrocyte
expressed periostin at 24 hrs after tMCAo. As expected, intracerebroventricular
injection of periostin before tMCAo and immediately after reperfusion reduced
infarct volume and prevented the worsening of neurological deficits at 24 hrs after
tMCAo. Conclusions: Since exogenous periostin showed neuroprotective effects in
vitro and vivo, periostin might be one of key molecules, which protect neurons
from ischemic injury. Further studies on periostin might shed light on new treatment
in cerebral ischemia.
大阪大学健康発達医学講座、2大阪大学臨床遺伝子治療学講座、3東京大学先端臨床
医学開発講座
1
Role ofintravenous administration of Hybrid StapleModified NFkB Decoy ODNson Inflammatory Bowel
Disease Model Mice.
79
18：00〜
島村 宗尚1、谷山 義明2、中神 啓徳1、里 直行2、鯉渕 信孝3、若山 幸示3、
モハマド アラタバコリ2、森下 竜一2
Pivotal roles of periostin in cerebral ischemia
78
ポスターセッション：その他
（1）
Poster Session：Others(1)
12月1日
（水）
／Wednesday, December 1
Program
Aⴊ▤↢‛කቇળKPFD
180
背景・目的：腎肥大は有効ネフロン数減少時の代償機転であり、糖尿病な
どによる腎障害の最早期病態であると同時に、その破綻が腎障害進行の要
因と考えられる。実際、高血圧などの動脈硬化性疾患を有するdonorにおい
て 術 後 に 腎 機 能 低 下 を 来 す。 我 々 は 動 脈 硬 化 性 疾 患 で は 内 皮 型NOS
(endothelial NOS; eNOS)の活性低下が生じることから、
「eNOS活性の低下が
代償性腎肥大の破綻を惹起する」との仮説をたて研究を行った。 NOS阻害
薬により代償性腎肥大が抑制され、NOの関与が示唆されている。しかし代
償性腎肥大におけるNO産生経路、
詳細な分子メカニズムは不明である。我々
は代償性腎肥大過程におけるeNOS 由来のNO産生亢進の役割と臓器肥大に
つながる細胞内シグナル伝達経路を解析した。方法：動物は野生型マウス
(C57BL/6; WT)、 内 皮 特 異 的eNOS過 剰 発 現 マ ウ ス (ECeNOS-TG) お よ び
eNOS欠損マウス (eNOS-KO) を使用した。右腎臓摘出術 ( UNx)あるいは偽
手術（Sham）を行い2週間後に対側腎代償性肥大を評価した。UNｘ直後の
対側腎血流変化をドップラー法にて評価した。次にWT-sham、WT-UNx、
ECeNOS-TG-sham、ECeNOS-TG-UNx、eNOS-KO-sham、eNOS-KO-UNx、の6
群に分け、腎重量/体重比などを検討した。NO下流の細胞内シグナルを検
討 す る た め 培 養 ヒ ト 近 位 尿 細 管 細 胞 に、NOド ナ ー
（S-Nitrosoglutathione(GSNO))、sGC stimulator (BAY 41-2272 ) を 添 加 し、
Protein kinase G (PKG) 活性、Akt pathway活性化について検討した。さらに
代償性腎肥大におけるsGCの役割をin vivoで確認するためeNOS-KO-UNxに
対しBAY 41-2272を投与し、代償性腎肥大が再現実験を行った。 結果：
WTではUNx後に腎重量は有意に増大したが、eNOS-KOでは代償性腎肥大
が抑制された。逆にECeNOS-TGでは代償性腎肥大が増強したIn vitroでは培
養尿細管細胞においてGSNO、BAY 41-2272の投与によりPKG活性上昇を認
め、Akt、s6Kまた４EBP のリン酸化亢進を確認した。またeNOS-KO-UNx
にBAY 41-2272を投与すると、代償性腎肥大を誘導された。結語：eNOS活
性は代償性腎肥大に重要な役割を果たしており、NO-sGC-PKG系が腎肥大
の細胞内シグナル伝達に重要であることが示された。
中枢神経傷害により失われた神経機能は、時間経過に伴い、部分的に自然
回復する。神経症状の改善は神経回路の修復に基づくと考えられているが、
神経回路の自発的な再生機構は明らかではない。我々は、多発性硬化症モ
デルマウス（EAEマウス）を用いて、傷害後に形成する新生血管が神経回
路の再生を導くことを見出した。EAEマウスでは、中枢神経系において血
管新生と神経回路の再生が生じることが、それぞれ独立して報告されてい
る。はじめに、本モデルマウスにおける血管新生と神経回路の再生の経時
変化を検討した。血管新生は、新生血管マーカーであるCD105陽性血管数
で評価した。四肢の運動機能を担う皮質脊髄路を可視化して観察するため、
大脳皮質第5層に順行性トレーサーのビオチン化デキストランアミンを注入
し、神経回路の再生を観察した。その結果、EAEマウスでは、血管新生の
後に神経回路の再生が生じる様子が認められた。このことから、中枢神経
障害後に形成する新生血管が神経回路の再生を促す可能性が示唆された。
続いて、新生血管の神経突起伸長への効果を検討するため、血管内皮細胞
と神経細胞との共培養実験を行った。大脳皮質神経細胞を、血管内皮細胞
存在下あるいは非存在下で培養したのち、神経細胞マーカーである抗Tuj1
抗体ならびに皮質脊髄路マーカーである抗Ctip2抗体を用いた免疫染色を行
い、Ctip2/Tuj1陽性突起長を計測した。その結果、血管内皮細胞との共培養
により、神経突起の伸長が促進された。また薬理学的検討から、神経突起
の伸長を促す血管由来因子の同定に成功した。最後に、同定した神経突起
伸長促進因子がEAEマウスにおける神経回路の自然回復に寄与するかを遺
伝学的に検証した。皮質脊髄路に同定因子のsiRNAを導入したマウスを用
いて、組織学的・行動学的解析を行った。その結果、同定因子欠損状態では、
時間経過に伴う神経回路の再生と麻痺症状の改善が抑制された。以上の結
果から、傷害後に形成する新生血管が神経回路の再生を導くことが示唆さ
れた。
大阪大学大学院医学系研究科分子神経科学
村松 里衣子、山下 俊英
川崎医科大学 腎臓・高血圧内科学
長洲 一、佐藤 稔、城所 研吾、冨田 奈留也、柏原 直樹
中枢神経傷害後に形成する新生血管による神経回路再生
の促進
83
代償性腎肥大の破綻とNO-sGC-PKG系の役割
82
17：30〜
Index
Aⴊ▤↢‛කቇળKPFD
Poster
181
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
【目的】動脈硬化の危険因子は血管内皮細胞の老化を誘導し、血管新生能を
低下させることが知られているが、独立した危険因子であるlipoprotein(a)
（Lp(a)）の血管内皮細胞系への老化に対する作用は明らかでない。これま
でに我々は、肝細胞増殖因子（HGF）がLp(a)TGマウスにおいて障害された
血管新生能を回復させること(Circ. 2002)、Angiotensin IIによる血管内皮前
駆 細 胞（EPC） の 老 化、 酸 化 ス ト レ ス を 抑 制 す る こ と を 報 告 し て き た
(Hypertens. 2009)。本研究ではこれらの報告をもとに、Lp(a)に起因する血管
内皮細胞の老化及び機能低下に対するHGFの作用について検討した。
【方法】
培養系内皮細胞（EC）の老化をSA-㱎 Galactosidase染色、酸化ストレスを
dihydroethidium（DHE）染色、管腔形成をMatrigel assayで評価し、タンパク
発現はwestern blotで確認した。EPCはDiIで標識、HAECとMatrigel上で共培
養し、incorporation能力を評価した。一方、同条件で培養したEPCを、大腿
動脈を結紮したヌードマウスに移植し、7日後の下肢血流、及び障害部位に
おける血管新生能を免疫染色（CD31）で確認した。
【結果】In vitroにおいて、
Lp(a)はECのSA-㱎 Galactosidase陽性細胞数の増加、superoxide産生を誘導し、
かつ管腔形成を抑制した。また、Lp(a)存在下ではp53、p21の発現上昇がみ
られた。EPCのECへのincorporation能力はLp(a)投与により有意に低下した(P
＜0.01)。下肢虚血マウスへのEPC移植後、7日目において新生血管の増加と
ともに下肢血流が回復していたが、Lp(a)を投与したEPCを移植した群では
回復の低下を認めた。上記のとおりに培養系及び動物モデルでみられた
Lp(a)による作用は、HGF投与により抑制される事が示された(P＜0.01)。【結
語】Lp(a)は、血管内皮細胞の老化、ROS産生やEPCの機能低下を介して動
脈硬化病態に関与している可能性が示唆された。一方、HGFはこれらLp(a)
誘導性病変に対する抑制作用を有することが明らかとなった。
大阪大学大学院医学系研究科臨床遺伝子治療学
岩林 正明、谷山 義明、眞田 文博、岡山 慶太、楠 博、家串 和真、東 純哉、
久徳 真梨子、清水 一賢、Amarnath Chatterjee、森下 竜一
Symposium
Background: Unopposed RANKL (receptor activator of NF㱖B ligand) activation
causes osteoporosis and vascular calcification in mice, and both phenotypes
coincide in postmenopausal women. RANKL inhibition by antibody decreases
osteoporosis and calcium deposition in mice. Estrogen reverses osteoporosis, and
clinical trials show its beneficial effect in vascular calcification. We assessed the
hypothesis that under estrogen deficiency, RANKL system regulates osteoporosis
and vascular calcification, and we propose the molecular mechanism underpinning
vascular calcification under RANKL activation. Methods and Results: RANKL
increased the expression of the calcification inducer BMP-2 in human aortic
endothelial cells (HAEC), and decreased the inhibitor MGP in human aortic smooth
muscle cells (HASMC), as quantified by real-time PCR and western blot analysis.
RANKL induced bone-related gene mRNA expression and calcium deposition
(Alizarin Red staining) followed by the osteogenic differentiation of HASMC.
Using specific estrogen receptor (ER) agonists, we found that estrogen inhibited
RANKL signaling in HAEC and HASMC via ER㱍. ApoE-/- mice fed with western
diet for 3 months presented atherosclerotic calcification (Oil Red and Alizarin Red
staining) and osteoporosis (㱘CT analysis) after ovariectomy, and increased
expression of RANKL, RANK and osteopontin in atherosclerotic lesion as detected
by in situ hybridization. Estrogen inhibited osteoporosis and BMP osteogenic
pathway in aorta by decreasing phospho-smad-1/5/8 and increasing MGP mRNA
expression. Conclusion: RANKL leads to an imbalance of pro- and anticalcification factors by regulating BMP, MGP, and bone-related protein expression.
Estrogen decreases BMP signaling and counteracts the effect of RANKL in an ER
㱍 dependent way.
1
大阪大学大学院医学系研究科臨床遺伝子治療学、2大阪大学大学院医学系研究科内
科系臨床医学老年腎臓内科学、3大阪大学大学院 大阪大学・金沢大学・浜松医科
大学連合小児発達学研究科 健康発達医学寄附講座
今日美1,2,3、中神 啓徳3、楽木 宏美2、森下 竜一1
How to write
Plenary Lecture
Nature Medicine
大窄 マリアナ
87
Special Talk
肝細胞増殖因子
（HGF）はLp(a)による内皮細胞の機能低下
を抑制する
Special
Lecture
Estrogen Prevents Aortic Calcification by Inhibiting
Vascular RANKL System in an Estrogen Receptor
Alpha Dependent Way
[Background] We recently reported that testosterone (T) rapidly activates eNOS via
a direct interaction between androgen receptor (AR) and the p85㱍 subunit of PI3
kinase. In this study, we further investigated the role of membrane AR in eNOS
activation using human aortic endothelial cells (HAEC). [Methods and Results]
Both T and membrane-impermeable bovine serum albumin-coupled T (T-BSA)
induced Akt/eNOS phosphorylation at ~30 min. In contrast, T, but not T-BSA,
transactivated the androgen-responsive element at 2 hr, indicating the non-genomic
activation of eNOS by T-BSA. AR was colocalized with caveolin-1, c-Src and
eNOS in the membrane fraction of HAEC. Imunoprecipitation assay revealed the
T-dependent association of AR with caveolin-1 an d c-Src. T-induced
phosphorylation of Akt/eNOS was abolished by PP2, a c-Src kinase inhibitor, and
by c-Src siRNA as well as by AR siRNA, suggesting that the membranous
interaction of AR with c-Src leads to eNOS activation. T and T-BSA also elicited a
rapid rise of intracellular Ca 2+ within a few minutes as demonstrated by fura-4
fluorescence. The rise of Ca2+ was blocked by an AR antagonist and by AR siRNA.
Finally, T-induced eNOS phosphorylation was prevented by the intracellular Ca 2+
chelator and by an endoplasmic reticulum Ca 2+ -ATPase pump inhibitor.
[Conclusions] We newly found that the membrane AR mediates the rapid activation
of eNOS through the regulation of intracellular Ca2+ and the interaction with c-Src.
President
Lecture
86
【背景・目的】摂取カロリーを食餌自由摂取時(AL)よりも30-50％制限する
カロリ―制限(CR)は、多面的な心血管保護効果をもたらし、ヒトにおいて
も有望な抗老化療法と期待されている。我々はCRにより中年齢ラットの虚
血再灌流(I/R)心筋傷害は軽減し、その心保護効果は、一酸化窒素合成酵素
(NOS)非特異的阻害薬、L-NAME長期投与により消失することを報告した
(Am J Physiol Heart Circ Physiol 2008;295:H2348)。CRによりさまざまな臓器
でendothelial NOS(eNOS)発現が亢進し、eNOS依存的にミトコンドリアの生
合 成 が 亢 進 す る こ と が 報 告 さ れ て い る(Nisoli F, et al。Science
2005;310:314)。そこで本研究では、CRによる心保護効果発現におけるそれ
ぞれの特異的NOS isoformの役割を明らかにする目的で、NOS isoform選択
的欠損マウスにCRを実施し、心保護効果誘導の有無を検討した。【方法・
結果】3ヶ月間ALまたはCRを行ったマウスから心臓を摘出し、Langendorff
手技によりI/R実験を行った。I/R傷害は左室機能の回復と再灌流時に灌流液
中に放出されたLDH活性により評価した。野生型(Wt)マウスとinducible
NOS(iNOS) -/-マウスではCRによりI/R傷害がALよりも軽減し、心保護効果が
誘導された。一方、eNOS-/- マウスとneuronal NOS(nNOS)-/- マウスではALと
CRとのI/R傷害の程度は同等であり、心保護効果は誘導されなかった。Wt、
iNOS-/-、nNOS-/- のI/R傷害は同等であったが、eNOS-/- ではWtと比べI/R傷害
は重篤で、CR後もWtと比べて血圧が高く、心臓は肥大していた。eNOS欠
損による高血圧がCRによる心保護効果誘導を妨げた可能性を検討する目的
で、一部のeNOS-/-ではALまたはCR実施期間にHydralazineによる降圧療法を
併用した。降圧治療によりeNOS-/- のI/R傷害はWtと同等まで回復したが、
CRによる追加的な心保護効果は観察されなかった。【結語】CRによる心保
護効果誘導においては、iNOSは不要であるが、eNOSとnNOSの両者が必須
である。
1
東京大学大学院医学系研究科漢方生体防御機能学、2東京大学大学院医学系研究科
加齢医学講座、3東京大学大学院医学系研究科医用生体工学講座システム生理学、4
東京大学大学院医学系研究科循環器内科
喩 静1、秋下 雅弘2、江頭 正人2、山本 希美子3、小泉 英樹1、橋本 亮1、
田中 君枝4、大内 尉義2、岡部 哲郎1
慶應義塾大学医学部内科（老年）
新村 健
Membrane androgen receptor mediates the rapid
activation of endothelial nitric oxide synthase by
testosterone
85
カロリー制限による心血管保護効果発現における特異的
一酸化窒素合成酵素の役割
84
ポスターセッション：性ホルモン・アンチエイジング
Poster Session：Anti-aging
12月2日
（木）
／Thursday, December 2
Program
-高
Aⴊ▤↢‛කቇળKPFD
182
【目的】当教室はレニン・アンジオテンシン(RA)系をはじめとして様々な遺
伝的負荷が高血圧や心血管疾患と関連があること、および病理学的・分子
生物学的アプローチよりRA系が動脈硬化発症・進展に寄与していることを
報告してきた。これら研究の集大成としてRA系遺伝子多型(AGT M235T多
型・ACE I/D多型・AT1 A1166C多型)が心血管イベント発症のリスクとなり
うるかを本態性高血圧症患者を対象としたコホート研究で検証した。
【方法】
本研究はNOn-invasive Atherosclerotic evaluation in Hypertension (NOAH)研究
のサブ解析として行われ、1998年1月から2004年6月にエントリーした515名
の高血圧患者(男性:女性=293：222、平均年齢=61.3±11.8歳)を対象とし、
AGT M235T多型・ACE I/D多型・AT1 A1166C多型)をTaqman-PCR法で同定
した。脳卒中、
心血管疾患(CVD; 虚血性心疾患・心不全)等の予後調査行った。
【結果】追跡期間中(平均7.6年間)、脳卒中=53,心血管疾患=41,死亡=46が認め
ら れ た。 遺 伝 子 多 型 頻 度 は(AGT: MM/MT/TT=354, ACE: II/ID/
DD=207/244/64, AT1: AA/AC/CC=438/70/7)であった。脳卒中,CVD発症複合
一次エンドポイントに対するKaplan Meier解析でACE I/D多型のDD型がリス
クとなった。CVD発症に対してはACE I/D多型およびM235T多型のDD型お
よびTT型がリスクとなったが、脳卒中発症に対しては各多型で有意差は認
められなかった。Cox-proportional hazard modelではACE遺伝子多型 DD型は
その危険因子(年齢・性別・喫煙・血圧・糖尿病・脂質異常・心筋梗塞や脳
卒中の既往・左室肥大)を交絡因子として補正しても有意な危険因子として
選択された。また、降圧薬服用の有無やACE阻害薬・ARBの使用の有無な
どを交絡因子として追加をしてもACE遺伝子多型DD型は有意な危険因子と
し て 採 択 さ れ た。 さ ら に 年 齢 別 解 析 で はKaplan Meier解 析 お よ びCoxproportional hazard modelにて65歳未満においてCVDおよび脳卒中のリスク
比はより高かった。
【結論】ACE I/D遺伝子多型のDD多型は本態性高血圧症
患者において心血管イベント及び脳心血管イベントの遺伝的危険因子とな
りうることが示唆された。
大阪大学大学院医学系研究科老年腎臓内科
加藤 のぞみ、多田羅 雄之、大石 充、楽木 宏実
RA系遺伝子多型の心血管病発症に対する遺伝的負荷
血圧患者を対象としたコホート研究-
88
医学部
老年腎臓内科学
Objectives: We recently reported that macrophage inflammatory protein-1β
(MIP-1β) induces monocyte adhesion to endothelial cells via NADPH-dependent
ROS generation. Although there is no expression of L-type Calcium channel in
monocyte, treatment of monocyte with certain calcium channel blocker (CCB) was
reported to inhibit adhesion to endothelial cells. Thus, the objective of the present
study is to elucidate the role of MIP-1β-CC chemokine receptor 5 (CCR5) systems
in inhibition of monocyte adhesion by CCBs.Methods: Human umbilical vein
endothelial cells (HUVECs) and THP-1 cells were used as endothelial cells and
monocytic cells, respectively. THP-1 cells were incubated in the presence of
Tempol(0.5mM), azelnidipine (100μmol/L) or amlodipine (10μmol/L) for 24
hours and then stimulated by Ang II (1μM) for 4 hours. The mRNA levels of
MIP-1β and CCR5 were determined by RT-PCR. Intracellular ROS was
determined fluorometrically with the use of dihydroethidium (DHE). We evaluated
adhesion of labeled THP-1 cells stimulated by AngII (1μM) to confluent HUVEC
monolayers.Results: Tempol, azelnidipine or amlodipine significantly inhibited the
expression of mRNA of MIP-1β and CCR5 in AngII-stimulated THP-1, but there
were no additional inhibitive effect of tempol when combined with azelnidipine or
amlodipine. Tempol, azelnidipine or amlodipine reduced intracellular ROS in
stimulated THP-1, and prevented the cell adhesion to endothelial cells treated with
Ang II, but there were no additional inhibitive effects of tempol when combined
with azelnidipine or amlodipine. Conclusion: These results suggested that
azelnidipine or amlodipine might inhibit cell adhesion by reduction of intracellular
ROS, possibly through MIP-1β and CCR5-dependent pathway.
大阪大学
岩本 義広、大石 充、多田羅 雄之、山本 浩一、塩田 敦、武田 昌生、
加藤 のぞみ、楽木 宏実
Calcium channel blocker prevents monocyte adhesion
to endothelial cells via MIP-1β and oxidative stress
system
89
17：30〜
Poster
Index
Aⴊ▤↢‛කቇળKPFD
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
183
Prox1 transcription factor plays pivotal roles during embryonic lymphatic
development by modulating the expression of various lymphatic endothelial cell
(LEC) markers such as VEGFR3. However, the molecular mechanisms by which
Prox1 transactivates its target genes remain largely unknown. Here we identified
Ets-2 as a candidate molecule that regulates the functions of Prox1 through yeast
two-hybrid screening. Ets-2 is a prototype of Ets transcription factor family and has
been implicated in angiogenesis. However, its roles during lymphangiogenesis (LA)
have not yet been elucidated. Expression of Ets-2 was observed in both embryonic
and adult lymphatic vessels as well as in blood vessels. In accordance with the
results of two-hybrid assays, we found endogenous Prox1 interacts with Ets-2 in
LEC. Ets-2 enhanced the expression of VEGFR3 in collaboration with Prox1 in
HUVEC. Enhanced expression of VEGFR3 by Ets-2 was also observed in LEC.
Additionally, Ets-2 enhanced Prox1-induced chemotaxes of HUVEC toward
VEGF-C, ligands for VEGFR3. Furthermore, ChIP assays demonstrated
endogenous Prox1 and Ets-2 bound to the VEGFR3 promoter. We next examined
the effects of Ets-2 on inflammatory LA in murine diaphragms. Intraperitoneal
administration of adenovirus encoding Ets-2 enhanced LA whereas expression of
dominant-negative Ets mutant (TM-Ets-1) suppressed it. We also examined the
possible involvement of other Ets family members in LA. IP assays showed Prox1
was able to bind other Ets family members. Inhibiting the activities of Ets family
members by TM-Ets-1 in HUVEC suppressed Prox1-induced expression of
multiple pro-lymphangiogenic factors including VEGFR3 and integrin 㱍9. These
findings suggest Ets family members function as transcriptional co-factors that
enhance Prox1-induced LA.
Symposium
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
＜目的＞腫瘍の増殖のためには酸素や栄養分の供給が不可欠であり、増殖
の現場では血管の新生や退縮が盛んに行われている。その腫瘍血管の新生
パターンを把握することは、腫瘍血管を制御するという治療概念に結びつ
く。今回われわれは、
小腸上皮の悪性化に伴い、
血管やリンパ管の新生パター
ンが どの様 に変化するのかを検索した。＜ 材料 と方 法＞12週齢、雄の
APC Min/+マウスに、高脂肪食や硫酸デキストランナトリウムを摂食させ、腸
に腺腫、腺癌を誘導させた。経時的にトマトレクチンを静注し、灌流固定
を行った。各種マーカーによる蛍光免疫染色を重ねて、共焦点レーザー顕
微鏡で観察し、ヒト手術標本と比較した。また、電子顕微鏡による超微形
態学的観察も行った。さらに、ウェスタンブロットなどを行い腫瘍内での
タンパク発現の変化を観察した。＜結果と考察＞小腸上皮が腫瘍化してい
く段階で、良性腫瘍（腺腫）においては、新生血管は著しく形態的変化を
認め、密度を増し、基底膜の不安定化が起こっていた。微細構造では、絨
毛先端の血管の内腔表面に微絨毛様の突起が出現するなどの変化が認めら
れた。また、血管特有のタンパクレベルの変化も見られた。さらに、上皮
が悪性化するにつれて、新生血管は密度をさらに増し、形態的変化が著し
く異型性を伴った。特に走行が無秩序になり、基底膜は多層化し、内腔側
が不整形を呈した。また、新生したリンパ管は大小不同であった。これら
の結果より、上皮の悪性化に伴い、腫瘍血管やリンパ管の新生パターンにも、
段階的な変化が現れることが示唆された。このように、腫瘍の悪性化を局
所の微小循環系の変化との関連でとらえることができれば、臨床診断の上
でも重要な指標となり、新たな治療戦略の開発につながるかもしれない。
1
東京大学大学院医学系研究科分子病理学、2東京大学先端科学技術研究センターシ
ステム生物医学
吉松 康裕1、伊藤 太智1、三平 元1、結城 圭子1、原田 香織1、森川 真大1、
山崎 智子1、岩田 要1、森下 保幸1、南 敬2、児玉 龍彦2、宮園 浩平1、
渡部 徹郎1
東京女子医科大学医学部解剖学・発生生物学講座、2東北大学加齢医学研究所腫瘍
循環研究分野
1
北原 秀治1、森川 俊一1、佐藤 靖史2、江崎 太一1
How to write
Plenary Lecture
Nature Medicine
Ets family members induce lymphangiogenesis via
physical and functional interaction with Prox1
Special Talk
消化管腫瘍における微小循環系の変化は上皮の悪性化と
相関するか？
Special
Lecture
93
Interaction between tumor endothelial cells (TECs) and tumor cells plays a key role
in the early stage of hematogenous metastasis. TECs provide the principal route by
which tumor cells exit the primary tumor site and enter the circulation. We have
reported TECs are different from normal endothelial cells (NECs) in various
aspects, such as chromosomal abnormality and gene expression profiles. In this
study, we isolated two types of TECs from different human tumor xenografts in
nude mice to analyze interaction between TECs and tumor cells. One is HMTEC
isolated from highly metastatic tumor and the other is LMTEC from low metastatic
tumor. HMTEC expressed higher levels of mRNA of angiogenesis-related genes
than LMTEC or NEC. We hypothesized TECs may promote metastasis, in
particular, intravasation at the primary site. We investigated the roles of TECs in
tumor metastasis; 1) migration of tumor cells towards TECs, 2) adhesion to
endothelial layer, 3) crossing the endothelium. Tumor cells migrated towards
conditioned-media (CM) from HMTEC more than LMTEC-CM or NEC-CM in
vitro. Tumor cells were more adhesive to HMTEC than to LMTEC. They migrated
through the HMTEC-monolayer most among all ECs in transendothelial assay.
These results suggested that HMTEC may help tumor cells to metastasize. Finally,
we analyzed the effects of CM from highly metastatic tumor cells on NECs. Tumor
CM induced mRNA expressions of several genes, such as cytokine and extracellular
matrix in NEC. Tumor cells adhered more to the tumor CM- treated NEC than to
non-treated NEC. These results suggest that TECs “educated” in tumor
microenvironment may be collaborating with tumor cells for metastasis.
1
北海道大学大学院歯学研究科血管生物学、2北海道大学大学院歯学研究科高齢者歯
科学、3北海道大学大学院医学研究科腫瘍外科学、4北海道大学大学院医学研究科病
態医科学、5北海道大学大学院歯学研究科口腔病理学
間石 奈湖1,2、大賀 則孝1、樋田 泰浩3、大場 雄介4、秋山 廣輔1、北山 和子1、
近藤 美弥子1、川本 泰輔1、大澤 崇宏1、山本 和幸1、井上 農夫男2、
進藤 正信5、樋田 京子1
Analysis of interaction between tumor endothelial
cells and tumor cells
91
President
Lecture
92
Angiogenesis is essential for the growth, progression, and metastasis of solid
tumors, in addition to various angiogenic diseases. Therefore, angiogenesis
inhibitors have been focused as agents having a potential therapeutic value.
Extracellular proteases have been identified to be involved in angiogenesis,
however, the participation in this process of intracellular proteases has not been
clarified. One of intracellular cysteine proteases, calpain, recently has been shown
to activate NF-㱖B through degradation of its endogenous inhibitor, I㱖B. We
found that the treatment of calpain inhibitor resulted in increase of I㱖B㱍 in
vascular endothelial cells and inhibited angiogenesis. This result demonstrates that
calpain degrades I㱖B in endothelial cells and contributes to
angiogenesis. Calpain degrades I㱖B phosphorylated by casein kinase (CK) 2
which shows elevated activity in human cancer, but not by I㱖B kinase (IKK)
which is a key in one pathway of NF-㱖B activation. In this experiment, we
concentrated our interest on the function of CK2 in angiogenesis to analyze the
involvement of another pathway of NF-㱖B activation. We examined using CK2
specific inhibitors on whether the suppression of NF-㱖B activation by blocking
CK2 causes anti-angiogenesis. CK2 inhibitor suppressed tube formation of bovine
pulmonary microvascular endothelial cells cultured on matrigel. Moreover, CK2
inhibitor reduced VEGF and bFGF induced-angiogenesis in implantation of gelling
basement membrane into mouse dorsal pocket. Additionally, we have found a result
that control of polyamine which activates CK2, regulates angiogenesis. These
results suggest a possibility that the suppression of NF-㱖B activation by blocking
CK2 inhibits angiogenesis.
1
（財）東京都医学研究機構東京都臨床医学総合研究所がん治療研究室、2東京都健
康安全研究センター・疫学情報室
芦野 洋美1、島村 眞里子2
Regulation of angiogenesis by CK2 inhibitor
90
ポスターセッション：血管細胞の転写調節
Poster Session：Transcriptional Regulation
12月2日
（木）
／Thursday, December 2
Program
広島国際大学薬学部薬学科生化学教室
Aⴊ▤↢‛කቇળKPFD
184
TGF-㱎シグナルは、血管内皮細胞上のI型受容体を介して血管新生を正負両
面から調節している。これまでにTGF-㱎シグナルの細胞内シグナル伝達分
子Smad2, Smad3を血管内皮細胞特異的に欠損したマウスの解析から、TGF㱎シグナルが血管新生抑制に関与しているシグナルではなく、血管の成熟
過程に必須のシグナル系であることを明らかにしてきた。しかしながら、
Smad2/3はTGF-㱎シグナル以外にアクチビンやNodalシグナル伝達の細胞内
シグナル伝達分子であるため、これまでに観察された表現系が、TGF-㱎シ
グナル欠損によるものであることを検証する必要があった。さらに、静脈
に由来するリンパ管形成におけるTGF-㱎シグナル系の役割に関して、生体
における検証は不十分であった。そこで、血管内皮細胞特異的またはリン
パ管内皮細胞特異的にTGF-㱎シグナルを欠損したマウスを作製し、血管・
リンパ管新生について検証を行った。
TGF-㱎I型及びII型受容体を血管内皮細胞特異的に欠損させたところ
（ALK5fl/fl; Tie2-Cre, T㱎RIIfl/fl; Tie2-Cre）、血管内皮細胞特異的Smad2/3欠損マ
ウス(Smad2fl/fl; Smad3-/-; Tie2-Cre(CDKO))マウスと同様に全身から出血して
胎生致死となり、CDKOマウスで得られた表現系は確かにTGF-㱎シグナル
欠損に依存することが示された。さらに非常に面白いことに、ALK5fl/fl;
Tie2-Cre, TβRIfl/fl; Tie2-Creマウス胚は、CDKOマウスよりも約半日遅い胎
生12〜14日で致死となり、Smad2/3の代償シグナルの存在が示唆された。
また、TGF-㱎I型及びII型受容体を、リンパ管内皮細胞特異的に欠損させ
たところ（ALK5fl/fl; Prox1-CreERTM, T㱎RIIF/F; Prox1-CreERTM）、リンパ管
形成は阻害されていなかったが、胎生14.5日に浮腫が確認できた。そこで
リンパ管内皮細胞のマーカーであるLYVE1で染色したところ、血管でみら
れた異常と同様な未成熟なリンパ管が形成されていることがわかったこれ
らの結果より、TGF-㱎シグナルは血管形成維持だけでなくリンパ管形成維
持にも必須のシグナルである可能性が示された。
遺伝子の発現調節領域の
血球細胞と血管内皮細胞は、ヘマンジオブラストと呼ばれる幹細胞から分
化することが知られているが、分化の方向性をどのようにして決定してい
るのかは分かっていない。これまでに、アフリカツメガエル胚の未分化領
域の細胞に対して低濃度のアクチビンとアンジオポエチン２を作用させる
ことにより、血管内皮細胞に分化させることに成功している。この系をも
ちいてDNAマイクロアレイによるディファレンシャルスクリーニングおよ
びツメガエル胚を用いたwhole-mount in situ hybridizationを行った結果、血管
内皮細胞で発現が見られる新規遺伝子xrasgrp2を同定した。現在までに、こ
の遺伝子の過剰発現およびモルフォリノオリゴによる発現抑制実験から、
XRASGRP2は血管形成に重要な役割を果たしていることが分かってきてい
る。今回、ヒト血管内皮細胞におけるrasgrp2遺伝子の発現調節領域の解析
を行った。ヒトrasgrp2遺伝子の第1エキソンには、３種類のオルタナティ
ブスプライシングされるエキソンが存在しているが、血管内皮細胞ではこ
れらのうち1種類 (D1E) しか使われていないことがRT-PCR解析により判明
した。そこで次に、ルシフェラーゼ遺伝子とヒトrasgrp2遺伝子の第1エキソ
ン (D1E) の5ʼ上流領域の配列を融合させたコンストラクトを構築し、ルシ
フェラーゼアッセイによる転写活性部位の同定を行なった。この結果、ヒ
ト血管内皮細胞におけるrasgrp2遺伝子の発現調節は、転写活性領域と転写
抑制領域の両方によって行われていることが示唆された。さらに、抗体を
用いたゲルシフトアッセイにより、転写抑制領域に結合する転写因子が
OCT1/POU2F1であることを明らかにした。一方、転写活性領域にはSP1結
合配列が有り、その配列に結合する転写因子の存在を確認することができ
た。今回の結果から、ヒト血管内皮細胞におけるrasgrp2遺伝子の発現は、
転写の活性と抑制のバランスにより調節されていることが示唆された。
長嶺 憲太郎、松田 明、堀 隆光
1
生化学研究室
伊東 史子1、伊東 進1,2
筑波大学大学院人間総合科学研究科実験病理学、2昭和薬科大学
血管内皮細胞における
解析
95
血管・リンパ管の安定性維持におけるTGF-βシグナルの
役割
94
17：30〜
Index
185
Poster
The 8th Korea-Japan Joint Symposium on Vascular Biology
Selected Oral
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Y.I.A.
Presentation
Aⴊ▤↢‛කቇળKPFD
生体機能薬理学
[目的]アンギオテンシンII受容体遮断薬(ARB)であるテルミサルタンはAT1
受 容 体 を 阻 害 す る だ け で な く、 選 択 的Peroxisome Proliferator-activated
Receptor-㱏(PPAR㱏)作用を有することが報告されている。しかし、テルミ
サルタンのもつ選択的PPAR㱏作用の糖尿病性血管障害における意義につい
ては不明である。我々はテルミサルタンのもつ選択的PPAR㱏作用を介した
血管保護の意義について検討を行った。[方法]2型糖尿病モデルマウス(db/
dbマウス)を、(1)Vehicle群、(2)テルミサルタン単独群(10mg/kg/day)、(3)テ
ル ミ サ ル タ ン(10mg/kg/day)+GW9662 (PPAR㱏 antagonist, 10mg/kg/day)併 用
群、(4)GW9662単独群(10mg/kg/day)、(5)ロサルタン単独群(10mg/kg/day)に
分け、各薬剤を8週間投与し、血管の内皮機能、血管リモデリング、炎症(TNF
㱍)、eNOS、NF㱖B活性化(リン酸化I㱖B㱍抗体を用いた蛍光免疫染色法に
よる定量化)等に対する効果について比較検討を行った。[成績]非糖尿病の
db/mマウス(コントロール)と比べて、db/dbマウスは内皮依存性血管弛緩反
応の障害、冠動脈壁肥厚、冠動脈周囲のマクロファージ浸潤と線維化が著
明であった。テルミサルタンはdb/dbマウスの上述の血管障害を有意に改善
し、その効果はロサルタンよりも強力であった。コントロールマウスと比
べてdb/dbマウスの血管eNOSリン酸化は著明に低下していたが、テルミサ
ルタンはロサルタンよりもリン酸化を有意に改善した。さらに、コントロー
ルマウスと比べてdb/dbマウスでは大動脈と冠動脈内皮・中膜でNF㱖B活性
およびTNF㱍発現が著明に増加していたが、テルミサルタンはいずれも有
意に抑制したのに対して、ロサルタンは抑制しなかった。上記のテルミサ
ルタンによる効果はいずれもPPAR㱏 antagonistの同時投与により抑制され
た。[結論](1)テルミサルタンは2型糖尿病の血管内皮機能障害、冠動脈リモ
デリング、炎症をロサルタンと比べて有意に改善した。(2)テルミサルタン
の血管保護作用には、選択的PPAR㱏作用を介するeNOS活性化とNF㱖B活
性化抑制が関与している。(3)PPAR㱏作用をもつARBは、糖尿病性血管障害
に対する有用な治療戦略と考えられる。
生命科学研究部
Symposium
AMP-activated protein kinase (AMPK) is an energy sensing kinase activated in
response to various cellular stresses inducing deprivation of AMP. Recently AMPK
is also implicated in key cellular pathways, including polarity establishment and
cell division. This suggests that in addition being a key regulator of physiological
energy dynamics, AMPK affects basic cellular functions by sensing various energy
consuming stresses, however downstream target of these functions is still unclear.
Here we identified microtubule plus end protein CLIP-170 as a novel AMPK
substrate by de novo screening method and showed the important role of
phosphorylated CLIP-170 in cell polarity and migration. Phosphorylation status of
CLIP-170 affected the dynamism of microtubule polymerization. Phosphorylated
CLIP-170 located at the more distal end of microtubules and had faster tuned over
rate on microtubules than un-phosphorylated CLIP-170. Inhibition of AMPK and
transfection of CLIP-170 non-phosphorylated dominant negative mutant reduced
the speed of tubulin polymerization with large amount of unphosphorylated CLIP170 left over on microtubule by in vivo molecular imaging. Reduced speed of
tubulin polymerization impaired establishment of cell polarity and inhibited
directional migration of cells. These results demonstrate that AMPK directly
regulates microtubule dynamics and structure by phosphorylation of CLIP-170. We
anticipate that this finding leads to the better understanding the effect of energy
status to microtubule mediated cell functions such as vesicle transport, cell division,
and establishment of cell polarity.
熊本大学大学院
外山 研介、中村 太志、片岡 恵一郎、光山 勝慶
高島 成二1、中野 敦1、望月 直樹3、北風 政史2
1
大阪大学大学院医学系研究科分子心血管医学、2国立循環器病研究センター心臓血
管内科、3国立循環器病研究センター研究所 細胞生物学部
99
How to write
Plenary Lecture
Nature Medicine
テルミサルタン有する選択的Peroxisome Proliferatoractivated Receptor-γ作用の糖尿病性血管障害における
役割
Special Talk
Phosphorylation of CLIP-170 by AMPK regulates
microtubule polymerization and cell polarity
Special
Lecture
98
【目的】近年の疫学研究から高血圧や糖尿病などの血管性危険因子がアルツ
ハイマー病（AD）の独立した危険因子でもあることが明らかにされ注目を
浴びている。一方で、βアミロイド蛋白（Aβ）を標的としたADの根本的
治療法の開発に伴い、ADを早期診断するためのバイオマーカーの必要性が
高まっている。血中Aβ値は非侵襲的に評価可能なマーカーとして期待さ
れているが、その臨床的有用性・妥当性は確立されていない。我々は特に
糖尿病病態とアルツハイマー病との病態関連の解析から、全身のグルコー
ス代謝がAβの血中濃度に影響を与えている可能性を見出し、この現象の
アルツハイマー病診断指標としての応用について検討を行った。【方法】2
種のADモデルマウス（APP23、APP/PS1マウス）を使用し、空腹・随時・
糖負荷（2g/kg i.p.）後の血中Aβ値をELISAにて測定した。またヒトAD患
者及び非AD認知症患者を対象とし、経口ブドウ糖負荷試験（75gOGTT:Oral
Glucose Tolerance Test）を行い血中Aβ量の変動を評価した。【結果】APP/
PS1Tgでは随時採血の血中Aβ40及びAβ42値は空腹時より有意に高値を示
し、食事・血糖状態が血中Aβ値に影響を与えることが示唆された。また
糖負荷後にTgマウスでは血中Aβ値の急速かつ有意な上昇が観察され、こ
の変化は若齢マウスに比し高齢マウスで有意に大きかった。野生型マウス
ではこのような増加は見られなかった。APP23マウスでも同様に血中Aβの
増加が観察された。ヒトAD患者では、空腹時採血における血中Aβ値（A
β40、42、42/40比）は非AD患者と有意な差は無かったが、75gOGTT後急
性期（〜30分値）にAβ42の有意な上昇を認めた。【考察】ADマウスを用
いた解析から、糖負荷後の血中Aβ値の増加量が診断及び病態重症度のバ
イオマーカーになり得る可能性が示唆された。AD患者では糖負荷後に血中
Aβ値が特異な変動を示すことが明らかとなり、非侵襲的な診断的指標と
して応用できる可能性が示唆された。
1
大阪大学大学院医学系研究科臨床遺伝子治療学、2大阪大学大学院医学系研究科老
年腎臓内科、3 医薬基盤研究所生物資源研究部実験動物開発研究室、4阪和第二泉北
病院
武田 朱公1,2、里 直行1,2、内尾-山田 こずえ3、湯 久浩4、守口 篤4、
楽木 宏実2、森下 竜一1
糖代謝が血中β・アミロイド蛋白
（Aβ）
量に与える影響の
検討：アルツハイマー病の新規診断指標への応用
97
President
Lecture
【背景】2型糖尿病患者では毛細血管ならびに細動脈での血管新生能が低下
していることが知られている。この血管新生障害は冠側副血行路の発達不
良、創傷治癒の遷延、糖尿病足壊疽など種々の糖尿病合併症の発症に寄与
する。糖尿病患者では、VEGF遺伝子導入、EPC移植などの治療的血管新生
に対して抵抗性があることが知られており、その要因のひとつにVEGF抵抗
性の存在が報告されているが、その分子メカニズムは完全には理解されて
ない。我々は、ヒト肝臓発現遺伝子情報を用いて、肝由来分泌タンパクセ
レノプロテインP（以下SeP）の産生が2型糖尿病状態で亢進していること、
SePが肝および筋細胞にインスリン抵抗性を誘導することで高血糖発症に寄
与することを報告してきた (Cell Metabolism 2010 in press)。今回このSePが
血管新生へおよぼす影響を検討した。【方法・結果】(1)マウス傍大動脈臓
側板中胚葉(P-Sp)培養系において、精製SePタンパク処置は血管内皮細胞の
発生を有意に抑制した。(2)精製SePタンパク処置は、ヒト臍帯静脈血管内
皮細胞（以下HUVEC）においてVEGF依存性の細胞増殖、遊走、管腔形成
を そ れ ぞ れ-17%、-26%、-25%と 有 意 に 抑 制 し た。 (3)SePを 前 処 置 し た
HUVECでは、VEGF刺激によるVEGFR2およびERK1/2のリン酸化が有意に
低下した。(4)C57B6Jマウスに、SePタンパク含有マトリゲルを皮下移植し
たところ、10日後の新生血管内皮細胞数は-84%と著明に低下した。(5)プラ
スミド尾静注による肝選択的SePタンパク過剰発現マウスは、皮膚潰瘍の閉
鎖速度が-18%と有意に低下した。(6)SePヘテロノックアウトマウスは、下
肢虚血術からの血流回復が、正常マウスに比し有意に早期であった。【考察】
本研究は、肝由来分泌タンパクSePの過剰産生が、血管内皮細胞にVEGF抵
抗性を誘導することで、2型糖尿病患者における血管新生障害発症の原因と
なることを示唆する。肝臓は分泌タンパクSePの産生を介して全身での血管
新生ネットワークに参加している可能性がある。各種成長因子の導入によ
る既存の治療的血管新生に対して反応性の乏しい2型糖尿病患者において
は、肝でのSeP産生抑制によるVEGF抵抗性の軽減が有効な治療となる可能
性がある。
金沢大学大学院医学系研究科環境医科学専攻恒常性制御学、2金沢大学大学院医学
系研究科循環医科学専攻血管分子生理学、3大阪大学大学院医学系研究科微生物研
究所情報伝達分野
1
石倉 和秀1、御簾 博文1、熊崎 雅史1、岡本 安雄2、多久和 陽2、木戸屋 浩康3、
山田 賢裕3、高倉 伸幸3、金子 周一1、篁 俊成1
肝由来分泌タンパクセレノプロテインPはVEGF抵抗性の
誘導を介して２型糖尿病における血管新生障害の発症に
寄与する
96
ポスターセッション：メタボ・糖尿病
（2）
Poster Session：Metabolic syndrome / Diabetes(2)
12月2日
（木）
／Thursday, December 2
Program
Aⴊ▤↢‛කቇળKPFD
186
背景：イコサペント酸エチルはイコサペンタエン酸(EPA)/アラキドン酸(AA)
比を改善することが報告されているが、EPA/AAと血管内皮機能改善効果と
の関連性については充分に検討されていない。目的：イコサペント酸エチ
ル内服によるEPA/AA比改善と血管内皮機能改善効果との関連性につき検討
すること。方法：高トリグリセライド血症例連続15例（平均年齢60.7±
13.2、男性7名）を対象とし、EPA/AA比を含む各種血液学的検査、血管機
能測定専用超音波装置（ユネクスEF）による前腕駆血法による血管内皮依
存性血管拡張反応評価を、イコサペント酸エチル投与前と投与後6ヵ月後に
施行した。前腕駆血法による血管内皮依存性血管拡張反応評価は、前腕動
脈駆血前上腕動脈血管径と一定時間駆血後開放し血流増加に伴い拡張した
上腕動脈血管径を計測し、血管径変化率（%FMD）を算出し、施行した。
結果：イコサペント酸エチル投与前EPA/AA比は、0.30±0.21であり、イコ
サペント酸エチル投与6ヵ月後のEPA/AA比は0.89±0.45であった。イコサペ
ント酸エチル投与前トリグリセライド値は252.7±90.4 mg/dl、投与6ヵ月後
トリグリセライド値は164.7±51.2 mg/dlであった。イコサペント酸エチル投
与前後のEPA/AA改善とイコサペント酸エチル投与前後%FMD改善の間には
正 の 相 関 を 認 め（r=0.525, p＜0.05）。 一 方、 ト リ グ リ セ ラ イ ド 値 改 善 と
%FMD改善の間には、相関を認めなかった。結語：イコサペント酸エチル
によるトリグリセライド値改善効果よりも、EPA/AA比改善効果が、血管内
皮機能改善効果により関連することが示唆された。
大阪市立大学大学院医学研究科循環器病態内科学、2西宮渡辺病院、3兵庫医療大学、
4
浅香山病院
【背景・目的】糖尿病合併症としての細小血管症の発症・進展には血管内皮
障害が基盤病態を形成している。微量アルブミン尿は糖尿病性腎症の最早
期病態であり、臨床的観察事実からアルブミン尿は内皮機能障害と関連す
ると想定されている。しかしながら糸球体内皮細胞は有窓性であり、アル
ブミンの透過性制御への関与は疑問視されていた。我々は、これまでに各
種糖尿病モデルラットの腎糸球体において内皮機能障害がアルブミン尿と
連関することを示してきた。高血糖によるNAD(P)H oxidase 活性化を起点
として、血管内皮細胞の endothelial nitric oxide (NO) synthase (eNOS) の補酵
素 で あ るtetrahydrobiopterin (BH4)が 酸 化 的 に 減 少 し、eNOS 機 能 異 常
（uncoupling）が生じる。以上より「血管内皮細胞内BH4の維持はeNOS recouplingを促進し、NO bioavailavilityを維持を介して糖尿病性腎症の発症・
進 展 を 抑 制 す る 」 と の 仮 説 を 立 て、BH4生 合 成 の 律 速 酵 素 で あ るGTPcyclohydrolase-I (GCH)を内皮特異的に高発現させたマウスを利用してこの
仮説を検証した。【方法】1型糖尿病モデルであるAKITAマウス (AKITA) に
内皮特異的GCH高発現マウス (Tg-GCH) を交配させた。野生型マウス (WT;
C57/BL6)、AKITA、Tg-GCH/AKITA の3群を作成し、20週まで飼育し、経時
的に尿中アルブミン排泄量を計測した後、20週後に腎組織を採取し検討し
た。【結果】血糖値はAKITA、Tg-GCH/AKITAで有意な上昇を認めたが、両
群間では有意差はなかった。15週時にはAKITA群では有意な尿中アルブミ
ン排泄増加を認めたが、Tg-GCH/AKITAで抑制効果を認めた。腎組織での
活性酸素種 (ROS）及びNOを検出するため、共焦点レーザー顕微鏡を用い
て可視化した。AKITA群では著明なROS増加とNO低下を認めたが、TgGCH/AKITAではこの不均衡は是正されていた。
【結語】糖尿病におけるア
ルブミン尿の発症には、BH4の減少及びeNOS uncouplingによる糸球体内皮
障害が関連しており、GCH-I過剰発現による内皮BH4レベルの増加はeNOS
uncouplingを是正しアルブミン尿を減少させることが示された。GCH-I活性
増加、BH4維持により糖尿病性腎症の発症・進展が抑制されることが示唆
された。
川崎医科大学腎臓・高血圧内科学
城所 研吾、佐藤 稔、長洲 一、冨田 奈留也、柏原 直樹
竹本 恭彦1、島田 健永1、井口 朋和1、中西 弘毅1、松本 健嗣1、花谷 彰久1、
中村 泰浩1、室生 卓1、吉川 純一2、東 純一3、田原 旭4、葭山 稔1
1
内皮GTP-cyclohydrolase-I過剰発現は糖尿病性腎症の発
症・進展を抑制する
101
イコサペント酸エチルによるEPA/AA比改善と血管内皮
機能改善効果との関連性
100
17：30〜
Index
Aⴊ▤↢‛කቇળKPFD
Poster
187
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Symposium
【背景】GLP-1は消化管から分泌されるホルモンであるインクレチンの１つ
で、インスリン分泌促進・グルカゴン分泌抑制などの作用がある。一方で
GLP-1受容体アゴニストの糖代謝改善以外の作用として、接着因子発現抑
制による動脈硬化病変減少という報告もされているが、そのメカニズムは
明らかでない。そこで、Wire Injury(WI)によるマウス大腿動脈傷害モデルを
用いて、GLP-1受容体アゴニストであるExendin-4(Ex-4)の血管リモデリング
への作用を明らかにするため、以下の実験を行った。
【方法】10週齢の野生型マウスを使用し、WIのみを行うcontrol群、WIを行
いOsmotic pumpでPBSを投与するPBS群、WIを行いpumpでEx-4(24nmol/kg/
day)を投与するEx-4群の3群に分け、それぞれに通常食(ND)と高脂肪食(HFD)
を与えて計6群で行った(各群n=5)。4週間経過を観察し、体重・血糖値、脂
質プロファイル、IPGTT後の血糖値・血清インスリン濃度、大腿動脈の新
生内膜増殖面積の測定を行った。また、mouse macrophage（RAW264.7cell）
にEx-4を添加した後LPSで刺激し、TNF-αとIL-6のmRNA発現量をリアルタ
イムPCR法を用いて測定した。 また、rat smooth muscle cell(SMC)を用いて
MTT assayを行った。
【結果】4週間後、HFDで血清脂質は上昇したがEx-4投与では低下しなかった。
IPGTTを行った結果、両食事群ともEx-4投与で血糖上昇は有意に抑えられ
たが血清インスリン濃度に差はなかった。新生内膜/中膜比の計測において、
ND・control群：3.3±0.1、ND・PBS群：3.4±0.8、ND・Ex-4群：0.9±0.1で
あり、Ex-4による新生内膜増殖抑制が認められ(p＜0.01)、HFDでも同様の
結果であった。RAW cellをLPSで刺激すると、TNF-αやIL-6の発現量が有
意に増加したが、Ex-4添加により濃度依存的に発現が抑制され、MTT assay
におけるSMCの増殖も、Ex-4添加により濃度依存的に抑制された。
【結論】以上からEx-4には、抗炎症・平滑筋細胞増殖抑制効果があると考え
ら れ、 そ れ がWI後 の 新 生 内 膜 増 殖 を 抑 制 し た と 考 え ら れ た。GLP-1R
agonistにはインスリン分泌促進作用とは独立した血管リモデリング抑制作
用を持つ可能性が示唆された。
(背景)ＡＣＥ２はアンジオテンシンＩＩ(ＡＩＩ)を分解しアンジオテンシン
１−７を産生することによりAIIシグナルを抑制する方向に働くことが明ら
かになってきている。我々はこれまでＡＣＥ２欠損マウスを用いて圧負荷
によりＡＣＥ２が心保護的に働くことや、糖尿病マウスにおいてＡＣＥ２
が腎保護的に働くことを報告してきた。一方、ＡＩＩをマウスに長期投与
するとインスリン抵抗性が悪化することや、アンジオテンシン(１−７)の
トランスジェニックラットでインスリン抵抗性が改善するとの報告がなさ
れておりACE2はインスリン抵抗性に影響を与えることが示唆される。今回
我々はＡＣＥ２欠損マウスを用いてACE2欠損がインスリン抵抗性に与える
影響を検討した。 (方法)普通食で飼育した１２週齢のオスのＡＣＥ２欠損
マウスとコントロールマウス(Ｃ５７ＢＬ/６Ｊ)に対して腹腔内糖負荷試験
(ipGTT),腹腔内インスリン負荷試験(ipITT)を施行しインスリン抵抗性を評
価した。また普通食で飼育した10週齢の各マウスにＡＩＩ(１００ｎｇ/ｍｉ
ｎ/ｋｇ)を浸透圧ポンプにより投与した２週間後にipGTT, ipITTを施行した。
また8週齢の各マウスに高脂肪高ショ糖食負荷を施行した2週間後から、７％
生理食塩水、オルメサルタン(3mg/kg/day)を浸透圧ポンプで2週間投与し
ipGTT, ipITTを施行した。(結果)普通食での飼育下でipGTTとipITTの結果に
はACE2欠損マウスとコントロールマウス間で差を認めなかった。普通食下
でAIIを投与すると各マウスともipGTT, ipITTでインスリン抵抗性の上昇を
認めたがACE2欠損マウスでより顕著であった。また高脂肪高ショ糖食負荷
により各マウスのインスリン抵抗性は上昇したがACE2欠損マウスでより顕
著であった。オルメサルタンを投与すると高脂肪高ショ糖食負荷下での
ACE2欠損マウスとコントロールマウス間のインスリン抵抗性の差は消失し
た。結語）ACE2欠損は高脂肪ショ糖負荷によるインスリン抵抗性の上昇を
増悪させた。ACE2欠損によるAIIシグナルの上昇が機序として考えられた。
大阪大学医学部老年.腎臓内科学
1
徳島大学医学部栄養学科、2徳島大学医学部医学科、3徳島大学大学院ヘルスバイオ
サイエンス研究部循環器内科学分野、4徳島大学大学院ヘルスバイオサイエンス研
究部心臓血管外科学分野
武田 昌生、山本 浩一、大石 充、神出 計、竹屋 泰、多田羅 雄之、塩田 敦、
小黒 亮輔、岩本 義広、楽木 宏実
西雄 千佳1、東田 真由子1、松岡 祐貴2、平田 陽一郎3、黒部 裕嗣4、中屋 豊1、
北川 哲也4、佐田 政隆3
How to write
Plenary Lecture
Nature Medicine
インスリン抵抗性に与えるアンジオテンシン変換酵素２
(ＡＣＥ２)欠損の影響に関する検討
Special Talk
GLP-1 receptor agonistの血管リモデリングに与える影
響の検討
Special
Lecture
105
高血圧および蛋白尿を伴う2型糖尿病での糖尿病性腎症患者において，テル
ミサルタンはロサルタンと比較し，降圧効果はほぼ同等だが，蛋白尿の減
少に関しては優れ、強い腎保護作用を有することが、大規模臨床試験
(AMADEO試験)により示されている。一方でテルミサルタンはアンジオテ
ンシン1型受容体(AT1R)阻害とは独立した機序でPPARγ活性化作用を有す
ることが示されており、PPARγアゴニストの腎線維化抑制作用の下流のエ
フェクターとして肝細胞増殖因子(HGF)が発現することが明らかになって
いる。ロサルタンと比較しテルミサルタンの腎保護作用が強い機序として
テルミサルタンのAT1-R阻害作用とは独立した作用であるPPARγアゴニス
ト作用により発現するHGFの抗線維化作用，抗炎症作用が存在するという
仮説を示すために以下の実験を行った。8週齢のAT1R欠損マウスに対し、
各薬剤(テルミサルタン、ロサルタン)投与を開始した1週間後に片則尿管結
札術を施行し、2週間後に腎臓の線維化、糸球体障害、炎症細胞の浸潤、組
織の上皮間葉転換(EMT)の評価を行った。テルミサルタン投与群では、ロ
サルタン投与群、および薬剤投与しないコントロール群と比較し、腎組織
の線維化部分(マッソントリクローム染色陽性部分)が有意に抑制されてい
た。また、免疫染色によるF4/80陽性部分、α-SMA陽性部分、TGF-β1陽性
部分もテルミサルタン投与群では減少しており、組織におけるマクロファー
ジの浸潤、及び上皮間葉転換(EMT)が抑制されていると考えられた。また
糸球体障害スコアもテルミサルタン投与群では有意に低値となっていた。
これらのテルミサルタンによる作用は、HGF中和抗体投与およびPPARγア
ンタゴニスト(GW9662)投与により打ち消されていた。一方で、血清および
腎組織の抽出液でのHGF濃度はテルミサルタン投与群で有意に上昇してい
た。テルミサルタンは、アンジオテンシンIIタイプ1受容体阻害作用と独立
したPPARγアゴニスト作用により、腎組織で肝細胞増殖因子（HGF)を介
して糸球体障害や腎線維化を抑制する作用を有している。また、このよう
な作用はロサルタンでは弱く、AMADEO試験での両薬剤の腎保護作用の差
異に影響を与えたと思われた。
President
Lecture
104
Objective ApolipoproteinCIII (apoCIII) in TG-rich lipoproteins is elevated in
patients with obesity, insulin resistance, or metabolic syndrome. Its level is also
associated with proinflammatory adipokines. We examined the direct effect of
apoCIII on adipokine expressions that are involved in these morbid conditions.
Methods and Results Fully differentiated mouse 3T3-L1 adipocytes were
incubated with apoCIII. ApoCIII activated NF-κB of 3T3-L1 adipocytes, and
induced the expression of MCP-1 and IL-6. ApoCIII also activated ERK and p38.
MEK1 inhibitor PD98059, but not p38 inhibitor SB203580, inhibited apoCIIIinduced up-regulation of MCP-1 and IL-6. We previously reported that apoCIII
activates proinflammatory signals through toll-like receptor 2 (TLR2). TLR2
blocking antibody abolished activation of NF-κB and ERK induced by apoCIII,
and inhibited apoCIII-induced up-regulation of MCP-1 and IL-6. ApoCIII also
reduced adiponectin expression of 3T3-L1 adipocytes, which was recovered by
TLR2 blocking antibody. ApoCIII induced the expression of MCP-1 and IL-6 in
TLR2-overexpressed HEK293 cells, but not wild-type HEK293 cells without
TLR2. ApoCIII induced the expression of MCP-1 and IL-6, and decreased
adiponectin expression in white adipose tissue of wild-type mice but not of TLR2deficient mice in vivo. Conclusion Our results suggest that apoCIII activates ERK
and NF-kB through TLR2, and induces proinflammatory adipokine expression in
vitro and in vivo. Thus, apoCIII links dyslipidemia to inflammation in adipocytes,
which in turn may contribute to atherosclerosis.
1
大阪大学大学院医学系研究科臨床遺伝子治療学、2大阪大学大学院医学系研究科老
年腎臓内科学
楠 博1,2、谷山 義明1,2、家串 和真1,2、真田 文博1、東 純哉1,2、岡山 慶太1,2、
久徳 真梨子1、岩林 正明1、アマルナス チャタジー1、楽木 宏実2、
森下 竜一1
東京医科歯科大学血流制御内科学、2東京医科歯科大学先進倫理医科学開発学
川上 明夫1、下門 顕太郎1、吉田 雅幸2
1
肝細胞増殖因子(HGF)はPPARγアゴニスト作用を持つ
ARBの腎保護作用のエフェクター分子として発現する
103
ApoCIII induces MCP-1 and IL-6 expression via TLR2
pathway in mouse adipocytes
102
ポスターセッション：メタボ・糖尿病
（3）
Poster Session：Metabolic syndrome / Diabetes(3)
12月2日
（木）
／Thursday, December 2
Program
循環病態
Aⴊ▤↢‛කቇળKPFD
188
The phenotypic change of smooth muscle cells (SMC) is critical in the pathogenesis
of atherosclerotic lesions. We cloned Mrf-2/ARID5B gene, a member of the AT-rich
interaction domain family of transcription factors, as a key regulator for the
phenotypic change of SMCs. Studies on gene-targeting mice suggested that this
factor is also involved in obesity and adipogenesis. Previously, our group reported
that genetic variations of Mrf-2 are the genetic risk factors for coronary artery
disease and diabetes mellitus (DM). And we found that expression level of Mrf-2 is
correlated with plasma level of adiponectin, which is secreted from the adipose
tissue and may suppress DM and atherosclerosis. Therefore, we hypothesize that
Mrf-2 may regulate the adiponectin expression.To reveal this mechanism, we use
the 3T3-L1 adipocyte differentiation model. Mrf-2 has two isoforms,α and β, that
differ in N-terminus. The expression of Mrf-2α preceded adiponectin up-regulation
during 3T3-L1 differentiation, and overexpression of Mrf-2α using retrovirus
construct resulted in several folds increase in adiponectin expression relative to the
control. We examine how Mrf-2α up-regulates adiponectin gene transcription
using human adiponectin promoter. As a result of luciferase reporter analysis, cotransfection of Mrf-2α with PPARγ2 and RXRα increase luciferase activity. And
the promoter analysis with deletion and mutation suggests that peroxisome
proliferator response element (PPRE) is important for the up-regulation of
adiponectin expression by Mrf-2α. In conclusion, our study suggests that Mrf-2α
may activate adiponectin transcription through PPRE site. This mechanism may be
valuable for increasing serum adiponectin levels and may affect susceptibility to
DM and atherosclerosis.
東京大学 循環器内科学、2東京大学 糖尿病・代謝内科学、3長崎大学
制御内科学、4東京大学 臨床疫学システム講座
1
大関 敦子1、渡辺 昌文1、眞鍋 一郎1、王 国琴1、今井 靖1、山内 敏正2、
原 一雄2、渡邉 綾1、河原崎 秀一1、前村 浩二3、門脇 孝2、山崎 力4、
永井 良三1
Mrf-2/ARID5B expression may affect susceptibility to
atherosclerosis and DM by regulating adiponectin
expression level
106
老年・腎臓内科学
【背景・目的】骨格筋のインスリン感受性に関して、近年骨格筋内脂肪蓄積
のインスリン抵抗性への関与について注目されているが、一定の見解が得
られていない。我々はこれまでに、
アンジオテンシンII受容体阻害薬（ARB）
の中でも選択性PPAR㱏作用を有するテルミサルタンが、高脂肪高炭水化物
食負荷ラットにおいて体重増加の抑制、エネルギー消費量の増大と全身の
インスリン感受性を改善すること、その機序として骨格筋ミトコンドリア
機能への影響を介することを報告してきた。今回は同薬の筋代謝に対する
影響について、骨格筋内脂肪蓄積と筋インスリン感受性に着目して検討を
行った。
【方法】8週齢のSDラットを対象に高脂肪高炭水化物食と同時にテ
ルミサルタン（Tel、3mg/kg）あるいはバルサルタン（Val、5mg/kg）対照）
を投与し、12週間後に骨格筋における各種遺伝子発現を検討、さらに骨格
筋内中性 脂肪（mTG）濃度を、トリグリセライドEテストワコーを用いて
測定した。またSDラットより摘出したヒラメ筋を薬剤存在下で培養し、β
酸化能と糖酸化能をCO2産生量により測定した。【結果】高脂肪高炭水化物
負荷SDラットにおいて、Tel投与群では骨格筋の脂肪酸取り込みに関連する
CD36遺伝子（p＜0.05）
、糖取り込みに関連するGLUT4遺伝子発現の増加（p
＜0.05）
、またエネルギー調節因子であるPDK4遺伝子の発現の増加（p＜0.05）
を認めた。しかし、Val投与群ではこれらの遺伝子変化を認めなかった。摘
出骨格筋の培養条件下の検討では、Tel群において脂肪酸β酸化能の増加（p
＜0.01）と、糖酸化能の増加（p＜0.03）を認めた（vs. 対照群）が、Val群
では変化を認めなかった。またmTG濃度はTel群で対照群、Val群に比し低
い傾向を示した。先の研究結果をふまえ、高脂肪高炭水化物負荷時の環境
下では、選択性PPAR㱏作用性ARB（SPPARM）の一つであるテルミサルタ
ンは、ミトコンドリア機能改善とPDK4発現増加による脂肪酸酸化の促進を
介して骨格筋内脂肪を減少させ、インスリン感受性を改善することが示唆
された。
【結語】SPPARMは、骨格筋の脂肪酸酸化促進による骨格筋内脂肪
の減少により、筋インスリン感受性を改善することが示唆された。
大阪大学
杉本 研、神出 計、大石 充、楽木 宏実
選択性PPARγ作動性アンジオテンシンII受容体拮抗薬
（ARB）
の筋インスリン感受性に対する影響
107
Index
Aⴊ▤↢‛කቇળKPFD
Poster
189
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Symposium
Anti-angiogenesis is an effective cancer treatment, however, tumor progression and
refractoriness to anti-angiogenesis are often observed in both clinical and preclinical
cancer models. We found that both hypoxic and low nutrition double-deprivation
stress (DDS) regions in tumor tissue were expanded following anti-VEGF
neutralization visualized by hypoxyprobe and a modified periodic-acid Schiff
staining. We hypothesized that cancer cells reside in the double-deprivation niche is
critical for the refractoriness to anti-angiogenic therapy. In agreement with our
hypothesis, cancer cells following a long term DDS increased migration, invasion
and anchorage independent growth in vitro, and demonstrated an increased tumor
progression and angiogenesis associated with an increase in CD31+ blood vessel
and infiltration of CD11b+ cells. We found that histone demethylase JHDM2A is
commonly up-regulated under DDS in human and mouse cell lines in vitro and in
vivo such as pre-angiogenic switch and “pre-refractory” phase of anti-VEGF
antibody treatment. Specific inhibition of JHDM2A, by siRNAs significantly
suppressed in vivo tumor growth. Importantly, suppression of JHDM2A
demonstrated synergetic effect in combination with anti-VEGF antibody therapy,
and improved an efficacy of anti-angiogenesis. Our results suggest that the targeting
of cancer cells residential in the double-deprivation niche by inhibition of JHDM2A
can be utilized for a novel treatment to overcome the refractoriness to antiangiogenic therapy.
Endothelial-Mesenchymal transition (EndMT) plays important roles in various
physiological and pathological processes. We previously reported that transforming
growth factor (TGF) -㱎2 induces mesenchymal differentiation of mouse embryonic
stem cell-derived endothelial cells (Kokudo et al.,2008). Furthermore, a recent
report showed that cardiac fibrosis and carcinoma associated fibroblast (CAF)
formation are related to EndMT (Zeisberg et al.,2007). However, the molecular
mechanisms that regulate EndMT remain to be elucidated. Here we show that TGF㱎2 induces EndMT of mouse pancreatic microvascular endothelial cells (MS-1).
MS-1 cells undergo morphological changes into spindle-like shape with the reorganization of actin stress fiber by the addition of TGF-㱎2. TGF-㱎2 induces the
expression of various mesenchymal markers including smooth muscle actin (SMA)
㱍, SM22㱍, fibronectin1 and collagen1㱍1. TGF-㱎2 also results in the
disappearance of VE-cadherin protein, an endothelial marker, from the surface of
cells expressing SMA㱍. These results suggest that TGF-㱎2 is capable of inducing
mesenchymal transition of MS-1 endothelial cells.
東京大学大学院医学系研究科病因病理学専攻分子病理学講座
東京医科歯科大学 大学院医歯学総合研究科 分子腫瘍医学、2独立法人国立長寿
医療研究センター研究所 遺伝子蛋白質解析、3上武大学副学長・東京医科歯科大学
客員教授
1
三平 元、吉松 康裕、渡部 徹郎、宮園 浩平
大澤 毅1、村松 昌2、土田 里香1、王 峰1、湯浅 保仁1、澁谷 正史3
111
How to write
Plenary Lecture
Nature Medicine
TGF-beta2 induces endothelial-mesenchymal transition
of MS-1 endothelial cells
Special Talk
Histone demethylase JHDM2A is a novel target to
overcome refractoriness of tumor cells to antiangiogenic therapy
Special
Lecture
110
老年・腎臓内科学、2大阪大学大学院先端移植基盤
The tissue-protective effects of erythropoietin (EPO) have been extensively
investigated, while EPO administration can raise the hemoglobin (Hb)
concentration. Recently, we reported that carbamylated erythropoietin (CEPO)
protected kidneys from ischemia reperfusion injury as well as EPO. To investigate
the clinical applications of CEPO, we next evaluated the long-term therapeutic
effect of CEPO using a tubulointerstitial model rat. We randomized subtotally (5/6)
nephrectomized rats to receive saline, EPO, or CEPO for 8 wk. CEPO- and EPOtreated rats improved serum creatinine compared with saline-treated control,
although Hb level was significantly increased in EPO-treated rats. Two-photon
microscopy revealed that EPO/CEPO significantly ameliorated tubular epithelial
cell damage assessed by endocytosis. In addition, CEPO/EPO protected endothelial
cells with a sustained blood flow rate. EPO/CEPO suppressed the number of
TUNEL-positive apoptotic cells with weak 㱍SMA staining. Furthermore, PCR
analysis demonstrated that TGF-㱎 and type I collagen expression was attenuated
in EPO or CEPO-treated rats, accompanied by a significant decrease in interstitial
fibrosis. In conclusion, we established a therapeutic approach to protect
tubulointersitial injury with CEPO, and thus, the therapeutic value of this approach
warrants further attention and preclinical studies.
1
大阪大学大学院医学系研究科
医療学
猪阪 善隆1、高原 史郎2、楽木 宏実1
President
Lecture
【目的】食塩感受性の腎障害に内皮障害がどのように関与しているのか、内
皮型NO合成酵素欠損マウス(eNOS-/-)を用いて検討した。
【方法】(1)野生型
マウス(WT)とeNOS-/-に通常食または高食塩食を与え、経時的にアルブミン
尿を測定し、食塩負荷1週と4週後に腎障害を組織学的に評価した。(2)高食
塩食で飼育したeNOS-/-を、ビークル群とARB(イルベサルタン)投与群、抗
酸化薬(テンポール)投与群に分け、RA系や酸化ストレスの関与について検
討した。
【成績】(1)WTでは、高食塩食により糸球体eNOSのリン酸化が亢
進し、尿中NO代謝産物(NOx)の排泄が著明に増加した。また、WTでは食塩
を負荷してもアルブミン尿の増加や糸球体障害は生じなかった。一方、
eNOS-/-では、食塩負荷により尿中NOxの排泄増加はみられず、急速に尿中
アルブミン値が増加した。また、負荷により糸球体スーパーオキシド(O 2 -)
の産生が増加し、負荷4週後には糸球体マクロファージの浸潤やMCP-1
mRNAの発現が有意に増強し、糸球体硬化も進行した。すなわち、eNOS-/では食塩負荷によるeNOS由来NO産生の代償がないために、アルブミン尿
増加や組織障害が出現することがわかった。また、eNOS-/-では、WTと違い、
負荷後早期より尿中アンジオテンシノーゲン(AGT)の排泄が増加し、炎症
惹起に先行して糸球体内皮細胞やメサンギウム細胞領域でAGT発現が有意
に増加することがわかった。さらに、Ang II産生も糸球体内で有意に増加し
ていることを免疫組織学的に確認した。 (2) 食塩負荷でみられたeNOS-/-で
の尿中AGT排泄や糸球体内のAGT発現とO2 - 産生の増加は、イルベサルタン
やテンポールの投与により血圧非依存性に抑制された。また、eNOS-/-でみ
られた食塩負荷によるアルブミン尿増加や糸球体炎症細胞の浸潤、MCP-1
発現の増加、糸球体硬化も両薬剤の投与ですべて有意に抑制された。eNOS
機能不全は、食塩過剰摂取により糸球体内でのAGT発現増強(RA系亢進)と
酸化ストレス増加(NO/ROS不均衡)を介し腎障害を進展することが示され
た。
【結論】eNOS機能不全による血管内皮障害は食塩負荷によりアルブミ
ン尿惹起を促進し、その機序に糸球体でのRA系活性化と酸化ストレス増加
が深く関与している。
1
熊本大学大学院 生命科学研究部 生体機能薬理学、2熊本大学大学院 生命科学
研究部 循環器病態学
中村 太志1、片岡 恵一郎1、名幸 久仁1、外山 研介1、董 一飛1、小川 久雄2、
光山 勝慶1
A nonerythropoietic derivative of erythropoietin
inhibits tubulointerstitial fibrosis in remnant kidney
109
17：30〜
eNOS機能不全による血管内皮機能障害は、
糸球体内のRA
系活性化と酸化ストレス増加を介し食塩感受性に腎障害
を進展する
108
ポスターセッション：その他（2）
Poster Session：Others(2)
12月2日
（木）
／Thursday, December 2
Program
Aⴊ▤↢‛කቇળKPFD
190
Hypoxic tumors are usually resistant to conventional chemotherapy and
radiotherapies, which typically target actively dividing cells. In contrast to blood
vessel networks in normal tissues, tumors contain a chaotic and abnormal
vasculature. These leaky vessels are unorganized, lack adequate pericyte coverage
which compromises delivery of anticancer drugs to tumors. How best to normalize
the aberrant tumor vasculature to maximize anti-cancer drug delivery comprises an
area of intensive investigation. Vasculogenic processes occur normally in adult
tissues to repair injured blood vessels, leading us to hypothesize that bone marrow
(BM) cells may be able to restore appropriate vessel function in tumor vasculature.
Culturing BM mononuclear cells with endothelial growth medium resulted in the
early outgrowth of spindle-shaped attached monocytic cells with pro-angiogenic
activity. Intravenous administration of these cultured proangiogenic monocytes into
nude mice bearing pancreatic cancer xenografts and genetically engineered mice
that develop pancreatic adenocarcinoma significantly reduced areas of hypoxia
without enhancing tumor growth. The resulting vasculature structurally mimicked
normal vessels with intensive pericyte coverage. Consistent with a marked
reduction in gene expressions involved in drug resistance such as MDR1 and
ABCG2 in proangiogenic monocytes-injected tumors, a combination of the
transplantation and chemotherapeutic agents reduced tumor size and significantly
increased areas of necrosis as compared to chemotherapy alone. Together, our
findings offer an alternate approach to improve delivery and efficacy of anti-cancer
drugs to hypoxic tumors through a remodeling of the abnormal tumor vessels.
1
旭川医科大学医学部内科学講座消化器・血液腫瘍制御内科学分野、2大阪医科大学
医学部薬理学講座
河本 徹1、水上 裕輔1、笹島 順平1、杉山 祥晃1、佐藤 一也1、伊井 正明2、
高後 裕1
Transplanting proangiogenic monocytes to tumorbearing mice attenuates innate resistance to
chemotherapy/radiotherapy
112
大阪大学大学院医学系研究科臨床遺伝子治療学
1
北海道大学 大学院歯学研究科 口腔病態学講座
血管生物学教室、2北海道大
学大学院歯学研究科 口腔病理病態学、3北海道大学大学院医学研究科 腫瘍外科学
Poster
Index
Aⴊ▤↢‛කቇળKPFD
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
191
Tumor angiogenesis is essential for tumor progression and metastasis. Traditionally,
it has been believed that tumor vessels are same to normal vessels, but many reports
suggested tumor vessels differ from its counterparts. For instances, tumor vessels
are tortuous in appearance and have loose endothelial connections. These
abnormalities resulted in leakiness of tumor blood vessels. It leads to the tumor
hypoxic and drug-resistible environment so that the cancer cells favorable to
survive. Recently, we have shown that tumor endothelial cells (TECs) express
elevated levels of several genes such as MDR1 and they are more resistant to
certain kinds of chemotherapeutic drugs compared to normal endothelial cells
(NECs).These abnormalities could be caused by the factors in tumor
microenvironment. In this study, we focused on microvesicles (MVs) shed by tumor
cells and analyzed the functions of MVs. MVs, 30 nm - a few um diameter
particles, are secreted from many types of tumor cells. MVs contain many kinds of
biologically active agents, such as growth factor receptor or transcription factor.
Thus, the MVs have been considered as signal transducer implicated in intercellular
communications. We examined whether NECs would acquire TEC specific
phenotypes by the treatment of MVs derived from human melanoma cells. NECs
were incubated with 50 ug/mL of the MVs for 24 hours. It was indicated that
PKH26 dye-labeled MVs were taken up into cytoplasm of NECs by
immunocytochemical study using confocal microscopy and FACS analysis. Several
gene expressions, such as SMA, snail, were up-regulated and VE-Cadherin was
down-regulated in NECs by tumor-derived MVs treatment. These results suggest
that tumor-the MVs might cause Endothelial-mesenchymal transition (EndMT) in
NECs.
Symposium
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
【背景と目的】Angiopoietins (Angs)/Tie-2 systemは、血管の成熟・安定化制御
に重要なシステムとして知られる。我々はこれまでに、血管内皮細胞にお
いて、受容体Tie-2がメタロプロテアーゼ活性依存性に75kDaと115kDaの可
溶型 (75-sTie-2と115-sTie-2)に変換されること、前者は MMP-14活性依存性
であることを見いだし、新規Angs/Tie-2 system制御系の存在の可能性を示唆
した。本研究では、
115-sTie-2産生機構に関する詳細な検討を行った。
【結果】
ヒト微小血管内皮細胞（HMEVCs）は、115-sTie-2や75-sTie-2を同程度に産
生しており、両者の産生はメタロプロテアーゼインヒビターGM6001処置で
抑 制 さ れ た。 一 方、MMP-14 siRNA処 置 やTIMP-2, TIMP-3処 置 に よ り75sTie-2産生は完全に抑制されるものの、115-sTie-2産生は抑制されなかった
ことから、115-sTie-2分泌にはTIMPsに非感受性で知られるADAM9の関与
が推測された。HMVECs へのADAM9 siRNA処置は75-sTie-2分泌に影響を
及ぼさない一方で、115-sTie-2分泌を有意に抑制した。HEK293細胞へTie-2
と luciferaseの共遺伝子導入を行った細胞（HEK293-Tie-2/luciferase）から
115-sTie-2が 産 生 さ れ る が、Tie-2とADAM9 の 共 遺 伝 子 導 入 し た 細 胞
（HEK293-Tie-2/ADAM9） か ら の115-sTie-2産 生 はHEK293-Tie-2/luciferaseの
それと比較して亢進していた。以上から、ADAM9はTie-2を115kDaの可溶
型へ変換する主要なメタロプルテアーゼであることが示唆された。TNF-α
はHMVECsに お け る115-sTie-2の 産 生 を 亢 進 し、 こ の 亢 進 効 果 はTIMP-2,
TIMP-3で抑制されず、ADAM9 siRNA処置で抑制された。さらにTNF-αは
HEK293-Tie-2/ADAM9における115-sTie-2産生を亢進すると同時に全長Tie-2
の発現を低下させた。マウス重症下肢虚血モデルにおいては、
正常と比較し、
虚血誘導一日目の大腿筋肉中においてTie-2 mRNAの発現が亢進している一
方で、全長Tie-2タンパクの発現は低下していた。さらに虚血誘導一日目の
大腿筋肉中ではTNF-α、115-sTie-2の発現が亢進していた。【結語】ADAM9
はTIMPsの有無にかかわらずTie-2を115kDaの可溶型に変換することで、血
管内皮細胞におけるTie-2シグナル調節に関与することが示唆された。
1
北海道大学大学院歯学研究科血管生物学教室、2北海道大学大学院歯学研究科口腔
病理病態学、3北海道大学大学院医学研究科腫瘍外科学
川本 泰輔1、大賀 則孝1、北山 和子1、秋山 廣輔1、近藤 美弥子1、間石 奈湖1、
大澤 崇宏1、山本 和幸1、進藤 正信2、樋田 泰浩3、樋田 京子1
九州大学大学院医学研究院病理病態学、2九州大学大学院薬学研究院 革新的バイ
オ医薬創成学、3国立病院機構福岡東医療センター
1
鬼丸 満穂1、米満 吉和2、居石 克夫3
116
How to write
Plenary Lecture
Nature Medicine
Tumor-derived microvesicles cause gene changes in
endothelial cells
Special Talk
血管内皮細胞におけるTie-2可溶型変換機構へのADAM9
の関与
Special
Lecture
115
Periostin (PN) is a secreted protein that has been suggested to be involved in cell
adhesion functions, however the molecular mechanism is still unknown. Moreover,
some papers report that PN is over-expressed in some tumors such as colon and
breast cancer, and is correlated with tumor growth and invasiveness. Since PN is
highly expressed in 4T1 mouse breast cancer cells, we made anti-PN antibody (PNAb) and investigated whether it could suppress the PN-induced adhesion and inhibit
the metastatic activity of the cells. We found that PN dose-dependently inhibited the
adhesion of 4T1 cells. Also, PN-Ab suppressed the migration and invasion of 4T1
cell.And we further assessed the role of PN in the lung-metastasized animal model,
in which BALB/c mice were injected with 4T1 cells in the foot pad, and received
intravenously control IgG or PN-Ab at the same time. The single PN-Ab injection
significantly suppressed the primary tumor growth after two weeks assessed in the
foot pad size, and decreased the number of metastasized colonies to the lung after
three weeks, compared to the control IgG injected group.Taken together, these
results show that the therapeutic potential of PN-Ab for breast cancer.
久徳 真梨子、谷山 義明、葛城 鳴門、楠 博、岡山 慶太、岩林 正明、
Amarnath Chatterjee、森下 竜一
大賀 則孝1、石川 修平1、秋山 広輔1、北山 和子1、近藤 美弥子1、間石 奈湖1、
川本 泰輔1、樋田 泰浩3、進藤 正信2、樋田 京子1
President
Lecture
It has been an important concept of tumor angiogenesis that tumor endothelial cells
(TECs) are genetically normal and homogenous.However, we have reported that
the TECs are different from normal endothelial cells (NECs). For example, TECs
up-regulate many genes and proliferate more rapidly and migrate more than normal
endothelial cells (NECs). Furthermore, TECs were cytogenetically abnormal.
However, it remains to be unclear whether TECs are different or not, depending on
phenotypes of tumors from which TEC are isolated.
In this study, we isolated two types of murine TECs from two different types of
tumor xenografts; highly metastatic melanoma (HMM) and low metastatic
melanoma (LMM) in nude mice and compared their phenotypic and genetic
characters between HMM-TEC and LMM-TEC.
HMM-TEC showed higher proliferative activity, motility and invasive activity than
LMM-TEC. In addition, HMM-TEC expressed higher levels of mRNAs of the
reported tumor endothelial-specific markers such as CD13, TEM-8 and
angiogenesis-related genes such as VEGF-R1, 2 than LMM-TEC. Furthermore,
HMM-TEC up-regulated the mRNA expression levels of stemness-related genes
such as Sca-1 and CD90 and they showed the spheroid morphologies with smooth
surface and high circularity in stem cell spheroid assay. In addition, an alkaline
phosphatase was activated after culture in osteogenic medium for 7 days in HMMTEC but not in LMM-TEC nor NEC, suggesting that HMM-TEC may have
potential to differentiate to osteoblast.
These results suggest that the phenotypes of TECs are different dependeing on
metastatic potential of tumors. The use of monomorphemic anti-angiogenic drugs
have limitations, thus studies on TEC heterogeneity will help us to develop ideal
antiangiogenic therapies.
Anti-Periostin Antibody Inhibits Tumor Progression
and Metastasis in Breast Cancer
114
17：30〜
Comparative characterization of tumor endothelial
cells isolated from highly and low metastatic tumors
113
ポスターセッション：その他
（3）
Poster Session：Others(3)
12月2日
（木）
／Thursday, December 2
Program
Aⴊ▤↢‛කቇળKPFD
192
【目的】
平滑筋細胞に発現していることが知られている Hic-5 は、
Paxillin ファ
ミリータンパクに属する接着斑タンパクであり、細胞接着斑の中で足場的
存在である。Hic-5 欠損型マウス（KO）にバルーン血管傷害術を行ったと
ころ、血小板凝集の形態変化が野生型マウス（WT）に比べ抑制されている
ことが明らかになった。本研究では血小板凝集における Hic-5 の役割につ
いて検討した。
【方法】まず WT、KO マウスの尾の先端を切断して生理食塩水の中に入れ、
止血するまでの時間を測定した。続いて WT 及び KO マウスより採取した
洗浄血小板を collagen、thrombin で刺激し、透過率および凝集塊数を測定し
た。さらに洗浄血小板液を thrombin で刺激し、
活性型 integrin 量を測定した。
【結果・考察】KO マウスで出血時間の延長が認められた。このことから
Hic-5 欠損により個体レベルで止血機能が低下していることが確認できた。
また、collagen 及び thrombin 刺激に対し、KO マウスの血小板では凝集抑制
が観察された。血小板凝集能の測定では洗浄血小板を使用しているので、
血小板機能そのものに Hic-5 が関与していることが確認できた。さらに KO
マウスでは WT マウスと比較して integrin 㱍IIb㱎3 の活性化が低下していた。
integrin 㱍IIb 㱎3 は血小板に発現している integrin の9割を占め、血小板凝集
に最も重要な役割を担っている。KO マウスの血小板凝集に抑制が見られ
たのは、integrin 㱍 IIb㱎3 の活性化が低下していたためであると考えられた。
今後 integrin 㱍IIb 㱎3 活性化における Hic-5 の役割を解析することにより、
ヒト血小板の病態解明につながるものと期待される。
重症虚血肢は、臨床症状として虚血性潰瘍や安静時疼痛を呈する慢性動脈
閉塞症の最も重篤な病態である。治療の第一選択は血行再建術であるが、
その適応が困難な症例では下肢切断リスクを低下させる確実な手段は存在
せず、新たな治療法が望まれている。
そのような病態に対し、近年血管新生因子を用いた遺伝子治療が多数試み
られている。我々は日本で発見された肝細胞増殖因子（HGF）に早期より
着目し、HGFに血管内皮細胞に特異的な増殖作用、血管新生作用等の血管
新生療法ツールとして有用な作用があることを見出した。2001年から臨床
研究を大阪大学病院において開始し、血行再建術の適応が無く内科的治療
にも反応しない22例の閉塞性動脈硬化症あるいはビュルガー病患者におい
て、6割以上の患者で明らかな症状の改善が認められた。
この成果を引き継ぐ形で、薬事承認取得を目的として、重症虚血肢を有す
る閉塞性動脈硬化症患者を対象としたプラセボ対照無作為化二重盲検比較
試験が、2004年から多施設共同治験として開始された。その結果、プライ
マリーエンドポイントである初回遺伝子導入3ヵ月後の虚血性潰瘍あるいは
安静時疼痛の改善において、HGF遺伝子治療群のプラセボ群に対する優越
性が確認され（p=0.014、Mantel Haenszel検定）、かつ安全性にも大きな問題
は 認 め ら れ な か っ た（Shigematsu H et al, Gene Therapy advance online
publication, April 15, 2010 (DOI 10.1038/ gt.2010.51)）
。さらに本試験では遺伝
子導入3年後までの長期フォローアップが行なわれている。これまで2年後
までの大切断発生率：5.3%、死亡率：2.6%という成績が得られており、こ
れはTASCIIで報告されている1年後の大切断発生率：30%、死亡率：25％、
欧州で実施されたFGF1プラスミドの第II相試験のプラセボ群における1年後
の大切断発生率：33.9%、死亡率：23.2％と比較すると良好な成績であると
いえる。今回は、長期データを含めたHGF遺伝子治療の成績を報告する。
大阪大学大学院医学系研究科臨床遺伝子治療学、2東京医科大学 血管外科、3北海
道中央労災病院せき損センター、4医療法人慶友会つくば血管センター、5旭川医科
大学 外科学第一講座、6戸田中央総合病院 血管内治療センター、7東京大学大学
院医学系研究科 生物統計学／疫学・予防保健学、8東北大学病院 東北大学未来
医工学治療開発センター、9大阪府立急性期・総合医療センター
1
森下 竜一1、重松 宏2、安田 慶秀3、岩井 武尚4、笹嶋 唯博5、石丸 新6、
大橋 靖雄7、山口 拓洋8、荻原 俊男9
昭和大学大学院薬学研究科臨床毒性学、2昭和大学医学部生化学
1
重症虚血肢に対するHGF遺伝子治療の長期成績
宮内 彩1、吉田 武美1、金山 朱里2、宮崎 章2
118
血小板凝集における細胞接着斑分子 Hic-5 の機能解析
117
Index
193
Poster
The 8th Korea-Japan Joint Symposium on Vascular Biology
Selected Oral
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Y.I.A.
Presentation
Aⴊ▤↢‛කቇળKPFD
内科、3
【背景・目的】 EPCは、障害血管の修復や虚血臓器における血管新生に寄
与することが知られ、患者EPCを用いた臨床応用も検討されている。 し
かし、老化や糖尿病などの心血管リスクは、EPC機能を低下させ、心血管
系疾患病態や、EPC導入療法の臨床効果に大きく影響している。 Ape１は、
DNA修復や様々な機序を介して多角的に抗酸化ストレス作用を発揮する遺
伝子である。 我々は、Ape1発現EPCは、酸化ストレス下の障害血管での
EPC効果を亢進させるのか検証した。【方法・結果】 マウス大腿動脈ワイ
ヤー障害による血管リモデリング実験において、老化マウス（C57BL6 1.5
years-old）骨髄を移植した群では、成人マウス（10 weeks-old）骨髄移植群
にくらべ、新生内膜肥厚度が増加していた。 老化骨髄マウスにおける増
悪化血管リモデリングは、成人マウス由来y-EPC(lin-,ckit+,Flk+)導入により
改善した。 老化マウス由来aged-EPCは、y-EPCにくらべ、細胞接着能な
どの機能が低下しており、炎症サイトカインTNFaの刺激に反応して、
y-EPC内のApe1遺伝子発現が誘導されるが、aged-EPCにおいては減弱して
いた。精製したEPCをApe1発現用組み換えAdenovirusに感染させた後、血
管ワイヤー障害モデルに導入した。 対照としてGFP発現adenovirus感染
EPCを用いた。 GFP-EPCにおいても新生内膜肥厚低下効果を認めるが、
Ape1-EPCに お い て 著 明 な 血 管 リ モ デ リ ン グ 改 善 効 果 を 示 し た。
【結
論】 Ape発現EPCは、EPCの有する障害血管の修復機能を亢進させ
た。 老化などの心血管系リスクにより機能低下しているEPCを改善させ
る方法として期待される。
旭川医科大学内科学循環呼吸神経病態学、2旭川リハビリテーション病院
旭川医科大学 心血管再生先端医療開発講座
1
Symposium
Background: It was hypothesized that pericardial fat as local visceral fat depot with
close proximity to coronary arteries may serve as a source of inflammatory
cytokines and cells in coronary atherosclerotic lesions. Macrophage accumulation
enhances the chronic inflammation in adipose tissue, but the macrophage
phenotypic change in human pericardial adipose tissue and its role in atherogenesis
are unknown.
Methods and Results:Pare samples were obtained from epicardial and subcutaneous
adipose tissues during elective cardiac surgery (CAD, n=38; non-CAD, n=40).
Infiltration of M1/M2 macrophages was investigated by immunohistochemistry
using antibodies against CD11c and CD206, respectively. We analyzed the
expression of pro- and anti-inflammatory adipocytokines in adipose tissues by realtime qPCR. Infiltration of macrophages and expression of pro- and antiinflammatory cytokines were enhanced in epicardial fat of patients with coronary
artery disease, when compared with that of non-CAD patient (P＜0.05). The ratio
of M1/M2 macrophage was positively correlated with severity of coronary artery
disease (r=0.312, P=0.039). Furthermore, the expression of pro-inflammatory
cytokines was positively correlated, and the expression of anti-inflammatory
cytokines was negatively correlated, with the ratio of M1/M2 macrophages in
epicardial adipose tissue of CAD patients. On the other hand, there was no
significant difference between the CAD and non-CAD groups in macrophage
infiltration and cytokine expression in subcutaneous adipose tissue.
Conclusions:The ratio of M1/M2 macrophages changed in epicardial adipose tissue
of CAD patients compared to non-CAD patients. The macrophage polarization in
epicardial adipose tissue might influence the atherogenesis in human coronary
arteries.
徳島大学大学院ヘルスバイオサイエンス研究部循環器内科学分野、2徳島大学大学
院ヘルスバイオサイエンス研究部心臓血管外科学分野、3榊原記念病院心臓血管外
科
1
山内 敦司1,2、川辺 淳一1,3、浅野目 晃1、竹原 有史1,3、長谷部 直幸1,3
平田 陽一郎1、元木 達夫2、黒部 裕嗣2、赤池 雅史1、田端 実3、高梨 秀一郎3、
佐田 政隆1
122
How to write
Plenary Lecture
Nature Medicine
Apurinic/apyrimidinic Endonuclease1(APE1)遺伝子導
入した内皮前駆細胞
（EPC）による新生内膜肥厚改善効果
Special Talk
Macrophages “shift” toward inflammatory state in
epicardial fat of patients with coronary artery disease
Special
Lecture
121
【背景】糸球体濾過率(GFR)が同程度でも、加齢等による腎機能低下と糖尿
病 や 高 血 圧 な ど 血 管 障 害 に よ る 腎 機 能 低 下 で は 病 態 が 異 な る。 推 定
GFR(eGFR)は慢性腎臓病(CKD)患者においてもその腎機能を推定する有用
な指標であるが、eGFRのみでは個々の腎臓病の病態の評価や予後の予測を
正確に行うのは難しいと考えられる。腎血流ドプラにおいて腎区域動脈で
の(最高流速-拡張末期流速)/最高流速で定義されるResistive index(RI)は、腎
内血管抵抗・動脈硬化度を反映する指標とされる。これまで、RIにより評
価される腎内血管障害・血管抵抗と血圧日内変動の関連についての充分な
検討は報告されていない。我々は過去の学会においてその両者の関連性を
報告してきたが、今回さらに症例数を増やし詳細に検討した結果を報告す
る。
【方法】2009年2月以降に大阪大学医学部附属病院老年・高血圧内科病
棟にて腎血流ドプラを施行した、CKDの有無を問わない連続194例(平均年
齢66.2歳)において、RIと、自由行動下血圧測定(ABPM)により評価した血圧
日内変動、採血・尿検査を含む複数のパラメーターとの相関を検討した。
【結
果】RIは、年齢、eGFR、body mass index、拡張期血圧、脈圧、尿蛋白など
と有意に相関した。CKD stageI、II、III相当の軽度腎機能低下例では、同程
度のeGFRであっても、糖尿病群が非糖尿病群に比べて有意にRIが高かった。
このことは、糖尿病によってGFRに変化が現れてくる前の段階から腎内の
血管障害をきたしていること、またそれを腎血流ドプラにより評価しうる
ことを示していると考えられる。血圧日内変動との関連では、
日内サージ
（早
朝収縮期血圧―夜間最低収縮期血圧）の大小で2群に分けて比較すると、
サー
ジの大きい群で有意にRIの高値を認め、このことにより血圧日内変動と腎
内血管抵抗・動脈硬化度との関連性が示唆された。【考察】本研究により、
RIが腎障害の病態評価や予後予測に有用であることが示された。また、血
圧日内変動が腎内血管病変とも相関していることが示された。血圧日内変
動は、腎保護の観点からも重要な治療ターゲットとなりうると考えられる。
大阪大学大学院医学系研究科老年・腎臓内科学
河合 達男、神出 計、大西 美幸、竹屋 泰、花咲 博子、多田羅 雄之、馬場 義親、
島岡 泉、大石 充、楽木 宏実
腎評価軸としての腎血流ドプラの有用性 -自由行動下血圧
測定(ABPM)との相関からの検討-
President
Lecture
Background: Dietary cholesterol oxidation product, oxysterol, is known to be
absorbed from intestine and incorporate into atherosclerotic lesions, and accelerates
the development of atherosclerosis in animals. However, the mechanism of
dietary oxysterol-induced atherogenesis has not been explored. Hence we aimed to
investigate molecular and cellular mechanisms by which dietary oxysterol
accelerates atherogenesis in hypercholesterolemic mice.
Methods and Results: ApoE-deficient mice were fed with either a control high-fat
diet (control-HFD) or HFD containing oxysterol (oxysterol-HFD; 6.8 % of
cholesterol was oxidized) for 8 weeks and infused with angiotensin II. OxysterolHFD enhanced the formation of aortic atherosclerosis in en face analysis,
macrophage infiltration, and MCP-1 expression in atherosclerotic lesions without
affecting plasma total cholesterol levels. In addition, oxysterol-HFD did accelerate
plaque destabilization (macrophage infiltration, fibrous cap thinning) and the
incidence of plaque rupture (control-HFD=3.5±0.3; oxysterol-HFD=4.9±0.5/
mouse; P＜0.05) in the brachiocephalic arteries. Treatment with ezetimibe, a
cholesterol absorption inhibitor, significantly decreased plasma cholesterol levels
and suppressed the enhanced atherosclerosis formation, monocyte activation,
MMPs activation, and the incidence of plaque rupture induced by oxysterol-HFD.
Conclusions: Dietary oxysterol accelerated atherogenesis through monocytemediated inflammation and MMP activation in hypercholesterolemic mice, which
is prevented by the treatment with ezetimibe. Inhibiting absorption of dietary
oxysterol by ezetimibe may be a reasonable therapeutic approach in vulnerable
patients who overly intake oxysterol.
九州大学医学研究院循環器内科学
佐藤 敬、的場 哲哉、香月 俊輔、中野 覚、江頭 健輔
Dietary Oxysterol Accelerates Atherosclerosis and
Plaque Rupture Through Macrophage Mediated
Inflammation in Mice
119
120
18：00〜
ポスターセッション：動脈硬化（4）
Poster Session：Atherosclerosis(4)
12月2日
（木）
／Thursday, December 2
Program
Aⴊ▤↢‛කቇળKPFD
194
背景：イコサペント酸エチルは血管不全改善効果を有することが報告され
ているが、血管機能改善と同時に血管形態に対して生じうる影響について
は、充分に検討されていない。目的：イコサペント酸エチル内服による血
管不全改善効果を評価すると同時に、血管形態の改善効果の有無につき検
討すること。方法：高トリグリセライド血症例連続14例（平均年齢60.2±
13.6歳、男性7名）を対象とし、血液学的検査、血管機能測定専用超音波装
置（ユネクスEF）による前腕駆血法による血管内皮依存性血管拡張反応評
価、ニトログリセリン投与法による血管内皮非依存性血管拡張反応評価を、
イコサペント酸エチル投与前と投与後6ヵ月後に施行した。血管内皮依存性
血管拡張反応は、前腕動脈駆血前と一定時間駆血後開放し血流増加に伴い
拡張した上腕動脈血管径を計測し、血管径変化率（%FMD）を算出し評価
した。血管内皮非依存性血管拡張反応は、ニトログリセリンによる血流増
加刺激前とニトログリセリン投与で生じた血流増加に伴い拡張した上腕動
脈血管径を計測し、血管径変化率（%NMD）を算出し評価した。また、同
部位で同時に中内膜複合体厚（IMT）計測を施行した。結果：イコサペン
ト酸エチル投与前%FMDは4.81±1.41、イコサペント酸エチル投与6ヵ月後
%FMDは6.42±2.19であり、有意な上昇を認めた（P=0.046）。イコサペント
酸エチル投与前%NMDは15.88±4.96、イコサペント酸エチル投与6ヵ月後
%NMDは16.4±6.57であり、有意な変化を認めなかった。イコサペント酸
エチル投与前中内膜複合体厚（IMT）は0.295±0.052mm、イコサペント酸
エチル投与6ヵ月後IMTは0.304±0.074mmであり、有意な変化を認めなかっ
た。結語：イコサペント酸エチルの6ヶ月間の投与にて血管内皮機能改善を
認めるが、中内膜複合体厚の改善には至らないことが示唆された。
大阪市立大学大学院医学研究科循環器病態内科学、2西宮渡辺病院、3兵庫医療大学、
4
浅香山病院
1
竹本 恭彦1、島田 健永1、井口 朋和1、中西 弘毅1、松本 健嗣1、花谷 彰久1、
中村 泰浩1、室生 卓1、吉川 純一2、東 純一3、田原 旭4、葭山 稔1
高トリグリセライド血症におけるイコサペント酸エチル
の血管不全改善効果：機能形態同時評価による検討
123
Objective: In this study, we examined the chronic effects of LDL apheresis on
clinical parameters and endothelial cell functions in hemodialysis patients with
PAD. Methods and Results: The enrolled patients were classified into 2 groups
according to the response to LDL apheresis: patients with improved ABI (ABI
responders) and patients with worsened ABI (ABI nonresponders) after treatment.
In ABI responders, the absolute walking distance and the ABI were increased at the
10th session compared with baseline (from 118±26 m to 333±45 m, P＜0.05;
from 0.53±0.06 to 0.69±0.06, P＜0.05; absolute walking distance and ABI,
respectively) and even at 3 months after the end of treatment (from 118±26 m to
297±63 m, P＜0.05; from 0.53±0.06 to 0.69±0.05, P＜0.05). On the other hand,
neither absolute walking distance nor ABI showed any changes during the study
period in ABI nonresponders. In ABI responders, apheresis resulted in a long-term
reduction of circulating levels of oxidized LDL, C-reactive protein, and fibrinogen.
In HUVECs the serum from the ABI responders increased expression of activated
ecNOS protein, which is phosphorylated at Ser-1177, and BrdU incorporation
activity. Furthermore, there were significant correlation between ABI and activated
ecNOS protein level (R=0.43, P＜0.05), between walking distance and activated
ecNOS protein level (R=0.57, P＜0.05), and between walking distance and
oxidized LDL (R=-0.45, P＜0.05), in HUVECs treated with ABI responder serum.
Conclusion: These results demonstrate that LDL apheresis decreases oxidized LDL
and inflammation and improves endothelial cell function in the responders. This
may be one of the mechanisms involved in the long-term therapeutic effect of LDL
apheresis on peripheral circulation in hemodialysis patients.
横浜市立大学医学部循環器・腎臓内科学
田村 功一、池谷 裕子、涌井 広道、三橋 洋、前田 晃延、出島 徹、大澤 正人、
金岡 知彦、白 善雅、柳 麻衣、戸谷 義幸、梅村 敏
Sustained Inhibition of Oxidized LDL is Involved in
the Long-Term Therapeutic Effects of Apheresis in
Dialysis Patients
124
18：00〜
Index
Aⴊ▤↢‛කቇળKPFD
Poster
195
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Angiogenesis a complex process accomplished by reiteration of modules such as
sprouting, elongation, and bifurcation, but its cell-based mechanisms remain largely
unknown. Time-lapse imaging of aortic ring sprouting assay surprisingly revealed
that, vascular endothelial cells (ECs) move backwards and forwards, taking over
each other even at the tip, showing hitherto unknown mode of morphogenetic cell
movement. In order to further understand the events, nuclei of individual ECs were
tracked and parameters reflecting EC movement were set to represent and integrate
different molecular interventions. In response to vascular endothelial growth factor
(VEGF), velocity and orientation were increased, and these were associated with
increment in neo-sprouting and elongation capacity of tip cell, which in turn
coincided with the increase in vessel elongation and branching point. As for DeltaNotch signaling, Delta-like 4 antibody (Dll4-Ab) treatment also led to increase in
vessel elongation, velocity and neo-sprouting, which were partially recapitulated by
inhibition of Notch by 㱏-secretase inhibitor, DAPT. Part of the discrepancy
between both treatments (increase in taking-over frequency and retrograde
directional motility) was recapitulated by platelet-derived growth factor receptor-㱎
antibody treatment, suggesting the effect of mural cells (MCs), which express Jag1,
another Notch ligand. These observations may unravel how VEGF-mediated branch
elongation is accomplished through EC-EC and EC-MC interaction, partly via
Notch signaling. To understand the phenomenon we are establishing a theoretical
simulation model using cellular automata. Our effort shown here may lead to a
holistic understanding of angiogenic morphogenesis integrating molecular, cellular
and modular level.
東京大学医学系研究科代謝生理化学教室
Symposium
Tumor endothelial cells (TEC) are a therapeutic target of metronomic
chemotherapy. In this study, we investigate drug resistance of TEC. We also
investigate the mechanism how endothelial cells (EC) acquire drug resistance.
Mouse TEC and normal endothelial cells (NEC) were isolated and purified from
melanoma xenografts and the dermis, respectively. MTS assay showed that TEC
were resistant to 5-FU and paclitaxel. And, multi-drug resistance 1 (MDR-1)
mRNA level was upregulated in TEC compared to NEC (P＜0.01). Human NEC,
human microvascular endothelial cells (HMVEC) were cultured with melanoma
conditioned medium (tumor CM) to see if NEC acquire drug resistance. After fiveday treatment with tumor CM, HMVEC became more resistant to serum starvation
and paclitaxel with up-regulation of MDR-1 gene which encodes P-glycoprotein (Pgp) (P＜0.01). In addition, Y-box binding protein 1 (YB-1) which is a
transcriptional factor of MDR-1 was translocated to the nuclear by tumor CM
treatment (P＜0.01). In the presence of P-gp inhibitor, verapamil, HMVEC were
resensitized to paclitaxel even after tumor CM treatment. When tumor CM was
pretreated with heat, it did not upregulate MDR-1 in HMVEC. In conclusion, TEC
are resistant to 5-FU and paclitaxel. Tumor CM induce drug resistance to NEC.
Nuclear translocalization of YB-1 and upregulation of MDR-1 following tumor
exposure might be a mechanism of drug resistance acquired by TEC.
北海道大学大学院歯学研究科血管生物学、2北海道大学大学院医学研究科腫瘍外科、
3
北海道大学大学院歯学研究科口腔病理病態学
1
有馬 聡、西山 功一、候 聡志、有馬 勇一郎、小関 宏昭、内島 泰信、
栗原 由紀子、栗原 裕基
How to write
Plenary Lecture
Nature Medicine
樋田 京子1、秋山 廣輔1、大賀 則孝1、樋田 泰浩2、北山 和子1、近藤 美弥子1、
間石 奈湖1、川本 泰輔1、大澤 崇宏1、山本 和幸1、進藤 正信3
128
Special Talk
Novel insight into angiogenesis: In-depth analysis
through time-lapse imaging and quantification
Special
Lecture
Tumor induces drug resistance of endothelial cells by
nuclear translocation of YB-1 and up-regulation of
MDR-1
これまで報告されてきたVEGF (血管内皮細胞増殖因子)などの多くの血管新
生因子は、低酸素ストレス応答に必須な転写因子HIF（Hypoxia-Inducible
Factor）により誘導されていることが報告されている。私達はHIFのサブタ
イプの1つであるHIF2αに焦点を絞り、酵母Two-Hybrid法を用いて解析を進
めた結果、HIF2αの結合因子としてInt6/eIF3eを同定した。Int6はマウスお
よびヒトにて乳癌の癌抑制遺伝子として報告されているが、これまでの詳
細な解析により、Int6がHIF2αに直接結合し低酸素非依存的にHIF2αを分
解する新たな機序を発見した(Chen et al. JBC 2007)。次に、Int6-siRNAをマ
ウス皮下に導入したところ、5日間で面積、長さとも7−10倍の有為な増加
を示す正常な動脈・静脈からなる血管新生効果が認められた。動物モデル
を用いた実験では、(1) 正常および糖尿病マウス（db/db）による皮膚損傷、
(2) 左環状動脈結紮によるラット心筋梗塞、(3) マウス凍結脳損傷、(4)マウ
ス下肢動脈閉塞の各モデルで、顕著な改善効果が見られた。さらに、Int6お
よびHIF2αのプロモーター解析等により、Int6を介するHIF2αのNegative
Feedback制御機構が明らかになった。すなわちInt6はHIF2αの発現量を調整
しそのhomeostasisを維持する機能があり、Int6-siRNAがInt6の抑制機序破綻
させてしまうためHIF2αの過剰発現を惹起し、著明な血管新生を誘導して
いた。以上の結果から、
Int6はHIF2αを介した血管新生を調節する
「マスター
スイッチ」の一つであることが示唆された（Chen et al. Circulation 2010）
。
これらの結果から、Int6-siRNAが創薬の候補として虚血性疾患の治療のみ
ならず、再生医療や細胞移植時の細胞の生存効果を改善する補助療法とし
ても応用が期待される。
1
東京都臨床医学総合研究所分子医療プロジェクト、2がん感染症センター都立駒込
病院、3京都大学大学院付属病院乳腺外科
芝崎 太1、陳 リー1、Alexander Endler1、内田 和代1、堀口 慎一郎2、
飯島 修1、戸井 雅和3
新規HIF2α抑制因子Int6/eIF3eによる正常血管新生機序
とInt6-siRNAによる虚血性疾患治療への応用
126
President
Lecture
127
背景：血管新生は、個体発生や創傷治癒、動脈硬化、癌の発育、転移など、
生理的にも病的にも重要な役割を果たしている。血管内皮増殖因子
（VEGF）
は血管新生の強力な誘導因子であるが、VEGFによる血管新生の制御機構は
まだ十分には明らかとなっていない。そこで我々は、VEGFによる血管新生
における細胞接着分子であるネクチン様分子のNecl-5の役割について検討
した。方法、結果：免疫組織染色にて、Necl-5は、マウス下肢の毛細血管
の内皮に強く発現していた。マウスの大腿動脈の結紮により、下肢での
Necl-5の発現は増加した。Necl-5ノックアウトマウスに対して下肢血管新生
のモデルである大腿動脈結紮術を施行し、野生型と比較したところ、Necl-5
ノックアウトマウスでは下肢の血流の回復は、野生型のマウスと比べて遅
延しており、結紮4週間後の毛細血管密度は低下していた。また、マトリゲ
ルプラグアッセイを行ったところ、VEGFによる新生血管の形成は、野生型
のマウスに比べてNecl-5ノックアウトマウスでは減弱していた。次にNecl-5
の血管新生の制御の分子機構を検討するために、培養ヒト臍帯静脈内皮細
胞において、Necl-5をsiRNAを用いてノックダウンした。VEGFにより誘導
される管腔形成、遊走、増殖はNecl-5のノックダウンにより阻害された。
一方、血清飢餓により誘導されるアポトーシスはNecl-5のノックダウンに
より増強された。Necl-5のノックダウンによりVEGFにより誘導される、
VEGF受容体（VEGFR2）とインテグリン㱍v㱎3との相互作用は抑制され、
VEGFによるAktの活性化は抑制された。結語：以上より、Necl-5はVEGFR2
とインテグリン㱍v㱎3との相互作用に必要で、VEGFによるシグナル伝達を
制御することにより、血管新生を調節していることが明らかとなった。
神戸大学大学院医学研究科循環器内科学
衣笠 允雄、力武 良行、小林 成美、呉羽 布美恵、銕 佑介、宮田 宗明、
久保 嘉輝、平田 健一
VEGFによる血管新生におけるNecl-5の役割
125
ポスターセッション：血管・リンパ管新生
（5）
Poster Session：Angiogenesis / Lymphangiogenesis(5)
12月2日
（木）
／Thursday, December 2
Program
1
旭川医科大学医学部心血管再生先端医療開発講座、2旭川医科大学医学部内科学循
環呼吸神経病態学
Introduction; Vasa vasorum (VV), a microvasculature in the adventitial layer of
arteries have been implicate to play a role in the pathogenesis of atherosclerosis.
The mechanism of VV angiogenesis is still unclear because of lack of appropriate
histochemical analysis. Weʼve developed in vivo angiogenesis assay using
collagen-coating tube (CCT) to observe the microvasculature around the arterial
walls. In this study, we investigated whether peripheral nerve is crucial for VV
angiogenesis in injured vascular walls. Methods and Results; C57BL6 male mouse
femoral arteries were injured by wire and biodegradable gelatin hydrogels releasing
VEGF and nerve growth factor (NGF) were applied outside of injured arteries. In
vivo CCT-angiogenesis assay demonstrated that vascular injury-induced VV
angiogenesis was enhanced by treatment with VEGF and VEGF+NGF. In VEGF/
NGF group, the average size of microvessels was increased and vascular
permeability was attenuated compared to VEGF group. Immunohistochemial and
electron microscopic analyses showed that tyrosin hydrogenase-positive peripheral
nerve was already observed around the pre-mature microvasculature, which was
partially covered with pericytes. NGF enhanced the level of peripheral nerverelated proteins in vascular adventitial tissues. Peripheral nerve-free ex vivo aorta
ring angiogenesis assay demonstrated that the average length of sprouting vessels
was increased by VEGF or NGF, but %length of pericyte-covered large
microvessels was not altered by combination of VEGF and NGF compared to
VEGF or NGF alone. Conclusion; NGF induces the distribution of peripheral
nerve fiber around the newly formed VV microvasculature, and contribute to
vascular stabilization and maturation.
1
旭川医科大学内科学講座循環呼吸神経病態内科学分野、2旭川医科大学心血管再生
先端医療開発講座、3旭川リハビリテーション病院内科
【背景・目的】 EPCは虚血組織における血管新生に関与し、細胞導入によ
る末梢虚血臓器の血流改善効果は臨床的にも実証されている。しかし、そ
の作用機序の詳細はいまだ不明な点が多い。我々は、PGI2がEPC機能維持
に重要な役割を果たすことを明らかにした。本研究では、プロスタサイク
リン受容体IP欠損マウスを用いて、EPCが末梢虚血の血流改善にどのよう
な機序で関与しているのか解明する。【方法・結果】 野生株マウス群(wild
type)とIP欠損マウス（IP-KO）を用い下肢虚血モデルを作成し、下肢虚血改
善度を血流ドップラー法で評価した。IP-KOでは著明な虚血改善度の低下
を認めたが、さらに骨髄移植により（A）骨髄特異的にIP欠損させた、（B）
骨髄以外の臓器をIP欠損させたモデルで検討した。両群とも有意に虚血改
善度の低下を認めたが、
（B）では虚血後、初期の血流改善低下を認めたが、
（A）群では、長期にわたる血流改善遅延が認められた。さらに（A）群に
おいて、wild type EPC(lin-cKit+Flk+)の導入により、虚血組織内の新生血管
数増加と、低下した血流改善度の改善が認められたが、IP欠損EPCでは効
果がなかった。EPCで産生されるVEGFなどの血管新生因子は、IP agonistで
あるベラプロストにより増加したが、IP欠損EPCでは基礎産生量も低下、
ベラプロストによる反応も認めなかった。大動脈輪状組織をマトリゲル内
で培養したEx vivo血管新生アッセーで、GFP発現EPCを共培養すると、血
管新生が亢進し、さらに新生血管周囲にGFP発現EPCが集積しているのが
確認され、より太い新生血管が形成された。IP欠損EPCでは、新生血管周
囲に集積する細胞は少なく、血管新生効果も有意に低下していた。
【結
論】 骨髄由来EPCのプロスタサイクリンは、EPCの血管新生因子産生増強
や新生血管周囲細胞化を介することで血管新生を促進し、安定した末梢虚
血改善作用に寄与している。
Aⴊ▤↢‛කቇળKPFD
川辺 淳一1、浅野目 晃2、山内 敦司2、竹原 有史1、長谷部 直幸1,2
浅野目 晃1、川辺 淳一1,2、山内 敦司1,3、竹原 有史1,2、長谷部 直幸1,2
196
Nerve growth factor induces maturation of vasa
vasorum neovasculature in injured vascular walls
130
内皮前駆細胞(EPC)を介したプロスタサイクリンの下肢虚
血改善作用 −血管周細胞分化による血管新生効果−
129
18：00〜
1
徳島大学大学院 HBS研究部 心臓血管外科学分野、2徳島大学大学院 HBS研
究部 薬理学分野、3徳島大学 疾患ゲノム研究センター 遺伝子実験施設
神奈川歯科大学生体管理医学講座薬理学分野
Index
Aⴊ▤↢‛කቇળKPFD
Poster
197
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
【はじめに】近年、血中に存在するendothelial microparticles (EMP)が炎症性
疾患に伴い増加することが報告されている。またcardiovascular diseaseにお
いては、EMP量が予後に相関するとされている。EMPは炎症性病態で血中
に増加するとされるが、どの部位の血管から産生されるのか、どのような
メカニズムによって出芽し放出されるのか未解明な部分が多い。我々は、
EMPのin vitroでの効率的な産生系を確立し、さらにin vivo病態モデルとの
比較検討を行い、疾患バイオマーカーとしての可能性を検討した。
【方法】
脳血管内皮細胞株であるb.End5を用い、炎症性サイトカインおよびLPSを作
用させ、EMP量をFACSによって解析した。細胞の炎症状態は、各種炎症マー
カーを指標にして評価した。またin vivoの検討では、急性・慢性炎症マウ
スモデルを用い血中EMPをFACSによって解析した。さらにEMPの構成蛋白
質について、SDS-PAGEサンプルに銀染色を施し評価した後、MS解析を行っ
た。【結果】b.End5を用いたin vitroの系において、3種類のサイトカインコ
ンビネーションでは炎症は惹起されるものの、顕著なEMPの産生は確認さ
れなかった。一方、サイトカイン+LPS、LPS単独群では、EMPの産生が確
認されるとともに、各種炎症マーカーを指標にした細胞の炎症の度合いも
強かった。また、急性・慢性炎症マウスモデルにおけるin vivoの検討では、
いずれの場合においても血中EMP量の増加が観察され、鋭敏なバイオマー
カーとしてのポテンシャルを有することを確認した。さらに、銀染色によ
るEMP構成蛋白質の解析では、in vitro、in vivoで約60 kD蛋白質の著明な増
加が観察された。【考察】EMPの効率的なin vitro産生系の確立に初めて成功
した。また、銀染色やMS解析の結果から、炎症によって誘導されるEMPの
主要な構成蛋白質は、in vitro、in vivoで関連性がみられることが示唆された。
さらに、脳血管障害などの局所の炎症病態においてEMPは鋭敏なバイオマー
カーとなることが強く示唆された。
[Objective] Vascular inflammation constitutes one of the major components of
atherosclerosis. However, the presence of preceding inflammation of the atheroprone artery has not been demonstrated in vivo. We examined the leukocyte
recruitment at the athero-prone femoral artery in the absence of confirmed
atherosclerosis using an intravital microscopy system (IVM). [Methods and
Results] Femoral arteries of apolipoprotein E-deficient (apoE-/-) mice at 6w, 8w,
and 12w of age were subjected to IVM and compared to those of wild type mice.
IVM demonstrated a significant increase of leukocyte recruitment in the observed
artery in apoE-/- mice as early as 6w. Dynamic observation of oxidative stress in
situ revealed the massive accumulation of dihydroethidium-related oxidative stress
and the number of adherent leukocytes was increased at 8w and 12w when
compared to wild type mice. Oil red O staining of the observed artery of apoE-/mice, however, failed to detect lipid deposition in the arterial segment where IVM
was conducted. Further, treatment with rosuvastatin for 2 weeks significantly
diminished the observed leukocytes recruitment to the femoral artery without
changing plasma lipid level. [Conclusion] We were able to visualize leukocyte
recruitment prior to the development of atherosclerosis in vivo and its stabilization
by statin. Our observation confirms a prerequisite role of inflammation in the
development of atherosclerosis.
Symposium
1
富山大学医学薬学研究部分子医科薬理学講座、2金沢工業大学ゲノム生物工学研究
所、3独立行政法人国立長寿医療研究センター遺伝子蛋白質解析室、4富山大学大学
院免疫バイオ・創薬探索研究講座、5富山県薬事研究所、6独立行政法人国立長寿医
療研究センター加齢健康脳科学研究部
東京医科歯科大学大学院医歯学総合研究科先進倫理医科学開発学
大坂 瑞子、萩田 澄彦、吉田 雅幸
How to write
Plenary Lecture
Nature Medicine
山本 誠士1、東 英梨月1,2、村松 昌3、柳橋 努4、生谷 尚士4、長井 良憲4、
高津 聖志4,5、渡邉 淳6、堂本 光子2、新飯田 俊平3、服部 裕一1
134
Special Talk
Statin has anti-inflammation effect by reducing the
oxidant stress of leukocytes and the vessel wall .
Special
Lecture
133
President
Lecture
脳血管内皮細胞株を用いたendothelial microparticles
(EMP) のin vitro産生系の確立およびin vivo病態モデル
での解析
近年、低酸素応答性転写因子Hypoxia Inducible Factor (HIF)-1㱍が、低酸素
ストレスに対して、血管新生や細胞増殖、糖代謝、pH調節や細胞アポトー
シスなどに関与する様々な遺伝子の発現制御を介して、生体の恒常性を維
持する上で重要な役割を担っていることがわかってきた。例えば、エリス
ロポエチンや血管内皮増殖因子（VEGF）
、アドレノメデュリン、マトリッ
クスメタロプロテアーゼ（MMPs）、エンドセリン（ET）-1、一酸化窒素合
成酵素（NOS）2などの遺伝子発現を制御することが報告されている。
一方、動脈硬化の病態進展機序には局所の炎症が深く関与しており、と
りわけT細胞の活性化がその炎症進展の中心的な役割を担うことがわかっ
てきた。また、動脈硬化血管局所では、酸素飽和度の低下による低酸素環
境であることが報告されている。そこで、動脈硬化において血管局所の低
酸素環境がT細胞の免疫応答機能にもたらす影響を明らかにするために、T
細胞特異的にHIF-1㱍の遺伝子欠失マウスにおいて大腿動脈カフ障害性の血
管リモデリングモデルを作製してT細胞を介する免疫応答について検討し
た。
本変異マウスでは、野生型と同様に生育するが、末梢T細胞数の増加が観
られた。これらHIF-1㱍欠失T細胞の初期の活性化は障害されていなかった。
興味深いことには、本変異マウスにおける動脈硬化モデルでは、対照群に
比較して内膜増生の増強と外膜周囲の炎症細胞の浸潤の増強が観察された。
また、低酸素不応答性のHIF-1㱍欠失T細胞では、野生型T細胞に比較して
炎症性サイトカインの強発現、IL-2産生および末梢T細胞の増殖の増強が観
察された。更に、特異抗原による抗体産生応答についても対照群に比較し
て増強していることが観察された。
以上の結果より、本動脈硬化モデルにおけるT細胞のHIF-1㱍は、動脈硬
化に伴う炎症進展過程に抑制的に機能している可能性が示唆された。
黒部 裕嗣1、冨田 修平2、高浜 洋介3、北川 哲也1、玉置 俊晃2
徳富 文彬、高橋 聡子、高橋 俊介、杉山 秀太、李 昌一
【目的】歯周病と循環器系疾患との関連性を測定可能にするために，我々は
四肢の血流の反応性充血を測定するプレチスモグラフィーを口腔循環系に
適応することに成功した。そしてさらに我々は、歯周病原因菌を播種させ
たラットの口腔内反応性充血を測定した結果、歯周病感染モデルにおいて
血管内皮機能の低下を認めた。そこで、今回我々は歯周病が循環器系疾患
に影響を与えるメカニズムを解明するため、脳卒中易発症性高血圧自然発
症ラット (Stroke-Prone Spontaneously Hypertensive Rat: SHRSP)の歯肉微小血
管血流量を経日的に測定し、本モデルによる口腔内循環調節機能について
検 討 し た。
【 方 法 】SHRSP(6週 齢 〜24週 齢)を 用 い、 体 重 測 定 後、sodium
pentobarbital (45 mg/kg) 麻酔下で、ラット口蓋歯肉を1分間圧迫し、解放後
の反応性充血をレーザードップラー血流計により測定した。測定項目は、
血圧、基底血流量 (basal) 、に加え最大反応 (peak) 、最大反応が 1/2 に低下
するのに要する時間 (T1/2) 、増加血流総量 (mass) の3つのパラメーターとし、
局 所 循 環 の 変 化 を 経 日 的 に 評 価 し た。 コ ン ト ロ ー ル(Wistar Kyoto Rat:
WKY6週齢〜24週齢) でも同様に評価を行った。
【結果および考察】WKYに
比べSHRSPの血圧は経日的に増加した。口蓋歯肉での反応性充血のpeakは
上昇し、T1/2、massは減少傾向であった。これはSHRSPの口腔内循環調節機
能の変化と、それに伴う口腔内微小循環血流量の低下を示している。以上
の結果から、歯周病感染モデルと同様にSHRSPにおいて、血管内皮機能変
調による歯肉微小循環系の血流量低下が確認された。さらに、今後は歯周
病原因菌を播種させたSHRSPにおける口腔内循環調節機能を検討していき
たい。
血管リモデリングに対してT細胞のHIF-1αは抑制的に機
能する
132
口腔循環系における脳卒中易発症性高血圧自然発症ラッ
ト(SHRSP)の反応性充血の解析
131
ポスターセッション：血管炎症
（2）
Poster Session：Vascular Inflammation(2)
12月2日
（木）
／Thursday, December 2
Program
Aⴊ▤↢‛කቇળKPFD
198
Background: Though inflammation within the adipose tissues thought to play a role
in metabolic syndrome, a causative connection between inflamed adipose tissue and
atherosclerosis is not fully understood. The present study aimed to examine the
direct effect of adipose tissue on macro vascular inflammation by intravital
microscopic analysis of the femoral artery of mouse after perivascular adipose
tissue transplantation. Methods and Results 0.05g of subcutaneous (SQ) or visceral
(VIS) adipose tissue harvested from C57BL/6 mice was transplanted to the
perivascular area of the femoral artery of recipient C57/BL6 mice. A quantitative
intravital microscopic analysis was performed on the artery. The number of
adherent leukocyte was increased after adipose tissue transplantation in a timedependent manner (1day to 7 days). VIS transplantation induced significantly more
leukocyte accumulation than SQ. Simultaneous flow cytometry showed the
expression of CD11b on peripheral neutrophils and monocytes was significantly
increased after adipose tissue transplantation with prominent induction with VIS
compared to SQ. Induction of oxidative stress in leukocyte was observed after
transplantation. Moreover, the expression levels of MCP-1 and MIP-1b were
significantly increased in the adipose tissues as well as in the serum of the recipient
mice. Conclusion These data indicate that adipose tissue directly stimulates atheroprone vascular inflammation by inducing leukocyte accumulation. The underlying
mechanisms seem to involve induction of MCP-1 and MIP-1b
東京医科歯科大学大学院医歯学総合研究科血流制御内科学、2東京医科歯科大学大
学院医歯学総合研究科先進倫理医科学開発学
1
萩田 澄彦1,2、大坂 瑞子2、下門 顕太郎1、吉田 雅幸2
Direct observation of adipose tissue-induced leukocyte
recruitment to the artery wall via MCP-1 and MIP-1b
135
18：00〜
1
国立循環器病研究センター研究所細胞生物学部、2Korea Advanced Institute of
Science and Technology
Angiopoientin-1 (Ang1)/Tie2 signal regulates both vascular quiescence and
angiogenesis. Previously, we reported that Ang1 assembles distinct Tie2 signaling
complexes in the presence or absence of cell-cell contacts, thereby regulating these
distinct functions. In the presence of cell-cell contacts, Ang1 induces transassociation of Tie2, which actives phosphatidylinositol 3-kinase/Akt pathway and
upregulates the genes involved in vascular quiescence which include a Notch
ligand, delta-like 4 (Dll4). Dll4/Notch signal is known to restrict angiogenesis.
Thus, we investigated the mechanism how Ang1 upregulates Dll4 to clarify the role
of Dll4/Notch signal in trans-associated Tie2-mediated vascular quiescence. Ang1
upregulates Dll4 and increased notch intracellular domain (NICD) only under
confluent cells. Ang1 stimulated the transcriptional activity of 㱎-catenin (㱎-cat)
via Akt-mediated phosphorylation of glycogen synthase kinase 3㱎 (GSK3㱎). A
GSK3㱎 inhibitor induced Dll4 expression, while depletion of 㱎-cat by siRNA
blocked either Ang1- or GSK3㱎 inhibitor-induced Dll4 expression. In addition,
GSK3㱎 inhibitor-induced Dll4 expression depended on the cell-cell junctions and
was impeded by DAPT, a 㱏-secretase inhibitor, implying the requirement of Notch
signaling in b-cat-dependent Dll4 expression. Consistently, NICD promoted Dll4
expression through RBP-J binding site in the third intron. Active mutant of 㱎-cat
potentiated NICD-dependent transcription of Dll4. These findings indicate that
trans-associated Tie2 activates transcriptional activity of 㱎-cat via Akt-mediated
inhibition of GSK3㱎, which results in the expression of Dll4 cooperatively with
Notch signaling. Dll4 upregulated by trans-associated Tie2 triggers Notch signaling,
which may contribute to vascular quiescence.
1
大阪大学大学院医学系研究科臨床遺伝子治療学、2大阪大学大学院医学系研究科老
年腎臓内科学
Fibrosis is one of the common disorders, which is attributed to excess deposition of
extracellular matrix components, however the mechanism remains unclear. Recently
it is reported that myofibroblasts are primarily responsible for cardiac fibrosis
through cytokine and growth factor-dependent mechanism. Myofibroblasts are
differentiated from resident fibroblasts and circulating fibroblast-like cells called
fibrocytes derived from bone-marrow stem cells. On the other hand, vascular
endothelial cells can be transformed into myofibroblasts, well known as End-MT,
which is intimately associated with perivascular fibrosis. As previous studies have
shown that HGF attenuated tissue fibrosis through inhibiting TGF-㱎 expression,
HGF has not only strong angiogenetic effect but antifibrotic effect independently.
We hypothesized that HGF contributes to prevent cardiac fibrosis through
suppressing proliferation of myofibroblasts and End-MT. We analyzed pressure
overloaded HGF-Tg mouse to clarify how HGF works as an antifibrotic factor and
establish a new antifibrotic therapy for the patients suffering from heart failure.
HGF inhibited differentiation of HCAECs (human coronary artery endothelial cells)
into myofibroblasts induced by TGF-㱎 and suppressed proliferation of
myofibroblasts in vitro. In vivo study, mRNA expression of collagen I/III and TGF㱎 was significantly lower than that of non-Tg groups. Fibrotic area in the heart
decreased especially in perivascular region in HGF-Tg operated mice accompanied
by the reduction of myofibroblasts. In echocardiography, both FS and LVDD were
preserved compared with banded littermates and mortality rate after operation
reduced as well. We concluded HGF reduces cardiac fibrosis in terms of inhibiting
End-MT and proliferation of myofibroblasts.
Index
Aⴊ▤↢‛කቇળKPFD
Poster
199
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
アルドステロンの様々な心血管への直接作用が報告されている。アルドス
テロンには遺伝子発現を介する古典的ゲノム作用の他、刺激後数分で作用
発現する非ゲノム作用が存在し共に病態に関与している。非ゲノム作用の
分子機構の詳細は不明である。ミネラルコルチコイド受容体(MR)は細胞質
に お い て はHSP90と 結 合 し そ の 立 体 構 造 が 安 定 に 保 た れ て い る。 一 方
Caveolin-1はカベオラの基盤となる蛋白質でありシグナル伝達の制御に関与
する。今回我々は血管平滑筋細胞でのアルドステロンの非ゲノム作用の発
現におけるHSP90/MR/Tyrosine-kinaseおよびcaveolin-1の果たす重要な役割を
見出した。
【方法】マウスの大動脈より血管平滑筋細胞（VSMC）を培養し
アルドステロン（100nM）で刺激し、蛋白解析、蛋白相互作用を解析した。
【成績】１）WT-VSMCにおいてアルドステロン刺激後、5分以内にMRと
HSP90の結合が促進し、ひきつづいてHSP90をscaffoldingとしてPyk2，Srcが
結合、相互に活性化した。ERK1/2，JNKなどのMAPKsも早期に活性化され
た。MR阻害薬の同時処理で上記蛋白結合は抑制され、チロシンキナーゼ、
MAPKsの 活 性 化 も 抑 制 さ れ た。 ２）Srcの 阻 害 剤、Pyk2の 欠 損、HSP90SiRNA処理により、MAPKsの活性化は抑制された。３）免疫染色において
アルドステロン刺激後早期にHSP90/MR/Src/Pyk2が細胞膜で共局在した。５)
アルドステロン刺激後早期に、caveolin-1とHSP90,及びMRの結合が促進し
た。６）免疫染色においてアルドステロン刺激後細胞膜においてcaveolin-1
とHSP90,MRが共局在した。７）caveolin-1のSiRNA処理により、アルドス
テロン刺激後のSrc/Pyk2, MAPKsの活性化が抑制された。
【結論】血管平滑
筋細胞では、アルドステロンにより、数分以内の早期にカベオラにおいて
HSP90/MR/tyrosinekinaseがcomplexを形成し活性化され下流にシグナルを伝
えると考えられた。
Symposium
血管新生は既存の血管から新たな血管枝が分岐して血管網を構築する生理
的現象であり、成体においては創傷治癒等の過程で生じることが知られて
いる。また慢性炎症、網膜症及び、悪性腫瘍等の進展においても重要な役
割を担っていることが明らかになっている。現在、血管内皮細胞増殖因子
(VEGF)に対する中和抗体等を用いた抗血管新生治療が網膜症及び、悪性腫
瘍において臨床で使用されている。しかしながら抗血管新生治療は当初予
測されていたほど強い効果をしめさず、消化管出血等の強い副作用を生じ
ることも報告されている。またVEGF中和抗体に抵抗性を示す症例も少なか
らず存在することから、より効果が高く副作用の少ない治療標的の同定が
望まれている。このようなことから、血管新生のさらなる詳細な分子機構
を解明することは重要である。 ZF50は本研究室にて血管内皮細胞において
VEGFにより発現誘導される新規血管新生制御因子として同定された。誘導
されたZF50はE3ユビキチンリガーゼと複合体を形成し、CBF1をポリユビ
キチン化することにより分解する。その結果、Notch signalを負に制御し管
新生を誘導することを見いだした (Ohnuki et. al.)。 ZF50は血管新生におい
て非常に重要な機能を有することから、その発現制御のメカニズムを解明
できれば新規治療・診断標的につながる成果が期待される。本研究におい
て我々は、血管内皮細胞におけるVEGF刺激での ZF50 発現調整のメカニズ
ムに着目し研究を行った。まずVEGF受容体の主要な下流シグナルにおいて
活性化されるキナーゼに対する阻害剤で処理した血管内皮細胞における
ZF50の発現の変化を解析し、その発現に重要なキナーゼを同定した。次に
ZF50の発現誘導が転写活性の変化によるものではなかったことから、転写
後のmRNAの制御について検討した。以上の結果より内皮細胞において新
規の血管新生制御因子のレベル調節機構が存在する事を見いだしたので報
告する。
京都府立医科大学循環器内科
山口 真一郎、沖垣 光彦、松原 弘明
1
愛媛大学大学院医学系研究科生化学・分子遺伝学分野、2富士レビオ株式会社研究
開発部門研究推進部バイオ研究グループ、3愛媛大学プロテオ医学研究センター細
胞増殖・腫瘍制御部門
三輪 大輔1,2、井上 博文1,3、東山 繁樹1,3
How to write
Plenary Lecture
Nature Medicine
血管平滑筋細胞におけるアルドステロンの非ゲノム作用
のカベオリンを基盤とした分子機構の解明
Special Talk
新規血管新生制御因子ZF50の発現制御機構の解析
Special
Lecture
139
Jianghui Zhang1、福原 茂朋1、迫 圭介1、Gou Young Koh2、
望月 直樹1
岡山 慶太1、谷山 義明1,2、堂阪 宜雄1,2、東 純哉1,2、家串 和真1,2、眞田 文博1、
楠 博1,2、岩林 正明1、楽木 宏実2、森下 竜一1
President
Lecture
138
Trans-associated Tie2 regulates vascular quiescence
through β-catenin-mediated upregulation of delta-like
4
137
Prevention of End-MT and Myofibloblast
Proliferation Is a Therapeutic Target For Cardiac
Fibrosis
136
ポスターセッション：血管シグナル伝達（2）
Poster Session：Vascular Signal Transduction(2)
12月2日
（木）
／Thursday, December 2
Program
Aⴊ▤↢‛කቇળKPFD
200
Background: We have demonstrated in animals and humans that endotheliumderived H 2 O 2 is an endothelium-derived hyperpolarizing factor (EDHF) and that
eNOS-derived superoxide anions is a major precursor of EDHF/H2O2. We also have
demonstrated in mice deficient of the whole NOSs system that endothelial NOSs
system plays diverse roles depending on vessel size, mainly serving as a superoxide
generating system to cause EDHF/H2O2 responses in resistance arteries (mesenteric
arteries, MA) while serving as a NO generating system in conduit arteries (the
aorta, Ao). In this study, we aimed to elucidate the mechanisms involved in the
diverse roles of endothelial NOSs system in mice. Methods and Results: We used
male wild-type mice. Since we previously reported that pathological eNOS
uncoupling is not the mechanism for the divergent roles of eNOS between Ao and
MA, we further examined this point. The extent of eNOS phosphorylation at
Ser1177 was significantly greater in Ao than in MA, whereas that at Thr495 was
significantly greater in MA than in Ao. STO-609, an inhibitor of CaMKK㱎 that
lies upstream of eNOS Ser1177 phosphorylation pathway, significantly decreased
EDHF-mediated relaxations and hyperpolarizations to acetylcoline (ACh) in MA
but not in Ao. In response to ACh, eNOS Ser1177 phosphorylation was enhanced in
both MA and Ao, which was inhibited by STO-609 in MA more than in Ao. The
AMPK inhibitor compound C and the PI3K inhibitor wortmannin had no effects on
EDHF-mediated relaxations or hyperpolarizations to ACh in MA and Ao.
Conclusions: These results suggest that eNOS activity is significantly attenuated in
MA and that CaMKK㱎 plays a key role for the diverse functions of endothelial
NOSs system between conduit and resistance arteries.
東北大学大学院医学系研究科循環器病態学
大橋 潤子、澤田 鮎子、高木 文、野田 一樹、中嶋 壮太、細谷 真紀、
下川 宏明
CaMKKβ Plays a Key Role for the Diverse Functions
of eNOS between Conduit and Resistance Arteries in
Mice
140
Index
Aⴊ▤↢‛කቇળKPFD
Poster
201
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
【目的】高齢化社会においては、種々の疾患が複雑に絡みあい病態が多様化
する中で共通の病因背景を共有する場合も少なくなく、包括的な治療が望
まれている。高血圧と骨粗鬆症においては、交感神経刺激が骨吸収を促進
することが報告され、またβblockerが骨折頻度を減少させるという疫学解
析がなされている。しかしながら従来のCa拮抗薬が骨代謝を改善したとい
う疫学報告はみられない。シルニジピンは、従来のL型Ca Channelに加えて
N型Ca Channelの阻害作用を持ち合わせており、交感神経終末からのノルア
ドレナリンの放出を抑制することから、
骨代謝における効果を検証した。
【方
法】本実験では、高血圧自然発症ラットSHRに卵巣摘出術を施して高血圧・
骨粗鬆症誘発ラットを作製し、シルニジピンおよび対照としてアムロジピ
ンを経口投与して一ヶ月後の骨代謝マーカーおよび膝関節頚骨近位部での
骨稜解析を行った。【成績】血清Ca、Pについては各群間で有意差は認めな
かったものの、尿中Deoxypyridinoline は、シルニジピン、アムロジピンと
も非経口投与群に対して有意に減少していた。骨形成と骨吸収バランスを
指標とした組織ALP/TRAP比はシルニジピンにおいて有意に上昇しており、
組織中の破骨細胞数も有意に減少していた。またDEXAを用いた骨量解析
においてもシルニジピンは卵巣摘出で誘発される骨量の減少を有意に抑制
して骨粗鬆症の進展を阻害していた。
【結論】Ca拮抗薬シルニジピンは、そ
のN型Ca Channelの阻害作用により骨吸収抑制効果を兼ね備えていることが
判明し、特に閉経後骨粗鬆症を併せる高血圧患者への投与選択において有
用であると考えられる。
1
大阪大学大学院医学系研究科老年腎臓内科、2大阪大学大学院医学系研究科臨床遺
伝子治療
志水 秀郎1、安政 奈都記2、中神 啓徳2、楽木 宏実1、森下 竜一2
Symposium
To study endothelial injury in vitro, we established a three-dimensional (3-D) blood
vessel model in which human umbilical vein endothelial cells were grown in the
presence of basic fibroblast growth factor and vascular endothelial growth factor.
We then performed comparative studies on cisplatin (cis-platinum-diamminedichloride, CDDP)-induced endothelial injury in 3-D and monolayer cultures. In
3-D culture, CDDP induced cell death and tube breakdown without DNA damage,
whereas CDDP induced apoptosis accompanied by DNA damage in monolayer
culture. CDDP also induced caspase-3 activation in a concentration-dependent
manner in both cultures. A broad-spectrum caspase inhibitor, zVAD-fmk, failed to
prevent CDDP-induced cell death and tube breakdown in 3-D culture, whereas
zVAD-fmk suppressed CDDP-induced apoptosis in monolayer culture. A calpain
inhibitor, MDL28170, attenuated CDDP-induced cell death and tube breakdown in
3-D culture, but not apoptosis in monolayer culture. These results showed that
calpain is involved in CDDP-induced endothelial injury in 3-D culture and there are
significant differences in signaling pathways between 3-D and monolayer cultures.
兵庫医科大学胸部腫瘍学講座、2兵庫医科大学がんセンター、3静岡県立大学食品栄
養科学部、4武庫川女子大学国際健康開発研究所、5兵庫医科大学内科学呼吸器内科
1
江口 良二1、藤盛 好啓1,2、太田 敏郎3、国政 和宏4、中野 孝司1,2,5
144
How to write
Plenary Lecture
Nature Medicine
Ca拮抗薬シルニジピンのN型Caチャネル阻害作用による
骨吸収抑制効果について
Special Talk
143
Special
Lecture
Calpain is involved in cisplatin-induced endothelial
injury in an
three-dimensional blood vessel
model
【Objective】
In Japanese diet, coffee contributes to a large share of the consumption of
polyphenols, as antioxidants. Since endothelial dysfunction play an important role
in atherosclerosis, we investigated the effect of coffee and polyphenols included in
coffee on endothelial function.
【Method&Results】
First, we evaluated the effect of coffee on endothelial function in vivo. Twelve
healthy men (age 45±2 years, BMI 23.9±0.9 kg/m2) were recruited to take a cup
of decaffeinated coffee (polyphenol concentration 2.5 g). The endothelial function
was evaluated by flow-mediated dilatation (FMD) before and 30, 60, 120 min after
ingestion of coffee. Coffee ingestion significantly increased FMD at 30 min (p＜
0.05) when compared to baseline. Next, to understand molecular mechanisms, we
measured the direct effect of polyphenols included in coffee; chlorogenic acid
(CGA), caffeic acid (CA), and ferulic acid (FA), on the expression of adhesion
molecules in activated HUVECs in vitro. HUVECs were pretreated with CGA, CA,
and FA for 20 hr at 25㱘M. CGA, CA, and FA significantly inhibited the TNF-㱍
-induced expression of VCAM-1, ICAM-1, and E-selectin in HUVEC. These
polyphenols also significantly inhibited adhesion of THP-1 cells to HUVECs under
flow.
【Conclusion】
These results suggested that coffee polyphenol improve endothelial function via
stabilizing inflammation.
1
東京医科歯科大学先進倫理医科学開発学分野、2お茶の水女子大学生活環境教育研
究センター、3ネスレ日本株式会社
豊崎 美紅1,2、谷 真理子2、岸本 良美2、福島 洋一3、大坂 瑞子1、萩田 澄彦1、
近藤 和雄2、吉田 雅幸1
The effect of coffee polyphenol on endothelial
function
142
18：00〜
President
Lecture
Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor
(HGF) are strong neurotrophic factors, which function as antiapoptotic factors.
However, the neuroprotective effect of GDNF and HGF in ameliorating ischemic
brain injury via an antiautophagic effect has not been examined. Therefore, we
investigated GDNF and HGF for changes of infarct size and antiapoptotic and
antiautophagic effects after transient middle cerebral artery occlusion (tMCAO) in
rats. For the estimation of ischemic brain injury, the infarct size was calculated at
24 hr after tMCAO by HE staining. Terminal deoxynucleotidyl transferasemediated dUTP-biotin in situ nick end labeling (TUNEL) was performed for
evaluating the antiapoptotic effect. Western blot analysis of microtubule-associated
protein 1 light chain 3 (LC3) and immunofluorescence analysis of LC3 and
phosphorylated mTOR/Ser(2448) (p-mTOR) were performed for evaluating the
antiautophagic effect. GDNF and HGF significantly reduced infarct size after
cerebral ischemia. The amounts of LC3-I plus LC3-II (relative to beta-tubulin) were
significantly increased after tMCAO, and GDNF and HGF significantly decreased
them. GDNF and HGF significantly increased p-mTOR-positive cells. GDNF and
HGF significantly decreased the numbers of TUNEL-, LC3-, and LC3/TUNEL
double-positive cells. LC3/TUNEL double-positive cells accounted for about 34.3%
of LC3 plus TUNEL-positive cells. This study suggests that the protective effects of
GDNF and HGF were greatly associated with not only the antiapoptotic but also the
antiautophagic effects; maybe two types of cell death can occur in the same cell at
the same time, and GDNF and HGF are capable of ameliorating these two pathways
岡山大学医学部神経内科
阿部 康二
Antiapoptotic and antiautophagic effects of GDNF
and HGF after transient middle cerebral artery
occlusion in rats.
141
ポスターセッション：その他
（4)
Poster Session：Others(4)
12月2日
（木）
／Thursday, December 2
Program
Aⴊ▤↢‛කቇળKPFD
202
【目的】高齢化社会においては、種々の疾患が複雑に絡みあい病態が多様化
する中で、共通の病因背景を共有する場合も少なくなく、包括的な治療が
望まれている。高血圧と骨粗鬆症においては、以前よりサイアザイド系利
尿剤（TD）が骨折頻度を減少させることが報告されている。また近年、
我々
はアンジオテンシンIIが骨芽細胞を介して破骨細胞を活性化させる事、そ
してACEIやARBが骨粗鬆症を改善することをみいだした。今回、低用量
TDとARBの併用の骨代謝における作用をＡＲＢ単独使用効果と比較検証し
たので報告する。
【方法】生後１０週齢の高血圧自然発症ラットSHRに卵巣
摘出術を施して高血圧・骨粗鬆症誘発ラットを作製し、ARB（Valsartan;
1mg or 3mg/kg/d）単独群および低用量TD（0.2mg/kg/d）+ ARB（1mg/kg/d）
併用群とに分けて経口投与し、１ヶ月後の骨代謝マーカー、レニン活性お
よび膝関節頚骨近位部での骨梁解析を行った。【結果】血清Ca、Pについて
は各群間で有意差は認めなかった。TD単独投与もしくは低用量ARB（1mg/
kg/d）単独投与では明らかな骨代謝改善効果を認めなかった。しかし、高
用量ARB（3mg/kg/d）もしくは低用量ARB（1mg/kg/d）+ TD (0.2mg/kg/d)併
用群は、尿中deoxypyridinoline を有意に低下させ、また骨形成と骨吸収バラ
ンスを指標とした組織ALP/TRAP比においても両群において有意な上昇を
みた。さらにDEXAにおいても両群において有意に骨量の温存が認められ
た。しかしながらレニン活性においては、低用量併用群が高用量ARBと比
較して有意な抑制を認めた。
【考察】低用量のTD、ARB併用群は、高用量
ARB単独投与と同等以上に骨吸収抑制効果が認められた。レニン活性が低
用量併用群で有意に抑制されていることを考慮すれば臓器保護の側面から
も低用量併用群の使用が望ましいと考えられる。
1
大阪大学大学院医学系研究科老年腎臓内科、2大阪大学大学院医学系研究科臨床遺
伝子治療
志水 秀郎1、安政 奈都記2、中神 啓徳2、楽木 宏実1、森下 竜一2
低用量の利尿剤とＡＲＢ併用はＲＡＳ活性を抑制して骨
代謝を改善する
145
17：30〜
Index
Aⴊ▤↢‛කቇળKPFD
Poster
203
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Aims- Endothelial activation and dysfunction has been implicated in the
progression of chronic heart failure and development of pulmonary edema.
Resveratrol trans-3,4,5-trihydroxystiene have been suggested to have antioxidative
and anti-inflammatory effects. In the present study we will investigate the effect of
resveratrol, a phytopolyphenol isolated from the seeds and skins of grapes, on
endothelial activation in patients with CHF Methods-A total of 20 patients with
advanced CHF was included in the study. Patients will be included if they have
functional class III or IV symptoms of CHF and a left ventricular ejection fraction
of <35% by left ventriculography with radionuclide or contrast medium. Twenty
volunteers will provide blood samples for use as a control. We will determine
whether the increased expression of intercellular adhesion molecule-1 and vascular
adhesion molecule-1 , markers of endothelial activation, elicited by sera obtained
from patients with advanced CHF in cultured human aortic endothelial cells can be
modified by resveratrol. Human aortic endothelial cells be treated with 70% sera
obtained from patients with CHF and normal individuals, respectively, with or
without pretreatments with resveratrol. The endothelial expression of intercellular
adhesion molecule-1 and vascular adhesion molecule-1 will be confirmed by
mRNA expression and Western blot. Results-The stimulation of culturedhuman
aortic endothelial cells with sera obtained from CHF patients significantly increase
expression of vascular adhesion molecule-1Pretreatment of the human aortic
endothelial cells resveratrol can significantly suppreess vascular adhesion
molecule-1 expression Conclusions-The findings of this study highlight the
Resveratrol may have potential in treating CHF patients
The department of cardiology,Cheng Hsin General Hospital
National Research Laboratory for Cardiovascular Stem Cells, Seoul
national university hospital, Seoul, Korea
Symposium
Increased vascular permeability is the main pathology for myocardial damage after
ischemia/reperfusion. Angiopoietin-1 has been known as an endothelial specificvascular stabilizing factor. However, the effects of angiopoietin-1 on
cardiomyocytes and vascular cells following myocardial infarction are ill-defined
yet. Here, we propose a mechanism whereby angiopoietin-1 increases the integrity
of the endothelial lining and it exerts a direct anti-apoptotic effect on
cardiomyocytes. First, we found that angiopoietin-1 reduced vascular leakage
through regulating VE-cadherin phosphorylation. The membrane expression of VEcadherin was markedly decreased on hypoxia/reoxygenation, which was recovered
by angiopoietin-1. Interestingly, these effects were mediated by the facilitated
binding between SHP2 or PTPu tyrosine phosphatase and VE-cadherin, leading to
its de-phosphorylation. siRNA against SHP2 or PTPu abolished the effect of
angiopoietin-1 on VE-cadherin de-phosphorylation, and therebydecreased levels of
membrane-localized VE-cadherin. Second, angiopoietin-1 protected
cardiomyocytes lacking angiopoietin-1 receptor Tie2, via integrin-b1.
Angiopoietin-1 decreased cardiomyocytes apoptosis through ERK phosphorylation
and ERK-dependent decrease of active caspase-9 leading to reduction of active
caspase-3. Neutralizing antibody against integrin-b1 blocked these effects of
angiopoietin-1 on cardiomyocytes. In mouse myocardial infarction model,
angiopoietin-1 enhanced cardiac function. Collectively, our findings suggest that
angiopoietin-1 has cardioprotective effects which are achieved by reductions in
both vascular leakage and cardiomyocyte apoptosis after ischemia/reperfusion.
Jen Hsu-Lung、Yin Wei Hsian、Huang Wen Pin
Joo-Yun Won、Sae-Won Lee、Soo-Kyung Kang、Hyo-Soo Kim
How to write
Plenary Lecture
Nature Medicine
The Effect of Resveratrol on Endothelial Activation
in Patients with Chronic Heart Failure
Special Talk
Angiopoietin-1 as a cardiotropic factor in myocardial
infarction
Special
Lecture
E4
We investigated the possible cerebrovascular dysfunction response to isoproterenol
induced cardiac hypertrophy (ISO-CH) in rabbit cerebral arteries (CAs). Non
hypertensive cardiac hypertrophy was induced in male New Zealand white rabbit
by 7 days isoproterenol injection (300 microgram/kg/day). We investigated the
alteration of protein expression in CAs of ISO-CH using two dimensional
electrophoresis (2DE) and Matrix Assisted Laser Desorption/Ionization - Time Of
Flight (MALDI -TOF) mass analysis (n=3). ROS generation (n=6) and followed
DNA damage (n=4) were assessed to evaluate deteriorative effect of ISO-CH on
CAs. Vascular response to vasoactive drugs, acetylcholine and angiotensin II, were
assessed to evaluate functional alteration of CAs in ISO-CH. Protein-protein
network analysis was applied to determine proteome based systemic changes of CA
in ISO-CH. Proteomic analysis revealed altered expression level of 30 proteins
including 28 down-regulated and 2 up-regulated proteins in CAs of ISO-CH.
Among the identified proteins, expression of cytoskeleton organizing proteins,
including actin related protein 1A and 2, alpha-actin, capping protein Z beta, and
vimentin were the most markedly down-regulated in ISO-hypertrophied CAs. In the
vascular response analysis, decreased transient Ca2+ efflux and constriction
response to angiotensin II were found in ISO-hypertrophied CAs. In addition,
highly increased superoxide generation and DNA damage were detected with
down-regulated anti oxidative stress proteins e.g. heat shock protein 9A and stressinduced-phosphoprotein 1 in ISO-hypertrophied CAs. We conclude that
disorganization of actin cytoskeletal network has a pivotal role in cerebrovascular
dysfunction under the cardiac hypertrophy condition.
Department of Physiology,University of Inje, Busan, Korea、2Kangwon
National University School of Medicine,Chuncheon 200-701, Korea
President
Lecture
E3
Factor Xa and thrombin are involved not only in blood coagulation but also in
migration of endothelial cells and vascular smooth muscle cells through activation
of pro-matrix metalloprotease (MMP)-2 followed by subsequent extracellular
matrix degradation. In the present work, we have explored the biochemical roles of
proteolytic function of factor Xa and thrombin that are associated with MMP-2
regulation for extracellular matrix alteration. This study demonstrates that factor Xa
is directly involved in the activation process of pro-MMP-2 in MT1-MMPindependent manner in human vascular endothelial cells and vascular smooth
muscle cells. More importantly, it was interesting to find that factor Xa and
thrombin are also able to degrade MMP-2 to regulate the metalloprotease activity.
Experimental evidence led us to hypothesize that factor Xa and thrombin regulate
the functional activity of MMP-2 by activation and degradation of the
metalloprotease
1
Hyoung Kyu Kim1、Won Sun Park2、Hye-Jin Heo1、Nari Kim1、
Jin Han1
Department of Biochemistry, College of Life Science and Biotechnology,
Yonsei University, Seoul, Korea
Bon-Hun Koo、Harim Jeon、Ok-Hee Jeon、○Doo-Sik Kim
Cerebrovascular dysfunction in isoproterenol-induced
cardiac hypertrophy involves cytoskeletal
disorganization
E2
MT1-MMP-independent Regulation of MMP-2 Activity
by Factor Xa and Thrombin
E1
Poster Session：Vascular Metabolism and heart failure
12月2日
（木）
／Thursday, December 2
Program
1
1
Aⴊ▤↢‛කቇળKPFD
204
It has been shown that elevation of intracellular cAMP inhibits inflammationassociated gene expression in vitro, as well as promotes cell survival and cardiac
function and contractility; however, the functional effect of cAMP on cardiac
allograft rejection, and its action mechanism were not clearly elucidated. This study
was designed to investigate the effects of cAMP on immune regulation and
apoptosis during acute rat cardiac allograft rejection. We found that the production
of immune markers such as inflammatory cytokines (IL-1㱎, IL-6, and TNF-㱍),
iNOS expression, and nitric oxide (NO) production, was significantly increased in
the blood and transplanted hearts of allograft recipients, but not of isograft controls.
These increases were effectively suppressed by the administration of the membrane
permeable cAMP analog dibutyryl cAMP (db-cAMP). Administration of db-cAMP
reduced allograft-induced elevation of several biochemical markers, such as
adhesion molecule expression, iron-nitrosyl complex formation, caspase-3
activation, and apoptotic DNA fragmentation in an animal model. Furthermore,
treatment of allograft recipients with db-cAMP prolonged median graft survival to
11 days compared with a median graft survival time of 8 days in saline-treated
allograft recipients. These results suggest that db-cAMP exerts a beneficial effect
on murine cardiac allograft survival by modulating allogeneic immune response
and cytotoxicity.
Background: Heart failure (HF) is associated with decreased cardiac contractility
and ventricular remodeling, features which are partially reversed by angiotensin
converting enzyme inhibitors (ACEi). Since Rho kinase (ROCK) mediates cellular
contraction through effects on the actin cytoskeleton, we hypothesize that ACEi
may improve heart function through inhibition of ROCK activity.
Methods and Results: We enrolled 30 consecutive HF subjects (average age: 52.7
± 8.2, 60% male in gender, NYHA class 3.2 ± 1.1), with impaired left ventricular
ejection fraction (LVEF) of less than 40% by echocardiography (average
LVEF=32.1 ± 8.2%), and age- and sex-matched 30 subjects with preserved LVEF
function (average LVEF=65.4 ± 9.4%) as the control group. ROCK activity was
determined in peripheral leukocytes. Compared with the control group, the subjects
with impaired LVEF have higher ROCK activity (0.83 ± 0.38 vs. 0.36 ± 0.23,
p=0.01). After correcting the risk factors between groups, we found a tendency
between the higher ROCK activity and reduced LVEF (R 2 =0.381, p=0.06).
Treatment with ACEi, enalapril, decreased ROCK activity (0.50 ± 0.33), improved
LVEF (40.4 ± 6.2%) and NYHA class (2.1 ± 1.3) (p<0.05 compared to baseline).
ROCK activity, LVEF, and NYHA class was unaffected by ACEi treatment in
control subjects. The improvement in LVEF and NYHA class in HF subjects
correlated with the reduction in ROCK activity (R 2 =0.35 and 0.31, respectively,
p=0.04 for both), but did not correlate with changes in high sensitivity-C reactive
protein, or the reduction in systolic blood pressure.
Conclusion: The ROCK activity is elevated in HF subjects and that improvements
in LVEF and NYHA class by ACEi are correlated with decrease in ROCK activity.
The reduction in ROCK activity by ACEi was independent of changes in
inflammation and blood pressure, suggesting that ROCK activity may be a novel
marker of HF and its response to therapy with ACEi.
Division of Cardiology, Internal Medicine, National Cheng Kung
University Hospital, Taiwan, Tainan、2Department of Biochemistry and
Molecular Biology, National Cheng Kung University, Taiwan, Tainan、
3
Vascular Medicine Research, Division of Cardiology, Brigham &
Womens Hospital, Harvard Medical School, Boston, MA, USA
Ping-Yen Liu1,3、Yun-Lin Tai1、Chi-Hsing Hsu1、Liang-Miin Tsai1、
Jyh-Hong Chen1、Hua-Lin Wu2、JK Liao3
Kwang-Soon Lee1、Kwon-Soo Ha1、Young-Geun Kwon2、
Young-Myeong Kim1
Vascular System Research Center and Department of Molecular and
Cellular Biochemistry School of Medicine, Kangwon National University,
Chuncheon, Kangwon-do, Korea、2Department of Biochemistry, College
of Sciences, Yonsei University, Seoul, Korea
Decreased Rho Kinase Activity by Angiotensin
Converting Enzyme Inhibitor is Associated with
Improved LV Function in Patients with HF
E6
Cyclic AMP prolongs graft survival by suppressing
apoptosis and inflammatory gene expression in acute
cardiac allograft rejection
E5
17：30〜
Index
Aⴊ▤↢‛කቇળKPFD
Poster
205
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
High recurrence and metastasis are hallmarks of urinary bladder cancer, yet the
relationship between lymphangiogenesis and lymphatic metastasis in bladder cancer
is not well known. Here, we identified a profound lymphangiogenesis in orthotopic
urinary bladder cancer (OUBC) mice, a model generated by intravesical
implantation of MBT-2 cells. OUBC mice displayed massive infiltration of
CD11b+/CD68+ tumor-associated macrophages (TAM) cells in primary tumor
mass and lymphatic metastasis to lymph nodes. Interestingly, TAMs were sources
of lymphangiogenic growth factors such as VEGF-C and VEGF-D. The blockade
of VEGF-C/D with soluble VEGF receptor-3 or the depletion of TAMs with
clodronate liposome markedly inhibited lymphangiogenesis and lymphatic
metastasis in OUBC. Our results indicate that VEGF-C/D are the main factors of
lymphangiogenesis and lymphatic metastasis in bladder cancer. Moreover, CD11b+/
CD68+ TAMs could be responsible for lymphangiogenesis and lymphatic
m e t a s t a s i s b y p r o d u c i n g V E G F - C / D . We b e l i e v e t h a t i n h i b i t i o n o f
lymphangiogenesis could be a possible strategy to prevent the recurrence of bladder
cancer after curative surgery or progression of bladder cancer during palliative
chemotherapy.
National Research Laboratory of Vascular Biology, Graduate School of
Medical Science and Engineering, Korea Advanced Institute of Science
and Technology, Daejeon, Korea
Hanseul Yang、○Chan Kim、Jeonyeob Jang、Kyuho Kim、
Gou Young Koh
Symposium
Objectives We sought to evaluate the role of a novel surrogate, the ratio of levels of
advanced glycation endproducts (AGE) to its scavenger, the soluble form of the
receptor of AGE (sRAGE), in the association of occurrence of albuminuria in
hypertension patients.Background Activation of the RAGE pathway triggers
oxidative stress. The engagement of RAGE by AGE activated key cell signaling
pathways such as mitogen- activated protein kinases and nuclear factor-㱖B.
Previous studies showed that AGEs are positively associated with arterial stiffness
and maybe a target for cardiovascular disease, whereas its scavenger receptors,
serving as a decoy, play a beneficial role. Thus, our study aims to elucidate the
relationship of the ratio of AGE to sRAGE and albuminuria in hypertensive
patients.Methods A total of 125 hypertensive patients were enrolled. Baseline
characteristics were collected. Univariate analysis to determine the possible factors
associated with albuminuria were analyzed. Multivariate analysis by cox-regression
was performed afterwards.Results Between the patients without and with
albuminuria, the target ratio became a candidate factor (3.96±3.07 vs. 5.66±5.90,
p=0.19). Other factors included diabetes, systolic blood pressure, triglyceride,
creatinine, and high sensitive C-reactive protein. The result of multivariate analysis
showed that diabetes, Cr, and the ration of AGE to sRAGE remained to be
significant factors associated with albuminuria in hypertension (p=0.02, 0.01, and
0.05, respectively).Conclusions The ratio of AGE to sRAGE may reflect oxidative
stress and serves as a possible prognostic surrogate for atherosclerosis by the
finding that it may be one of the major determinants for albuminuria in
hypertension.
Division of Cardiology, Hsin-Chu General Hospital, Department of
Health, Executive Yuan, R.O.C.、2Division of Cardiology, Department of
Medicine, Taipei Veterans General Hospital, Taiwan, R.O.C.、
3
Cardiovascular Research Center, National Yang-Ming University, Taipei,
Taiwan, R.O.C.
1
Kuang-Hsing Chiang1,3、Po-Hsun Huang2,3、Shao-Sung Huang3、
Jaw-Wen Chen2,3、Shin-Jong Lin2,3
E10
How to write
Plenary Lecture
Nature Medicine
Lymphangiogenesis and lymphatic metastasis in
urinary bladder cancer
Special Talk
E9
Cilostazol is a selective inhibitor of phosphodiesterase 3 that increases intracellular
cyclic AMP (cAMP) levels and activates protein kinase A, thereby inhibiting
vascular smooth muscle cell (VSMC) proliferation. We investigated whether AMPactivated protein kinase (AMPK) activation induced by Heme Oxygenase-1 (HO-1)
is a downstream mediator of the beneficial effects of cilostazol on VSMC and
whether cilostazol may prevent cell proliferation by activating AMP-activated
protein kinase (AMPK) in VSMC. In the present study, we examined that cilostazol
were treated with various concentrations in VSMCs. Treatment of cilostazol
increased HO-1 and phosphorylation of AMPK dose- and time-dependently.
Cilostazol also significantly decreased PDGF-induced VSMC proliferation by
activating AMPK induced by HO-1. Pharmacological and genetic inhibition of
HO-1 and AMPK blocked the cilostazol-induced inhibition of cell proliferation and
ROS production. These data suggested that cilostazol-induced HO-1 and AMPK
activation might attenuate PDGF-induced VSMC proliferation and ROS production.
Special
Lecture
The ratio of levels of advanced glycation endproducts
to its soluble form of receptor associated with
albuminuria in hypertensive patients
Department of Pharmacology, Aging-associated Vascular Disease
Research Center, College of Medicine, Yeungnam University、
Department of Cardiothoracic Surgery, Aging-associated Vascular
Disease Research Center, College of Medicine, Yeungnam University
2
President
Lecture
Lectin-like oxidized low-density lipoprotein (oxLDL) receptor 1 (LOX-1) is highly
expressed in atherosclerotic lesions and plays a major role in mediating oxLDLinduced vascular inflammation. Chlamydia pneumoniae has been found in
atherosclerotic lesions and is related to atherosclerotic pathogenesis, although its
specific mechanism remains unknown. This study was conducted to investigate the
regulatory mechanisms of LOX-1 expression in GroEL1 (a heat shock protein from
C. pneumoniae)-administered hypercholesterolemic rabbits and in GroEL1stimulated human coronary artery endothelial cells (HCAECs). In the
hypercholesterolemic rabbit model, GroEL1 administration enhanced fatty streak
and macrophage infiltration in atherosclerotic lesions, which may be mediated by
elevated LOX-1 expression. In the in vitro study using HCAECs, stimulation with
GroEL1 protein significantly increased toll-like receptor 4 (TLR4) and LOX-1
expression. Increased LOX-1 expression was downregulated by Akt activation and
PI3K-mediated endothelial nitric oxide synthase (eNOS) activation. PI3K inhibitor
(LY294002) and NOS inhibitor L-Ng-nitro-L-arginine methyl ester induced LOX-1
mRNA production, while the nitric oxide (NO) donor S-nitrosocysteine ameliorated
the increasing effect of LOX-1 mRNA in GroEL1-stimulated HCAECs. LOX-1
expression was regulated by nicotinamide adenine dinucleotide phosphate
(NADPH) oxidase (including Rac1), which mediates reactive oxygen species (ROS)
production and intracellular mitogen-activated protein kinase signaling pathway
activation in GroEL1-stimulated HCAECs. Treatment with polyethylene-glycolconjugated superoxide dismutase, apocynin, or diphenylene iodonium significantly
decreased GroEL1-induced LOX-1 expression, as did the knockdown of Rac1 gene
expression by RNA interference. Our results demonstrated that the GroEL1 protein
of C. pneumoniae may induce LOX-1 expression in endothelial cells, which
mediate formation of the fatty streak in atherogenesis. The elevated level of LOX-1
may be mediated by the PI3K/Akt signaling pathway, eNOS activation, NADPH
oxidase-mediated ROS production, and MAPKs activation in GroEL1-stimulated
HCAECs. The GroEL1 protein of C. pneumoniae may contribute to vascular
inflammation and cardiovascular disorders.
Department of Internal Medicine, Taipei Medical University and
Hospital, Taipei, Taiwan、2Department of Cardiovascular surgery, Taipei
Medical University and Hospital, Taipei, Taiwan、3Division of Cardiology,
Taipei Veterans General Hospital, Taipei, Taiwan
1
Jung Eun Kim1、Jin Young Sung1、Dong Hyup Lee2、
Hyoung Chul Choi1
Chun-Yao Huang1、Chun-Ming Shih1、Nai-Wen Tsao2、
Shing-Jong Lin3、Jaw-Wen Chen3、Po-Hsun Huang3、
Feng-Yen Lin3
1
Cilostazol inhibits vascular smooth muscle cell
proliferation through activation of AMP-activated
protein kinase and Heme Oxygenase-1
E8
GroEL1, a Hsp60 of C. pneumoniae, Induces LOX-1
Expression and Enhances Atherogenesis in
Hypercholesterolemic Rabbits
E7
Poster Session：Angiogenesis / Lymphangiogenesis
12月2日
（木）
／Thursday, December 2
Program
Aⴊ▤↢‛කቇળKPFD
206
Anthocyanins play an important role on physiological functions related to human
health. Previously, we isolated and purified cyanidin-3-O-sambubioside from the
extract of Acanthopanax sessiliflous fruit using high-performance liquid
chromatography (HPLC). The purpose of this study was to investigate inhibitory
effect of cyanidin-3-O-sambubioside on cancer cell metastasis. To examine the
inhibitory effect of cyanidin-3-O-sambubioside on angiogenesis, we assessed with
the chorioallantoic membrane (CAM) and tube formation assay. Cyanidin-3-Osambubioside interrupted the formation of neo-vasculature and tube-like structure
in a dose dependent manner. The Gelatin zymography revealed the cyanidin-3-Osambubioside significantly decreased the secretion of matrix metalloproteinase-9
(MMP-9). Cyanidin-3-O-sambubioside also down-regulated the expression of
MMP-9 and MMP-2 in human breast cancer cell line, MDA-MB-231. Moreover,
the treatment of MDA-MB-231 cells with cyanidin-3-O-sambubioside decreased
cell motility by wound-healing assay. It is reported that protein kinases C (PKCs),
mitogen-activated protein kinases (MAPKs), and nuclear factor-kappa B (NFkappaB) are involved in the regulation of MMP-9 in the processes of tumor cell
invasion and metastasis. Our research observed that were mainly focused on
signaling pathway of PKCs and NF-kappaB and around signals. Taken together,
these results suggest that cyanidin-3-O-sambubioside inhibits metastasis such as
angiogenesis and invasion through regulation of MMP-9 activity in breast cancer
cells.
Department of Genetic Engineering, Sungkyunkwan University, Suwon
440-746, Republic of Korea、2Research Laboratories, Kooksoondang
Brewery Co. Ltd., Seongnam 462-120, Republic of Korea、3College of
Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of
Korea、4Faculty of Food Science and Technology, Sejong University,
Seoul, Republic of Korea
1
Guwon Jeong1、Sang-Jin Lee1,2、Yong-Woo Jung1、
Jae Cheol Lee3、Jeong-Whan han3、Sang-Ho You4、
Gye-Won Kim2、Sungyoul Hong1
Effect of Cyanidin-3-O-Sambubioside from
Acanthopanax sessiliflorus Fruit on Angiogenesis
through Regulation of Matrix Metalloproteinase-9
E11
Transcriptional factor, Sox17, is one of the Sox family members which have a highmobility-group (HMG) box as a DNA binding domain. Sox17 has been known to
be important for the generation of various tissues such as definitive endoderm and
hematopoietic cells during development. Despite its importance in vascular
morphogenesis, the expression of Sox17 is unclear within vasculatures. During
early vascular development, Sox17 expression is exclusive to endothelial cells
within vasculature regardless of their subtypes based on our studies. Endothelial
specific Sox17 expression persists in the adult vasculature while its vascular
expression is gradually restricted to arterial endothelial cells. Interestingly, Sox17
expression is activated in adult endothelial cells when angiogenesis is activated by
tumor growth. Related with its expression, Sox17 deficiency induces vascular
changes in shape and function during vascular development and tumor
angiogenesis. Taken together, our study demonstrates that Sox17 plays a crucial
role in vascular morphogenesis as well as function.
KAIST, Daejeon, Korea
Seunghun Lee、Hanseul Yang、Gou Young Koh、Injune Kim
Endothelial Sox17 Regulates Vascular Morphogenesis
and Function
E12
17：30〜
Index
Aⴊ▤↢‛කቇળKPFD
Poster
207
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Symposium
Institute of Cellular and System Medicine, National Health Research
Institutes, Zhunan, Taiwan
Huei-Hsuan Cheng、Bo-Rui Chen、Wei-Chung Lin、
Kenneth K. Wu
How to write
Plenary Lecture
Nature Medicine
Cytokines and phorbol ester stimulate p300 HAT and
inflammatory gene expression in a cell cycledependent manner
Special Talk
p300 is a transcriptional coactivator which mediates the expression of a multitude
of genes sharing common promoter features through binding to DNA-bound
transactivators, interaction with core transcription factors and modification of
chromatin. It plays an important role in cell proliferation, transformation and
inflammation. p300 is a histone acetyltransferase (HAT) which acetylates histones
to modify chromatin structure and acetylates transactivators to enhance their
binding activity. p300 HAT is essential for transcription of diverse genes such as
cyclooxygenase (COX-2). Molecular analysis reveals that HAT domain is situated
at the carboxy-terminal half of the molecule which is blocked by an intramolecular
autoinhibitory element. Little is known about p300 HAT activation by exogenous
stimuli. Since the basal p300 HAT is repressed by intramolecular autoinhibition, we
reasoned that the pro-inflammatory mediators are capable of activating p300 HAT
and thereby stimulating gene transactivation. To address this, we evaluated the
effects of interleukin-1㱎 (IL-1㱎), tumor necrosis factor 㱍 (TNF㱍) and phorbol
12-myristate 13-acetate (PMA) on p300 HAT activation in human fibroblasts. The
results show a very low basal p300 HAT activity in serum-starved cells which was
increased by PMA, IL-1㱎 and TNF㱍. The stimulatory effects of the proinflammatory mediators were repressed after serum repletion. PMA-induced p300
HAT activation and COX-2 expression were blocked by rottlerin and PD98059 in
serum-starved quiescent fibroblasts but not in serum-repleted proliferative cells.
Similarly, they were inhibited by protein kinase C 㱐 (PKC㱐) small interference
(si) RNA in quiescent and not in proliferative cells. Direct measurement of PKC㱐
activity reveals that PKC㱐 was stimulated by PMA in quiescent cells but became
unresponsive to PMA in proliferative cells. PMA activated extracellular signal
regulated protein kinases (ERK) in quiescent but not in proliferative fibroblasts, and
ERK activation in quiescent cells was blocked by rottlerin and PKC㱐 siRNA,
consistent with signal transmission from PKC㱐 to ERK.
Special
Lecture
E15
Myocardin play a pivotal role in the regulation of cardiac and smooth muscle gene
expression, and transactivates serum response factor (SRF) in cardiac and smooth
muscle cell. We recently demonstrated that Enhancer of Polycomb (Epc1), a
chromatin protein is required for skeletal muscle differentiation by recruiting both
serum response factor (SRF) and p300 to the SRE of muscle-specific gene
promoters. Here we investigated that Epc1 recruits both Myocardin and SRF to
induce smooth muscle cell (SMC) differentiation. Using RT-PCR and
immunohistochemistry, we observed that expression of Epc1 was up-regulated the
rat carotid artery after balloon injury. Epc1 physically interacted with Myocardin in
mammalian cells. In addition, Epc1 is highly expressed in the mouse smooth
muscle tissue including bladder, lung and esophagus. We also carried out
environmental cues which actively regulate SMC phenotypic switching, such as
platelet-derived growth factor-BB (PDGF-BB); we infused recombinant PDGF-BB
into rat SMC. Our data indicate that PDGF-BB suppress expression of Epc1 as well
as SMC-specifc gene, such as smooth muscle (SM) alpha-actin and SM22alpha in
vitro. Transfection of Epc1 to SMCs poteintiated the myocardin-induced expression
of smooth muscle-specific genes. Moreover, Epc1 enhance physical interaction
between Myocardin and SRF. Epc1 enhances the binding of SRF induced by
myocardin in the SM22alpha promoter. These results suggest that Epc1 plays a role
in the initiation of smooth muscle differentiation/proliferation and its interaction
with myocardin modulates the expression of SRF-dependent smooth musclespecific genes.
Department of Pharmacology, Chonnam National University Medical
School, Gwangju, South Korea、2Medical Research Center of Gene
Regulation, Chonnam National University Medical School, Gwangju,
South Korea、3Department of Anatomy, Chonnam National University
Medical School, Gwangju, South Korea、4Heart Research Center,
Chonnam National University Hospital, Gwangju, South Korea
1
Yoojung Kim1,2,4、Hosouk Joung1,2、Ju-Ryoung Kim1,2、
Sera Shin2,4、Eun Mi Kim1,2、Jin Sook Kwon4、
Gwang Hyeon Eom1,2、Nakwon Choe1,2、Hae Jin Kee1,2,4、
Kwang-Il Nam3、Hyun Kook1,2,4
Epc1 regulates smooth muscle cell gene expression by
interacting with myocardin
E14
President
Lecture
Thymosin beta-4 has multi-functional roles in cell physiology, but little is known
about its mechanism(s) of action. We previously reported that thymosin beta-4
stimulated angiogenesis through the induction of vascular endothelial growth factor
(VEGF). To identify the mechanism of VEGF induction by thymosin beta-4, we
have used a luciferase assay system with VEGF in the 5ʼ promoter region. We also
analyzed the effect of thymosin beta-4 on VEGF mRNA stability and on the
expression and stability of hypoxia-inducible factor (HIF)-1 alpha. We found that
thymosin beta-4 induces VEGF expression by an increase in the stability of HIF1alpha protein. Analysis of the expression patterns of thymosin beta-4 and HIF1alpha in colon cancer tissue microarray showed that thymosin beta-4 and HIF-1
alpha co-localized in these biopsies. These data show that thymosin beta-4 induces
the expression of VEGF indirectly by increasing the protein stability of HIF-1
alpha.
Department of Parasitology and Genetics, Kosin University College of
Medicine, Busan, Rep of Korea,、2Department of Physiology, School of
Dentistry, Pusan National University, Busan, Rep of Korea、3Craniofacial
Developmental Biology and Regeneration Branch, National Institute of
Dental and Craniofacial Research, National Institutes of Health,
Bethesda, MD, USA
1
Jin-Ok Jo1、Su-Ryun Kim2、Moon-Kyung Bae2、Yun-Jeong Kang1、
Mee Sun Ock1、Hynda K Kleinman3、Hee-Jae Cha1
Thymosin beta-4 induces vascular endothelial growth
factor (VEGF) in normoxic condition by stabilizing
hypoxia-inducible factor (HIF)-1 alpha protein
E13
Poster Session：Transcriptional Regulation and regeneration
12月2日
（木）
／Thursday, December 2
Program
Aⴊ▤↢‛කቇળKPFD
208
Men with diabetic erectile dysfunction (ED) often have severe endothelial
dysfunction and respond poorly to oral phosphodiesterase-5 inhibitors. This study
was performed to examine the effectiveness of adipose tissue-derived stromal
vascular fraction (AD-SVF) in promoting cavernous endothelial regeneration and
restoring erectile function in diabetic animals. Eight-week-old C57BL/6 mice were
used and diabetes was induced by intraperitoneal injection of streptozotocin. ADSVF was isolated from epididymal adipose tissues of GFP transgenic mice. At 8
weeks after induction of diabetes, the animals were divided into 6 groups: controls,
diabetic mice, and diabetic mice treated with a single intracavernous injection of
PBS or AD-SVF (1 x 10 4 cells, 1 x 10 5 cells, or 3 x 105 cells/20 㱘l, respectively).
Two weeks later, erectile function was measured by cavernous nerve stimulation.
The penis was stained with antibodies to CD31, CD34, phosphohistone H3,
phospho-eNOS, and VEGF-A. Penis specimens from a separate group of animals
were used for cGMP quantification. The highest erectile response was noted in
diabetic mice at a concentration of 1 x 10 5 cells, which reached up to 82% of the
control values. Local delivery of AD-SVF significantly increased cavernous
endothelial proliferation, eNOS phosphorylation, and cGMP expression compared
with that in untreated and PBS-treated diabetic group. Intracavernous injection of
AD-SVF increased cavernous VEGF-A expression and induced recruitment of
CD34(+)CD31(-) progenitor cells. Some AD-SVF underwent differentiation into
cavernous endothelial cells. These findings suggest that AD-SVF induces recovery
of cavernous endothelial integrity and erectile function predominantly by provision
of trophic factors, and in part by direct incorporation and differentiation of injected
AD-SVF. The results support the concept of cavernous endothelial regeneration by
use of an AD-SVF as a curative therapy for diabetic ED.Supported by a grant of the
Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family
Affairs, Republic of Korea (Jun-Kyu Suh, A084511).
Laboratory of Regenerative Sexual Medicine and Department of
Urology, Inha University School of Medicine, Incheon, Korea、
2
Department of Biological Sciences and Laboratory for Vascular Biology,
Korea Advanced Institute of Science and Technology (KAIST), Daejeon,
Korea
1
Young Jun Koh、Gou Young Koh
Ji-Kan Ryu1、Munkhbayar Tumurbaatar1、Hai-Rong Jin1、
Woo Jean Kim1、Mi-Hye Kwon1、Shuguang Piao1、Min Ji Choi1、
Guo Nan Yin1、Kang-Moon Song1、Young Jun Koh2、
Gou Young Koh2、Jun-Kyu Suh1
Tremendous efforts have been made to establish effective therapeutic
neovasculaization using adipose tissue-derived stromal vascular fraction (SVF), but
the efficiency is low, and underlying mechanisms and their interaction with the host
in a new microenvironment are poorly understood. Here we demonstrate that direct
implantation of SVF derived from donor adipose tissue can create a profound
vascular network through the disassembly and reassembly of blood endothelial cells
(BECs) at the site of implantation. This neo-vasculature successfully established
connection with recipient blood vessels to form a functionally perfused circuit.
Addition of vascular growth factors to the SVF implant improved the efficiency of
functional neo-vasculature formation. In contrast, spheroid culture of SVF prior to
implantation reduced the capacity of vasculature formation, possibly caused by
cellular senescence or alteration. Implanting SVF into the mouse ischemic hindlimb
induced the robust formation of a local neo-vascular network and salvaged the limb.
Moreover, the co-implantation of SVF prevented fat absorption in the subcutaneous
adipose tissue graft model. However, allograft of SVF failed to achieve proper new
vasculature formation. Taken together, freshly isolated SVF can effectively induce
new vessel formation through the dynamic reassembly of BECs and could be
applied to achieve therapeutic neovascularization for relieving ischemia and
preventing fat absorption in an autologous manner.
Graduate School of Biomedical Science and Engineering, KAIST, Korea
Freshly Isolated Stromal Vascular Fraction from
Adipose Tissue Forms Profound Vascular Network
through the Dynamic Reassembly of Blood
Endothelial Cells
E17
Intracavernous Delivery of Adipose Tissue-Derived
Stromal Vascular Fraction Rescues Erectile Function
by Enhancing Endothelial Regeneration in the
Streptozotocin-Induced Diabetic Mouse
E16
17：30〜
Index
Aⴊ▤↢‛කቇળKPFD
Poster
209
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Objective: Emerging evidence indicate that far infra-red (FIR) therapy exerts
beneficial effects in the cardiovascular system, but limited data are available on
potential mediating mechanisms. Recent insights suggest that the injured
endothelial monolayer is regenerated by circulating endothelial progenitor cells
(EPCs), but their functions are impaired in diabetes. We tested the hypothesis that
FIR radiation can improve high glucose-suppressed EPC functions and restores
collateral flow to ischemic tissue in diabetic mice.Methods and Results: Unilateral
hindlimb ischemia was induced in streptozotocine (STZ)-induced diabetic mice,
which were divided into control and FIR therapy groups. The latter mice were
placed in a far-infrared dry sauna at 34C for 30 min once daily for 5 weeks. Laser
Doppler perfusion imaging demonstrated that the ischemic limb/normal side blood
perfusion ratio in the thermal therapy group was significantly increased beyond that
in controls, and significantly greater capillary density was seen in FIR therapy
group. These beneficial effects were markedly abolished by an eNOS inhibitor
(L-NAME). Bone marrow-derived EPCs differentiated into endothelial cells
defined as GFP+/CD31+ double positive cells were significantly increased in
ischemic tissue around the vessels in diabetic mice received FIR radiation as
compared with those in control group. In in-vitro studies, treatment of cultured
EPCs with FIR radiation for 30 minutes promoted high glucose (25 mM)-impaired
EPC proliferation, migration and tube formation, and inhibited high glucoseinduced EPC senescence and reduced H2O2 production. Nude mice received
human EPCs treated with FIR in high glucose medium showed a significant
improvement in blood flow recovery in ischemic limb than those without FIR
therapy.Conclusions. FIR therapy promoted collateral flow recovery and new
vessels formation in STZ-diabetic mice. These beneficial effects may derive from
enhancement of EPC functions and homing process, which provide some novel
rational to the vascular protective properties of FIR therapy in diabetic patients.
Division of Cardiology, Taipei Veterans General Hospital
Po-Hsun Huang、Shing-Jong Lin、Jaw-Wen Chen
Symposium
Backgrounds Lentivirus vectors provide a delivery system that can both transduce
nondividing cells and integrate transgenes into the genome of target cells which
enable long-term tracing in vitro and in vivo. However, their transduction efficiency
and stability of gene expression has not been evaluated in murine embryonic stem
cells (ESC) or induced pluripotent stem cells (iPS). Therefore, we evaluated
lentiviral transduction into ESC or iPS and its gene expression. Methods Human
immunodeficiency virus type 1-based lentivirus vectors containing the green
fluorescent protein (GFP) gene were transduced into the cells with or without
coating the culture dishes with retronectin. The effieciency of repeated transduction
were compared with single transduction. Embryonic body (EB) from the transduced
ESC and iPS was formed by hanging drop method. Spontaneous differentiation was
induced by re-attaching the EB to gelatin-coated dish. GFP expression was
examined during differentiation. Results Following single transduction for 12h
without retronectin and 2 days of culture, 27.3% ± 6.7% of iPS were positive for
GFP. Retronectin significantly increased the positivity of GFP (42. 0% ± 5.5%.
n=4, p<0.001). Repeated transduction (3 times) elevated the positivity upto 95% ±
3.4%. There was no difference of the transduction rate between iPS and ESC.
Seven days after spontaneous differentiation of the >90% GFP-positive EB, GFP
positivity was significantly reduced to 39.3 ± 9.4%. CONCLUSIONSThese data
show that lentivirus with retronectin precoating and repreated transduction increases
the efficiency of gene transfer in murine ESC or iPS. This method, however, may
not be useful for the research in cardiovascular differentiation from iPS or ESC due
to the significant gene silencing after differentiation.
National Research Laboratory for Cardiovascular Stem cell, Seoul
National University College of medicine , Seoul, Korea、2Department of
Internal Medicine, Seoul National University Hospital, Seoul, Korea
1
How to write
Plenary Lecture
Nature Medicine
Seok-Jin Koh1、Chang-Hwan Yoon2、Young-Eun Choi1、
Hyun-Jae Cho2、Hyo-Soo Kim2
E21
Special Talk
Far infra-red therapy restores high glucosesuppressed endothelial progenitor cell functions and
promotes ischemia-induced angiogenesis in diabetic
mice
Special
Lecture
Efficienct lentiviral transduction of murine induced
pluripotent stem cells and its silencing during
differentiation
Objective: We investigated a new strategy that enhances cellular potency and
provides an enriched source of cardiac progenitor cells.
Background: Recent studies suggest that resident cardiac stem/progenitor cells
could be an optimal cell source for repairing a damaged heart. However, cardiac
progenitor cells are difficult to maintain in terms of purity and multipotency when
propagated in attached 2-dimensional culture systems in vitro.
Methods: We applied the repeated sphere formation strategy (that is, cardiac
explant ; migrating cells out of explant ; primary cardiosphere (CS) formation ;
cardiosphere-derived cells (CDCs) in adherent culture condition ; secondary CS
formation by 3-dimensional culture)
Results: Murine CSs were generated from cardiac explants of C57BL/6 mice. Cells
in secondary CS showed a higher potency and differentiation potentials.
Furthermore, when transplanted into the infarcted myocardium, secondary CSs
engrafted robustly, improved LV dysfunction, and reduced infarct sizes more than
CDCs did. In addition to the cardiovascular differentiation of transplanted
secondary CSs, paracrine effects, such as, robust VEGF secretion, were found to
enhance neovascularization in the infarcted myocardium. Regarding the molecular
mechanisms involved, ERK/E-selectin and ERK/Sp1/VEGF auto-paracrine loop
were responsible for sphere initiation and maturation, respectively. When we
applied this strategy to adult human cardiac tissues, secondary CS was readily
generated, and cellular characteristics and molecular pathways of sphere formation
were similar to murine findings.
Conclusion: These results provide a simple and reproducible strategy for enhancing
cellular potency for cardiac repair. Furthermore, this strategy may be applicable to
other types of stem/progenitor cells for cell-based therapy
Seoul National Univ. Hospital, Seoul, Korea
Ho-Jae Lee、Hyun-Jai Cho、Seock-Won Youn、Seok-Jin Koh、
Joo-Yun Won、Yeon Ju Chung、Jaewon Lee、Sooryeonhwa Jin、
Chang-Hwan Yoon、Sae-Won Lee、Eun Ju Lee、Hae-Young Lee、
Young-Bae Park、Hyo-Soo Kim
Secondary sphere formation enhances the
functionality of cardiac progenitor cells from murine
and human cardiac explants
E19
President
Lecture
E20
Background: Ultrasonic microbubble-mediated gene delivery has shown to be a
feasible method in the non-viral gene delivery. The responsiveness of ultrasonic
microbubble transfection (UMT) of VEGF gene into endothelial progenitor cells
(EPC) remained unclear. Methods: EPC originated from porcine peripheral blood
were cultured with medium containing constructed VEGF plasmid DNA (pDNA,
0.5 kb; CMV promoter) and air-gassed, hard shell microbubbles followed by UMT.
Expression of VEGF was evaluated by ELISA 48 hours post gene delivery. In
parallel, a series of parameters of ultrasound and functional evaluations were
conducted to investigate the effects of UMT of VEGF gene into EPC. Results: The
parameters of 1 W/cm 2 , duty cycle 5% and exposure time 30 seconds, which
yielded a relatively high luciferase expression around 5 LU/cell (n=4, p<0.01 vs
control) and VEGF protein expression around 2000 pg/ml (n=4, p<0.05 vs control)
with relatively low intensity of ultrasound, were selected as ultrasound parameters
of gene delivery. UMT with the optimized parameter did not degrade VEGF pDNA.
VEGF-containing supernatant originated from transfection of human embryonic
kidney 293 (HEK293) cells and UMT of EPC led to improvement of activities of
migration in human aortic endothelial cells (HAEC) around 50%, as well as of
proliferation in HAEC more than 20 % (n=4, p<0.05 vs control), respectively, with
no obvious adverse effect on transfected EPC. Furthermore, a duration of stable
secretion of VEGF protein in UMT-transfected EPC can be around 48-96 h (n=4,
p<0.05 vs control). Conclusions: UMT successfully delivers angiogenic gene into
porcine EPC and UMT-mediated VEGF gene transfer enhances the functional
activities of HAEC, while possesses no obvious adverse effect on viable transfected
cells. The results suggested that UMT could potentially be a safe and useful tool for
VEGF gene delivery into EPC. Further investigation is prerequisite to clarify the
clinical impact in peripheral artery disease.
Mackay Memorial Hospital, Cardiovascular division, Laboratory of Heart
and Circulatory Physiology, Taipei, Taiwan
Cheng-Huang Su、Chiung-Yin Chang、Sih-Wei Tseng、
Cheng-Ho Tsai、Hung-I Yeh
Ultrasonic microbubble-mediated angiogenic gene
delivery into endothelial progenitor cells
E18
Poster Session：Stem Cell / Progenitor Cells
12月2日
（木）
／Thursday, December 2
Program
Aⴊ▤↢‛කቇળKPFD
210
Aim: Plasma levels of adiponectin, an adipose-specific protein with putative antiatherogenic properties, could be down-regulated in obese and diabetic subjects.
Recent insights suggest that the injured endothelial monolayer is regenerated by
circulating endothelial progenitor cells (EPCs), but high glucose reduces number
and functions of EPCs. Here, we tested the hypothesis that globular adiponectin can
improve high glucose-suppressed EPC functions by restoration of endothelial nitric
oxide synthase (eNOS) activity.Methods: Late EPCs isolated from healthy subjects
appeared with cobblestone shape at 2-4 weeks. EPCs were incubated with high
glucose (25 mM) and treatment with globular adiponectin for functional study.
Migration and tube formation assays were used to evaluate the vasculogenetic
capacity of EPCs. The activities of eNOS, Akt and concentrations of nitric oxide
(NO) were also determined. Results: Administration of globular adiponectin at
physiological concentrations promoted EPC migration and tube formation, and
dose-dependently upregulated phosphorylation of eNOS, Akt and augmented NO
production. Chronic incubation of EPCs in high-glucose medium significantly
impaired EPC function and induced cellular senescence, but these suppression
effects were reversed by treatment with globular adiponectin. Globular adiponectin
reversed high glucose-impaired EPC functions through NO-, p38 MAPK- and
AMPK-related mechanisms. In addition, nude mice received EPCs treated with
adiponectin in high glucose medium showed a significant improvement in blood
flow than those received normal saline.Conclusion: The administration of globular
adiponectin improved high glucose-impaired EPC functions in vasculogenesis by
restoration of eNOS activity. These beneficial effects may provide some novel
rational to the vascular protective properties of adiponectin.
Division of Cardiology, Taiepi Veterans General Hospital
Po-Hsun Huang、Shing-Jong Lin、Jaw-Wen Chen
Globular adiponectin improves high glucosesuppressed endothelial progenitor cell function
through endothelial nitric oxide synthase dependent
mechanisms
E22
17：30〜
Index
Aⴊ▤↢‛කቇળKPFD
Poster
211
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Gomisin J (GJ) is a small molecular weight lignan contained in Fructus
Schisandrae, the dried seed of Schisandra chinensis (SC) which is a well-known
medical herb for the improvement of cardiovascular symptoms in Korea. In this
study, we investigated the vascular effects of Gomisin J from SC. Gomisin J (0.1 30 㱘g/ml) caused a concentration-dependent relaxation in both the endotheliumintact and -denuded rat thoracic aortas. The relaxant effect of Gomisin J was more
prominent in the endothelium-intact aorta than that in the endothelium-denuded
aorta. The endothelium-dependent vasodilatation by Gomisin J was markedly
attenuated by L-NAME (a nitric oxide synthase inhibitor) and ODQ (a soluble
guanyl cyclase inhibitor) but not by tetraethylammonium (a nonselective blocker of
K+ channels) and indomethacin (a cyclooxygenase inhibitor). And also, Gomisin J
induced NO production in a time- and concentration-dependent manner in
association with an enhanced phosphorylation of eNOS with an increased cytosolic
translocation of eNOS. In endothelium-denuded aorta, vasorelaxation by Gomisin J
was clearly inhibited by calyculin A (an inhibitor of MLC phosphatase), but not by
ML-9 (a MLC kinase inhibitor). Furthermore, phenylephrine-enhanced MLC
phosphorylation was significantly attenuated by Gomisin J. Based on these results,
it is suggested that vascular relaxation by Gomisin J is mediated not only by
endothelium-NO pathway via eNOS activation, but also by direct effect on vascular
smooth muscle via dephosphorylation of MLC.
Department of Pharmacology, School of Medicine, Pusan National
University、2College of Natural Resources & Life Sciences, Pusan
National University, Gyeongnam 627-706, Republic of Korea
1
Jin U. Bae1、Ji Y. Park1、Jung W. Yun1、Young W. Choi2、
Chi D. Kim1
Symposium
Farnesyltransferase (FTase) inhibitors also function to suppress the release of
vascular endothelial growth factor (VEGF) from tumor cells by inhibiting Ras
activation; however, the effects of FTase inhibitors on VEGF-induced angiogenesis
in endothelial cells have not been studied. We have investigated the antiangiogenic
effect and molecular mechanism of LB42708, a selective nonpeptidic FTase
inhibitor, using in vitro and in vivo assay systems. LB42708 inhibited VEGFinduced Ras activation and subsequently suppressed angiogenesis in vitro and in
vivo by blocking the MEK/ERK and PI3K/Akt/endothelial nitric-oxide synthase
pathways in endothelial cells without altering FAK/Src activation. In addition, this
inhibitor suppressed VEGF-induced endothelial cell cycle progression at the G1
phase by suppressing cyclin D1 expression and retinoblastoma phosphorylation as
well as up-regulating the cyclin-dependent kinase inhibitors p21 and p27.
Knockdown of Ras by short interfering RNA revealed similar inhibitory effects on
VEGF-induced angiogenic signal events compared with LB42708. Moreover, the
inhibitory effects of LB42708 were significantly higher than those of SCH66336, a
well known FTase inhibitor. LB42708 suppressed tumor growth and tumor
angiogenesis in both xenograft tumor models of Ras-mutated HCT116 cells and its
wild-type Caco-2 cells, indicating its potential application in the treatment of both
Ras-mutated and wild type tumors. These data indicate that the antitumor effect of
LB42708 can be associated with direct inhibition of VEGF-induced tumor
angiogenesis by blocking Ras-dependent MAPK and PI3K/Akt signal pathways in
tumor-associated endothelial cells.
Vascular System Research Center and Department of Molecular and
Cellular Biochemistry School of Medicine, Kangwon National University,
Chuncheon, Kangwon-do, Korea 、2Department of Biochemistry, College
of Sciences, Yonsei University, Seoul, Korea
1
Chun-Ki Kim2、Dong-Kwon Lee1、Kwon-Soo Ha1、
Young-Guen Kwon2、Young-Myeong Kim1
E26
How to write
Plenary Lecture
Nature Medicine
Gomisin J from schisandra chinensis induces
vasorelaxation in rat thoracic aorta
Special Talk
E25
Special
Lecture
LB42708, a farnesyltransferase inhibitor, suppresses
VEGF-induced angiogenesis by inhibiting Rasdependent MAPK and PI3K/Akt signal pathways
President
Lecture
Under normal culture conditions, rat aortic smooth muscle cells (RASMCs) display
their phenotypic modulation from a dedifferentiated (synthetic type) to a
differentiated (contractile type) state. Insulin-like growth factor 1 (IGF-1) is unique
among growth factors in promoting RASMCs differentiation via preferential
activation of phosphatidylinositol 3-kinase (PI3K) pathways. The differentiation of
synthetic type RASMC is achieved by repetitive subculture at high cell density.
Especially, plating the cells on fibronectin-coated dish enhanced the differentiation
of RASMCs. Both Akt and ERK were significantly activated by fibronectin.
Inhibition of PI3K/Akt signaling pathways blocked the differentiation of RASMC
whereas inhibition of ERK pathway did not affect. More importantly, silencing of
Akt1 abolished the differentiation of RASMCs whereas silencing of Akt2 did not
affect RASMC differentiation. Moreover, silencing of integrin-linked kinase (ILK)
completely blocked the differentiation of RASMCs, and reduced the
phosphorylation of Akt. In addition, Silencing of Akt1 impaired promoter activity
of smooth muscle cell marker genes such as SM22㱍 and SMA. Finally, mice
lacking Akt1 displayed enhanced neointima formation after carotid artery ligation.
Given these results, we suggest that Akt1 is involved in extracellular matrix
associated signaling pathways and regulates vascular integrity to contractile
phenotype.
Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear
hormone receptor superfamily of ligand-dependent transcription factors. PPARs
play an important role in the general transcription control of numerous cellular
processes, including lipid metabolism, glucose homeostasis, cell cycle progression,
cell differentiation, inflammation and extracellular matrix remodeling. Three PPAR
isoforms, designated PPAR㱍, PPAR㱐 and PPAR㱏, have been identified.
Previous studies have shown that agonists of PPAR㱍 and PPAR㱏 regulate
vascular smooth muscle cell (VSMCs) functions. We postulate that PPAR㱍
agonists protect VSMC against oxidant-induced apoptosis via upregulation of 14-33. PPAR㱍 agonists such as WY14643 and GW9518 inhibited VSMC capase 3
activation and PARP cleavage i n a concentration-dependent manner.
Carbaprostacyclin, an agonist of PPAR㱍 and PPAR㱐, completely abolished
caspase 3 activation and PPAR cleavage, while 15d-PGJ 2, a PPAR㱏 agonist had no
effect. Seven isoforms of 14-3-3 (14-3-3㱎, 㱏, 㱑, 㱓, 㱔, 㱟, and 㱒) have been
identified and characterized in mammalian cells. All isoforms except 14-3-3㱟 were
detected in VSCM at basal state. WY14643 (50 㱘M) or GW9578 (5 㱘M)
increased selectively 14-3-3㱑 expression. 14-3-3㱑 was not influenced by H 2 O 2
and was elevated by WY14643 in the presence of H2O2. 14-3-3㱑 overexpression
was previously reported to protect endothelial cells from H2O 2-induced apoptosis
by sequestering Bad and reducing Bad-mediated apoptosis. Our preliminary results
suggest that PPAR㱍 activation protects VSMC from apoptosis via the 14-3-3㱑
cytoprotective pathway.
Institute of Cellular and System Medicine, National Health Research
Institutes, Zhunan, Taiwan
Yen-Chung Chen、Shu-Fan Yang、Kenneth K Wu
Sung Ji Yun、Eun Kyoung Kim、Jung Min Ha、Young Whan Kim、
In Hye Jin、Dae Han Woo、Chi Dae Kim、Sun Sik Bae
MRC for Ischemic Tissue Regeneration, Medical Research Institute, and
Department of Pharmacology, Pusan National University School of
Medicine, Kyungnam, Republic of Korea
Protection of vascular smooth muscle cell survival by
peroxisome proliferator-activated receptor-α agonists
via 14-3-3 upregulation
E24
Regulation of vascular smooth muscle cells
phenotypes by matrix-mediated activation of PI3K/
Akt1 signaling pathways
E23
Poster Session：Vascular Signal Transduction
12月2日
（木）
／Thursday, December 2
Program
Aⴊ▤↢‛කቇળKPFD
212
Carbon monoxide (CO) plays a significant role in vascular functions. We here
examined the molecular mechanism by which CO regulates hypoxia-inducible
transcription factor-1 (HIF-1)-dependent expression of vascular endothelial growth
factor (VEGF), which is an important angiogenic factor. We found that astrocytes
stimulated with the CO-releasing molecule (CORM-2) promoted angiogenesis by
increasing VEGF expression and secretion. CORM-2 also induced
hemeoxygenase-1 (HO-1) expression and increased nuclear HIF-1㱍 protein level,
without altering its promoter activity and mRNA level. VEGF expression was
inhibited by treatment with HIF-1㱍 siRNA and a HO inhibitor, indicating that CO
stimulates VEGF expression via upregulation of HIF-1㱍 protein level, which is
partially associated with HO-1 induction. CORM-2 activated the translational
regulatory proteins p70S6k and eIF-4E, as well as phosphorylated their upstream
signal mediators Akt and ERK. These translational signal events and HIF-1㱍
protein level were suppressed by inhibitors of phosphatidylinositol 3-kinase (PI3K),
MEK, and mTOR, suggesting that the PI3K/Akt/mTOR and MEK/ERK pathways
are involved in a translational increase in HIF-1㱍. In addition, CORM-2 also
increased stability of the HIF-1㱍 protein by suppressing its ubiquitination, without
altering the proline hydroxylase-dependent HIF-1㱍 degradation pathway. CORM2 increased HIF-1㱍 /HSP90㱍 interaction, which is responsible for HIF-1㱍
stabilization, and HSP90 specific inhibitors decreased this interaction, HIF-1㱍
protein level, and VEGF expression. Furthermore, HSP90㱍 knockdown
suppressed CORM-2-induced increases in HIF-1㱍 and VEGF protein levels.
These results suggest that CO stimulates VEGF production by increasing HIF-1㱍
protein level via two distinct mechanisms, translational stimulation and protein
stabilization of HIF-1㱍.
Vascular System Research Center and Department of Molecular and
Cellular Biochemistry School of Medicine, Kangwon National University,
Chuncheon, Kangwon-do, Korea、2Department of Molecular and Cellular
Biochemistry School of Medicine, Kangwon National University,
Chuncheon, Kangwon-do, Korea、3Department of Biochemistry, College
of Sciences, Yonsei University, Seoul, Korea
1
Yoon Kyung Choi1,2、Kyu-Sun Lee1,2、Hansooo Lee1、
Kwon-Soo Ha2、Young-Guen Kwon3、Young-Myeong Kim1,2
Carbon monoxide promotes VEGF expression by
increasing HIF-1α protein level via translational
activation and stabilization of HIF-1α protein
E27
1
Chungbuk National University
Fight against Angiogenesis-Related Blindness (FARB) Laboratory,
Department of Ophthalmology, College of Medicine, Seoul National
University, Seoul, Korea
Index
Aⴊ▤↢‛කቇળKPFD
Poster
213
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Pyridoxal-5ʼ-phosphate (PLP), the active form of vitamin B6, was previously
studied in our laboratory as a potential anti-aggregative agent which has beneficial
effects in preventing cardiovascular diseases. However, the relationship between
the anti-aggregative effects and morphological changes of PLP is unclear. In this
study, platelet-rich plasma (PRP) was respectively activated by thrombin, ADP, and
collagen and co-incubated with the tetrapeptide, Arg-Gly-Asp-Ser (RGDS), or PLP.
Morphological changes in platelets were evaluated by scanning electronic
microscopy (SEM). The results indicated that optimal aggregative concentrations of
thrombin, ADP, and collagen were 0.5 units/ml, 25 㱘M, and 0.1 mg/ml,
respectively. Meanwhile, the optimal anti-aggregative concentrations of PLP and
RGDS to those platelet agonists were 1.5 and 0.23 mM, respectively. The SEM data
showed that morphological changes in platelet by RGDS or PLP treatment were
similar, including lower activation, being sustained in the pseudopod stages, and
having smaller fibrin spindles during thrombin induction. The smaller fibrin spindle
formation caused by PLP or RGDS might be helpful in preventing blood
coagulation. In this study, we provide morphological evidence that the antiaggregative effect of PLP is partially similar to that of RGDS in inhibiting
fibrinogen binding to GP23 receptors
Symposium
The hyaloid vessel is a transient intraocular circulatory system that undergoes a
complete regression as the retina becomes matured with retinal vascularization. If
the complete involution of the hyaloid vessels failes, the pathological persistence of
these vessels results in persistent hyperplastic primary vitreous (PHPV) associated
with severe ocular pathologies. Unfortunately despite its clinical significance,
cellular and molecular processes involved in hyaloid regression remain to be
elucidated. Herein, we for the first time demonstrated that autophagy could
contribute to the regression of hyaloid vessels in early developing retina. In
developing retina, hyaloid vessel regression coincided with retinal vascular
development; this occurred simultaneous with apoptotic and autophagic processes.
Moreover, in vascular endothelial cells under hypoxic conditions, LC3-II
conversion was detected along with caspase-3 activation. The autophagy inducer
rapamycin induced autophagy-mediated cell death of vascular endothelial cells in a
dose-dependent manner. Moreover, rapamycin significantly enhanced the involution
of hyaloid vessels in the early developing eye. Therefore, our results suggest that
the autophagy pathway would be involved in hyaloid regression that occurs during
early ocular development. Furthermore, activation of the autophagy pathway could
be considered for a therapeutic approach to PHPV.
Jin Hyoung Kim、Jeong Hun Kim
1
Dept. Biotechnology, HungKuang University、2Dept. Biology, National
Cheng Kung University
Wen-Jen Yu1、Ru-Chun Dai1、Sue-Joan Chang2
How to write
Plenary Lecture
Nature Medicine
Autophagy-induced regression of hyaloid vessels in
early ocular development
Special Talk
Anti-aggregative Effects of Pyridoxal-5ʼ-phosphate on
Platelet Morphological Changes
Special
Lecture
E32
Gomisin A is a small molecular weight lignan contained in Schisandra chinensis.
Previously, we demonstrated that Gomisin A induced vasodilation through
activation of endothelium-nitric oxide pathway, and partially dephosphorylation of
myosin light chain in smooth muscle. Thus, we investigated the preventive effect of
Gomisin A on angiotensin II (AII)-induced hypertension in mice. C57/BL6 mice
infused subcutaneously with AII (2 㱘g/kg/min, osmotic mini-pump) showed an
increase in blood pressure. To determine the single-dose effect, Gomisin A was
infused into femoral vein, and then blood pressure was monitored via carotid artery.
At concentrations of 10-100 㱘g/kg, blood pressure in hypertensive mice was
decreased by Gomisin A in a concentration-dependent manner. In the continuousdose test, Gomisin A (2 and 10 㱘g/kg/min) was infused subcutaneously for 2
weeks using osmotic pump. The increase in blood pressure in AII-infused mice was
significantly attenuated by infusion of Gomisin A in a dose-dependent manner. In
addition, both endothelium-dependent vasodilation and nitric oxide production in
aorta from Gomisin A-treated mice were significantly higher than those in control.
Based on these results, it was suggested that Gomisin A had preventive effects on
AII-induced hypertension via its effects on hypertensive vasculature.
President
Lecture
E31
It is well known that microarray and microfluidic systems have a number of distinct
advantages compared to macroscale instruments; high-throughput, high speed, low
reagent usage, low energy consumption and automation. Such systems provide new
tools and opportunities for pharmaceutical and biological fields to develop new
drugs and to solve problems in genomics, proteomic, metabolomics, cellomics, and
organomics. I will describe how such systems can be applied to vascular biology by
using the following biochips; Protein Microarray Chip: This system can be used
for screening new drugs for targeting protein-protein interactions, and for multipletarget antibody mimic peptides that can overcome the tolerance effect found in
present antibody therapy. Droplet-Based Microfluidics Chip: This system enable
the generation and manipulation of monodisperse liquid droplets in an immiscible
carrier fluid, and such encapsulated droplets can be used to mimic artificial cells or
isolated reaction vessels. It can be applied to high-throughput drug screening by
using pico-/femto-liter volumes of samples with high efficiency. Multiple
Microelectrode Array Cell Chip: This system features six individual electrodes,
thus enabling six simultaneous independent measurements. It can be used for realtime NO sensing in endothelial cells. I will report recent advances in this area with
an angiogenic factor angiogenin in particular.
Jung W. Yun1、Jin U. Bae1、Young W. Choi2、Chi D. Kim1
Soo-Ik Chang
Department of Pharmacology, School of Medicine, Yangsan, Pusan
National University、2College of Natural Resources & Life Sciences,
Pusan National University, Gyeongnam 627-706, Republic of Korea
Preventive effect of Gomisin A on angiotensin IIinduced hypertension in mice
E30
17：30〜
Application of Microarray and Microfluidic Systems
on Vascular Biology
E28
Poster Session：Others
12月2日
（木）
／Thursday, December 2
Program
18：00〜
Aⴊ▤↢‛කቇળKPFD
214
OBJECTIVESHere we investigated the effects of human resistin on atherosclerotic
progression and clarified its underlying mechanisms.BACKGROUNDResistin is an
adipokine first identified as a mediator of insulin resistance in murine obesity
models. But, its role in human pathology is under debate. Although a few recent
studies suggested the relationship between resistin and atherosclerosis in human,
the causal relationship and underlying mechanism have not been clarified.
METHODS We cloned rabbit resistin, which showed 78% identity to human
resistin at cDNA level, and its expression was examined in three different
atherosclerotic rabbit models. To evaluate direct role of resistin on atherosclerosis,
collared rabbit carotid arteries were used. Histological and cell biologic analyses
were performed.RESULTS Rabbit resistin was expressed by macrophages of the
plaque in the three different atherosclerotic models. Peri-adventitial resistin gene
transfer induced macrophage infiltration and expression of various inflammatory
cytokines, resulting in the acceleration of plaque growth and instabilization. In vitro
experiments elucidated that resistin increased monocyte-endothelial cell adhesion
by up-regulating VLA-4 on monocytes and their counterpart VCAM-1 on
endothelial cells. Resistin augmented monocyte infiltration in collagen by direct
chemoattractive effect as well as by enhancing migration toward MCP-1.
Administration of Connecting Segment-1 peptide, which blocks VLA-4-VCAM-1
interaction, ameliorated neointimal growth induced by resistin in vivo.
CONCLUSIONSOur results indicate that resistin aggravates atherosclerosis by
stimulating monocytes, endothelial cells and vascular smooth muscle cells to induce
vascular inflammation. These findings provide the first insight on the causalrelationship between resistin and atherosclerosis.
Department of Internal Medicine, Seoul National University College of
Medicine, Seoul, Korea
Hyun-Chae Lee、Youngjin Cho、Sang Eun Lee、Jin Hur、
Sahmin Lee、Seock-Won Youn、Jaewon Lee、Sooryeonhwa Jin、
Yoo-Wook Kwon、Hyun-Jai Cho、Byung-Hee Oh、Young-Bae Park、
Hyo-Soo Kim
Human Resistin is an Inflammatory Cytokine that
Aggravates Atherosclerosis
E36
The pathological angiogenesis in the retina is the major cause of vision loss at all
ages. In particular, retinopathy of prematurity (ROP) is a leading cause of blindness
in children. This study was to investigate whether gold nanoparticle (GNP) could
inhibit retinal neovascularization in the animal model of ROP. Intravitreal injection
of GNP significantly inhibited retinal neovascularization in the mouse model of
ROP. In addition, GNP effectively suppressed VEGF-induced in vitro angiogenesis
of retinal microvascular endothelial cells including proliferation, migration and
capillary-like networks formation. GNP blocked VEGF-induced autophosphorylation of VEGFR-2 to inhibit consequently ERK-1/2 activation. GNP
never affected on the cellular viability of retinal microvascular endothelial cells and
induced no retinal toxicity. Our data suggests that GNP could be a potent inhibitor
to retinal neovascularization without retinal toxicity. Furthermore, GNP could be
extensively applied to variable vaso-proliferative retinopathies mediated by VEGF.
Coronary artery calcium (CAC) and high sensitive C-reactive protein (hsCRP) have
been developed to predict the cardiovascular events in coronary artery disease
(CAD) patients. However, the application of these markers in predicting
cardiovascular events in different genders and age subgroups needs more
clarification. The study aim was to investigate the hypothesis that combined use of
hsCRP level and CAC score can increase the predict value of future cardiovascular
events in male subjects suspected with CAD. We included 90 male patients with
typical stable angina symptoms. We measured the serum hsCRP and CAC score by
electron beam computerized tomography. These baseline parameters were
correlated to the clinical cardiovascular events within 12 months follow-up period.
During the follow-up period up to 50 months (median 27 months), 13 major
cardiovascular events were recorded. In multivariate regression analysis, after
adjusted with conventional risk factors, hsCRP and CAC score, hsCRP level was
the only independent predictor of cardiovascular events in male subjects. (Relative
risk of patients with hsCRP >1 mg/dl as compared to those with hsCRP <1 mg/dl
was 4.52; 95% confident interval [CI], 1.139 to 17.97; p = 0.032). Further analysis
was performed among 4 groups classified by CAC score (CAC score > 100 or
<100) and hsCRP (>1 or <1 mg/L). The relative risk for hsCRP >1mg/L and CAC
score>100 group compared with hsCRP <1 mg/L and CAC score <100 increased
markedly to 7.50 (95% CI:1.42 to 39.61, p=0.018) for the cardiovascular events. In
conclusion, the risk stratification based on both hsCRP and CAC score might be
benefit to male subjects with suspected coronary artery disease, since combined use
of these 2 markers contributed significantly to the incidence of cardiovascular
events.
Cardiology division, Taipei Medical University WanFang Hospital,
Taiwan, Republic of China、2Division of Cardiology, Department of
Medicine, Taipei Veterans General Hospital、3Cardiovascular Research
Center, National Yang-Ming University
1
Jong Shiuan Yeh1、Po Hsun Huang2,3、Hsin Bang Leu2,3、
Tao Chen Wu2,3、Jaw Wen Chen3
The Role of High-sensitive C-reactive Protein and
Coronary Artery Calciumin in the Prognosis of Male
Subjects with Coronary Artery Disease
E37
Accumulation of ROS in aerobic organisms is thought to cause oxidative damage
which is believed to be strongly associated with certain human diseases such as
aging, carcinogenesis and atherosclerosis. Antioxidants that can neutralize free
radicals may be used to protect human body from diseases and retard aging. In our
previous studies, 50% ethanol extracts from red bean non-fermented (RBE) or
fermented by Bacillus subtilis IMR-NKI (RBNE) had showed antioxidant activities.
The main purpose of this study was to investigate the effects of RBE or RBNE
administration on antioxidant-oxidant status of aged ICR mouse. The 12-monthold ICR mice were divided into 7 groups and orally treated with distilled water,
RBE (0.15, 0.3, 0.6 g /100g BW/day) or RBNE (0.15, 0.3, 0.6 g 100g BW/day)
over 30 consecutive days. At the end of animal feeding experiment, the plasma was
analyzed for total antioxidant capacity (TAC) while the tissues cytosols were
analyzed for lipid peroxidation (MDA), antioxidants levels and enzymes activities.
In addition, results were compared with those of 8-week-old ICR mice which were
treated with distilled water. The results showed that there was an age-related
increase in MDA levels in tissues, however, the TAC in plasma and the antioxidants
and antioxidative enzymes in tissues showed an age-related decrease. Orally
administration of RBE or RBNE exhibited significantly a decrease in the tissues
MDA levels and an increase in the plasma TAC and 㱍-tocopherol and GSH
concentrations in brain tissues of aged mice. In conclusion, the administration of
RBE or RBNE may increase invivo antioxidant defense system and have some
protective effects in decreasing oxidative stress in aged mice.
Department of Food and Nutrition, Providence University, Taiwan, R. O.
C.
Su-Tze Chou、Yun-Chin Chung、Hsin-Yi Peng、Jen-Chieh Ho、
I-Ping Shih
Jeong Hun Kim
Fight against Angiogenesis-Related Blindness (FARB) Laboratory,
Department of Ophthalmology, College of Medicine, Seoul National
University, Seoul, Korea
Effect of red bean and the bean fermented by Bacillus
subtilis extracts on antioxidative status in aged mice
E34
Gold Nanoparticle Inhibits Retinal Neovascularization
via Suppression of VEGFR-2 Activation
E33
Poster Session：Anti-aging and atherosclerosis
12月2日
（木）
／Thursday, December 2
18：00〜
Dept. of Biotechnology, Translational Research Center for Protein
Function Control, College of Life Science & Biotechnology, Yonsei
University, Seoul, Korea
1
Index
Aⴊ▤↢‛කቇળKPFD
Poster
The 8th Korea-Japan Joint Symposium on Vascular Biology
Selected Oral
Presentation
215
Taurine, a non essential sulfur-containing amino acid, directly interacts with
endothelial cells and transports into the cells. We examined the effect of taurine on
angiogenesis and its underlying signal pathway. Taurine increased angiogenesis in
vitro and in vivo, which is followed by activation of the phosphatidylinositol
3-kinase (PI3K)/Akt, MEK/ERK, and Src/FAK signaling pathways. Taurine
promoted endothelial cell cycle progression to S- and G2/M-phases by upregulating the positive cell cycle proteins, particularly cyclins D1 and B, as well as
down-regulating the negative cell cycle proteins, p53 and p21WAF1/CIP1, resulting in
the phosphorylation of Rb at Ser-780 and Ser-807/811. This angiogenic event was
inhibited by inhibitors of PI3K and MEK. In addition, PI3K inhibitor blocked the
activation of Akt and ERK, while Akt knockdown did not affect taurine-induced
ERK activation, indicating that PI3K is upstream mediator of both MEK and Akt.
Endothelial cell migration was suppressed by inhibitors of PI3K, MEK, and Src,
suggesting that taurine-induced cell migration is regulated by the Src-dependent
and -independent pathways. Moreover, inhibition of cellular taurine uptake by 㱎
-alanine and taurine transporter knockdown promoted taurine-induced cell
proliferation and ERK and Akt activation, suggesting that extracellular taurine
elicits angiogenesis. Taurine did not induce vascular inflammation and permeability
in vitro and in vivo. These data demonstrate that extracellular taurine promotes
angiogenesis by Akt- and ERK-dependent cell cycle progression and Src/FAKmediated cell migration without vascular inflammation, indicating that it is valuable
to use for treatment of vascular dysfunction-associated human diseases.
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Forskolin, a potent activator of adenylyl cyclases, has been implicated in
modulating angiogenesis, but the underlying mechanism has not been clearly
elucidated. We investigated the signal mechanism by which forskolin regulates
angiogenesis. Forskolin stimulated angiogenesis of human endothelial cells and in
vivo neovascularization, which was accompanied by phosphorylation of CREB,
ERK, Akt, and endothelial nitric oxide synthase (eNOS) as well as NO production
and VEGF expression. Forskolin-induced CREB phosphorylation, VEGF promoter
activity, and VEGF expression were blocked by the PKA inhibitor PKI. Moreover,
phosphorylation of ERK by forskolin was inhibited by the MEK inhibitor PD98059,
but not PKI. The forskolin-induced Akt/eNOS/NO pathway was completely
inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, but not
significantly suppressed by PKI. These inhibitors and a NOS inhibitor partially
inhibited forskolin-induced angiogenesis. The exchange protein directly activated
by cAMP (Epac) activator, 8CPT-2Me-cAMP, promoted the Akt/eNOS/NO
pathway and ERK phosphorylation, but did not induce CREB phosphorylation and
VEGF expression. The angiogenic effect of the Epac activator was diminished by
the inhibition of PI3K and MEK, but not by the PKA inhibitor. Small interfering
RNA-mediated knockdown of Epac1 suppressed forskolin-induced angiogenesis
and phosphorylation of ERK, Akt, and eNOS, but not CREB phosphorylation and
VEGF expression. These results suggest that forskolin stimulates angiogenesis
through coordinated cross-talk between two distinct pathways, PKA-dependent
VEGF expression and Epac-dependent ER Kactivation and PI3K/Akt/eNOS/NO
signaling.
Vascular System Research Center and Department of Molecular and
Cellular Biochemistry School of Medicine, Kangwon National University,
Chuncheon, Kangwon-do, Korea、2Department of Molecular and Cellular
Biochemistry School of Medicine, Kangwon National University,
Chuncheon, Kangwon-do, Korea、3Department of Biochemistry, College
of Sciences, Yonsei University, Seoul, Korea
Vascular System Research Center and Department of Molecular and
Cellular Biochemistry School of Medicine, Kangwon National University,
Chuncheon, Kangwon-do, Korea、2Department of Biochemistry, College
of Sciences, Yonsei University, Seoul, Korea.
1
Kyu-Sun Lee1、Yi-Young Baek1、Young-Geun Kwon2、
Young-Myeong Kim1
Symposium
1
Dong-Kwon Lee1,2、Chun-Ki Kim1,2、Hansooo Lee1、
Kwon-Soo Ha1,2、Young-Guen Kwon3、Young-Myeong Kim1,2
E41
How to write
Plenary Lecture
Nature Medicine
Extracellular taurine elicits angiogenesis by activating
the ERK-, Akt-, and FAK-dependent signal pathways
Special Talk
Forskolin increases angiogenesis through the
coordinated cross-talk of PKA-dependent VEGF
expression and Epac-mediated PI3K/Akt/eNOS
signaling
Special
Lecture
E40
Autophagy is an intracellular catabolic mechanism that contributes to cell
homeostasis by removing unnecessary components (Cho YS and Kwon HJ, ARPR,
2010 in press). Dynamic membrane redistribution during autophagy results in
autophagosome formation, which is the double membrane that surrounds
unnecessary components, and is followed by fusion with the lysosome, creating the
autophagolysosome (Ohsumi Y, Nat Rev Mol Cell Biol, 2001). The components that
are degraded within the autophagolysosome are reused as energy source in the cell.
We recently discovered autophagonizer, a novel synthetic small molecule with
unique structure that induces autophagy in mammalian cells. Autophagonizer
induces autophagy and inhibits cell proliferation, but its effect was not inhibited by
known autophagy inhibitors (Choi, IK, Cho, YS, Jung HJ, and Kwon HJ, BBRC
2010). Furthermore, autophagonizer induces apoptosis independent autophagic cell
death, which makes it possible to become a potential drug candidate for diseases
such as cancer and artheroscerlosis. Here, the anti-angiogenic effect of
autophagonizer was tested with tube formation and invasion assay. These results
suggest that autophagonizer also inhibits angiogenesis, and that autophagonizer
may be a potential anti-cancer agent. However the direct target of autophagonizer
still remains to be uncovered. The target protein identification using the ORFeome
system which is the collection of the overexpression of all the open reading frames
(ORFs) of Schizosaccharomyces pombe is underway to reveal the signaling
pathway behind autophagonizer induced autophagy. By examining the cell growth
with the entire ORFeome pool, autophagonizer resistant and sensitive strains can be
identified. These strains can provide clues to autophagonizer ʻ s target protein and
related signaling pathway to clarify the link between autophagy and angiogenesis.
President
Lecture
Angiogenesis is the new blood vessels formation from existing blood vessels.
Although this process is regulated strictly and is involved in normal physiological
condition, it usually occurs in development of fertilized egg or healing of wound.
This process is also induced in hypoxic conditions such as tumor growth. During
hypoxia, cells secrete several angiogenesis factors like VEGF (Vascular endotherial
growth factor), which are induced by HIFs (Hypoxia inducible factors). In the
course of our screening for small molecule targeting HIF, tyrosine kinase inhibitor
was identified as the specific inhibitor of HIF that induces HIF-1/2㱍 degradation
during hypoxia. This activity leads to inhibition of angiogenesis both in vitro and in
vivo angiogenesis assays including tube formation, chemoinvasion and CAM
(Chick chorioalantoic membrane) assay. Collectively, these data demonstrated that
tyrosine kinase inhibitor exhibits anti-angiogenic activity via the induction of HIF1/2㱍 degradation.
Ju̲yearl Park、Ki Hyun Kim、Ho Jeong Kwon
Yoon Sun Cho1、Minoru Yoshida2、Ho Jeong Kwon1
Department of Biotechnology, Translational Research Center for
Protein Function Control, College of Life Science & Biotechnology,
Yonsei University, Seoul, Korea、2Chemical Genetics Laboratory, RIKEN,
Wako, Saitama 351-0198, Japan
Discovery and characterization of bioactive small
molecule targeting HIF, a hypoxia inducible factor
E39
Autophagonizer, a novel autophagy inducer exhibits
anti-angiogenic effects
E38
Poster Session：Angiogenesis / Lymphangiogenesis
12月2日
（木）
／Thursday, December 2
Program
Aⴊ▤↢‛කቇળKPFD
216
The natural product sesamin has been known to act as a potent antioxidant and
prevent endothelial dysfunction. We here found that sesamin increased in vitro
angiogenic processes, such as endothelial cell proliferation, migration, and tube
formation, as well as neovascularization in an animal model. This compound
elicited the activation of multiple angiogenic signal modulators, such as ERK, Akt,
endothelial nitric oxide synthase (eNOS), NO production, FAK, and p38 MAPK,
but not Src. The MEK inhibitor PD98059 and the PI3K inhibitor Wortmannin
specifically inhibited sesamin-induced activation of the ERK and Akt/eNOS
pathways. These inhibitors reduced angiogenic events, with high specificity for
MEK/ERK-dependent cell proliferation and migration and PI3K/Akt-mediated tube
formation. Moreover, inhibition of p38 MAPK effectively inhibited sesamininduced cell migration. The angiogenic activity of sesamin was not associated with
VEGF expression. Furthermore, this compound did not induce vascular
permeability and upregulated ICAM-1 and VCAM-1 expression, which are
hallmarks of vascular inflammation. These results suggest that sesamin stimulates
angiogenesis in vitro and in vivo through the activation of MEK/ERK-, PI3K/Akt/
eNOS-, p125FAK-, and p38 MAPK-dependent pathways, without increasing vascular
inflammation, and may be used for treating ischemic diseases and tissue
regeneration.
Vascular System Research Center and Department of Molecular and
Cellular Biochemistry School of Medicine, Kangwon National University,
Chuncheon, Kangwon-do, Korea、2Department of Molecular and Cellular
Biochemistry School of Medicine, Kangwon National University,
Chuncheon, Kangwon-do, Korea、3Department of Biochemistry, College
of Sciences, Yonsei University, Seoul, Korea
1
Ok-Won Seo1,2、Hansoo Lee1、Kwon-Soo Ha1,2、
Young-Guen Kwon3、Young-Myeong Kim1,2
Angiogenic activity of sesamin through the activation
of multiple signal pathways
E42
LIM domain binding protein 2 (Ldb2/CLIM1) is a nuclear protein that is known for
its interaction with the LIM domains of the LIM homeodomain (LIM-HD) and
LIM-only (LMO) families of proteins. Although its mammalian paralogue, Ldb1
has been suggested to be essential for development, the role of Ldb2 has not been
clearly determined. Here, we showed that Ldb2 is highly and specifically expressed
in endothelial cells, and is detected in the blood vessels during development of
zebrafish. Interestingly, morpholino against ldb2 resulted in abnormal
intersegmental vessel (ISV) formation, which is reminiscent of dll4 mutant
zebrafish vessels. In the search of target genes of Ldb2, we found that the
expression of DLL4 is reduced in LDB2 knockdown HUVECs. DLL4 promoter
analysis revealed the existence of conserved LMO2 complex (LMO2COM) binding
sites. Both deletion and site mutation indicated that these binding sites are required
for the transcriptional regulation of DLL4 by LDB2. These findings suggested that
LDB2 may play an important role in angiogenic sprouting via transcriptional
regulation of DLL4 in endothelial cells.
Department of Biochemistry, Yonsei University, Seoul, Korea、2School
of medicine, Kangwon National University, Chuncheon, Korea、3National
Cardiovascular Center Research Institute, Osaka University, Osaka,
Japan
1
Hyun-Jung Choi1、Seung-sik Rho1、Dong-hoon Choi1、
Naoki Mochizuki3、Young-Myeong Kim2、Young-Guen Kwon1
LDB2 regulates angiogenic sprouting via DLL4
E43
18：00〜
Index
Aⴊ▤↢‛කቇળKPFD
Poster
217
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Thrombomodulin (TM) is a glycoprotein originally found on the surface of vascular
endothelial cells. TM has five domains that exert multiple functions. It has recently
been reported that the extracellular N-terminal lectin-like domain (D1) of TM
possesses anti-inflammatory properties. Our previous results showed that
recombinant TM domain 1 (rTMD1) could suppress lipopolysaccaride-induced
acute inflammatory responses. However, the detailed mechanisms of rTMD1 in
chronic inflammation, such as atherosclerosis, are still unknown. To further
investigate the role of rTMD1 in atherosclerosis, we prepared and purified rTMD1
wild-type (rTMD1 wt) protein and its mutants, rTMD1 Ala25Thr and rTMD1
Gly61Ala, using Pichia pastoris expression system. These two mutants are naturally
occurring polymorphisms in TMD1 and have been found in patients with
thromboembolic diseases, such as sagittal sinus thrombosis or myocardial
infarction. Our previous studies showed that rTMD1 has a specific carbohydrate
ligand, Lewisy (Ley). Here, we found that these two rTMD1 mutants had lower
binding affinity with Ley than rTMD1 wt. Further, wt rTMD1 blocked the adhesion
and the transendothelial migration of monocytes through Ley which decorates
adhesion molecules on endothelial cells. However, the inhibitory effects of rTMD1
mutants were reduced compared with rTMD1 wt. In vivo, the formation of
atherosclerotic plaque in apolipoprotein E knockout mice was significantly reduced
in the treatment of rTMD1 wt and rTMD1 Gly61Ala compared with saline only.
Moreover, the plaque formation was significantly lower in rTMD1 wt than both
mutants treatment. In summary, rTMD1 inhibited inflammatory reactions by
interfering with the interaction of monocytes and endothelial cells through binding
to Ley on endothelial cells; however, the polymorphisms of TMD1 had decreased
anti-inflammatory effects.
Department of Biochemistry and Molecular Biology, College of Medicine,
National Cheng Kung University, Tainan, Taiwan
Symposium
Insulin resistance and metabolic syndrome have become the major health problems
in recent years. Activation of peroxisome proliferators-activated receptor gamma
(PPAR㱏) was reported to be effective in improving insulin resistance and
regulating blood glucose in diabetes. Toona sinensis Roem leaves (TSL) extracts
were documented to exert the hypoglycemic effect, however, the mechanisms
underlying and its relationship to the PPAR㱏 is still unknown. Our previous study
has been indicated that TSL-E6 is the effective extract for PPAR㱏 activation and it
may result in hypoglycemic effects. In this study, the PPAR㱏 activities of TSL-E6
were evaluated using 3T3-L1 adipocytes and high fat diet (HFD, 54 % energy by
fat) induced hyperglycemic mice. The regulation of TSL-E6 extract on the PPAR㱏
gene and protein expression, and its downstream aP2 gene expression were also
investigated using the Western blotting and RT-PCR, respectively. The TSL-E6
treatment significantly increased the PPAR㱏 gene and protein expressions in 3T3L1 adipocytes. Moreover, the PPAR㱏 gene and protein expressions, and its
downstream aP2 gene expression were elevated in the adipose tissue of
hyperglycemic mice with TSL-E6 treatment for 8 weeks. The analysis of HPLC
and LC-MS identified that gallic acid was the major compounds in TSL-E6. Our
results suggested that TSL-E6 might be the PPAR㱏 ligand, which is involved in
the regulation of glucose homeostasis. Gallic acid may be the possible active
compound in TSL-E6.
Department of life science, National Cheng Kung University,Taiwan,
R.O.C、2Graduate School of Biotechnology, HungKung University,
Taiwan, R.O.C
1
Guey-Yueh Shi、Wei-Ling Lin、Hua-Lin Wu
Wen-Cheng Huang1、Bu-Chin Yu1、Tsung-Chi Tsai1、
Wen-Jen Yu2、Sue-Joan Chang1
E47
How to write
Plenary Lecture
Nature Medicine
Effect of the polymorphism of thrombomodulin lectinlike domain on anti-inflammation
Special Talk
Study of
extracts on the PPARγ
activation in the hyperglycemic mice and adipocytes.
Special
Lecture
E46
Metabolic syndrome is a combination of medical disorders that increase the risk of
cardiovascular disease and diabetes. Type 2 diabetes is a heterogeneous disorder,
afflicting an estimated 6-8% of the adult population in Korea; its worldwide
frequency is expected to continue to grow by 6% per annum, potentially reaching a
total of 300-400 million cases in 2020. The main force driving this increasing
incidence is an overwhelming increase in obesity, the single most important
contributor to the pathogenesis of diabetes.
The increasing prevalence of obesity, a main causative factor in metabolic
syndrome, has become a worldwide epidemic. Obesity reduces life expectancy and
increases health problems, particularly heart disease, type 2 diabetes, sleep apnea,
certain types of cancer, and osteoarthritis. Although lifestyle modification is
considered the first line therapy for obesity, it is often ineffective by itself. Two
anti-obesity medications are currently approved by the Food and Drug
Administration (FDA) for long term use; Orlistat and Sibutramine. Orlistat reduces
intestinal fat absorption and Sibutramine decreases appetite. However, there are
several limitations of these drugs in terms of efficacy and side effects.
㱍-Lipoic acid (ALA), a naturally occurring short chain fatty acid containing sulfurhydryl groups, has potent antioxidant capacities. ALA is an essential cofactor for
mitochondrial respiratory enzymes and improves mitochondrial function. ALA is
widely used in clinical practice to treat diabetic polyneuropathy, without serious
adverse effects. Additionally, ALA has many beneficial therapeutic potentials for
human diseases like primary biliary cirrhosis, atherosclerosis, Alzheimer disease,
cataract, glaucoma and insulin resistance in type 2 diabetes. Particularly, we
previously reported that chronic ALA treatment profoundly reduces body weight
gain in rodents. However, it is not known whether it is also effective in human
obesity. In this study, we present the efficacy of ALA by conducting a randomized,
double blind, placebo-controlled study in obese subjects.
President
Lecture
Background: Thiazide diuretics are one of the usually recommended first-line
therapies for hypertensive patients. However, there are concerns that it may
potentially deteriorate glucose and lipid metabolisms. This study aimed to elucidate
their potential effects on metabolic profiles and serum biomarkers in addition to
blood pressure (BP) reduction in patients with and without metabolic syndrome
(MS). Methods: Non-diabetic hypertensive patients were evaluated if they did not
take diuretics before and if their office systolic BP > 140 mmHg or diastolic BP >
90 mmHg. A diet and a modification of the previous life style were advised for the
period of 2 weeks. Additional hydrochlorothiazide 50 mg was given once every
morning for another 2 weeks. Patients were divided into 2 groups according to
whether they had MS. MS was defined according to the National Cholesterol
Education Program Adult Treatment Panel III guidelines. Results: A total of 101
patients were treated for two weeks, including 49 patients with MS and 52 patients
without MS. The patients with MS had more male gender (p= 0.018) and higher
body mass index (p< 0.001), waist-hip ratio (p< 0.001), fasting blood sugar (p<
0.001), cholesterol (p= 0.017), triglyceride (p< 0.001), and homeostasis model
assessment for insulin resistance (HOMA-IR, p< 0.001) at baseline. After 2 weeks
of treatment, both groups of patients had significant BP reduction (p< 0.001). In the
patients with and without MS, there were significantly decreased of asymmetric
dimethylargine (ADMA) (p< 0.001 and p= 0.003, respectively) and increased
adiponectin (p< 0.001 and p= 0.004, respectively). However, only patients with MS
had significantly decreased fasting blood sugar (p= 0.015) and cholesterol (p=
0.007) after 2 weeks of thiazide. Conclusions: Hydrochlorothiazide is appropriate
for nondiabetic essential hypertension patients. In addition to BP reduction,
metabolic profiles were improved, especially in the patients with MS.
Department of Internal Medicine, Kyungpook National University, College
of Medicine, Daegu, Republic of Korea
In-Kyu Lee
Chin-Chou Huang、Hsin-Bang Leu、Tao-Cheng Wu、
Shing-Jong Lin、Jaw-Wen Chen
Taipei Veterans General Hospital
Mutiple Role of Alpha Lipoic Acid; New Look at an
Old Drug in Metabolic Syndrom
E45
Thiazide Diuretics in Nondiabetic Essential
Hypertension with and without Metabolic Syndrome
E44
Poster Session：Metabolic syndrome / Diabetes and vascular inflammation
12月2日
（木）
／Thursday, December 2
Program
Aⴊ▤↢‛කቇળKPFD
218
Ninjurin1 (nerve injury-induced protein, Ninj1), a homophilic adhesion molecule,
is essential for cell-to-cell interaction, however little known about the function in
the central nervous system. Here we found that Ninj1 is expressed in three major
compartments of normal adult rat brain - meninges, choroid plexus, and
parenchymal perivascular space. In addition, Ninj1-expressing round shaped cells
are recruited into these three regions of experimental autoimmune
encephalomyelitis (EAE) rat brain. We further identified that Ninj1 is expressed in
the myeloid lineage cells (macrophages/monocytes and neutrophils) and endothelial
cells of the region of blood-brain barrier (BBB) distruption in EAE rat brain.
Moreover, Ninj1 mediates homophilic binding between BV2 (monocyte lineage
murine microglia) and human brain microvascular endothelial cells. Altogether,
these results suggest that Ninj1 may be a potential therapeutic target to regulate
monocyte/neutrophil trafficking across BBB in the inflammatory diseases such as
multiple sclerosis.
1
NeuroVascular Coordination Research Center, College of Pharmacy and
Research Institute of Pharmaceutical Sciences, Seoul National
University, Seoul, Korea.、2Department of Anatomy and Neurobiology,
Biomedical Science Institute, School of Medicine, Kyung Hee University,
Seoul, Korea、3WCU Department of Molecular Medicine and
Biopharmaceutical Sciences, Graduate School of Convergence Science
and Technology, College of Pharmacy, Seoul National University, Seoul,
Korea
Bum Ju Ahn1、Hyo-Jong Lee1、Min Wook Shin1、
Jeong-Hyun Choi1、Hoang Le1、Jong-Ho Cha1、Ji-Hyeon Park1、
Hyun Ho Kim1、Joo-Won Jeong2、Kyu-Won Kim1,3
Role of ninjurin 1 in the migration of myeloid cells
into inflamed CNS
E48
Vascular inflammation plays an important role in the pathogenesis of human
diseases, including cardiovascular diseases and sepsis. Keap1 is a cytoplasm
repressor of the transcription factor Nrf2. resulting in the expressional regulation of
phase II genes, such as NQO1, 㱏-GCLC, 㱏-GCLM, GST-㱘1, GST-A4 and HO1, which are involved in cell survival and immune suppression. We here
investigated the regulatory effects of keap1 knockdown using a siRNA approach on
inflammatory gene expression in endothelial cells. Keap1 knockdown increased
Nrf2 activation and phase II gene expression, as well as suppressed TNF-㱍
-induced NF-㱖B activation and expression of adhesion molecules, such as ICAM
and VCAM, resulting in the suppression of interaction between endothelial cells
and monocytes. Moreover, keap1 knockdown suppressed TNF-㱍-induced ROS
production and GSH oxidation, which are important cellular processes for NF-㱖B
activation. The effects of keap1 siRNA on TNF-㱍-induced cellular events were
effectively reversed by HO-1 inhibitor (SnPP) and GCL inhibitor (BSO), but not
significant by other phase II enzyme inhibitors, indicating that the antiinflammatory effect of keap1 siRNA in endothelial cells is associated with
expression of HO-1 and GCL. Furthermore, intravenous injection with keap1
siRNA diminished the production of the LPS-induced cytotoxic mediators, TNF-㱍
and NO, in a mouse model, as well as protected mice from LPS-induced tissue
damage and mortality. These results indicate that keap1 knockdown increased
expression of phase II genes, particularly HO-1 and GCL, responsible for inhibiting
NF-㱖B-dependent inflammatory gene expression and septic mortality.
Vascular System Research Center and Department of Molecular and
Cellular Biochemistry School of Medicine, Kangwon National University,
Chuncheon, Kangwon-do, Korea、2Department of Molecular and Cellular
Biochemistry School of Medicine, Kangwon National University,
Chuncheon, Kangwon-do, Korea、3Department of Biochemistry, College
of Sciences, Yonsei University, Seoul, Korea.
1
Ji-Hee Kim1,2、Dong-Kwon Lee1,2、Hansooo Lee1、
Kwon-Soo Ha1,2、Young-Guen Kwon3、Young-Myeong Kim1,2
Keap1 knockdown inhibits vascular inflammation
through the up-regulation of Nrf2-dependent phase II
gene expression
E49
18：00〜
1
The Cardiovascular Departemtn of Internal Medicine, Cheng Hsin
General Hospital,Taipei,Taiwan、2The Taipei Veteranʼs General Hospital,
Taipei, Taiwan、3The Clinical Institute, National Yang-Ming University,
Taipei, Taiwan
Background: Endothelial progenitor cells provide an endogenous repair mechanism
for endothelial dysfunction, which is implicated in chronic heart failure. In this
study, the potential effect of peroxisome proliferator activated receptor-㱍 activator;
fenofibrate on the function of endothelial progenitor cells was investigated in vitro.
Hypothesis: Fenofibrate could improve endothelial progenitor cells function
through phosphatidylinositol-3ʼ-kinase/protein kinase B and adenosine
monophosphate activated protein kinase (AMPK)signaling and nitric oxide
production.
Methods: Cultured endothelial progenitor cells collected from 20 patients (aged 65
±8 years) with symptomatic chronic heart failure were characterized by antihuman CD-34, CD-133 monoclonal antibody. The inhibitory effects of fenofibrate
at 5, 10, 20, and 50 㱘M on the cultured endothelial progenitor cells were tested.
Fibronectin-coated plate adhesion, modified Boyden chamber assay and in vitro
angiogenesis assay kit were used to assess the adhesion, migration and tube
formation ability of the endothelial progenitor cells. Furthermore, the signal
transduction pathways of the drug effects were studied using Western blot analysis.
Result: Fenofibrate significantly increased endothelial progenitor cells adhesion to
fibronectin-coated plates, migration, and tube forming function dose-dependently
(P<0.05, P<0.05, and P<0.05, respectively). Western blot analysis showed that
protein kinase B and endothelial nitric oxide synthase phosphorylation were
significantly increased by 87% and 38%, respectively (P<0.05 and P<0.05,
respectively) by fenofibratre. Small interfering RNA of endothelial nitric oxide
synthase and phosphatidylinositol 3ʼ-kinase inhitor LY294002 and adenosine
monophosphate kinase blocking agent (compound-C) significantly inhibited the
fenofibrate-induced endothelial progenitor cell adhesion, migration and tube
formation (P<0.05, P<0.05, and P<0.05, respectively).
Conclusion: We demonstrated the fenobibrate can improve the adhesion, migration,
and tube formation function of the endothelial progenitor cells collected from
chronic heart failure patients through phosphatidylinositol 3ʼ-kinase/protein kinase
B and adenosine monophosphate activated protein kinase signaling and nitric oxide
production.
1
School of Life Sciences and Biotechnology, College of Natural
Sciences, Kyungpook National University,Korea、2CHA University, Dep.
Biomedical Science, Lab. Vascular Medicine & Stem Cell Biology、
3
RIKEN Center for Developmental Biology, Kobe, Japan
Endothelial progenitor cells (EPCs) contribute to tumor vasculature during tumor
progression. Recently, we reported that decursin is a novel candidate for
angiogenesis inhibitor (Jung et al., Carcinogenesis 2009). Here we have investigated
whether decursin regulates EPC differentiation and function for the inhibition of
tumor vasculogenesis. Decursin significantly decreased number of EPC colony
forming unit of human cord blood-derived AC133 + cells. Decursin dosedependently (5~100 µM) decreased cell number of EPC committing cells
demonstrated by EPC expansion study. Decursin inhibited EPC differentiation from
committing cells into large EPC colonies. And decursin inhibited migration and
tube formation of cultured EPCs isolated from mouse bone marrow. Decursin (4
mg/kg) inhibited tumor-induced mobilization of circulating EPCs (CD34 + /
VEGFR2 + cells) from bone marrow and incorporation of Dil-Ac-LDL labeled or
GFP+ EPCs into the neovessels of xenograft Lewis lung carcinoma tumor in wild
type or bone-marrow transplanted mice. Moreover, decursin suppressed that of
Ang-2, angiopoietin receptor Tie-2, Flk-1 (KDR, VEGFR-2), and endothelial nitric
oxide synthase (eNOS) in cultured EPCs in a dose dependent manner. Therefore,
decursin clearly attenuated the contribution of EPC populations in neovessels of
tumor mass grown in mouse. Taken together, these results suggest that decursin
affects EPC differentiation and function, and thereby inhibits tumor vasculogenesis
in early tumorigenesis.
Index
Aⴊ▤↢‛කቇળKPFD
Poster
The 8th Korea-Japan Joint Symposium on Vascular Biology
Selected Oral
Presentation
219
- Background and aims
Stem cells are multipotent cells with the ability to self-renew and differentiate into
specialized cells in response to appropriate signals. The development of new
approaches for the directed differentiation of ESCs into cardiomyocytes will likely
provide therapeutic application of ES cells in heart disease. Recently, a number of
small molecules have been shown to important for modulating specific
differentiation pathway of ESCs. Our research goal is to identify crucial factors and
target genes involved in cardiomyogenesis using small molecules as cardiomyocyte
differentiation factors.
- Methods and Reslts
We generated mESCs expressing enhanced green fluorescence protein (eGFP)
under direction of a
α-myosin heavy chain (α-MHC) gene promoter that
specifically works in cardiomyocytes. After 750 mESCs were cultured in hanging
drop for three days, the resultant individual embryoid bodies (EBs) was treated with
10μM small molecule. Then GFP positive area scoring and FACS analysis were
used for the selection of most effective compound. And large-scale expression
studies were performed using Illumina expression microarray.
Comprehensive transcriptome analysis of cardiomyocytes in comparison with
undifferentiated mESCs led to identification of 895 up-regulated probe sets and 819
down-regulated probe sets. Among them, cardiomyocyte genes including ANP (1.6
fold) and cTnT (1.5 fold) are up-regulated. And the effect of hit compound is
confirmed by real-time qPCR. Moreover, myotubule formation was slightly
increased in hit compound treated cells in comparison with vesicle treated C2C12
myoblast cells.
- Conclusions
Though multiple screening steps, we obtained hit compound to accelerate
cardiomyocyte differentiation. These results may involve in molecular mechanisms
related to cardiomyogenesis and it will be ultimately facilitate the application of
developments of stem cell therapy and related to small molecule therapeutics.
Y.I.A.
Presentation
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
ObjectiveEndothelial heme oxygenase (HO)-1 expression accelerate vascular repair
by enhancing the recruitment and migration of circulating EPCs. The goal of this
study was to investigate the role of HO-1 in the direct effects of Ginkgo biloba
extract 761 (as a HO-1 inducer) on vascular remodeling in response to mechanical
vascular injury, the biologic impact on vascular neointimal growth process.
MethodsMice pretreated GBE for 14 days and underwent wire-induced femoral
artery injury. Masson trichrome staining for neointimal hyperplasia and confocal
microscopy for re-endothelialization were performed. The characteristics of
mobilized peripheral blood mononuclear cells and human vascular progenitor cells
functional effect by GBE were also determined. ResultsAt day 28 after wireinduced femoral, the tendency of reduction neointimal hyperplasia was observed at
GBE treated group. Intriguingly, the delayed endothelial recovery and enhanced
smooth muscle progenitor cells on wire-induced femoral artery were restored after
GBE treatment. GBE reduction neointima hyperplasia was losing in present
ZnPPIX (HO-1 inhibitor), suggestion HO-1-induced involved this processing. In
addition, chronic culture incubation with GBE resulted in activation endothelial
progenitor cell (EPC) and impaired smooth muscle progenitor cell (SMPC)
migration, as well as HO-1 protein accumulation. GBE-stimulated late EPC and
-suppressed SMPC migration were abolished by HO-1 siRNA suggested that GBEinduced HO-1 involved activation EPCs and suppression SMPCs. Conclusions Our
findings demonstrate that HO-1 involves GBE-induced mobilization putative
vascular progenitor cells in peripheral blood, which reduces neointimal hyperplasia
at wire-induced femoral artery injury.
Lab of cardiovascular genomics, Division of life and pharmaceutical
science, Ewha womans university, Seoul, Korea
Central Research Institute, Choongwae Pharma Corp., Hwasung-Si,
Korea
Sejin Jeon、Chae-ji Lim、Eun Jung Lee、Bora Kim、Se-Woong Oh、
Goo Taeg Oh
Symposium
Institute and Department of Pharmacology, National Yang-Ming
University, Taipei, Taiwan
Jia-Shiong Chen
How to write
Plenary Lecture
Nature Medicine
Identification of Novel Differentiation Factors during
Cardiomyogenesis in mouse embryonic stem cells
(mESCs)
Special Talk
Ginkgo biloba extract 761 activates endothelial
progenitor cells and suppresses smooth muscle-like
cells after mechanical-injury: Role of heme oxygenase
1
Special
Lecture
E53
Wen-Pin Huang1,3、Wei-Shian Yin1,3、Jaw-Wen Chen2,3、
Shing-Jong Lin2,3
Seok Yun Jung1、Jin Hwa Choi1、Sang-Mo Kwon2、
Takayuki Asahara3、You Mie Lee1
President
Lecture
E52
Fenofibrate improves endothelial progenitor cell
function in vitro through phosphatidylinositol-3ʼkinase/protein kinase B and nitric oxide production
E51
Decursin inhibits tumor vasculogenesis by suppression
of differentiation and mobilization of endothelial
progenitor cells from bone marrow
E50
Poster Session：Stem Cell / Progenitor Cells
12月2日
（木）
／Thursday, December 2
Program
Aⴊ▤↢‛කቇળKPFD
220
Not only the proliferation of endothelial cells but also the differentiation of
endothelial progenitor cells (EPCs) involve in neovascularization and
vasculogenesis. Chlamydia pneumoniae is a genus of obligate bacteria in the family
of Chlamydiaceae. High proportions of atherosclerosis sufferer hold the Chlamydia
pneumoniae in their plasma. The electronic microscopy showed that GroEL of
Chlamydia pneumoniae existence in atherosclerotic plaque. Whether GroEL of
Chlamydia pneumoniae influences EPCs function remains to be elucidated. In our
present study, the MTT assay shows that GroEL inhibits EPC proliferation in
concentration dependent manner. Long-term incubation of GroEL (100ng/ml) for 5
days decreases the number of early EPCs. 24-hours GroEL treatment diminishes
early and late EPCs adhesiveness; and GroEL could also reduce the EPC migration
and the tube formation capacity. Furthermore, GroEL enhances the percentage of
senescence-associated beta-galactosidase-positive EPCs. In addition, GroEL
inhibits eNOS activation, in contrast with activated ERK1/2 and p38 MAPK,
increased NF-kappa B translocation and ICAM-1 expression mediated by TLR4. In
conclusion, GroEL of Chlamydia pneumoniae could cause inflammation and
dysfunction through TLR4 activation and eNOS inactivation, and impairs the
vasculogenesis ability in late EPCs. We expect that the results of present study will
provide insights into the development of therapeutic strategies for the prevention of
Chlamydia pneumoniae-induced atherogenesis.
Institute of Clinical Medicine, National Yang-Ming University, Taipei,
Taiwan、2Cardiovascular Research Center, National Yang-Ming
University, Taipei, Taiwan、3Department of Pharmacology, National
Yang-Ming University, Taipei, Taiwan、4Division of Cardiology and
Department of Medical Research and Education Taipei Veterans
General Hospital, Taipei, Taiwan、5Department of Internal Medicine,
Taipei Medical University, Taipei, Taiwan.
1
Tsai Hsiao-Ya1,2、Chen Jaw-Wen2,3,4、Lin Feng-Yen5、
Lin Shin-Jong1,2,4
GroEL, a heat shock protein 60 of
, attenuates human endothelial progenitor
cells function
E54
◆no data◆
◆no data◆
Hsueh-Hsiao Wang
◆no data◆
E55
Poster
Index
Aⴊ▤↢‛කቇળKPFD
Selected Oral
Presentation
The 8th Korea-Japan Joint Symposium on Vascular Biology
Y.I.A.
Presentation
221
Homocysteine (HCY), an amino acid derived from methione is an independent risk
factor for coronary artery disease. The essential oil (AM-EO), extracted by steam
distillation from the flowers parts of Achillea millefolium has reported to exhibit
antioxidative and antibacterial activities. However, little is known about the
protective role of AM-EO in oxidative stress. In the present study, we investigated
whether AM-EO could provide protection against HCY-induced oxidative damage
in cultured rat vascular smooth muscle cells (VSMC). Cultured VSMC were
exposed to HCY (100 㱘M) treatment in the absence or presence of AM-EO (2.5-20
㱘g/mL). The results indicated that the AM-EO treatment alone did not affect the
cell viability of VSMC. The treatment with 100 㱘M HCY significantly elevated
the levels of malondialdehyde (MDA), a product of lipid peroxidation, and
superoxide anion production in cells. In addition, HCY treatment alone caused an
increase in glutathione levels and antioxidative enzymes activities. However, the
treatment of VSMC with AM-EO significantly decreased HCY-induced superoxide
anion production, reduced MDA accumulation and regulated antioxidant status in
cells. In conclusion, these results suggest that Achillea millefolium essential oil
may ameliorate the HCY-induced oxidative stress in cultured rat vascular smooth
muscle cells.
Backgrounds Recently peroxisome proliferator-activated receptor-gamma
coactivator-1alpha (PGC-1㱍), a master regulator of mitochondrial biogenesis and
cellular energy metabolism, has been shown to control angiogenic process to fulfill
oxygen and substrate requirements in tissue. Even though intact vascular barrier
function is necessary for new vessels to supply sufficient oxygen and substrate to
tissue, yet, the role of PGC-1㱍 in vascular barrier function has not been elucidated.
Therefore, in this study we investigated whether PGC-1㱍 plays a role in regulating
endothelial cell barrier function.Methods and Results In C57/BL6 mouse, local
PGC-1㱍 over-expression reduced vascular leakage at the ears of the mice, as
evidenced by both Evans blue dye and TRITC-dextran exclusion test. In vitro,
PGC-1㱍 overexpression at human umbilical vein endothelial cells reduced
starvation induced endothelial barrier dysfunction by 83%, as assessed by the
passage of FITC-dextran through human umbilical vein endothelial cell
monolayers. This reduction of barrier dysfunction was accompanied by reduction in
Angiopoietin-2 expression induced by starvation of the endothelial cells. PGC-1㱍
overexpression attenuated FOXO1㱍 nuclear translocation and decreased
Angiopoietin-2 transcription which is regulated by FOXO1㱍.Conclusions These
data demonstrate that PGC-1㱍 suppresses the expression of angiopoietin-2 in
endothelial cell and enhances vascular barrier function both in vitro and in vivo and
thus supports the function of PGC-1㱍 as a molecular regulator of tissue metabolic
demand and supply.
Symposium
The 18th Annual Meeting of the Japanese Vascular Biology and Medicine Organization
Department of Food and Nutrition, Providence University, Taichung,
Taiwan, R.O.C、2Department of Cosmetic Science, Providence
University, Taichung, Taiwan, R.O.C.
1
National Research Laboratory for Cardiovascular Stem cell, Seoul
National University College of medicine , Seoul, Korea、2Department of
Internal Medicine, Seoul National University Hospital, Seoul, Korea
1
Hsin-Yi Peng1、Jen-Chieh Ho1、Ying Shih2、Chia-Pei Lai1、
○Su-Tze Chou1
How to write
Plenary Lecture
Nature Medicine
Tae-Kyu Lee1、Sang-Eun Lee2、Jin Hur1、Baek-Kyung Kim1、
Sooryeonhwa Jin1、Soo-Kyung Kang1、Yoo-Wook Kwon1、
Byung-Hee Oh1、Young-Bae Park1、Hyo-Soo Kim1,2
E59
Special Talk
The effects of Achillea millefolium essential oil on
homocysteine-induced oxidative stress in cultured rat
vascular smooth muscle cells
Special
Lecture
PGC-1alpha regulates endothelial cell barrier function
by suppressing angiopoietin-2 expression
BackgroundCardiovascular safety of rosiglitazone has been challenged. On the
other hand, many basic researches have suggested consistently that rosiglitazone
had various beneficial effects on vascular cells. However, its potential role in
expression of TF, a primary molecule for coagulation, has not been studied. We
investigated the effects of rosiglitazone on tissue factor (TF) expression and
elucidated its underlying mechanisms.Methods and ResultsRosiglitazone inhibited
TF expression in response to tumor necrosis factor-㱍 (TNF-㱍) in human umbilical
vein endothelial cells (HUVECs), human acute monocytic leukemia cell line (THP1) and human umbilical vein smooth muscle cells (SMCs). The overexpression of
TF was mediated by increased phosphorylation of mitogen-activated protein (MAP)
kinase, which was blocked by rosiglitazone. The main MAP kinase differed
depending on each cell type. Luciferase and ChIP assays showed that transcription
factor, activator protein-1 (AP-1), was a pivotal target of rosiglitazone to lower TF
transcription. Intriguingly, two main drugs for drug-eluting stent, paclitaxel or
rapamycin, significantly exaggerated thrombin-induced TF expression, which was
also effectively blocked by rosiglitazone in all cell types. In contrast to its effect on
TF, rosiglitazone did not impair TF pathway inhibitor (TFPI) in three types of cells.
In rat carotid artery balloon injury model with paclitaxel infusion, rosiglitazone
attenuated TF expression in injured vasculature.ConclusionsRosiglitazone reduced
TF expression in vascular cells via MAP kinase and AP-1 inhibitions. Also,
rosiglitazone reversed the paclitaxel-induced aggravation of TF expression, which
suggests that it could be a potential therapeutic option to prevent stent thrombosis
after paclitaxel-eluting stents implantation.
National Research Laboratory for Cardiovascular Stem Cell, Seoul
National University College of medicine, Seoul, Korea、2Department of
Internal Medicine, Seoul National University Hospital, Seoul, Korea
1
Baek-Kyung Kim1、Jun-Bean Park2、Yoo-Wook Kwon1、
Hyun-Chae Lee1、Seok-Won Yoon1、Young-Eun Choi1、
Tae-Kyu Lee1、Jaewon Lee1、Byung-Hee Oh2、Young-Bae Park1、
Hyo-Soo Kim1,2
President
Lecture
E58
Background: Previous studies showed that microalbuminuria is an index of
hypertensive organ damage. Given the role of inflammation and oxidative stress in
the pathogenesis of microalbuminuria, and the remarkable antioxidant and antiinflammatory properties of bilirubin, this study was undertaken to evaluate the
relationship between serum bilirubin concentrations and the degree of urinary
albumin excretion in patients with essential hypertension. Methods: Totally, 120
subjects with essential hypertension were enrolled in the study, in which 80 patients
(67%) with normoalbuminuria (albumin excretion rate [AER] of < 20 㱘g/min,
overnight urine samples), 30 patients (25%) with microalbuminuria (an AER of 20
to 200 㱘g/min), and 10 patients (8%) with macroalbuminuria (an AER > 200 㱘g/
min). Serum bilirubin level was determined by an enzymatic method with bilirubin
oxidase on an autoanalyzer. Results: Compared to hypertensive patients with
normoalbuminuria, patients with micro- or macroalbuminuria had significantly
higher plasma high-sensitivity C-reactive protein (hs-CRP) levels (P < 0.001),
higher serum creatinine levels (P < 0.001), and lower serum bilirubin concentrations
(P = 0.029). In addition, there was an inverse correlation between serum bilirubin
concentrations and plasma hs-CRP levels (P = 0.011). By multivariate regression
analysis, serum bilirubin concentrations were an independent determinant of and
had an inverse correlation to log (urinary albumin excretion) in hypertensive
patients (㱎 = -0.164, P = 0.042). Conclusions: In patients with essential
hypertension, serum bilirubin levels are inversely associated with albuminuria. The
development of interventions that promote bilirubin levels may be a potential target
to reduce vascular damage in hypertensive patients.
Division of Cardiology, Taipei Veterans General Hospital, Taipei, Taiwan
Shao-Sung Huang、Po-Hsun Huang、Kuang-Hsing Chiang、
Jaw-Wen Chen、Shing-Jong Lin
Rosiglitazone Suppresses Paclitaxel-induced Tissue
Factor Overexpression in Vascular Cells: A Promising
Answer to Rosiglitazone Controversy
E57
18：00〜
Association of serum bilirubin levels with albuminuria
in patients with essential hypertension
E56
Poster Session：Others
12月2日
（木）
／Thursday, December 2
Program
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Background: Extracellular superoxide dismutase (EC-SOD) is potentially more
suitable for vascular gene transfer than other SODs because it is a secreted enzyme
and has a remarkably long half-life. It has been reported that oxidative stress
stimulate vascular smooth muscle cell proliferation through MEK/ERK 1/2
pathway. We hypothesize that EC-SOD overexpression will have a protective effect
against oxidative stress in proliferation or migration of the rat vascular smooth
muscle cells (VSMC) and MEK/ERK 1/2 pathway is involved.Methods and
Results: EC-SOD was constructed in the pcDNA 3.1-TOPO mammalian expression
vector and the hemagglutinating virus of Japan envelope (HVJ-E) was used for
gene delivery. VSMC transfected with EC-SOD by HVJ-E were used for
subsequent assays 48 hrs after transfection. The control groups included nontransfected cells and pcDNA 3.1-TOPO vector-transfected cells. The inhibition
effects of cell proliferation by MTT assay and cell migration by wound healing
assay were assessed, respectively. Additionally, for determining proliferationassociated pathway, VSMCs were serum starved for 24 hrs and stimulated by 10%
fetal bovine serum for 10 min. These VSMCs were then lysed and prepared for
examining MEK/ERK 1/2 pathway using Western blot. Our data showed that the
SOD-overexpressed VSMCs revealed more decrease in proliferation and migration,
compared to the control groups. The MEK/ERK 1/2 activity of SOD-overexpressed
VSMCs was decreased compared to those of the control groups.Conclusions: The
results suggest that EC-SOD gene transfer may potentially reduce VSMCs
proliferation and migration, which may involve the MEK/ERK 1/2 pathway.
Division of Cardiology, Kaohsiung Veterans General Hospital,
Kaohsiung City, Taiwan, R.O.C、2Department and Graduate Institute of
Pharmacology, College of Medicine, Kaohsiung City, Taiwan, R.O.C.
1
Shoa L Lin1、Li J Ou1、Jwu L Yeh2
Impact of Extracellular Superoxide Dismutase for
inhibition of the smooth muscle cell proliferation and
migration
E60

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