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VASCULAR BIOLOGY
HDAC5 is a repressor of angiogenesis and determines the angiogenic gene
expression pattern of endothelial cells
*Carmen Urbich,1 *Lothar Rössig,1 David Kaluza,1 Michael Potente,1 Jes-Niels Boeckel,1 Andrea Knau,1 Florian Diehl,1
Jian-Guo Geng,2 Wolf-Karsten Hofmann,3 Andreas M. Zeiher,1 and Stefanie Dimmeler1
1Molecular
Cardiology, Department of Medicine III, University of Frankfurt, Frankfurt, Germany; 2Division of Hematology, Oncology and Transplantation,
University of Minnesota Medical School, Minneapolis; and 3Department of Hematology, Oncology and Transfusion Medicine, Charité, University Hospital
Benjamin Franklin, Berlin, Germany
Class IIa histone deacetylases (HDACs)
are signal-responsive regulators of gene
expression involved in vascular homeostasis. To investigate the differential
role of class IIa HDACs for the regulation
of angiogenesis, we used siRNA to specifically suppress the individual HDAC isoenzymes. Silencing of HDAC5 exhibited a
unique pro-angiogenic effect evidenced
by increased endothelial cell migration,
sprouting, and tube formation. Consistently, overexpression of HDAC5 decreased sprout formation, indicating that
HDAC5 is a negative regulator of angiogenesis. The antiangiogenic activity of
HDAC5 was independent of myocyte enhancer factor-2 binding and its deacetylase activity but required a nuclear localization indicating that HDAC5 might affect
the transcriptional regulation of gene
expression. To identify putative HDAC5
targets, we performed microarray expression analysis. Silencing of HDAC5 increased the expression of fibroblast
growth factor 2 (FGF2) and angiogenic
guidance factors, including Slit2. Antago-
nization of FGF2 or Slit2 reduced sprout
induction in response to HDAC5 siRNA.
Chromatin immunoprecipitation assays
demonstrate that HDAC5 binds to the
promoter of FGF2 and Slit2. In summary,
HDAC5 represses angiogenic genes, such
as FGF2 and Slit2, which causally contribute to capillary-like sprouting of endothelial cells. The derepression of angiogenic
genes by HDAC5 inactivation may provide a useful therapeutic target for induction of angiogenesis. (Blood. 2009;113:
5669-5679)
Introduction
Histone deacetylases (HDACs) are cofactors for the regulation of
gene transcription. Human HDACs are grouped into 3 classes
based on their similarity to known yeast factors: class I HDACs are
similar to the yeast transcriptional repressor yRPD3, class II
HDACs to yHDA1, and class III HDACs to ySIR2.1 Nonselective
inhibitors of class I and class II HDACs reduce tube formation of
endothelial cells in vitro,2 inhibit postnatal neovascularization in
response to hypoxia,3 and block tumor angiogenesis.4 Moreover,
the enzymatic activity of class I and class II histone deacetylases is
essential for endothelial commitment of progenitor cells.5 Mice
deficient for the class IIa HDAC isoform HDAC7 fail to maintain
proper endothelial cell-cell adhesion and have a leaky, ruptureprone vasculature, leading to embryonic lethality.6 Recently, it has
been shown that members of class IIa HDACs are modulators of
angiogenesis in vitro.7-10 In particular, protein kinase D–dependent
phosphorylation of HDAC5 and HDAC7 plays an important role in
vascular endothelial growth factor (VEGF)–induced angiogenesis
in vitro.9,10 However, beyond the characterization of HDAC7 as an
essential regulator of embryonic blood vessel development, the
individual functions of class IIa HDAC isoforms during postnatal
angiogenesis and the underlying mechanisms remain poorly defined.
Angiogenesis requires the migration of endothelial cells from
preexisting vessels.11 Paracrine secretion of angiogenic growth
factors creates a milieu that regulates directed movement of
endothelial cells.12 Among these growth factors, fibroblast growth
factor 2 (FGF2) released from endothelial cells potently stimulate
migration and capillary-like growth of these cells via an autocrine
action.13-16 Beyond growth factors, so-called axon guidance factors
known for their role in neuronal wiring have recently been
identified as essential regulators of capillary sprout formation.17
These factors provide guidance cues for the directional movement
of specialized endothelial “tip” cells at the forefront of navigating
blood vessels similar to axonal growth cones.18 Guidance factors
are families of surface membrane receptors and their ligands
that can be secreted (ie, Slit2) or membrane-bound ligands (ie,
EphrinB2). After ligand-receptor interaction, both partners enable
bidirectional communication between cells, which includes reverse
signaling via the membrane-bound ligand to the ligand-bearing cell
in addition to the outside-in signaling that arises from ligandmediated activation of the receptor.19 Signaling via the guidance
factor EphB4 is crucial for both endothelial cell migration20,21 and
the formation of capillary-like structures.22 Likewise, Slit2 acts as
molecular guidance cue in cellular migration, which is mediated by
its interaction with one of 4 different roundabout (robo) receptors.
Depending on the differentiation stage and the individual robo
receptor subtype involved, Slit2 can act as a chemorepellent or
attractant.23 In a model of tumor angiogenesis, Slit2 attracts
endothelial cells and promotes tube formation, and its interaction
with Robo1 is essential for tumor vascularization.24
Because angiogenic activation of endothelial cells to migrate
and to form sprouts is associated with characteristic changes in
gene expression profiles,25 the identification of upstream regulatory
Submitted January 13, 2009; accepted February 11, 2009. Prepublished online as
Blood First Edition paper, April 7, 2009; DOI 10.1182/blood-2009-01-196485.
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.
*C.U. and L.R. contributed equally to this study.
The online version of this article contains a data supplement.
BLOOD, 28 MAY 2009 䡠 VOLUME 113, NUMBER 22
© 2009 by The American Society of Hematology
5669
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BLOOD, 28 MAY 2009 䡠 VOLUME 113, NUMBER 22
URBICH et al
mechanisms that coordinate pro-angiogenic activation is crucial.
Here, we assessed the role of individual HDAC isoenzymes for
endothelial cell migration and capillary-like sprout-forming capacity and explored the role of HDAC5 as an essential repressor of a
gene expression pattern required for angiogenic functions in
endothelial cells.
Spheroid-based angiogenesis assay
Methods
Cell-cycle analysis
Cell culture
Twenty-four hours after transfection, adherent cells were incubated with
bromodeoxyuridine (BrdU; 10 ␮M). Cells were detached with trypsin,
washed in phosphate-buffered saline, and incubated with 20 ␮L anti–BrdUfluorescein isothiocyanate for 20 minutes and with 2.5 ␮L 7-aminoactinomycin D for 15 minutes according to the manufacturer’s protocol
(BrdU Flow Kit; BD Biosciences). Analysis was performed using a FACS
SCAN flow cytometer and CellQuest software (BD Biosciences).
Pooled human umbilical vein endothelial cells (HUVECs) were purchased
from Lonza Verviers (Verviers, Belgium) and cultured in endothelial basal
medium (EBM; Lonza Verviers SPRL) supplemented with hydrocortisone,
bovine brain extract, gentamicin, amphotericin B, epidermal growth factor,
and 10% fetal calf serum (Invitrogen, Karlsruhe, Germany) until the third
passage. After detachment with trypsin, cells were grown in 6-cm culture
dishes for at least 18 hours. Neutralizing FGF2 antibody (clone bFM-1) was
bought from Upstate Biotechnology (Lake Placid, NY). RoboN and R5
were kindly donated by Jian-Guo Geng (Division of Hematology, Oncology
and Transplantation, University of Minnesota Medical School, Minneapolis, MN).
Plasmids and transfection
HUVECs (3.5 ⫻ 105 cells/6-cm well) were grown to 60% to 70%
confluence and then transfected with 3 ␮g plasmid DNA. Suk-Chul Bae
kindly provided the wild-type human HDAC5 construct (HDAC5 in
pCS4-3myc) as described previously.26 The HDAC5 S259/498A construct
was engineered by stepwise site-directed mutagenesis. The HDAC5
⌬myocyte enhancer factor-2 (MEF2) mutant was constructed by sitedirected mutagenesis as described for the homologous isoenzyme HDAC4.27
Eric N. Olson (Department of Molecular Biology, University of Texas
Southwestern Medical Center, Dallas, TX) kindly provided the HDAC5
1-767aa construct.28 Constitutively active PKD1 and HDAC7 wt were
kindly provided by Franck Dequiedt (Cellular and Molecular Biology Unit,
FUSAGx, Gembloux, Belgium).29 pcDNA3.1-myc-His served as empty
vector control (mock). Transfection was performed using Superfect transfection reagent (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol.
RNA interference
For siRNA-mediated silencing, HUVECs were grown to 60% to 70%
confluence and transfected with GeneTrans II (MoBiTec, Göttingen,
Germany) according to the manufacturer’s protocol. siRNAs were synthesized by Eurogentec (Cologne, Germany) or QIAGEN targeting the
sequences given in Table S1 (available on the Blood website; see the
Supplemental Materials link at the top of the online article). All other
siRNA oligonucleotide sequences are available on request.
Western blot analysis
For Western blot analysis, HUVECs were lysed with radio-immunoprecipitation
assay (RIPA) buffer (ready-to-use solution containing 150 mM NaCl,
1.0% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl
sulfate [SDS], 50 mM Tris, pH 8.0; Sigma-Aldrich, Munich, Germany) for
20 minutes on ice. After centrifugation for 15 minutes at 20 000g (4°C), the
protein content of the samples was determined according to the Bradford
method. For immunoprecipitation, cells were lysed with RIPA buffer for
15 minutes on ice. Equal amounts of protein were loaded onto SDSpolyacrylamide gels and blotted onto polyvinylidene difluoride membranes.
Western blots were performed using antibodies directed against HDAC5
(Cell Signaling Technology, Danvers, MA), topoisomerase Ia (Santa Cruz
Biotechnology, Heidelberg, Germany), c-Myc (Santa Cruz Biotechnology),
MEF2 (Santa Cruz Biotechnology), protein kinase D (Cell Signaling
Technology), or tubulin-␣ (Lab Vision, Fremont, CA).
Endothelial cell spheroids of defined cell number were generated as described
previously.30 Further details are provided in Document S1.
Scratched wound assay
Migration of HUVECs was detected using a “scratched wound assay.”31
Further details are provided in Document S1.
MTT viability assay
Assessment of cell viability was performed using the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. At 24 hours and
48 hours after transfection, 0.5 mg/mL MTT was added to each well, and
cells were incubated for 4 hours at 37°C. Cells were washed with
phosphate-buffered saline and lysed 30 minutes at room temperature with
lysis buffer (40 nM HCl in isopropanol). Absorbance was photometrically
measured at 550 nm.
Tube formation assay
Twenty-four hours after transfection, HUVECs (5 ⫻ 104) were cultured in a
12-well plate (Greiner Bio-One, Frickenhausen, Germany) coated with
200 ␮L Matrigel Basement Membrane Matrix (BD Biosciences). Tube
length was quantified after 24 hours by measuring the cumulative tube
length in 5 random microscopic fields with a computer-assisted microscope
using the program KS300 3.0 (Carl Zeiss, Jena, Germany).
Matrigel plug assay
HUVECs transfected with siRNA against HDAC5 or scrambled siRNA
were suspended in Matrigel, which was then subcutaneously injected into
the backs of 8- to 12-week-old nude mice.32 After 7 days, blood vessel
infiltration in Matrigel plugs was quantified by analysis of hematoxylin and
eosin staining using microscopy. To analyze perfused capillaries, 200 ␮L
fluorescein isothiocyanate–conjugated lectin (1 mg/mL; Sigma-Aldrich)
was injected intravenously 30 minutes before the mice were killed. To
visualize capillaries, plug sections were stained with an antibody against
mouse CD31. Nuclei were stained with 4,6-diamidino-2-phenylindole. For
hemoglobin analysis, the Matrigel plug was removed after 7 days and
homogenized in 130 ␮L deionized water. After centrifugation, the supernatant was used in the Drabkin assay (Sigma-Aldrich) to measure hemoglobin
concentration. Stock solutions of hemoglobin are used to generate a
standard curve. Results are expressed relative to total protein in the
supernatant. Animal studies were performed with the permission of the
State of Hesse, Regierungspraesidium Darmstadt, and conform to the Guide
for the Care and Use of Laboratory Animals.
RT-PCR
Total RNA was isolated using the RNeasy Mini Kit (QIAGEN); 2 ␮g RNA
from each sample was reverse-transcribed (RT) into cDNA and subjected to
conventional PCR using the oligonucleotide primers summarized in Table
S2. Semiquantitative analysis was performed densitometrically using the
Scion Image software (version 4.0.2; Scion, Frederick, MD).
Real-time PCR (Lightcycler)
Total RNA was isolated using the RNeasy Mini Kit; 2 ␮g RNA from each
sample was reverse-transcribed into cDNA and subjected to real-time PCR
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BLOOD, 28 MAY 2009 䡠 VOLUME 113, NUMBER 22
using the LightCycler FastStart DNA MasterPlus SYBR Green I Kit (Roche
Applied Science, Indianapolis, IN). Lightcycler reactions were performed
with the LightCycler 1.2 System and LightCycler 3.5 quantification
software (Roche Applied Science). Oligonucleotides used for real-time
PCR are summarized in Table S3. All other oligonucleotide sequences are
available on request.
Oligonucleotide array
Total RNA was isolated from 3 independently transfected HUVECs; 10 ␮g
total RNA was hybridized to the HG U133 Plus 2.0 microarray (Affymetrix,
Santa Clara, CA). The standard protocol used for sample preparation and
microarray processing is available from Affymetrix. To detect differentially
expressed genes, data analysis was performed as described previously.33
The microarray data have been deposited into the GEO database under
accession number GSE15499.34
Boyden chamber migration
Transwell membranes (8 ␮m; Corning Life Sciences, Schiphol-Rijk, The
Netherlands) were coated on both sides with fibronectin (2.5 ␮g/mL;
Sigma-Aldrich) overnight at 4°C; 600 ␮L serum-free EBM with 1% bovine
serum albumin containing VEGF, FGF2, or 600 ␮L conditioned medium of
siRNA-transfected HUVECs were placed in the lower chambers. Then
5 ⫻ 104 HUVECs in 100 ␮L serum-free EBM containing 1% bovine serum
albumin were incubated in the upper chamber at 37°C in 5% CO2 for
5 hours. Cells remaining on the upper surface of the filters were mechanically removed, and HUVECs that had migrated to the lower surface were
fixed with 4% formaldehyde and counted in 5 fields using a fluorescence
microscope (Axiovert 100; Carl Zeiss).
ChIP
HUVECs were harvested by scraping and cross-linked for 5 minutes by
directly adding 1% formaldehyde to the culture medium. The fixed cells
were washed and lysed for 5 minutes on ice (50 mM Tris pH 8.0, 2 mM
ethylenediaminetetraacetic acid, 0.1% NP-40, 10% glycerol) to obtain cell
nuclei. The nuclei were pelleted and lysed for another 5 minutes on ice
(50 mM Tris pH 8.0, 5 mM ethylenediaminetetraacetic acid, 0.1% SDS).
The chromatin was sheared by sonication (Branson Sonifier 450; Branson
Ultrasonics, Danbury, CT). Sheared chromatin samples were taken as input
control or used for immunoprecipitation with an antibody directed against
c-Myc (Santa Cruz Biotechnology). After immunoprecipitation, the samples
were washed 5 times and subjected to either Western blot analysis or PCR
using primer pairs directed against promoter regions within the FGF2 or
Slit2 genes (Table S4).
Statistical analysis
Data are mean plus or minus SEM. Two treatment groups were compared
with the independent samples t test or Mann-Whitney U test, and 3 or more
groups by one-way analysis of variance followed by post hoc analysis
adjusted with a least significant difference correction for multiple comparisons (SPSS, Chicago, IL). Results were considered statistically significant
when P value was less than .05.
Results
Effects of HDAC isoenzymes on in vitro angiogenesis
To investigate the individual contribution of class IIa HDAC isoenzymes to angiogenic functions of endothelial cells in vitro, we transfected HUVECs with siRNA oligonucleotides targeting the individual
HDAC isoenzymes (Table S1). siRNA transfection caused a significant
suppression of individual HDAC isoenzymes after 24 hours (Figures 1A, S1A). Silencing of the isoforms HDAC7 and HDAC9 blocked
endothelial sprouting in a 3-dimensional spheroid assay and endothelial
cell migration in a scratched wound assay (Figure 1B,C). In contrast,
HDAC5 AND ANGIOGENESIS
5671
surprisingly, siRNA directed against HDAC5 profoundly increased
sprout length, the number of branch points, and the number of sprouts
per spheroid (Figures 1B, S2) and stimulated endothelial cell migration
(Figure 1C). Because HDACs may affect cell cycle progression and cell
death,35 we measured proliferation by BrdU incorporation and viability
by MTT assay. However, transfection of siRNA against HDAC5,
HDAC7, or HDAC9 did not significantly affect endothelial cell
proliferation (Figure 1D). In addition, knockdown of HDAC5 and
HDAC9 did not affect viability, whereas siRNA against HDAC7
slightly but significantly decreased viability after 48 hours (Figure 1E).
To further investigate toxic effects of the siRNA transfection, we used
2 additional siRNA oligonucleotides (siRNA I and II) against HDAC5,
HDAC7, and HDAC9. As shown in Figure S1, HDAC5 and HDAC9
siRNA did not affect endothelial cell viability, whereas siRNA against
HDAC7 (siRNA II) significantly reduced cell viability after 48 hours.
These data indicate that, among the class II HDACs, HDAC5 exhibits a
specific and unique antiangiogenic activity without affecting proliferation or survival.
Antiangiogenic effect of HDAC5
Based on our finding that HDAC5 is a repressor of endothelial cell
migration and sprouting, we focused our following study on the
role of HDAC5 for angiogenesis in vitro and vivo. Transfection of
endothelial cells with 2 different siRNA oligonucleotides against
HDAC5 profoundly decreased HDAC5 protein expression (Figure
S3). To confirm the proangiogenic effect of HDAC5 silencing, we
used an additional siRNA-targeting HDAC5 and demonstrated that
the second oligonucleotide sequence also effectively increased
capillary sprout length (Figure S4). Moreover, HDAC5 siRNA also
significantly enhanced tube formation in an additional in vitro
angiogenesis assay (Figure 1F). To determine whether HDAC5
silencing also increase angiogenesis in vivo, HDAC5 siRNAtransfected HUVECs were mixed with Matrigel and subcutaneously implanted in nude mice in vivo. After 7 days, the number of
invaded cells as determined by hematoxylin and eosin staining was
significantly increased in HDAC5 siRNA-treated Matrigel plugs
compared with scrambled controls (Figure 1G,H). To analyze
vascularization, we stained the Matrigel plugs for mouse CD31 and
detected fluorescent lectin, which was intravenously infused
30 minutes before the mice were killed. As shown in Figure 1I and
J, the number of CD31⫹ structures as well as the number of lectin⫹
structures were significantly enhanced in response to HDAC5
siRNA. Moreover, the hemoglobin content, indicative of Matrigel
plug perfusion, was significantly augmented in HDAC5-siRNA
plugs (Figure 1K). Together, these data document that HDAC5 is a
negative regulator of angiogenesis in vitro and in vivo.
Mechanism underlying the antiangiogenic function of HDAC5
To analyze the mechanism underlying the antiangiogenic function
of HDAC5, we transfected endothelial cells with plasmids encoding wild-type HDAC5 (HDAC5 wt) or different mutants (Figure
2A). The expression of HDAC5 wt and the HDAC5 mutants is
shown by RT-PCR (Figure 2B) and Western blot (Figure 2C).
Overexpression of HDAC5 wt significantly inhibited sprout length
(Figure 2D). Moreover, transfection of an HDAC5 S259/498A
mutant, which is preferentially localized in the nucleus,36 significantly suppressed sprout length (Figures 2D, S5b), suggesting that
the nuclear localization of HDAC5 is important for its repressive
effect. In addition, overexpression of HDAC5 wt and the HDAC5
S259/498A mutant profoundly decreased the number of branch
points and the number of sprouts per spheroid (Figure S5). PKD is
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BLOOD, 28 MAY 2009 䡠 VOLUME 113, NUMBER 22
URBICH et al
B
A
scrambled
HDAC5 siRNA
HDAC7 siRNA
*
*
*
*
*
*
siRNA
siRNA
D
C
*
*
*
siRNA
siRNA
E
*
F
24 h
48 h
scrambled
*
HDAC5 siRNA
siRNA
siRNA
G
H
*
I
*
scrambled
K
J
HDAC5 siRNA
*
Figure 1.
*
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BLOOD, 28 MAY 2009 䡠 VOLUME 113, NUMBER 22
A
MEF2
HDAC5 AND ANGIOGENESIS
NLS
Histone deacetylase
S
NH2
MEF2
NLS
A
259
A
498
NH2
MEF2
NLS
A
AA
184/186 259
A
498
MEF2
NH2
NES
S
NH2
Histone deacetylase
Histone deacetylase
NLS
S
259
S
498
5673
COOH
HDAC5 wt
COOH
HDAC5 S259/498A
COOH
HDAC5 ∆MEF2
NES
NES
COOH
HDAC5 1-767aa
C
B
HDAC5
myc
GAPDH
mock
1-767aa
wt
S259/498A ∆MEF2
Tubulin
HDAC5
mock
wt
S259/498A ∆MEF2 1-767aa
HDAC5
Sprout length (% mock)
D
E
*
Whole cell lysate
*
*
*
IP á myc
WB:
HDAC5-myc
WB:
MEF2
Plasmid
mock
wt ∆MEF2
HDAC5
mock
wt ∆MEF2 Ab
HDAC5
Figure 2. Mechanism underlying the antiangiogenic function of HDAC5. (A) Scheme of different HDAC5 constructs. (B) RT-PCR analysis of HUVECs overexpressing
different HDAC5 constructs after 14 hours. (C) Western blot analysis of HUVECs overexpressing different HDAC5 constructs after 14 hours. (D) Capillary-like sprout formation
from spheroid cultures of HUVECs overexpressing different HDAC5 constructs (n ⫽ 3-10; *P ⬍ .05 vs mock). (E) Lysates of HUVECs overexpressing different HDAC5
constructs were immunoprecipitated with an antibody against c-myc. Western blots were performed with an anti-c-myc or an anti-MEF2 antibody in whole cell lysates or after
immunoprecipitation. Ab indicates antibody control.
known to phosphorylate HDAC5 leading to its nuclear export.37
Therefore, as a control, we cotransfected endothelial cells with
constitutively active PKD and HDAC5 wt or HDAC5 S259/498A
mutant (Figure S6). PKD overexpression reversed the repressive
effect of HDAC5 wt on endothelial cell sprouting (Figure S6). In
contrast, overexpression of the nonphosphorylatable HDAC5 mutant is refractory to PKD and still repressed sprouting (Figure S6).
Because MEF2 interacts with HDAC5 in cardiac myocytes and is
also crucial for the embryonic development of the vasculature38 as
well as for postnatal endothelial cell signaling,39 we tested whether
Figure 1. Role of class IIa HDAC isoenzymes for angiogenic function and cell-cycle progression in endothelial cells. (A-F) Effects of siRNA against individual class IIa HDAC
isoenzymes in HUVECs on (A) HDAC mRNA expression as measured by quantitative PCR. Data (delta Ct) are mean ⫾ SEM (n ⫽ 3, *P ⬍ .05 vs scrambled). (B) Capillary-like sprout
formation from spheroid cultures (n ⱖ 4; *P ⬍ .05). (C) Scratched wound endothelial cell migration (n ⱖ 3; *P ⬍ .05). (D) Proliferation rate measured by flow cytometric detection of
BrdU-labeled HUVECs in the S-phase (n ⫽ 7-11). (E) Viability measured by MTT assay (n ⫽ 3). (F) Tube formation in a Matrigel assay (n ⫽ 6; *P ⬍ .05). (G-K) Biochemical and histologic
analysis of Matrigel plugs containing scrambled versus HDAC5 siRNA-transfected HUVECs at day 7 after subcutaneous implantation (n ⱖ 3 per group). (G) Representative hematoxylin
and eosin–stained plug sections. Pictures were taken using an Axiovert 100M microscope, an AxioCam camera, a Plan-NEOFLUAR 10⫻/0.30⬁/0.17 objective, and the AxioVision Rel.
4.6.3 Sp1 software (Carl Zeiss). (H) Number of invaded cells per hematoxylin and eosin–stained plug section (*P ⬍ .05). (I) Number of CD31⫹ structures per plug section. (J) Number of
lectin⫹ structures per plug section. (K) Hemoglobin content of Matrigel plugs (* P ⬍ .05).
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5674
URBICH et al
MEF2 is required for HDAC5 function in endothelial cells.
Therefore, cells were transfected with an HDAC5 mutant construct
(HDAC5 ⌬MEF2), which does not bind to MEF2 as shown by
immunoprecipitation (Figure 2E). Transfection of endothelial cells
with HDAC5 ⌬MEF2 still significantly decreased sprout length
(Figure 2D), indicating that binding of HDAC5 to MEF2 is
dispensable for the repressive effect of HDAC5 on endothelial cell
sprouting. Having shown that the nuclear localization of HDAC5 is
important and MEF2 is not required for its function, we investigated whether the enzymatic activity of HDAC5 is involved in the
antiangiogenic effect. Transfection of an HDAC5 mutant lacking
the deacetylase domain (HDAC5 1-767aa28) also significantly
diminished sprout formation (Figure 2D), suggesting that the
repressive effect of HDAC5 is independent of its deacetylase
activity and is mediated by the N-terminal part of the protein.
Identification of target genes regulated by HDAC5 in
endothelial cells
To identify target genes regulated by HDAC5 in endothelial cells,
we profiled the transcriptome of cells transfected with HDAC5
siRNA. Analysis of the different HDAC isoenzymes revealed that
the siRNA against HDAC5 used for this experiment selectively
suppressed HDAC5 but none of the other class IIa HDAC isoforms
(Figure S7). Bioinformatic analysis showed that 2.0% of analyzed
genes were up-regulated and 1.1% were down-regulated in response to HDAC5 siRNA (⬎ 1.5-fold vs scrambled siRNA, 3P
level indicating relevant expression; Figure 3A). Among the
significantly regulated genes with altered RNA levels were genes
involved in angiogenesis, including angiogenic growth factors and
receptors (eg, FGF2, neuropilin 2, VEGFR2, TGFBR2), guidance
molecules (eg, Slit2), and homeodomain transcription factors (eg,
HOXA9; Figure 3B). The expression of selected angiogenesisrelated genes was further analyzed by real-time PCR. As shown in
Figure 3C, FGF2, Slit2, and EphB4 were time-dependently,
significantly up-regulated in HDAC5 siRNA-transfected HUVECs.
Moreover, FGF2 and Slit2 mRNA expression was increased in vivo
in Matrigel plugs containing HUVECs transfected with HDAC5
siRNA compared with scrambled-transfected cells (data not shown).
In contrast, other genes involved in angiogenesis, such as the
VEGF isoforms A to D, angiopoietins, neuropilin 1, TGFBR1, and
TGFBR3, were not significantly regulated in response to HDAC5
inhibition (Figures S8, S9).
Contribution of FGF2 and Slit2 regulation for the repressive
effect of HDAC5
Because HDAC5 siRNA induced the expression of several secreted
gene products known for their proangiogenic effects, such as FGF2
or Slit2, we investigated whether the secretion of angiogenic
factors might contribute to the proangiogenic effect of HDAC5
silencing. Indeed, incubation of endothelial cells with conditioned
medium from HDAC5 siRNA-transfected HUVECs increased
capillary-like sprout formation (Figure 4A) and stimulated endothelial cell migration in a Boyden chamber assay (Figure 4B),
indicating that HDAC5 silencing derepresses secreted
proangiogenic and chemoattractive stimuli for endothelial cells.
Because FGF2 is a secreted gene product repressed by HDAC5,
which stimulates angiogenesis in vivo,16 we assessed the functional
contribution of FGF2 regulation for the repressive effect of
HDAC5. Measurements of FGF2 using enzyme-linked immunosorbent assay revealed that the up-regulation of FGF2 mRNA is
associated with an increased release of FGF2 protein (scrambled
BLOOD, 28 MAY 2009 䡠 VOLUME 113, NUMBER 22
24 ⫾ 1 pg/mL vs HDAC5 siRNA 33 ⫾ 4 pg/mL and Figure 4C).
Supplementation of spheroid cultures with exogenous FGF2 stimulates sprouting under control conditions, whereas increased sprout
formation in HUVECs transfected with HDAC5 siRNA was not
further augmented by the addition of FGF2 (Figure 4D). To test
whether an increased FGF2 secretion essentially underlies the
observed increase in capillary-like sprout formation in HDAC5
siRNA-transfected HUVECs, secreted FGF2 was blocked by a
neutralizing antibody.30 HDAC5 siRNA-enhanced sprouting was
significantly reduced when FGF2 was neutralized (Figure 4E).
Nevertheless, in the presence of the neutralizing antibody against
FGF2, HDAC5 suppression still increased sprouting compared
with control albeit at a lower level (Figure 4E). Thus, we conclude
that FGF2 release contributes to increased sprout formation in
response to HDAC5 siRNA, whereas a part of the effect of HDAC5
siRNA is mediated via FGF2-independent mechanisms, suggesting
the contribution of additional effector genes. Beyond FGF2,
HDAC5 siRNA modulated the expression of the axon guidance
factor Slit2 (Figure 3B,C). So-called guidance molecules, known
for their role in neuronal wiring, have recently been identified as
essential regulators of capillary sprout formation.17 These factors
control the guided and directional movement of specialized endothelial cells situated at the tips of the vascular sprouts similar to
axonal growth cones.18 Guidance factors are families of surface
membrane receptors and their ligands. The secreted ligand Slit2
acts as a guidance cue in cellular migration and promotes tube
formation and tumor vascularization via its receptor Roundabout 1
(Robo1),24 whereas another receptor Robo4 was shown to block
angiogenesis40 but stabilizes the vasculature in vivo.41 Because
Slit2 is a ligand for Robo1, which is expressed in HUVECs
(0.91 ⫾ 0.05 normalized expression, Affymetrix oligonucleotide
microarray), we assessed the contribution of Slit2 in HDAC5regulated endothelial cell sprouting. siRNA oligonucleotides against
Slit2 inhibited the increase in sprout formation in HUVECs
cotransfected with HDAC5 siRNA (Figure 4F). To assess whether
signaling via the Slit2 receptor Robo1 mediates the HDAC5
siRNA-induced increase in sprout formation, we used receptor
antagonists for Robo1-mediated Slit2 signaling.24 Robo1 neutralization with R5, an IgG2b monoclonal antibody to the first immunoglobulin domain of Robo1, significantly reduced the capacity of
HDAC5 siRNA to increase sprout formation (Figure 4G). Likewise, another antagonist of Robo1-mediated signaling, Robo1-Fc,
also reduced the HDAC5 siRNA-induced increase in sprout
formation by 57% plus or minus 25%. Of note, the inhibition of
Robo1 activation with either of the 2 antagonists only partially
suppressed, whereas Slit2 siRNA entirely blocked HDAC5-siRNA
mediated increase in sprout formation, indicating that Slit2 exhibits
effects beyond Robo1 activation.
Mechanism of the regulation of FGF2 and Slit2 by HDAC5
Having identified FGF2 and Slit2 as HDAC5 targets relevant for
the suppression of angiogenic endothelial cell activity by HDAC5,
we investigated the mechanism by which HDAC5 controls FGF2
and Slit2 expression. Therefore, we performed chromatin immunoprecipitation (ChIP) assays in HUVECs overexpressing HDAC5
wt and the nuclear mutant (S259/498A) to investigate the binding
of HDAC5 to the promoter of FGF2 and Slit2 (Figure 5A). The
immunoprecipitation of HDAC5 wt and the S259/498A mutant is
shown in Figure 5B. The nuclear localized HDAC5 S259/498A
mutant, and, to a minor extent, the HDAC5 wt was bound to the
promoter of FGF2 and Slit2 (Figure 5C). The quantitative analysis
of HDAC5 binding to the FGF2 and Slit2 promoter regions is
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BLOOD, 28 MAY 2009 䡠 VOLUME 113, NUMBER 22
HDAC5 AND ANGIOGENESIS
A
B
Symbol
Product
Genbank
Affymetrix
x-fold
P-value
LIPG
Lipase, endothelial
NM_006033
219181_at
2.24
0.0009
SLIT-2
Slit (Drosophila) homolog 2
AF055585
209897_s_at
2.15
0.0363
DCN
Decorin
AF138300
201893_x_at
2.10
0.0004
HOXA9
Homeobox A9
AI246769
209905_at
2.08
0.0016
EDG1
Endothelial differentiation,
sphingolipid G-protein-coupled
receptor, 1
NM_001400
204642_at
2.03
0.0501
HOX1,
HOX1G
Homeo box A9
U41813
214651_s_at
2.00
0.0201
BFGF, FGFB,
HBGH-2
Fibroblast growth factor 2
(basic)
NM_002006/
M27968
204422_s_at/
204421_s_at
1.94*
0.0275
HOX1,
HOX1E
Homeo box A3
NM_030661
208604_s_at
1.92
0.0564
ITGAV
Integrin alpha V
AI093579
202351_at
1.73
0.0040
PLD1
Phospholipase D1,
phophatidylcholine-specific
AJ276230
215723_s_at
1.69
0.0224
Neuropilin 2
AF280545/
AF280546
223510_at/
210841_s_at
1.63*
0.0000
TGFBR2
Transforming growth factor,
beta receptor II (70-80kD)
NM_003242/
D50683
207334_s_at/
208944_at
1.61*
0.0000
FLT1
fms-related tyrosine kinase 1
(vascular endothelial growth
factor/vascular permeability
factor receptor)
AA058828
222033_s_at
1.59
0.0321
SIR2L1
Sirtuin 1
NM_012238.
3
218878_s_at
1.58
0.0310
hPLD1
Human ARF-activated
phosphatidylcholine-specific
phospholipase D1a (hPLD1)
mRNA
U38545
177_at
1.57
0.0223
ETS2
V-ets avian erythroblastosis
virus E26 oncogene homolog 2
NM_005239
201329_s_at
1.55
0.0030
HTK, MYK1,
TYRO11
EphB4
NM_004444
202894_at
1.52
0.0288
NSF
N-ethylmaleimide-sensitive
factor
NM_006178
202395_at
1.51
0.0256
NPN2,
VEGF165R2,
VEGF1265R2
scrambled
5675
HDAC5 siRNA
C
mRNA expression (% scr)
*
24h
48h
*
*
*
*
Figure 3. Identification of target genes regulated by HDAC5 in endothelial cells. (A) Bioinformatic analysis of the RNA profile of HUVECs transfected with HDAC5 siRNA
after 24 hours (n ⫽ 3 each). Up- and down-regulated genes in response to HDAC5 siRNA versus scrambled siRNA as a proportion of all analyzed genes are shown in a gene
tree analysis. The color scale is shown on the right. The brightness indicates the trust. Blue represents low expression; red, high expression. (B) Summary of selected genes
relevant to angiogenesis, which were up-regulated in response to HDAC5 siRNA transfection in the oligonucleotide array. *In case of 2 different oligonucleotides representing
one gene, the data were pooled. Because we focused in our following study on Slit2 and FGF2, both targets are highlighted in gray. (C) Quantitative analysis of HDAC5 and
selected target gene expression in response to HDAC5 siRNA versus scrambled siRNA after 24 hours and 48 hours using real-time PCR (n ⫽ 3; *P ⬍ .05 vs scrambled).
shown by quantitative PCR in Figure S10. Consistent with the
ChIP analysis, HDAC5 wt as well as the nuclear localized HDAC5
mutant decreased the expression of FGF2 and Slit2 (Figure 5D,E).
Discussion
Based on the cumulative evidence for an essential requirement of
class I and II histone deacetylase enzymatic activity for angiogenic
functions of endothelial cells, we focused our interest on the
contribution of the individual mammalian HDAC isoenzymes to
angiogenic signaling in endothelial cells. Our data show that, in
contrast to other HDAC isoforms required for angiogenesis,
HDAC5 is a negative regulator of angiogenic functions in endothelial cells. This is consistent with a study by Ha et al, which was
published during the preparation of the manuscript, showing that
VEGF-induced angiogenesis depends on the nuclear export of
HDAC5.9 The present study, for the first time, describes the
HDAC5-dependent transcriptional profile in endothelial cells and
provides novel insights into the HDAC5-dependent regulation of
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BLOOD, 28 MAY 2009 䡠 VOLUME 113, NUMBER 22
URBICH et al
Sprout length (% scr)
B
scrambled
HDAC5
Conditioned medium from HUVEC
transfected with siRNA
*
Migrated cells per field
A
VEGF
-
+
-
-
Conditioned medium from HUVEC
transfected with scrambled siRNA
-
-
+
-
Conditioned medium from HUVEC
transfected with HDAC5 siRNA
-
-
-
+
C
FGF2 release (% scr)
Sprout length (µm)
D
scrambled
HDAC5
scrambled
siRNA
E
HDAC5
siRNA
F
P < .005
Sprout length
(% Iso-IgG / scr)
Sprout length (% scr)
P < .005
antibody
Iso-IgG
FGF2
Iso-IgG
FGF2
siRNA
siRNA
scr
scr
HDAC5
HDAC5
Iso-IgG+scr
G
*
Sprout length
(% Iso-IgG / scr)
5676
R5+scr
Iso-IgG+HDAC5 siRNA
antibody
Iso-IgG
R5
Iso-IgG
R5
siRNA
scr
scr
HDAC5
HDAC5
R5+HDAC5 siRNA
Figure 4.
scr
Slit2
HDAC5
HDAC5
scr
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BLOOD, 28 MAY 2009 䡠 VOLUME 113, NUMBER 22
Figure 5. Binding of HDAC5 to the promoter of FGF2 and Slit2.
(A) Overview of primer pairs amplifying the indicated promoter
regions relative to the transcription start within the FGF2 and Slit2
genes. (B) Western blot analysis was performed to confirm the
immunoprecipitation of myc-tagged overexpressed HDAC5 wt and
HDAC5 S259/498A mutant. (C) The ChIP was performed in
HUVECs overexpressing myc-tagged HDAC5 wt or HDAC5 S259/
498A mutant using an antibody against c-myc. Representative PCR
with primers detecting the indicated promoter regions of the FGF2
and Slit2 genes are shown (n ⫽ 3). Ab indicates antibody control.
(D) Expression of FGF2 mRNA in HUVECs transfected with
HDAC5 wt or HDAC5 S259/498A mutant after 48 hours (n ⫽ 4).
(E) Expression of Slit2 mRNA in HUVECs transfected with HDAC5
wt or HDAC5 S259/498A mutant after 48 hours (n ⫽ 3).
HDAC5 AND ANGIOGENESIS
5677
A
FGF2 promoter
-1000
CUG
CDS
-809/-613
-1800
-365/-181
Slit2 promoter
AUG
CDS
-663/-508
B
Input
ChIP à myc
HDAC5
mock S259/498A
wt
mock S259/498A wt
HDAC5
C
Ab
HDAC5
Input
ChIP à myc
FGF2 promoter region -365/-181
FGF2 promoter region -809/-613
Slit2 promoter region -663/-508
D
mRNA expression
(% empty vector)
HDAC5
wt
mock
S259/498A wt
Ab
HDAC5
E
mRNA expression
(% empty vector)
mock S259/498A
Figure 4. Role of HDAC5 for sprout formation and secretion of angiogenic factors. (A) Sprout formation from endothelial cell spheroid cultures incubated with conditioned
medium from HUVECs transfected with scrambled siRNA or HDAC5 siRNA for 42 hours (n ⫽ 9). (B) Boyden chamber migration of nontransfected HUVECs toward a
conditioned medium. The medium was derived from HUVECs transfected with scrambled siRNA or HDAC5 siRNA for 48 hours. The number of migrated cells per field after
5 hours is given. Addition of exogenous VEGF (50 ng/mL, 5 hours) served as control. *P ⬍ .05 versus conditioned medium of scrambled siRNA-transfected HUVECs (n ⫽ 4).
(C) Enzyme-linked immunosorbent assay measurement of FGF2 release into medium incubated for 2 days with HUVECs transfected with siRNA against HDAC5 (n ⫽ 4).
(D) Capillary-like sprout formation from HUVEC spheroid cultures stimulated with exogenous FGF2 (30 ng/mL) after transfection of scrambled siRNA or HDAC5 siRNA for
24 hours (n ⫽ 4). (E) Capillary-like sprout formation from HUVEC spheroid cultures in the presence of a neutralizing FGF2 antibody (4 ␮g/mL) or IgG control after transfection
of scrambled siRNA or HDAC5 siRNA for 24 hours (n ⫽ 5-7). (F) Sprout formation from spheroid cultures of scrambled versus HDAC5 siRNA-transfected HUVECs
cotransfected with either of 2 independent Slit2 siRNA oligonucleotides (n ⫽ 2). (G) Capillary-like sprout formation from HUVEC spheroid cultures in the presence of R5, an
IgG2b monoclonal antibody to the first immunoglobulin domain of Robo1, which neutralizes activation by Slit2, or 12CA5 IgG control antibody with no antagonistic effect at the
Robo1 receptor after transfection of scrambled siRNA or HDAC5 siRNA for 24 hours. *P ⬍ .05 versus HDAC5 siRNA with 12CA5 IgG control antibody (n ⫽ 4-10).
Representative images are shown on the right. Pictures were taken using an Axiovert 100M microscope, an AxioCam camera, a Plan-NEOFLUAR 10⫻/0.30⬁/0.17 objective,
and the AxioVision Rel. 4.6.3 Sp1 software (Carl Zeiss).
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5678
BLOOD, 28 MAY 2009 䡠 VOLUME 113, NUMBER 22
URBICH et al
genes, which are well known to control angiogenesis. From the list
of putative targets obtained by the expression array, we here
confirmed the regulation of FGF2, a potent and well-established
pro-angiogenic growth factor, and the guidance factor Slit2.
Blockade of FGF2 and Slit2 both significantly reduced the HDAC5
siRNA-mediated up-regulation of sprout formation. In addition, the
increase of other axon guidance factors, such as the receptor
EphB4, or homeobox genes, such as HOXA9, which are crucial for
migration and angiogenesis,20,42,43 may contribute to the promigratory effect observed in HDAC5-siRNA-treated endothelial
cells. In addition, we found that silencing of the other class IIa
HDAC isoforms HDAC7 and HDAC9 blocked angiogenesis. The
essential requirement of HDAC7 for angiogenesis shown in this
study is consistent with a previous report demonstrating an
embryonic lethal phenotype of HDAC7-deficient mice.6 However,
the role of HDAC9 for angiogenesis has not yet been addressed.
Thus, this study describes a novel proangiogenic function of
HDAC9 in endothelial cells.
Having shown that HDAC5 is a repressor of angiogenic gene
expression in endothelial cells, we addressed the underlying mechanism
and hypothesized that HDAC5 may exert its function in the nucleus.
Indeed, the repressive function of HDAC5 required its nuclear localization. A recent study demonstrated that nitric oxide stimulates nuclear
shuttling of HDAC4 and HDAC5 and deacetylation in endothelial
cells.44 Because nitric oxide exerts pro- or antiangiogenic activities
depending on the concentration,45-48 the findings of Illi et al44 do not
argue against the requirement of a nuclear localization for the antiangiogenic effect of HDAC5. Consistently, preferentially nuclear localized
HDAC5 was shown to bind to the promoters of the 2 novel targets
identified in the present study, namely, FGF2 and Slit2. However, it is
known that class II HDACs do not directly bind DNA; therefore, the
question remains how HDAC5 can bind to the FGF2 and Slit2
promoter. Using the ChIP assay, we cannot distinguish between a direct
and indirect binding to the respective promoters. Thus, we hypothesize
that HDAC5 is indirectly associated with the FGF2 and Slit2 promoter,
eg, via binding to transcription factors. The regulation of endothelial cell
functions by HDAC7 has been attributed to the repression of the
transcription factor MEF2.6 HDAC5 also is well established to bind
MEF2 and inhibit MEF2-dependent transcription in muscle differentiation.36,49 However, the antiangiogenic function of HDAC5 in endothelial cells is independent of MEF2 binding because a mutant unable to
bind MEF2 still efficiently blocked endothelial sprout formation.
Recently, Ha et al have shown that the nuclear export of HDAC5
regulates VEGF-induced MEF2 transcriptional activation.9 Based on
our data, we cannot exclude an HDAC5-dependent mechanism involving MEF2 transcriptional regulation.
The repressive function of HDAC5 was also independent of the
direct deacetylase activity of the enzyme. These data do not
exclude a role of deacetylation for the antiangiogenic effect of
HDAC5 because class II HDACs can interact with other HDAC
isoenzymes (class I and II). Because broad-spectrum HDAC
inhibitors block angiogenesis, one would expect a reduction of
angiogenesis rather than a stimulation of angiogenesis in response
to HDAC5 silencing if other HDACs might contribute to HDAC5
function. In general, class II HDACs can interact with a variety of
proteins, for example, by recruiting a multiprotein complex
containing the transcriptional corepressors SMRT (silencing
mediator for retinoic acid receptor and thyroid-hormone receptor) and N-CoR (nuclear hormone receptor corepressor) and
HDAC3 to their C-terminus as it has been shown for HDAC4
and HDAC7, respectively.50,51 In addition, the C-terminus of
HDAC4 and HDAC5 interacts with Smad3, which leads to the
transcriptional repression of Runx2 during osteoblast differentiation.52 However, in our study, the deacetylase-deficient HDAC5
mutant (1-767 aa), which lacks the C-terminus, still repressed
endothelial cell sprouting, indicating that other mechanisms
account for the repressive effects of HDAC5 on angiogenesis
signaling. Indeed, the N-terminal part of class IIa HDACs might
also repress transcriptional activity through the interaction with
corepressors, such as HP1 or CtBP.53-55 Thus, further studies are
required to address the specific underlying mechanism of
HDAC5 function in endothelial cells.
The identification of a specific control of angiogenesis in vitro
and in vivo by HDAC5 may have potential therapeutic implications. One may consider using HDAC5 inhibitors to improve
therapeutic angiogenesis (eg, after ischemia) or to use HDAC5
activators to block pathologic angiogenesis. Interestingly, a recent
publication demonstrates that inhibition of HDACs increases
dendritic sprouting, learning, and memory.56 A putative regulation
of axon guidance molecules by HDAC5, if confirmed in neuronal
cells, may also have an impact on neuronal cell functions.
Acknowledgments
The authors thank Dorit Lüthje and Nicole Konecny for their expert
technical assistance.
This work was supported by the Deutsche Forschungsgemeinschaft (grant SFB/TR 23, subproject B5, C.U.; grant PO1306/1-1,
M.P.; and grant Exc 147/1 Excellence Cluster Cardiopulmonary
System), and by the Deutsche Krebshilfe (grant no. 107154; L.R.).
Authorship
Contribution: C.U. and L.R. designed research, collected data, and
wrote the paper; D.K. performed research and collected and
analyzed data; M.P. designed research and edited the paper; J.-N.B.
performed research; A.K. and F.D. performed research and collected data; J.-G.G. provided antibodies and designed research;
W.-K.H. collected and analyzed data; A.M.Z. wrote and edited the
paper; and S.D. designed research and wrote and edited the paper.
All authors checked the final version of the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Stefanie Dimmeler, Molecular Cardiology,
Department of Internal Medicine III, University of Frankfurt,
Theodor Stern-Kai 7, 60590 Frankfurt, Germany; e-mail:
[email protected].
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From www.bloodjournal.org by guest on July 28, 2017. For personal use only.
2009 113: 5669-5679
doi:10.1182/blood-2009-01-196485 originally published
online April 7, 2009
HDAC5 is a repressor of angiogenesis and determines the angiogenic
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Carmen Urbich, Lothar Rössig, David Kaluza, Michael Potente, Jes-Niels Boeckel, Andrea Knau,
Florian Diehl, Jian-Guo Geng, Wolf-Karsten Hofmann, Andreas M. Zeiher and Stefanie Dimmeler
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