From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
VASCULAR BIOLOGY
Vascular endothelial growth factor-D transgenic mice show enhanced blood
capillary density, improved postischemic muscle regeneration, and increased
susceptibility to tumor formation
Anna-Mari Kärkkäinen,1 Antti Kotimaa,1 Jenni Huusko,1 Ivana Kholova,1 Suvi Elina Heinonen,1 Anna Stefanska,1
Marike Hinke Dijkstra,1 Hanna Purhonen,1 Eveliina Hämäläinen,1 Petri Ilmari Mäkinen,1 Mikko Petri Turunen,1 and
Seppo Ylä-Herttuala1
1A.I.
Virtanen Institute, Department of Biotechnology and Molecular Medicine, University of Kuopio, Kuopio, Finland
Vascular endothelial growth factor-D
(VEGF-D) has angiogenic and lymphangiogenic activity, but its biologic role
has remained unclear because knockout
mice showed no clear phenotype. Transgenic (TG) mice expressing the mature
form of human VEGF-D (hVEGF-D) were
produced by lentiviral (LV) transgenesis
using the perivitelline injection method.
Several viable founders showed a macroscopically normal phenotype and the
transgene transmitted through germ line.
Expression of hVEGF-D mRNA was high
in skeletal muscles, skin, pancreas, heart,
and spleen. A significant increase was
found in capillary density of skeletal
muscles and myocardium, whereas no
changes were observed in lymphatic capillary density. After induction of hindlimb
ischemia, the TG mice showed enhanced
capacity for muscle regeneration. However, on aging the TG mice had significantly increased mortality from malignant tumors, of which half were breast
adenocarcinomas characterized with the
absence of periductal muscle cells. Some
tumors metastasized into the lungs. In
addition, lung and skin tumors were
found, but no blood- or lymphatic vessel–
derived malignancies were detected. We
conclude that in mice hVEGF-D is an
angiogenic factor associated with improved muscle regeneration after ischemic injury but also with increased
incidence of tumor formation with a preference for mammary gland tumors.
(Blood. 2009;113:4468-4475)
Introduction
Vascular endothelial growth factors (VEGFs) are key mediators of
blood and lymphatic vessel formation during embryonic development and in adults. Blood vessel formation occurs either by
vasculogenesis, when a new network of capillaries is formed from
endothelial precursor cells differentiating in situ, or by angiogenesis, when new capillaries invade the organ by forming vascular
sprouts that originate from pre-existing vessels.1 The lymphatic
system comprises a separate vascular system that also permeates
most organs. Lymphatic vessels are essential for immune surveillance, tissue fluid homeostasis, and fat absorption. Defects in
formation or function of lymphatic vessels cause lymphedema, and
lack of lymphatic vessels result in fluid accumulation, which causes
prenatal death.2 Lymphangiogenesis occurs either by local de novo
differentiation of lymphatic endothelium from lymphangioblasts or
by sprouting from preexisting lymphatic veins.3
Currently, 7 members have been identified in the VEFG family:
VEGF-A, -B, -C, -D, -E (viral VEGF analogs), -F (snake venom
VEGFs), and placental growth factor (PlGF). Of these, VEGF-E
and -F represent exogenous VEGFs.4
Several VEGF receptors (VEGFRs) have been identified for VEGFs:
VEGFR-1 (flt-1; fms-induced tyrosine kinase receptor), VEGFR-2
(KDR; kinase insert domain–containing receptor), and VEGFR-3 (flt-4)
belong to the superfamily of receptor tyrosine kinases (RTKs). They
share a common structure, yet they bind VEGFs in distinct affinities and
specificities. In addition, Neuropilins 1 and 2 (Nrp-1, Nrp-2) bind some
specific VEGFs. Nrps are non-RTKs that are believed to serve as
coreceptors for certain VEGFs and their isoforms.5
VEGF-D (also known as c-fos–induced growth factor, FIGF) is a
secreted growth factor consisting of a central VEGF-homology domain
(VHD), receptor binding domains, and propeptides in both termini.
VEGF-D is secreted into the extracellular space as a full-length
VEGF-D homodimer. After secretion, proprotein convertases cleave the
C- and N-terminal propeptides from the VHD to form the mature
VEGF-D.6 The mature hVEGF-D binds to hVEGFR-2 and -3 with
higher affinities than the full-length unprocessed hVEGF-D. Numerically, the mature hVEGF-D has approximately 290-fold higher affinity
to hVEGFR-2 and approximately 40-fold higher affinity to hVEGFR-3
than the unprocessed full-length hVEGF-D, which mainly binds to
hVEGFR-3.7 In this article hVEGF-D refers to mature hVEGF-D⌬N⌬C.
Cellular effects of hVEGF-D depend on the receptor and target cell type.
Vascular endothelial cells express VEGFR-2, and the expression is
up-regulated inter alia by angiogenesis.8 hVEGF-D exerts angiogenic
effects on binding to VEGFR-2, whereas when bound to VEGFR-3 on
lymphatic endothelium it stimulates lymphangiogenesis. In addition to
VEGFR-2 and -3, hVEGF-D is able to interact with Nrp-2, which has a
role in lymphangiogenesis.5 VEGF-D probably possesses differing
functions in different species; in mouse hVEGF-D binds to both
VEGFR-2 and -3, whereas mVEGF-D binds only to VEGFR-3.9,10
We used lentiviral (LV) perivitelline injection technique for the
generation of transgenic (TG) mice and followed them up to
Submitted July 31, 2008; accepted November 14, 2008. Prepublished online as
Blood First Edition paper, December 10, 2008; DOI 10.1182/blood-2008-07-171108.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
An Inside Blood analysis of this article appears at the front of this issue.
© 2009 by The American Society of Hematology
4468
BLOOD, 30 APRIL 2009 䡠 VOLUME 113, NUMBER 18
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 30 APRIL 2009 䡠 VOLUME 113, NUMBER 18
HUMAN VEGF-D TRANSGENIC MICE
4469
Hindlimb ischemia
Figure 1. Schematic presentation of the third generation lentiviral vector used
in the transgenesis studies and Southern blot image of a copy number
analysis. (A) CMV indicates cytomegalovirus promoter; ⌿, packaging signal; cPPT,
central polypurine tract. The TG cassette contains the human phosphoglycerate
kinase (hPGK) promoter, human VEGF-D cDNA truncated from C- and N-termini
(hVEGF-D), and woodchuck hepatitis virus PRE element (wPRE). SIN indicates that
the lentiviral vector is self-inactivating because of the deletion in U3 LTR to form ⌬U3;
U5, 5⬘ LTR; RRE, rev-responsive element. (B) Analysis of the 4 hVEGF-D TG founder
mice (1, 2, 3, and 4) and 3 F1 mice (2a, 2b, and 2c) from founder number 2. The copy
numbers of standards (left half of the image) are indicated (copies/15 ␮g of mouse tail
genomic DNA). wPRE was used as a probe sequence. Molecular weight marker
(MW) between the standard and sample lanes is 2.5 kb. A vertical line has been
inserted to indicate a repositioned gel lane; 1, 1 to 5 copies; 2, 7 to 10 copies; 3, 5 to
7 copies; 2a, 5 to 7 copies; 2b, 7 to 10 copies; and 2c, 7 to 10 copies.
Hindlimb ischemia model has been described.14 Briefly, mice were
anesthetized with Rompun-Ketalar mixture (10 mg/kg xylazine [Rompun],
80 mg/kg ketamine), and ligation of the arteria femoralis superficialis was
performed. Animals were killed by CO2 inhalation 1, 2, and 3 weeks after
the operation. The organs were perfused with 1 ⫻ PBS via left ventricle.
After perfusion, samples from all major tissues were collected for molecular biological and histological analyses. Histologic samples were fixed in
4% PFA–15% sucrose for 4 hours, rinsed in 15% sucrose, processed, and
embedded in paraffin. Slides (5 ␮m thick) were cut and routinely stained
with hematoxylin-eosin (HE).
Copy number and integration site analyses
Mouse genomic DNA was digested with AflII or SacII restriction endonucleases for the copy number analysis and for the determination of the number
of integration sites, respectively. Prehybridization of the membranes was
performed before hybridization with 32P-CTP–labeled wPRE probe. For the
copy number analysis, a standard curve was generated by adding the
plasmid amount equal to copy numbers of 1, 5, 10, 20, and 30 into 15 ␮g of
TG-negative mouse genomic DNA.
Reverse transcriptase–PCR
generation F3. The LV transgenesis method is still rarely used
despite its versatility and advantages over traditional transgenesis
methods.11 In this study, LV transgenesis was an effective method
for producing TG offspring. We found that hVEGF-D TG mice
showed no major changes in lymphatic capillary density. Instead,
they showed enhanced angiogenesis as well as improved muscle
regeneration after injury. Unexpectedly, the TG mice possessed an
increased susceptibility to tumor formation.
Methods
Lentiviral vector construction
Vesicular stomatitis virus G-protein (VSV-G)–pseudotyped, third generation LVs were constructed to directly express the mature form of hVEGF-D
under the human phosphoglyserate kinase (hPGK) promoter. Woodchuck
hepatitis virus pre-element (wPRE) and central polypurine tract (cPPT)
were used in the vector backbone. The virus production, concentration, and
p24 assays were performed as described earlier.12 Titers of the concentrated
LVs ranged from 8.5 ⫻ 108 to 1.9 ⫻ 109 TU/mL. A schematic representation of the LV vector is shown in Figure 1A.
Experimental animals
TG mice were produced by LV transgenesis with the use of the perivitelline
injection method as described.11,13 Briefly, after breeding, fertilized 1-cell–
stage oocytes were collected from oviducts of superovulated CD2F1 donor
females weighing 9 to 12 g. Approximately 100 pL of 109 TU/mL
hVEGFD-LV was injected into the perivitelline space of the oocytes. After
overnight recovery and growth in vitro, the 2-cell–stage embryos were
transferred to pseudopregnant CD2F1 female mice. Pups were weaned and
genotyped at 3 weeks of age. The material for genotype polymerase chain
reaction (PCR) was skin attained from earmarking. The genotyping was
performed by PCR with the use of the following primers: forward primer,
5⬘-TTGCCAGCTCTACCACCAG-3⬘; reverse primer, 5⬘-TTCATTGCAACAGCCACCAC-3⬘. The primers were specific for hVEGF-D cDNA.
Animals used in the study were from F0 to F3 generations. TG-negative
littermates (the same genetic and epigenetic background lacking hVEGF-D)
were used as controls. All animal studies had the approval of the
Experimental Animal Committee of Kuopio University.
Expression of hVEGF-D mRNA in tissues of TG mice was analyzed by
real-time reverse transcriptase–PCR (RT-PCR). Total RNAs of different
tissues were extracted by RNeasy kit (QIAGEN, Valencia, CA) according
to the manufacturer’s instructions. cDNA synthesis was performed with the
use of random hexamer primers and PCR by predesigned hVEGF-D
primers and probe (Applied Biosystems, Foster City, CA). Normalization
was performed with the use of mouse ␤-actin control kit (Applied
Biosystems).
Enzyme-linked immunoabsorbent assays and serum clinical
chemistry
hVEGF-D and mVEGF-A concentrations were analyzed with the use of
Quantikine Immunoassays (Human VEGF-D or Mouse VEGF-A; R&D
Systems, Minneapolis, MN). Immunoassays were performed from tissue
lysates and from serum samples. Tissues were homogenized in T-PER
buffer (Tissue Protein Extraction Reagent; Pierce Chemical, Rockford, IL),
and lysates were prepared according to the manufacturer’s instructions.
Mouse serum samples from TG-negative and hVEGF-D TG mice were
analyzed with the use of standard clinical chemistry methods at the
Department of Clinical Chemistry, Kuopio University Central Hospital.
Immunohistochemistry
Paraffin-embedded sections (5 ␮m thick) were immunostained with the
following antibodies: CD31 (platelet endothelial cell adhesion molecule
1[PECAM-1], 1:50; BD Biosciences PharMingen, San Diego, CA), CD34
(1:20; Hycult Biotechnology, Uden, The Netherlands), LYVE-1 (1:1000,
microwave pretreatment in citrate buffer; ReliaTech, Braunschweig, Germany), hVEGF-D (1:100, microwave pretreatment in citrate buffer; R&D
Systems), VEGFR-2 (1:100; eBiosciences, San Diego, CA), VEGFR-3
(1:50; R&D Systems), cytokeratin (CK, 1:50; Affinity BioReagents,
Golden, CO), cytokeratin 7 (CK7, 1:50, microwave pr-treatment in citrate
buffer; Santa Cruz Biotechnology, Santa Cruz, CA). Immunohistochemical
reactions were developed with the use of ABC Vectastain Elite staining kit
(Vector Laboratories, Burlingame, CA) and DAB (Invitrogen Zymed, San
Francisco, CA).
Light microscopic analysis
Histologic sections were assessed with an Olympus AX-70 microscope
(Olympus, Tokyo, Japan) with AnalySIS software (Soft Imaging System,
Muenster, Germany). Paraffin slices of all major organs (liver, spleen,
pancreas, heart, lungs, salivary glands, testes/ovaries, aorta, limb skeletal
muscles, small intestine, brain, and skin) of TG-positive and TG-negative
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
4470
BLOOD, 30 APRIL 2009 䡠 VOLUME 113, NUMBER 18
KÄRKKÄINEN et al
Figure 2. Human VEGF-D expression in TG mice.
Results of one representative real-time RT-PCR run with
3 mice were chosen to show the general pattern of TG
expression in different tissues. F1-1 and F1-2 are mice of
generation F1; F3-1, -2 and -3 are TG mice of generation
F3. (A) All analyzed tissues are included in the same
histogram. Because the scale is wide, the tissues that
showed moderate to very low TG expression are shown
with a lower scale in panel B. (C,D) Representative
ELISA assays for hVEGF-D protein content in TG mice.
(C) hVEGF-D protein in tissue lysates of F1 and F3 TG
mice. (D) hVEGF-D protein in plasma of 5 F1 mice. Error
bars represent ⫾ SD.
mice were stained with HE, and their histologies were observed for major
phenotypic changes. Hindlimb skeletal muscle (musculus rectus and
musculus caput gastrocnemius) and myocardium slices were stained with
CD34 immunohistochemistry and photographed under 20⫻ objective.
Capillary densities (capillaries/myocyte in skeletal muscles, capillaries/
mm2 in myocardium) were determined as described.14 Hindlimb skeletal
muscles with ischemic injury were stained with HE. Areas of regeneration
and different manifestations of ischemic injury were measured with the use
of AnalySIS software (Soft Imaging System). In assessing capillary density
and morphometry, 8 randomly selected fields were analyzed blindly.
Statistical methods
Statistical analyses were performed with GraphPad Prism version 4.00
(GraphPad Software, San Diego, CA). Results of the TG-positive and
TG-negative groups were analyzed with the use of unpaired, 2-tailed t test
with confidentiality of 95%, considering P values less than .05 as
statistically significant.
Results
Lentiviral transgenesis
We produced hVEGF-D mice with the use of LV transgenesis. The
amount of LV injected into one oocyte was 103 TU. Ninety-one
percent of all injected embryos survived viral microinjection, and
on the average 70% of the born founders were TG positive.
Southern blot analysis was used to analyze copy numbers of the TG
mice (Figure 1B). The copy numbers in all founders were between
1 and 10 copies/genome, showing a moderate variation between
the mice. Either the same or a lower copy number was detected in
F1 offspring. In consecutive generations the copy numbers remained similar to the copy numbers of F1 mice.
The number of chromosomal integrations was determined from
F1, F2, and F3 TG mice. Results showed that the provirus was
integrated into one chromosome in most mice, whereas in one F1
mouse and one F3 mouse, derived from the same founder,
2 chromosomal integration sites were found (data not shown).
Thus, copy numbers of more than one in most mice are probably
due to concatamerization of the provirus.
In F1 generations the litters were small (2-3 pups), but 90% to
100% of the pups were positive for the transgene. As the number of
generation advanced, the litter sizes grew, but the number of
TG-positive pups decreased. Nevertheless, the transgene was
transmitted through the germ line from one generation to another.
hVEGF-D mRNA
Expression levels of hVEGF-D mRNA varied between mice, as
well as between tissues in each mouse. However, each RT-PCR
run showed that the same tissues had highest hVEGF-D
expressions in all generations. The tissues of high expression
were skeletal muscle, skin, pancreas, and heart (Figure 2A).
Most of the other tissues fell into the category of moderate
expression (spleen, lung, kidney, and gonads; Figure 2B),
whereas each analysis showed very low or no TG expression in
liver and brain (Figure 2B). The expression was easily detectable in TG mice in all generations.
hVEGF-D protein
Protein expression of hVEGF-D in TG mice is shown in Figure 2C
and D. Concentrations were measured from tissue lysates (Figure
2C) and serum (Figure 2D). No hVEGF-D was detected in
TG-negative mouse samples. Similar to mRNA levels, the number
of generation of the TG mice did not affect TG protein expression
levels. The hVEGF-D concentration was slightly lower in the F3
mice than in the F1 mice (Figure 2C). However, the difference
between the generations was not significant. In serum samples the
hVEGF-D protein concentration varied between 20 and 700 pg/mL
(Figure 2D).
The results of mVEGF-A enzyme-linked immunoabsorbent
assays (ELISAs) showed that hVEGF-D did not affect endogenous
expression of mVEGF-A in TG mice with various copy numbers
(data not shown).
Clinical chemistry
Markers of lipid metabolism, liver and kidney function, and tissue
damage were measured from serum of TG-positive and TGnegative mice. Blood for these measurements was collected on
killing. The concentration of total serum cholesterol (Chole),
high-density lipoprotein (HDL), low-density lipoprotein (LDL),
triglycerides (Trigly), aspartate aminotransferase (ASAT), creatinine (Crea), lactate dehydrogenase (LD), and creatine kinase (CK)
were measured with the use of standard clinical chemistry methods.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 30 APRIL 2009 䡠 VOLUME 113, NUMBER 18
HUMAN VEGF-D TRANSGENIC MICE
4471
Table 1. Serum clinical analysis of hVEGFD TG mice
Mouse group
Chole,
mmol/L
HDL,
mmol/L
LDL,
mmol/L
Trigly,
mmol/L
ASAT,
U/mL
Crea,
␮mol/L
LD, U/mL
CK, U/mL
23.5 ⫾ 5.2
Controls F1
2.0 ⫾ 0.4
1.6 ⫾ 0.3
0.13 ⫾ 0.04
1.5 ⫾ 0.2
1.00 ⫾ 0.23
12.3 ⫾ 1.5
2.5 ⫾ 0.5
Controls F3
0.9 ⫾ 0.1
0.7 ⫾ 0.1
0.13 ⫾ 0.02
1.2 ⫾ 0.1
1.12 ⫾ 0.35
12.3 ⫾ 1.5
1.6 ⫾ 0.3
16.2 ⫾ 2.4
Controls all
1.4 ⫾ 0.3
1.1 ⫾ 0.2
0.13 ⫾ 0.02
1.3 ⫾ 0.1
1.08 ⫾ 0.44
12.3 ⫾ 0.1
2.3 ⫾ 0.3
21.2 ⫾ 3.1
VEGF-D TG F1
1.9 ⫾ 0.3
1.5 ⫾ 0.4
0.24 ⫾ 0.02
1.1 ⫾ 0.3
0.61 ⫾ 0.14
11.7 ⫾ 0.7
1.5 ⫾ 0.2
11.9 ⫾ 3.1
VEGF-D TG F3
1.3 ⫾ 0.3
1.1 ⫾ 0.2
0.10 ⫾ 0.02
0.8 ⫾ 0.2
0.37 ⫾ 0.03
9.0 ⫾ 1.2
0.7 ⫾ 0.1
8.5 ⫾ 1.9
VEGF-D TG all
1.5 ⫾ 0.2
1.2 ⫾ 0.2
0.15 ⫾ 0.02
1.3 ⫾ 0.2
0.47 ⫾ 0.06
10.9 ⫾ 0.7
1.2 ⫾ 0.07
11.3 ⫾ 1.6
Chole indicates cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; Trigly, triglycerides; ASAT, aspartate aminotransferase; Crea, creatinine; LD,
lactate dehydrogenase; and CK, creatine kinase.
Results of clinical analyses are summarized in Table 1. No
significant differences were observed between the groups.
mature form of hVEGF-D is a highly potent angiogenic factor
but not a lymphangiogenic factor in mice.
Vascularity
Regeneration after hindlimb ischemic injury
The effect of the transgene on vascular density was measured
from intact hindlimb skeletal muscles and myocardium of F0 to
F3 mice by CD34 immunohistochemistry. The angiogenic effect
was also determined in mice aged 1 to 2 years to study whether
the transgene was functional also in aged mice. A representative
image of CD34 immunostained skeletal muscle is shown in
Figure 3A and B. For methodologic comparison, we also show a
representative image of a serial section immunostained for
CD31 in both TG-positive and TG-negative mice (Figure 3C,D,
respectively). Results show that constitutively expressed hVEGFD⌬N⌬C is a potent angiogenic factor in mice. The effect of the
transgene on the density of lymphatic capillaries was studied
with LYVE-1 immunohistochemistry. These stainings showed a
similar number of lymphatic capillaries in TG mice and control
mice. Thus, the hVEGF-D⌬N⌬C transgene has no lymphangiogenic effect in the TG mice (Figure 3E,F). Difference in the
capillary count of hindlimb skeletal muscles of nonoperated
TG-negative and TG-positive mice was statistically significant
(P ⬍ .05; Figure 3G). The number of capillaries in the myocardium was also significantly higher in the TG-positive mice than
in the TG-negative littermates (Figure 3H). No major effect was
found on lymphatic capillary density in skeletal muscles or other
tissues. These results suggest that the constitutively expressed
To study the effect of the transgene expression on mouse muscle
biology in pathologic conditions, we induced hindlimb ischemia
using a surgical operation. Signs of tissue damage and regeneration
were studied by morphometrical measurements of the injured
muscles and by measuring the areas of active regeneration.
HE-stained musculus caput gastrocnemius of the TG mice and the
TG-negative littermates 7 days after the ischemic injury are shown
in Figure 4. When the ischemic area (apoptosis/necrosis, fibrosis,
fat atrophy) was measured, 4% to 15% of the total muscle areas in
the hVEGF-D TG mice was damaged, whereas the same range in
the TG-negative mice was 35% to 89%. Signs of active regeneration were found in larger areas of ischemic limb muscles in the
hVEGF-D TG mice compared with the TG-negative control mice
(data not shown).
Spontaneous tumors
On aging the hVEGF-D TG mice showed high frequency of
malignant tumors (Figure 5). Mice obtained from LV transgenesis
with the same vector backbone and the same promoter but a
different transgene (human Nrf2) have not developed any tumors
(data not shown). In addition, the TG-negative littermates never
developed tumors during the study. Mice with tumors originated
A
C
E
B
D
F
G
H
Figure 3. Blood and lymphatic capillary density in TG and control mice as shown by CD34, CD31, and LYVE-1 stainings. Images were taken from
immunohistochemically stained 5-␮m paraffin sections of mouse tissues and analyzed with Olympus Provis AX70 Microscope attached to Olympus ColorView 12 Camera. The
software for imaging and analyses was AnalySIS (Soft Imaging System). (A) Representative figure from a TG mouse showed an increase in capillary density (CD). (B) In
comparison, a normal capillary density was present in control mouse (CD34). (C) Representative figure from a TG mouse showed an increase in capillary density (CD31). (D) In
comparison a normal capillary density was present in control mouse (CD31). (E) Lymphatic vessels were detected only in the interstitium accompanied by venules and
arterioles in TG mice (LYVE-1). (F) Control muscles showed a similar pattern and number of lymphatic vessels (LYVE-1). Bars in panels A through F ⫽ 100 ␮m.
(G) Capillaries/myocyte of nonoperated musculus rectus of the hVEGFD TG mice and the TG-negative controls. The mice were from generations F1, F2, and F3. (H) Capillary
density in myocardium of the hVEGF-D TG-positive and the TG-negative littermates. The mice were from generations F1, F2, and F3. Error bars represent ⫾ SD.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
4472
BLOOD, 30 APRIL 2009 䡠 VOLUME 113, NUMBER 18
KÄRKKÄINEN et al
A
B
from 2 different founders. This implies that the integration site is
not involved in the tumor formation. Unexpectedly, half of the
tumors were mammary adenocarcinomas, and one of the mice with
mammary gland tumor was a male (aged 9 months). In addition,
2 lung adenocarcinomas and 2 skin carcinomas were found (Table 2).
No lymphomas or other types of lymphatic malignancies were found in
the TG mice.
Characterization of tumors
Most of the mammary gland tumors were poorly differentiated
(grade 3) with a solid and comedo growth pattern. One of the mice
with tumors was lactating when the tumor was noticed. In one of
the mice the mammary gland tumor metastasized into the lungs.
hVEGF-D was expressed in the surface layer of the ductal
component, but not in the solid parts of the tumor. Active
angiogenesis was detected in most tumors. Well-differentiated
multifocal adenocarcinomas in the lungs, as well as skin tumors
(basal cell carcinoma and anaplastic carcinoma) were found in
some mice. In the lung tumor, the papillary and stroma of the
papilla stalk were hVEGF-D positive. Anaplastic skin carcinoma
showed focal hVEGF-D positivity and intratumoral angiogenesis.
A representative panel of the immunostained tumor samples is
shown in Figure 6. Detailed characterization of the tumors is
presented in Table 2. All tumors showed detectable amounts of
hVEGF-D mRNA with high or moderate expression levels.
Figure 5. Kaplan-Mayer survival curve showing tumor mortality of hVEGF-D TG
mice and TG-negative controls. This curve presents all hVEGF-D TG mice that
died of cancer, regardless of their original background or generation. There were
2 high-expressing founders and 1 founder with lower level of expression. First
4 points represent mice that died of mammary gland tumor before the age of 1 year.
The TG-negative controls had no tumors in any generation during the follow-up.
Figure 4. Morphologic characteristics in musculus
caput gastrocnemius 7 days after hindlimb ischemia
operation. Images were taken from HE-stained 5-␮m
paraffin sections of mouse tissues and analyzed with
Olympus Provis AX70 Microscope attached to Olympus
ColorView 12 Camera. The software for imaging and
analyses was AnalySIS (Soft Imaging System). (A) Increased regeneration shown in a hVEGF-D TG mouse on
day 7 after hindlimb ischemia operation. Whole area
showed late regeneration features as eosinophilic cytoplasm and internalization of nuclei. Bar, 200 ␮m.
(B) Control group showed large area of necrosis with pale
flocculated cytoplasm (*). Early stage of regeneration is
represented by basophilic small myocytes with internalized nuclei (#). Bar, 200 ␮m.
Discussion
The high success rate of the LV transgenesis is most likely due to
the LV perivitelline injection method, compared with the traditional transgenesis method based on plasmid microinjection into
the oocyte pronucleus. In contrast to the perivitelline injection, in
the traditional plasmid microinjection method the oocyte membrane must be pierced to bring the plasmid into the pronucleus. In
an alternative LV transgenesis method, the zona pellucida must be
removed, whereas in the LV perivitelline injection technique it is
left in place to cover the oocyte during migration through the
oviduct into the uterus and during implantation of the embryo.
Thus, the LV perivitelline injection TG method results in a
significantly higher number of implanted embryos and transgenic
founders.11 Another advantage of the perivitelline injection method
over the zona removal is that the fertilized oocytes are transduced
at the 1-cell stage. Thus, the transgene is incorporated into each cell
of the developing embryo, and mosaicism is usually avoided.
To our knowledge all published studies of LV transgenesis to
date were performed only up to the F1 generation. Therefore, we
also wanted to study whether the transgene still was functional in
the TG mice of consecutive generations and in TG mice aged from
1 to 2 years. Our results suggest that the hPGK-driven transgenes
were integrated into genomic sites, where they most probably were
not subjected to methylation,15 and that the hPGK promoter can be
used to achieve constitutive long-term expression of transgenes.
Our results also suggest that LV transgenesis leads to a long-term
and effective expression of transgenes in successive generations.
All TG mice with constitutive hVEGF-D expression showed
normal growth, were fertile, and appeared generally healthy with
no obvious macroscopic phenotype. This implies that constitutive
hVEGF-D expression is well tolerated during embryonic
development.
Overexpression of VEGFR-2–specific ligands promotes angiogenesis and lymphatic vessel enlargement but no lymphatic vessel
sprouting.16 Therefore, we hypothesized that the hVEGF-D TG
mice could have a proangiogenic phenotype. Indeed, increased
blood capillary density was found in skeletal muscles and myocardium of the TG-positive mice compared with the TG-negative
littermates. Moreover, after ischemic hindlimb injury, muscle
regeneration was faster, and injured areas were significantly
smaller, suggesting that hVEGF-D overexpression improves healing capacity of muscle tissue. These results are consistent with our
previous findings that intramuscular and intracardiac adenoviral
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 30 APRIL 2009 䡠 VOLUME 113, NUMBER 18
HUMAN VEGF-D TRANSGENIC MICE
4473
Table 2. Histopathologic and immunohistochemical characterization of tumors
Organ and tumor type
Histology
Immunohistochemistry
Metastases
Generation, sex,
comment
Mammary gland
Adenocarcinoma
Adenocarcinoma
Solid, comedo, trabecular growth
CD31, CD34, LYVE-1 negative in tumor
structures, necrosis, grade 3
cells
Solid, comedo growth pattern,
necrosis
Mammary gland preserved at the
edge, grade 3
Adenocarcinoma
Tubular, nestal, papillar, and
hVEGF-D positive in ductal part; CK7
⫺
F1, male, left cervical
mammary gland
⫺
F3, female, left inguinal
positive; CD31, CD34, LYVE-1 negative
mammary gland,
in tumor cells; increased number of
lactating, skin
ulcerated
vessels in tumor; angiogenic features
hVEGF-D positive in ductal part (surface);
Lung metastases
F1, female, right
trabecular growth pattern;
CK7 positive; CD31, CD34, LYVE-1
inguinal mammary
necrosis
negative in tumor cells; increased
gland
Mammary gland preserved at the
edge, grade 3
Adenocarcinoma
hVEGF-D positive in ductal part (surface);
pattern, single acinar/ductal
Tubular growth pattern, in situ
number of vessels in tumor; angiogenic
features
hVEGF-D positive in ductal part (surface);
⫺
F1, female, right
component,mammary gland
CD31, CD34, LYVE-1 negative in tumor
abdominal
largely preserved, grade 1
cells; increased amount of vessels in
mammary gland
tumor; angiogenic features
Lung
Adenocarcinoma
Multifocal, foci of
hVEGF-D negative
⫺
F1, female
hVEGF-D positive in stroma of papilla stalk;
⫺
F1, male
⫺
F3, female
⫺
F1, male
bronchioloalveolar
proliferation, grade 1
Papillary adenocarcinoma
Multifocal, papillary growth
pattern, grade 1
CD31, CD34, LYVE-1 negative in tumor
cells
Skin
Basal cell carcinoma
Predominantly solid growth of
round basal cells, necrosis
Anaplastic carcinoma
Solid infiltrative growth of spindle
hVEGF-D negative; CD31, CD34, LYVE-1
negative in tumor cells
hVEGF-D positive in some tumor cells;
and round cells, necrosis,
CD31, CD34, LYVE-1 negative in tumor
infiltrating soft tissues (muscle,
cells; increased amount of vessels in
bone)
tumor; angiogenesis features
hVEGF-D gene transfers increased vascularity and improved blood
flow in transduced tissues.14,17,18 Thus, mature hVEGF-D is primarily a proangiogenic growth factor, whether used as a transgene in
mice, or applied with the use of local gene therapy in larger
animals, implying that the effects are mostly mediated by VEGFR-2.
Until now, only mice with skin-specific overexpression of
full-length hVEGF-D have been generated, whereby the human
keratin 14 (hK14) promoter was used to drive the expression of the
transgene.9 The hK14 promoter directs the TG expression into the
basal cells of the epidermis. These mice showed strong and
selective hyperplasia of skin lymphatic vessels but not of skin
blood vessels. Furthermore, the lymphatic capillaries in the skin
were dilated and 3-fold larger than those of the wild-type mice.
However, in the hK14–hVEGF-D study,9 a different form of
hVEGF-D was used than in the present study. The processed form
of hVEGF-D–hVEGF-D⌬N⌬C (formed by proteolytical procession
of N- and C-terminal ends of the hVEGF-D prepropeptide) almost
exclusively binds to VEGFR-2, leading to angiogenesis.6,7,17 The
unprocessed or full-length hVEGF-D, which was used as the
transgene in the hK14–hVEGF-D study,9 equally stimulates phosphorylation of both VEGFR-2 and VEGFR-3.6
In the present study we have produced TG mice by using a LV
that, when integrated into the host cell genome, directly produces
the hVEGF-D⌬N⌬C and does not increase the amount of full-length,
unprocessed hVEGF-D. In addition, the difference in the VEGFR
binding capabilities between human and mouse further explains the
angiogenic phenotype of the TG mice. In mouse, both proteolytically processed and unprocessed mVEGF-D almost solely bind to
mVEGFR-3, leading to enhanced lymphangiogenesis.6,7,10 In addi-
tion, it has been shown that the hVEGF-D⌬N⌬C in mouse binds to
the angiogenic mVEGFR-2 and not the lymphangiogenic mVEGFR3.10,17 These findings most probably explain the different phenotypes regarding the blood and lymphatic vasculature in the
2 studies, as well as offer a logical explanation for the absence of
lymphangiogenesis in the present study.
The role of VEGF-D in vivo has also been studied by creating
VEGF-D knockout (KO) mice.19 These mice showed a normal
phenotype, including normal lymphatic vasculature, and the researchers thus concluded that either the mVEGF-D does not have a
major role in lymphangiogenesis during the embryonic development or that it is compensated by VEGF-C or some other, yet
unknown, VEGFR-3–activating agents.19 Because mVEGF-D is
capable of binding only to mVEGFR-3, it is not surprising that
these KO mice did not have an angiogenic phenotype.
In addition to increased capillary density and improved muscle
regeneration after injury, the phenotype of hVEGF-D TG mice also
included a tendency to form tumors. In addition, half of the tumors
were primary mammary gland adenocarcinomas. In the present
study, we observed hVEGF-D expression in the well-differentiated
ductal part of the mammary adenocarcinomas, in lung papillary
adenocarcinomas, and in skin anaplastic carcinomas, suggesting a
pathogenetic role of hVEGF-D in tumorigenesis. However, blood
and lymphatic vessel–derived malignancies, such as hemangioma,
lymphangioma, angiosarcoma, and lymphangiosarcoma, were not
found. There was no expression of CD31, CD34, or LYVE-1
outside the vasculature. Nevertheless, we observed intratumoral
angiogenesis, but not lymphangiogenesis, in the samples of mammary adenocarcinomas and skin anaplastic carcinomas. The mouse
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
4474
BLOOD, 30 APRIL 2009 䡠 VOLUME 113, NUMBER 18
KÄRKKÄINEN et al
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Figure 6. Representative histology of tumors in hVEGF-D TG mice. Images were taken from HE-stained or immunohistochemically stained paraffin sections of mouse tissues and
analyzed with Olympus Provis AX70 Microscope attached to Olympus ColorView 12 Camera. The software for imaging and analyses was AnalySIS (Soft Imaging System). (A) Mammary
adenocarcinoma with a predominantly solid growth pattern showed focal ducts. Bar, 200 ␮m. The objective lens used was UPlanApo 4⫻/0.16 ⬁/⫺ (Olympus). (B) Higher magnification of
the solid part of mammary carcinoma shown in panel A with atypical cells and a few mitotic figures. Bar, 50 ␮m. The objective lens used was UPlanApo 20⫻/0.70 (Olympus). (C) Mammary
adenocarcinoma with a solid growth pattern displaying only single remnants of ducts. Bar, 200 ␮m. The objective lens used was UPlanApo 4⫻/0.16 ⬁/⫺ (Olympus). (D) Higher
magnification of the solid part of mammary carcinoma shown in panel C with atypical cells and a few mitotic figures. Bar, 50 ␮m. The objective lens used was UPlanApo 20⫻/0.70
(Olympus). (E) Mammary adenocarcinoma showing a tubular growth pattern. Bar. 200 ␮m. The objective lens used was UPlanApo 4⫻/0.16 ⬁/⫺ (Olympus). (F) Higher magnification of the
carcinoma shown in panel E showing both tubular and solid areas. Bar, 50 ␮m. The objective lens used was UPlanApo 20⫻/0.70 (Olympus). (G) Well-differentiated mammary
adenocarcinoma showing a tubular growth pattern. Bar, 200 ␮m. The objective lens used was UPlanApo 4⫻/0.16 ⬁/⫺ (Olympus). (H) Higher magnification of the carcinoma shown in
panel G. Bar, 50 ␮m. (I) Immunostaining for hVEGF-D was positive in the surface of ductular structures. Picture from carcinoma is shown in panels E and F. Bar, 100 ␮m. The objective lens
used was UPlanApo 10⫻/0.4 Ph1 ⬁/0.17 (Olympus). (J) Solid parts of tumor shown in panels E and F showed CK7 immunopositivity. Bar, 200 ␮m. The objective lens used was UPlanApo
4⫻/0.16 ⬁/⫺ (Olympus). (K) Increased number of vessels with angiogenic features in mammary adenocarcinoma shown in panels C and D. Bar, 200 ␮m. The objective lens used was
UPlanApo 4⫻/0.16 ⬁/⫺ (Olympus). (L) Lung with metastases of mammary adenocarcinoma. Original tumor shown in panels E and F. Bar, 50 ␮m. The objective lens used was UPlanApo
20⫻/0.70 (Olympus). (M) Higher magnification of the lung metastasis shown in panel L. Note dilated ducts. Bar, 200 ␮m. The objective lens used was UPlanApo 4⫻/0.16 ⬁/⫺ (Olympus).
(N) Lung papillary adenocarcinoma. Bar, 50 ␮m. (O) Higher magnification of the adenocarcinoma papillae in tumor shown in panel N. Bar, 50 ␮m. The objective lens used was UPlanApo
20⫻/0.70 (Olympus). (P) Positive immunostaining for hVEGF-D in papilla stalk. Note positivity also in vessels at the border of the tumor. Bar, 100 ␮m. The objective lens used was
UPlanApo 10⫻/0.4 Ph1 ⬁/0.17 (Olympus). (Q) Multifocal foci of bronchioloalveolar proliferation in lung adenocarcinoma. Bar, 200 ␮m. The objective lens used was UPlanApo 4⫻/0.16
⬁/⫺ (Olympus). (R) Higher magnification of the bronchioloalveolar proliferation in adenocarcinoma shown in panel R. Bar, 50 ␮m. The objective lens used was UPlanApo 20⫻/0.70
(Olympus). (S) Solid growth in skin basal cell carcinoma. Note necrosis. Bar, 200 ␮m. The objective lens used was UPlanApo 4⫻/0.16 ⬁/⫺ (Olympus). (T) Higher magnification of the basal
cell carcinoma. Note mitosis. Bar, 50 ␮m. The objective lens used was UPlanApo 20⫻/0.70 (Olympus). (U) Skin anaplastic carcinoma consists of spindle cells. Bar, 200 ␮m. The objective
lens used was UPlanApo 4⫻/0.16 ⬁/⫺ (Olympus). (V) Higher magnification of the anaplastic carcinoma. Note atypia. Bar, 50 ␮m. The objective lens used UPlanApo 20⫻/0.70 (Olympus).
(W) Some tumor cells were positive for hVEGF-D immunostaining in an anaplastic carcinoma. Bar, 100 ␮m. The objective lens used was UPlanApo 10⫻/0.4 Ph1 ⬁/0.17 (Olympus).
(X) Increased number of vessels with angiogenic features in an anaplastic carcinoma shown in panels U and V. Bar, 100 ␮m. The objective lens used was UPlanApo 10⫻/0.4 Ph1 ⬁/0.17
(Olympus).
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 30 APRIL 2009 䡠 VOLUME 113, NUMBER 18
HUMAN VEGF-D TRANSGENIC MICE
strain used in this study (CD2F1) is not known for spontaneous
tumor formation. However, LVs could be connected to tumorigenesis. Therefore, we compared the hVEGFD TG mice with other
LV-produced TG mice, namely hPGK-GFP and hPGK-hNrf2. In
these TG mice, no malignancies or early deaths have been recorded
(data not shown).
In line with our findings, tumor angiogenesis has been shown to
be induced by hypoxia, estrogens, VEGF-A, and VEGF-D in
primary breast carcinomas.20,21 Because myoepithelia plays an
important role in mammary gland tumorigenesis, one possible
mechanistic explanation could be the overexpression of VEGF-D
in these cells. In previous studies, the role of VEGF-D was seen
mainly in the context of lymph node metastasis and intratumoral
lymphangiogenesis.20,22 Nevertheless, in breast cancer cultures, an
autocrine role of hVEGF-D and other members of the VEGF
family has been implicated.22,23
In conclusion, the hVEGF-D TG mice showed a significantly
increased angiogenesis in skeletal muscles and myocardium, and
less muscle damage after hindlimb ischemic injury compared with
the TG-negative controls. However, the incidence of tumor formation was significantly increased in the TG mice, with a preference
for mammary gland adenocarcinoma formation.
Acknowledgments
We thank the technical staff of the Department of Biotechnology
and Molecular Medicine for their expert contribution to the study.
4475
This work was supported by the Academy of Finland, Finnish
Funding Agency for Technology and Innovation, Kuopio University Foundation, European Union (Lymphangiogenomics: ISLH2007-00284/Ha-7), and Leducq Foundation/Fondation Leducq (06
CVD 04).
Authorship
Contribution: A.-M.K. designed, coordinated and performed research, analyzed data, and wrote the paper; A.K. performed
histologic analyses, analyzed data, contributed to the animal work,
and prepared the images; J.H. performed transgeneses; I.K. analyzed all tumor data and reported the results of the analyzed
tumors; S.E.H. performed each hindlimb ischemia surgical operation; A.S. performed most of the additional immunohistochemistry;
M.H.D. and E.H. contributed to molecular biology analyses; H.P.
set up analysis for expression levels; P.I.M. originally cloned the
LV backbone vector; M.P.T. performed ELISA assays; and S.Y.-H.
contributed to conception and design of the research, critically
revised the manuscript for important intellectual content, and
supervised the research.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Seppo Ylä-Herttuala, University of Kuopio,
A.I.Virtanen Institute, Department of Biotechnology and Molecular Medicine, Neulaniementie 2, 70210 Kuopio, Finland; e-mail:
[email protected].
References
1. Millauer B, Wizigmann-Voos S, Schnürch H, et al.
High affinity VEGF binding and developmental
expression suggest Flk-1 as a major regulator of
vasculogenesis and angiogenesis. Cell. 1993;72:
835-846.
2. Karkkainen MJ, Haiko P, Sainio K, et al. Vascular
endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic
veins. Nat Immun. 2003;5:74-80.
3. Tammela T, Saaristo A, Holopainen T, et al.
Therapeutic differentiation and maturation of lymphatic vessels after lymph node dissection and
transplantation. Nat Med. 2007;13:1458-1466.
4. Yamazaki Y, Morita T. Molecular and functional
diversity of vascular endothelial growth factors.
Mol Div. 2006;10:515-527.
5. Kärpänen T, Heckman CA, Keskitalo S, et al.
Functional interaction of VEGF-C and VEGF-D
with neuropilin receptors. FASEB J. 2006;20:
1462-1472.
6. McColl BK, Paavonen K, Karnezis T, et al. Proprotein convertases promote processing of
VEGF-D, a critical step for binding the angiogenic
receptor VEGFR-2. FASEB J. 2007;21:10881098.
7. Stacker SA, Stenvers K, Caesar C, et al. Biosynthesis of vascular endothelial growth factor-D involves proteolytic processing which generates
non-covalent homodimers. J Biol Chem. 1999;
274:32127-32136.
8. Dvorak HF, Nagy JA, Feng D, Brown LF, Dvorak
AM. Vascular permeability factor/vascular endothelial growth factor and the significance of microvascular hyperpermeability in angiogenesis. Curr
Top Microbiol Immunol. 1999;237:97-132.
9. Veikkola T, Jussila L, Makinen T, et al. Signalling
10.
11.
12.
13.
14.
15.
16.
17.
via vascular endothelial growth factor receptor-3
is sufficient for lymphangiogenesis in transgenic
mice. EMBO J. 2001;20:1223-1231.
Baldwin ME, Catimel B, Nice EC, et al. The specificity of receptor binding by vascular endothelial
growth factor-d is different in mouse and man.
J Biol Chem. 2001;276:19166-19171.
Huusko J, Mäkinen PI, Alhonen L, Ylä-Herttuala
S. Generation of transgenic and knockdown mice
with lentiviral vectors and RNAi techniques. In:
Latterich M eds. RNAi. BIOS advanced methods.
Boca Raton, FL: Taylor & Francis Group; 2008:
91-108.
Kankkonen HM, Turunen MP, Hiltunen MO, et al.
Feline immunodeficiency virus and retrovirusmediated adventitial ex vivo gene transfer to rabbit carotid artery using autologous vascular
smooth muscle cells. J Mol Cell Cardiol. 2004;36:
333-341.
Tiscornia G, Tergaonkar V, Galimi F, Verma IM.
CRE recombinase-inducible RNA interference
mediated by lentiviral vectors. Proc Natl Acad Sci
U S A. 2004;101:7347-5731.
Kholová I, Koota S, Kaskenpää N, et al. Adenovirus-mediated gene transfer of human vascular
endothelial growth factor-d induces transient angiogenic effects in mouse hind limb muscle. Hum
Gene Ther. 2007;18:232-244.
Hofmann A, Kessler B, Ewerling S, et al. Epigenetic regulation of lentiviral transgene vectors in a
large animal model. Mol Ther. 2006;13:59-66.
Wirzenius M, Tammela T, Uutela M, et al. Distinct
vascular endothelial growth factor signals for lymphatic vessel enlargement and sprouting. Exp
Med. 2007;204:1431-1440.
Rissanen TT, Markkanen JE, Gruchala M, et al.
VEGF-D is the strongest angiogenic and lymphangiogenic effector among VEGFs delivered
into skeletal muscle via adenoviruses. Circ Res.
2003;92:1098-1106.
18. Rutanen J, Rissanen TT, Markkanen JE, et al.
Adenoviral catheter-mediated intramyocardial
gene transfer using the mature form of vascular
endothelial growth factor-D induces transmural
angiogenesis in porcine heart. Circulation. 2004;
109:1029-1035.
19. Baldwin ME, Halford MM, Roufail S, et al. Vascular endothelial growth factor D is dispensable for
development of the lymphatic system. Mol Cell
Biol. 2005;25:2441-2449.
20. Currie MJ, Hanrahan V, Gunningham SP, et al.
Expression of vascular endothelial growth factor
D is associated with hypoxia inducible factor
(HIF-1alpha) and the HIF-1alpha target gene
DEC1, but not lymph node metastasis in primary
human breast carcinomas. J Clin Pathol. 2004;
57:829-834.
21. Van den Eynden GG, Van Laere SJ, Van der
Auwera I, et al. Differential expression of hypoxia
and (lymph)angiogenesis-related genes at different metastatic sites in breast cancer. Clin Exp
Metastasis. 2007;24:13-23.
22. Akahane M, Akahane T, Shah A, Okajima E,
Thorgeirsson UP. A potential role for vascular endothelial growth factor-D as an autocrine growth
factor for human breast carcinoma cells. Anticancer Res. 2005;25:701-707.
23. Weigand M, Hantel P, Kreienberg R,
Waltenberger J. Autocrine vascular endothelial
growth factor signalling in breast cancer. Evidence from cell lines and primary breast cancer
cultures in vitro. Angiogenesis. 2005;8:197-204.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2009 113: 4468-4475
doi:10.1182/blood-2008-07-171108 originally published
online December 10, 2008
Vascular endothelial growth factor-D transgenic mice show enhanced
blood capillary density, improved postischemic muscle regeneration,
and increased susceptibility to tumor formation
Anna-Mari Kärkkäinen, Antti Kotimaa, Jenni Huusko, Ivana Kholova, Suvi Elina Heinonen, Anna
Stefanska, Marike Hinke Dijkstra, Hanna Purhonen, Eveliina Hämäläinen, Petri Ilmari Mäkinen, Mikko
Petri Turunen and Seppo Ylä-Herttuala
Updated information and services can be found at:
http://www.bloodjournal.org/content/113/18/4468.full.html
Articles on similar topics can be found in the following Blood collections
Vascular Biology (499 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.