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Angiogenesis: The role of PDGF-BB on adipose-tissue derived stem cells (ASCs)

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Recently, it was shown that mesenchymal stem cells (MSCs) are capable of differentiating into endothelial cells which highlights the potential role of MSCs in neovascularization. In the present study, we investigated the paracrine factors responsible for tube formation in human adipose-tissue derived stem cells (ASCs). Moreover, we analyzed ASC's migration towards PDGF-BB and altered levels of proteins involved in different pathways. Freshly isolated human adipose tissue-derived stem cells were seeded onto wells coated with Matrigel and cultured in endothelial growth medium. Capillary-like tube formation was observed after 18 hours culture. Tube formation was significantly reduced in the presence of antibodies against platelet-derived growth factor receptor beta (PDGF) or basic fibroblast growth factor (bFGF). Reverse phase proteomic assay (RPPA) was used to interrogate the expression of 139 phosphorylated or native proteins after incubation with PDGF-BB protein for 24 hours. The present data suggest, that freshly isolated ASCs contain a subpopuplation of stem cells that can form capillary like tubes which is dependent on PDGF and bFGF signaling pathway. Furthermore, Migration of human ASCs significantly increased in response to increased concentrations of PDGF-BB. In addition, incubation of ASCs with PDGF-BB altered phosphorylation of several transcription proteins that are widely expressed throughout the hematopoietic system, targeting genes that have been associated with proliferation, anti-apoptosis or differentiation.
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Clinical Hemorheology and Microcirculation 48 (2011) 5–13
DOI 10.3233/CH-2011-1397
IOS Press
5
Angiogenesis: The role of PDGF-BB on
Adiopse-tissue derived Stem Cells (ASCs)
Sebastian Gehmert
a,b,d
, Sanga Gehmert
c,d
, Mulyadi Hidayat
a,b
, Maitham Sultan
a,b
,
Arne Berner
a,b
, Silvan Klein
a,b,d
, Johannes Zellner
a,b
, Michael M
¨
uller
a,b
and Lukas Prantl
a,b,d,
a
Center of Plastic and Reconstructive Surgery, University Medical Center Regensburg,
Regensburg, Germany
b
Department of Trauma Surgery, University Medical Center Regensburg, Regensburg, Germany
c
Department of Obstetrics and Gynecology, University Medical Center Regensburg,
Regensburg, Germany
d
Department of Molecular Pathology, The University of Texas, MD, Anderson Cancer Center,
Houston, TX, USA
Abstract. Recently, it was shown that mesenchymal stem cells (MSCs) are capable of differentiating into endothelial cells which
highlights the potential role of MSCs in neovascularization. In the present study, we investigated the paracrine factors responsible
for tube formation in human adipose-tissue derived stem cells (ASCs). Moreover, we analyzed ASC’s migration towards PDGF-
BB and altered levels of proteins involved in different pathways. Freshly isolated human adipose tissue-derived stem cells were
seeded onto wells coated with Matrigel and cultured in endothelial growth medium. Capillary-like tube formation was observed
after 18 hours culture. Tube formation was significantly reduced in the presence of antibodies against platelet-derived growth
factor receptor beta (PDGF) or basic fibroblast growth factor (bFGF). Reverse phase proteomic assay (RPPA) was used to
interrogate the expression of 139 phosphorylated or native proteins after incubation with PDGF-BB protein for 24 hours. The
present data suggest, that freshly isolated ASCs contain a subpopuplation of stem cells that can form capillary like tubes which is
dependent on PDGF and bFGF signaling pathway. Furthermore, Migration of human ASCs significantly increased in response
to increased concentrations of PDGF-BB. In addition, incubation of ASCs with PDGF-BB altered phosphorylation of several
transcription proteins that are widely expressed throughout the hematopoietic system, targeting genes that have been associated
with proliferation, anti-apoptosis or differentiation.
Keywords: Angiogenesis, adipose-tissue derived stem cells, PDGF-BB, endothelial differentiation, migration
1. Introduction
Angiogenesis is a very tight controlled process governed by a fine balance between angiogenetic and
antiangiogenetic factors [3, 17, 22]. To date migration and proliferation of endothelial progenitor cells has
been shown to be involved in this process. Furthermore, motility of pericytes is enhanced during vascular
development due to endothelial cells that secret proteins and form a chemotaxis-like gradient in order to
recruit pericyctes [11, 14]. PDGF-BB/PDGF- signaling plays an important role in vascular maturation
and has been well-documented by genetic studies. Recently, several studies have correlated PDGF-BB
signaling with vascular remodeling and shown that transgenic PDGF-BB expression can increase pericyte
Corresponding author. E-mail: lukas.prantl@klinik.uni-regensburg.de.
1386-0291/11/$27.50 © 2011 – IOS Press and the authors. All rights reserved
6 S. Gehmert et al. / The role of PDGF-BB on ASCs
density [18, 23]. However, the origin of these pericytes remains unknown. Recent work has shown that
altering culture conditions could render mesenchymal stem cells (MSCs) capable of differentiating into
endothelial cells [15, 25], and this further highlights the potential role of MSCs in neovascularization.
Platelet-derived growth factor (PDGF) is important for tissue repair following injury in adult tissues
[9]. But little is known about ASC’s cellular responses to PDGF-BB activation including migration,
differentiation, and survival.
In the present study, we investigated the paracrine factors responsible for tube formation in human ASCs.
Moreover, we analyzed ASC’s migration towards PDGF-BB and altered levels of proteins involved in
different pathways.
2. Materials and methods
2.1. Cell lines
Human dermal microvascular endothelial cells (DMVC) were obtained from LONZA and cultured
in EGM 2 (LONZA; DMVC) at 37
Cina5%CO
2
-containing chamber. For isolation of human ASCs,
unprocessed subcutaneous adipose tissue was obtained from patients undergoing elective body-contouring
procedures, which was in accordance with guidelines of the Declaration of Helsinki for biomedical
research. The study was performed in accordance with the ethical guidelines of the journal [2]. Briefly,
subcutaneous fat tissue was washed in phosphate-buffered saline, and minced into pieces of <2 mm
3
.
Serum-free MEM (1 ml/1 g tissue) and Liberase Blendzyme 3 (Roche Diagnostics) (2 U/1 g tissue)
were added and incubated under continuous shaking at 37
C for 45 min. The digested tissue was sequen-
tially filtered through 100- and 40-m filters (Fisher Scientific) and centrifuged at 450g for 10 min. The
supernatant was discarded and pelleted cells were washed twice with Hanks’ balanced salt solution (Cell-
gro) and finally resuspended in MEM growth medium containing 20% FBS, 2 mM L-glutamine 100
U/ml penicillin, 100 g/ml streptomycin or LONZA EGM 2 (only for tube formation assay). Plastic-
adherent passage 0 cells were then grown in culture vials (Greiner) at 37
C in a humidified atmosphere
containing 5% CO
2
followed by daily washes to remove red blood cells and non-attached cells. After the
passage 0 cells reached 80% confluence, they were seeded at a density of 3000 cells/cm
2
.
2.2. Tube formation assay
To investigate the role of paracrine factors on tube formation of ASCs, 96-well plates were coated with
50 L growth factor reduced phenol red-free Matrigel (BD Bioscience, Franklin Lakes, NJ). Supplement-
free EGM2 and 50 L serum were added to each well. The medium was incubated with antibodies against
platelet derived growth factor (PDGF)-receptor alpha, beta, or bFGF (4 g/mL, abcam). The antibodies
were added 30 minutes prior to the seeding of cells (5 × 10
4
cells/well). After 18 hours of incubation,
tube formation was observed under a microscope and quantified by tube length and number of tubes per
microscopic field. The measurements were taken with 10× magnifications in randomly selected fields.
2.3. Migration assays
The Transwell Migration System (BD Biosciences) with a 3-m pore size was used for the migration
experiments. ASCs were plated in the upper chamber of the system. The lower chamber was filled
S. Gehmert et al. / The role of PDGF-BB on ASCs 7
with 1 ml of medium containing different concentrations of PDGF-BB protein. After 8 h of migration
through the membrane, cells were fixed and stained with calcein. To block the PDGF receptors, we added
neutralization antibodies against either PDGF- or PDGFR- (R&D Systems) 1 hour prior the migration
assays. Migrated cells were quantified in randomly selected fields (n = 3) per condition (triplicate, n =3)
using fluorescence microscope (Nikon TE-2000U) with a Cascade camera (Photometrics) and Image J
software [1].
2.4. RNA extraction and real-time PCR
RNA was extracted from human ASCs or DMVC, using RNAqueous kit (Ambion, Applied Biosys-
tems, Carlsbad, CA) according to the manufacturer’s instructions and reverse transcribed (iScript,
Bio-Rad). PDGF-Receptor beta mRNA expression level was determined with quantitative real-time
PCR, using SYBR Green assay (forward primer 5
-TGTGACGGAGAGTGTGAATGAC-3
; reverse
primer 5
-AGGGTGCGGTTGTCTTTGAAC-3
). GAPDH was used as a reference gene (forward primer
5
-GAAGGTGAAGGTCGGAGTC-3
; Reverse primer 5
-GAAAGATGGTGATGGGATTTC-3
).
2.5. Western blot analysis
Cells were lysed in radioimmunoprecipitation assay lysis buffer (Upstate Biotechnology) that included
protease inhibitor cocktail (Roche Diagnostics), and Western blot analysis was performed as we described
previously [4].
2.6. RPPA analysis
Reverse phase proteomic assay (RPPA) was used to interrogate the expression of 139 phosphorylated
or native proteins. Three samples of ASCs were incubated with 50 ng PDGF-BB or in serum free medium
for 24 hours. The cells were washed twice in ice-cold PBS and lysed in RPPA lysis buffer and prepared
as previously described [24]. Lysates were two-fold-serial diluted for 5 dilutions (from undiluted to
1 : 16 dilution) and arrayed on nitrocellulose-coated slide in 11 × 11 format. Samples were probed with
antibodies by CSA amplification approach and visualized by DAB colorimetric reaction. Slides were
scanned on a flatbed scanner and spots from tiff images were quantified by MicroVigene. Relative
protein levels for each sample were determined by interpolation of each dilution curves. All the data
points were normalized for protein loading and transformed to median centered values for hierarchical
cluster analysis.
2.7. Statistical analysis
Results are shown as means ± SD. Continuous variables were compared by means of one-way ANOVA
with Scheffe post hoc correction using SPSS statistical software package (SPSS Inc., IL, USA). Values
at p < 0.05 were considered as statistically significant.
8 S. Gehmert et al. / The role of PDGF-BB on ASCs
3. Results
3.1. Characterization of human ASCs
ASCs are characteristically plastic adherent, spindle-shaped cells. They are positive for CD44, CD90,
CD105 and negative for CD11b, CD14, CD34, CD45, as we have described previously. ASCs were further
evaluated regarding their multipotent differentiation capacity by incubation in adipogenic and osteogenic
induction medium. ASCs that were maintained in adipogenic induction medium displayed characteristic
multiple intracellular bright white oil droplets. These droplets showed red vesicles when stained with Oil
Red O stain. Cells cultured in osteogenic medium showed black regions within the monolayer, indicating
calcification deposits from differentiated osteoblasts. Calcification of extracellular matrix was assessed
by Alizarin Red S stain and showed red staining of mineralized deposits. No lipid droplets or calcification
was observed in cells cultured in control medium.
3.2. PDGFR-b is involved in endothelial differentiation of human ASCs
To evaluate the endothelial differentiation potential of human ASCs morphologically, freshly isolated
ASCs were seeded onto Growth Factor Reduced Matrigel in EGM2 culture medium, and tube formation
was analyzed. Our data provide evidence that ASCs form tube-like structures in EGM2 medium. In order
to investigate the role of angiogenic factors in ASC’s tube formation, we added antibodies against PDGF
receptor alpha or beta and found that an antibody against PDGF receptor beta antibody but not alpha
receptor abolished the previously seen effect (Fig. 1). In addition, neutralizing antibody against bFGF
significantly reduced tube formation of freshly isolated ASCs. Quantitative analysis of total tube length
confirmed that both bFGF and PDGFR beta antibodies significantly reduced tube formation. Moreover,
our data showed that antibody against PDGF beta is more efficient in blocking tube formation than that
of bFGF antibody.
3.3. PDGFR-b is expressed on human ASCs
To confirm that ASCs express PDGF beta receptor, we performed real-time PCR and Western blot
analysis. Our data show that human ASCs express mRNA for PDGF beta receptor at significantly higher
levels (
∗∗
p < 0.001) compared with endothelial cells (Fig. 2). Furthermore, PDGF-beta receptor expression
on ASCs was confirmed by Western blot (Fig. 3).
3.4. PDGF-BB dependent migration of ASCs
Migration of human ASCs significantly increased in response to increased concentrations of PDGF-BB
(Fig. 4). The migration was completely blocked by the presence of PDGFR- antibody, whereas it was
only slightly reduced by the presence of PDGFR- antibody. It is known that PDGF-BB’s action is mainly
mediated via the PDGF- receptor. Western blot analysis revealed a very weak expression of PDGFR-
on human ASCs in comparison with control cell lines, whereas PDGFR- was solely expressed on human
ASCs (Fig. 3).
S. Gehmert et al. / The role of PDGF-BB on ASCs 9
Fig. 1. Quantitative analysis of total tube length. After 18 hours of incubation, tube formation was observed and quantified by tube
length per microscopic field. The measurements were taken with 10× magnifications in randomly selected fields. AB indicates
antibody. Antibodies against bFGF and PDGF-BB significantly decreased tube formation in ASCs (
∗∗
p < 0.001). PDGF-BB AB
blocks more efficient tube formation in ASCs than bFGF-AB (p = 0.002).
3.5. PDGF-BB activates mTOR, PI3, Akt and STAT pathway in ASCs
PDGF-BB stimulation induced an increase in phosphorylation of Akt at threonine 308 (T308), serine
473 (S473) and increased phosphorylation of 70-kDa ribosomal protein S6 kinase (p70S6 K) at T389 as
well as ribosomal protein S6 at S235 and S240 site (Fig. 5). Furthermore, we detected significant higher
phosphorylation for TSC2 at T1462 site in ASCs incubated with PDGF-BB when compared to control
ASCs. Noteworthy, ASCs treated with PDGF-BB demonstrated phosphorylation and activation of signal
transducers and activators of transcription, especially for STAT3 pY705, STAT 5 pY694 and STAT 6
pY641.
4. Discussion
The aim of this study was to identify and analyze specific factors that are involved in ASC’s migrations
and endothelial differentiation. Here, we show that PDGF-BB attracts ASCs in a dose dependent manner
and confirmed that ASCs express PDGFR-b. We further demonstrate that ASCs have the capacity to form
capillary-like tube structures which were abolished by blocking PDGF receptor beta on ASCs. Moreover,
RPPA analysis showed that mTOR, PI3, Akt and STAT pathway is activated in ASCs after PDGF-BB
incubation. These results offer insight into a new role for PDGF in promoting angiogenesis associated
with wound healing and tumor growth.
10 S. Gehmert et al. / The role of PDGF-BB on ASCs
Fig. 2. Relative expression of PDGFR-. PDGF beta receptor expression in human dermal microvascular endothelial cells
(DMVEC) and adipose-tissue derived stem cells (ASCs) was analyzed by real-time PCR with normalization to GAPDH
expression.
Fig. 3. Western blot showed that only ASCs express PDGFR- but not control cell line (human breast cancer cell line). PDGFR-
was expressed on both cell lines but very weak by ASCs when compared to control.
PDGF-BB is a well-characterized growth factor displaying potent biological effects on mural cells
including pericytes. Simultaneous overexpression of PDGF-BB and FGF2 in fibrosarcomas led to the
formation of high-density primitive vascular plexuses [21]. Our data that antibodies against either PDGFR
beta or bFGF inhibit tube formation are in line with these previous reports. Our data provide a novel
mechanistic insight on the regulation of angiogenic responses and a potential role for ASCs in angio-
genesis. Diminished peripheral blood flow and decreased local neovascularization are critical factors
that contribute to the delayed or nonhealing wounds in patients with diabetes or radiation exposure. The
correction of impaired local angiogenesis is a key component in developing therapeutic protocols for
treating chronic wounds. Endothelial progenitor cells (EPCs) are the key cellular effectors of postnatal
neovascularization and play a central role in wound healing, but their circulating and wound-level num-
bers are decreased in diabetes, implicating an abnormality in EPC mobilization and homing mechanisms
S. Gehmert et al. / The role of PDGF-BB on ASCs 11
Fig. 4. Migration of ASCs is mediated by PDGFR-. Migration of ASCs was significantly increased by PDGF-BB protein
(
∗∗
p < 0.001). Migration was inhibited by the presence of PDGFR- neutralizing antibodies and did not show significant difference
when compared to serumfree control (N.S.).
[12, 19]. ASCs represent an alternative cellular source for the treatment of nonhealing wound because
it have the ability to differentiate into endothelial cells and form capillary networks which is necessary
for wound healing. Our data provide evidence that ASCs can form capillary like tubes when cultured in
3-D environment. More importantly, we showed that tube formation is dependent on PDGF signaling
pathway.
In addition, we investigated PDGF-BB effects on ASCs and found that PDGF-BB increased phospho-
rylation of Akt (at T308 and S473), p70S6 K (at T389) and S6 (at S235 and S240). This is of importance
since T308 and S473 phosphorylation site of Akt are required for maximal activation of Akt [7, 10].
Moreover, p70S6K is known to promote protein synthesis and cell survival due to phosphorylation of S6
an important downstream effector. Akt was shown to phosphorylate and inactivate TSC2 [16, 20] thereby
activating mTOR.
We found significant increase of STAT3 Y705 phosphorylation that is requisite for nuclear translocation
of the STAT3 transcription factor [13]. Furthermore, we detected phosphorylation of STAT5 which is
widely expressed throughout the hematopoietic system, targeting genes that have been associated with
proliferation, anti-apoptosis or differentiation [5, 6, 8]. Peripheral arterial disease (PAD) is a common
manifestation of systemic atherosclerosis associated with a significant limitation in limb function due to
ischemia. In the absence of effective pharmacological, interventional or surgical treatment, amputation is
undertaken at the end-stage as a solution to unbearable symptoms. The concept of therapeutic angiogenesis
has become widely accepted during the past few years. Adipose tissue consists of multiple cell populations,
including mesenchymal stem cells, which are able to migrate and differentiate into endothelial cells as
we have shown in the present work. The promising results from various preclinical studies provide the
basis for clinical trials using ASCs. Our findings in this study provide new mechanistic insight into the
regulation of angiogenic responses especially of PDGF-BB and open up the possibility of ASCs in tissue
engineering and reconstruction.
12 S. Gehmert et al. / The role of PDGF-BB on ASCs
Fig. 5. Heat map represents unsupervised hierarchical clustering of ASCs and corresponding protein data after incubation for
24 h with 50 ng PDGF-BB or in serum free medium (A, ASCs in serumfree medium; B, ASCs incubated with PDGF-BB). The
columns represent proteins and the rows represent samples. Each cell is colorized based on the level of protein expression in that
sample. PDGF-BB stimulation induced an increase in phosphorylation of Akt and activation of signal transducers and activators
of transcription (STAT).
Acknowledgments
The authors would like to thank Yiling Lu and Nancy Shi, Department of Systems Biology, UT MD
Anderson Cancer Center, for her professional assistance in Reverse Phase Proteomic Assay (RPPA). In
addition, authors are grateful to Dr. Alt for helpful comments on this study. This work was supported in
part by the Alliance of Cardiovascular Researchers.
References
[1] M.D. Abramoff, P.J. Magelhaes and S.J. Ram, Image processing with image, J Biophotonics International 11 (2004),
36–42.
[2] Anonymous, Ethical guidelines for publication in Clinical Hemorheology and Microcirculation, Clin Hemorheol Microcirc
44 (2010), 1–2.
[3] A. Armulik, A. Abramsson and C. Betsholtz, Endothelial/pericyte interactions, Circ Res 97 (2005), 512–523.
[4] X. Bai, J. Ma, Z. Pan, Y.H. Song, S. Freyberg, Y. Yan, D. Vykoukal and E. Alt, Electrophysiological properties of human
adipose tissue-derived stem cells, Am J Physiol Cell Physiol 293 (2007), C1539–C1550.
S. Gehmert et al. / The role of PDGF-BB on ASCs 13
[5] H.L. Bradley, T.S. Hawley and K.D. Bunting, Cell intrinsic defects in cytokine responsiveness of STAT5-deficient hemato-
poietic stem cells, Blood 100 (2002), 3983–3989.
[6] H.L. Bradley, C. Couldrey and K.D. Bunting, Hematopoietic-repopulating defects from STAT5-deficient bone marrow are
not fully accounted for by loss of thrombopoietin responsiveness, Blood 103 (2004), 2965–2972.
[7] D.P. Brazil, J. Park and B.A. Hemmings, PKB binding proteins. Getting in on the Akt, Cell 111 (2002), 293–303.
[8] K.D. Bunting, H.L. Bradley, T.S. Hawley, R. Moriggl, B.P. Sorrentino and J.N. Ihle, Reduced lymphomyeloid repopulating
activity from adult bone marrow and fetal liver of mice lacking expression of STAT5, Blood 99 (2002), 479–487.
[9] J.R. Crosby, K.A. Tappan, R.A. Seifert and D.F. Bowen-Pope, Chimera analysis reveals that fibroblasts and endothelial
cells require platelet-derived growth factor receptorbeta expression for participation in reactive connective tissue formation
in adults but not during development, Am J Pathol 154 (1999), 1315–1321.
[10] S.R. Datta, A. Brunet and M.E. Greenberg, Cellular survival: A play in three Akts, Genes Dev 13 (1999), 2905–2927.
[11] M. Enge, M. Bjarnegard, H. Gerhardt, E. Gustafsson, M. Kalen, N. Asker, H.P. Hammes, M. Shani, R. Fassler and C.
Betsholtz, Endothelium-specific platelet-derived growth factor-B ablation mimics diabetic retinopathy, EMBO J 21 (2002),
4307–4316.
[12] G.P. Fadini, S. Sartore, C. Agostini and A. Avogaro, Significance of endothelial progenitor cells in subjects with diabetes,
Diabetes Care 30 (2007), 1305–1313.
[13] P.C. Heinrich, I. Behrmann, G. Muller-Newen, F. Schaper and L. Graeve, Interleukin-6-type cytokine signalling through
the gp130/Jak/STAT pathway, Biochem J 334(Pt 2) (1998), 297–314.
[14] M. Hellstrom, M. Kalen, P. Lindahl, A. Abramsson and C. Betsholtz, Role of PDGF-B and PDGFR-beta in recruitment
of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse, Development 126
(1999), 3047–3055.
[15] R. Huss, C. Lange, E.M. Weissinger, H.J. Kolb and K. Thalmeier, Evidence of peripheral blood-derived, plastic-adherent
CD34(-/low) hematopoietic stem cell clones with mesenchymal stem cell characteristics, Stem Cells 18 (2000), 252–260.
[16] K. Inoki, Y. Li, T. Zhu, J. Wu and K.L. Guan, TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR
signalling, Nat Cell Biol 4 (2002), 648–657.
[17] S. Kaessmeyer and J. Plendl, Angiogenesis and vasculogenesis in the corpus luteum in vitro, Clin Hemorheol Microcirc
41 (2009), 83–101.
[18] P. Lindahl, B.R. Johansson, P. Leveen and C. Betsholtz, Pericyte loss and microaneurysm formation in PDGF-B-deficient
mice, Science 277 (1997), 242–245.
[19] C.J. Loomans, E.J. de Koning, F.J. Staal, M.B. Rookmaaker, C. Verseyden, H.C. de Boer, M.C. Verhaar, B. Braam, T.J.
Rabelink and A.J. van Zonneveld, Endothelial progenitor cell dysfunction: A novel concept in the pathogenesis of vascular
complications of type 1 diabetes, Diabetes 53 (2004), 195–199.
[20] B.D. Manning, A.R. Tee, M.N. Logsdon, J. Blenis and L.C. Cantley, Identification of the tuberous sclerosis complex-2
tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway, Mol Cell 10 (2002),
151–162.
[21] L.J. Nissen, R. Cao, E.M. Hedlund, Z. Wang, X. Zhao, D. Wetterskog, K. Funa, E. Brakenhielm and Y. Cao, Angiogenic
factors FGF2 and PDGF-BB synergistically promote murine tumor neovascularization and metastasis, J Clin Invest 117
(2007), 2766–2777.
[22] W. Risau, Mechanisms of angiogenesis, Nature 386 (1997), 671–674.
[23] P. Soriano, Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice, Genes Dev 8
(1994), 1888–1896.
[24] R. Tibes, Y. Qiu, Y. Lu, B. Hennessy, M. Andreeff, G.B. Mills and S.M. Kornblau, Reverse phase protein array: Validation
of a novel proteomic technology and utility for analysis of primary leukemia specimens and hematopoietic stem cells, Mol
Cancer Ther 5 (2006), 2512–2521.
[25] M. Wosnitza, K. Hemmrich, A. Groger, S. Graber and N. Pallua, Plasticity of human adipose stem cells to perform
adipogenic and endothelial differentiation,
Differentiation 75 (2007), 12–23.
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