Clinical Hemorheology and Microcirculation 48 (2011) 5–13
Angiogenesis: The role of PDGF-BB on
Adiopse-tissue derived Stem Cells (ASCs)
, Sanga Gehmert
, Mulyadi Hidayat
, Maitham Sultan
, Silvan Klein
, Johannes Zellner
, Michael M
and Lukas Prantl
Center of Plastic and Reconstructive Surgery, University Medical Center Regensburg,
Department of Trauma Surgery, University Medical Center Regensburg, Regensburg, Germany
Department of Obstetrics and Gynecology, University Medical Center Regensburg,
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
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: firstname.lastname@example.org.
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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
. 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
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
-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 . Brieﬂy,
subcutaneous fat tissue was washed in phosphate-buffered saline, and minced into pieces of <2 mm
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
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. 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
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
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
). GAPDH was used as a reference gene (forward primer
; Reverse primer 5
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
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 . 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
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
3.1. 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
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
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
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 . 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
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
We found signiﬁcant increase of STAT3 Y705 phosphorylation that is requisite for nuclear translocation
of the STAT3 transcription factor . 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).
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.
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