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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
Angiogenesis: The role of PDGF-BB on
Adiopse-tissue derived Stem Cells (ASCs)
Sebastian Gehmert
, Sanga Gehmert
, Mulyadi Hidayat
, Maitham Sultan
Arne Berner
, Silvan Klein
, Johannes Zellner
, Michael M
and Lukas Prantl
Center of Plastic and Reconstructive Surgery, University Medical Center Regensburg,
Regensburg, Germany
Department of Trauma Surgery, University Medical Center Regensburg, Regensburg, Germany
Department of Obstetrics and Gynecology, University Medical Center Regensburg,
Regensburg, Germany
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:
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
-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
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
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
; reverse
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
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
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
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
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).
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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... In addition, only PI3K inhibitor (LY294002) but not the MAPK inhibitor (PD98059) was associated with a significant decrease of transmigration for ASCs towards breast cancer cells (Fig. 3b). Western blot analyses of ASCs revealed strong expression of PDGFR-␤ and only weak expression of PDGFR-␣ when compared with control cell line (Fig. 3c) as previously reported by our group [25]. ...
Introduction: Mesenchymal stem cells (MSCs) have been described in breast cancer models to migrate towards carcinoma and integrate into tumor associated stroma supporting tumor growth, increasing their metastatic potency and contributing to tumor-angiogenesis. Platelet-derived growth factor (PDGF) isoforms (AA, BB, CC) stimulate growth, survival and motility of MSCs and certain other cell types. Noteworthy, breast carcinomas are known to express PDGF. We aim to further shed light on i) the relevance of the different PDGF isoforms on adipose tissue derived stem cells (ASCs) migration and ii) the underlying pathway dependent on PDGF stimulation. Materials and methods: Breast cancer cell lines were purchased and ASC?s were isolated from murine subcutaneous adipose tissue. The transmigration of ASC?s towards the PDGF-isoforms was assessed by using recombinant human PDGF-AA, PDGF-BB and PDGF-CC in a trans-well culture dish system. Transmigrated ASC?s were quantified in 5 randomly selected fields per condition using fluorescence microscopy after calcein-staining. PDGF-BB depended transmigration of ASC?s was verified by downregulation and overexpression of PDGF-BB in breast cancer cell line using shRNA-technique. In addition, a PI3-kinase inhibitor (LY294002) and a MAP-kinase inhibitor (PD98059) were used to identify the pathway involved in the PDGF-BB mediated migration of ASC?s towards tumor. Results: ASC?s transmigration significantly increased towards PDGF AA at 50 ng and only showed further increase by 500 ng which was similar to cell behavior when exposed to PDGF CC. In comparison, PDGF-BB significantly increased ASC?s transmigration already at a low level of 5 ng with further significant increase for 20 ng and 40 ng. Cell transmigration was blocked with PDGFR-α antibodies but only for PDGF-AA and PDGF-CC whereas PDGFR-β blockage showed a significant effect on transmigration for PDGF-BB and PDGF-CC but not for PDGF-AA. Neutralizing antibodies in combination with PDGF receptor blockage confirmed findings. In addition, only PI3-kinase inhibitor but not the MEK-1 selective inhibitor caused a significant decrease of transmigration for ASCs towards breast cancer cells. Discussion: The transmigration of ASC's is most significantly enhanced by PDGF-BB via the PI3-kinase pathway. This data support that PI3-kinase is an important key player for MSC migration towards malignancy which need further research to prevent tumor progression in early disease stage.
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In plastic surgery, lipofilling is a frequent procedure. Unsatisfactory vascularization and impaired cell vitality can lead to unpredictable take rates in the fat graft. The proliferation and neovascularization inducing properties of adipose tissue-derived stem cells may contribute to solve this problem. Therefore, the enrichment of fat grafts with stem cells is studied intensively. However, it is difficult to compare these studies because many factors-often not precisely described-are influencing the results. Our study summarizes some factors which influence the cell yield like harvesting, isolation procedure and quantification. Stem cells were isolated after liposuction. Quantification was done using a cell chamber, colony counting, or flow cytometry with changes to one parameter, only, for each comparison. Quantification of cells isolated after liposuction at the same harvesting site from the same patient can vary greatly depending on the details of the isolation protocol and the method of quantification. Cell yield can be influenced strongly by many factors. Therefore, a comparison of different studies should be handled with care.
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Platelet-derived growth factor-BB (PDGF-BB) is a potent mitogenic, angiogenic and chemoattractant, and is one of the most abundant growth factors in platelet-derived products. The goal of the present study was to examine the effects of PDGF-BB on cellular morphology and cellular viability using 3D stem cell cultures. On day 1, spheroids formed well in silicon-elastomer-based concave microwells. The addition of 10 or 100 ng/ml PDGF-BB did not affect the morphology of the cell spheroids. During longer periods of incubation, the cell spheroids maintained their shape without noticeable alterations. The majority of cells in the spheroids exhibited green fluorescence when analyzed using a live/dead assay, indicative of live cells. On day 1, the Cell Counting Kit-8 (CCK-8) assay values for PDGF-BB at 0, 10 and 100 ng/ml were 0.241±0.003, 0.227±0.001 and 0.241±0.004, respectively; on day 3, the CCK-8 assay values for PDGF-BB were 0.233±0.005, 0.278±0.001 and 0.194±0.003, respectively; and on day 7, they were 0.248±0.014, 0.293±0.031 and 0.346±0.034, respectively. The 100 ng/ml group showed significantly higher values compared with the control group on day 7. Together, the results of the present study showed that the addition of 10 and 100 ng/ml PDGF-BB increased cellular viability, suggesting that PDGF-BB may be usable in cell therapy.
Radiation therapy has been increasingly employed as a tool to cure and palliate majority of solid tumors. Although radiotherapy has shown promising results in preserving structure and function of organs, it is associated with late side effects mainly manifested in the form of tissue fibrosis. Recent advances in molecular biology techniques has helped better understand the molecular mechanisms involved in radiation induced fibrosis. Currently, very few treatment modalities are available to treat the condition with moderate success rate. Stem cell therapies and particularly adipose tissue and adipose derived stem cells therapies have shown promising results in clinical applications. Identification of the key factors involved in the mitigation process will help to enhance the beneficial effects and develop new therapy approaches.
Background: Autologous fat transfer in breast reconstruction has become increasingly important in breast reconstructive surgery. Although the indication to obtain fat, the various operative procedures, and the risks for the patient have been addressed in a large number of studies, detailed information on the everyday use of autologous lipotransfer in breast units in Germany is still lacking. Methods: The objective of the study was to obtain primary data on the use of autologous lipotransfer to treat breast cancer patients in Germany and to determine measures for quality assurance in the daily practice. An online questionnaire concerning breast cancer and lipofilling was sent to specialists in gynecology and plastic surgery. Results: Two-thirds of the specialists who responded to the questionnaire use autologous lipotransfer for breast reconstruction and did not report an increase of local recurrence following lipotransfer. There were only small differences between gynecologists and plastic surgeons regarding the procedure and indication for lipotransfer. The method is highly accepted by patients and physicians, and both gynecologists and plastic surgeons rated the improvement achieved through lipofilling as 'high'. Conclusions: The lack of randomized controlled data, especially in high-risk patients, demonstrates the necessity for a registry study on this topic. Our survey describes, in detail, the indications for lipofilling as well as its appropriate application in breast cancer patients in Germany and may thereby reduce the present therapeutic uncertainties.
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Adipose-derived stem cells (ASCs) are multipotent mesenchymal progenitor cells that have functional and phenotypic overlap with pericytes lining microvessels in adipose tissue. The role of CD140b [platelet-derived growth factor receptor- β (PDGFR-β)], a constitutive marker expressed by ASCs, in the angiogenic behavior of human retinal endothelial cells (HREs) is not known. CD140b was knocked down in ASCs using targeted siRNA and lipofectamine transfection protocol. Both CD140b+ and CD140b- ASCs were tested for their proliferation (WST-1 reagent), adhesion (laminin-1 coated plates), and migration (wound-scratch assay). Angiogenic effect of CD140b+ and CD140b- ASCs on HREs was examined by co-culturing ASCs:HREs in 12:1 ratio for 6 days followed by visualization of vascular network by Isolectin B4 staining. The RayBio® Membrane-Based Antibody Array was used to assess differences in human cytokines released by CD140b+ or CD140b- ASCs. Knockdown of CD140b in ASCs resulted in a significant 50% decrease in proliferation rate, 25% decrease in adhesion ability to Laminin-1, and 50% decrease in migration rate, as compared to CD140b+ ASCs. Direct contact of ASCs expressing CD140b+ with HREs resulted in robust vascular network formation that was significantly reduced with using CD140b- ASCs. Of the 80 proteins tested, 45 proteins remained unchanged (>0.5-<1.5 fold), 6 proteins including IL-10 downregulated (<0.5 fold) and 29 proteins including IL-16 & TNF-β were upregulated (>1.5 fold) in CD140b- ASCs compared to CD140b+ ASCs. Our data demonstrate a substantial role for CD140b in the intrinsic abilities of ASCs and their angiogenic influence on HREs. Future studies are needed to fully explore the signaling of CD140b in ASCs in vivo for retinal regeneration.
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New insights in facial aging made it apparent that not only sagging/ptosis but also facial volume loss is a major factor of this process. Classic facial rejuvenating procedures that only lift or reposition tissue in a vertical vector did not correct for facial volume loss. Since the (re-)introduction of lipofilling it appeared that this procedure is a well-tolerated natural alternative to correct for volume loss due to facial aging. The success and extent of the effect of lipofilling, however, is plagued by a vast number of variables, like the technique of harvesting, the fat processing and injection of the lipograft; factors that all influence the lipograft viability and the ASC. Platelet Rich Plasma (PRP) has been suggested to increase the lipograft viability and retention Moreover, PRP could also increase wound healing resulting in a faster recovery. With our studies we have attempted to investigate and clarify the role of lipofilling and lipofilling with the additional use of PRP in facial rejuvenation with regard to the aesthetic outcome, possible regenerative skin effects and recovery time. Also, we have tried to unravel the effects of PRP on the ASC itself. This thesis concludes that lipofilling is a valuable additive to lifting procedures in order to maximize rejuvenating effect. PRP proves a powerful additive when looking at patient recovery, but its effect on rejuvenation remains uncertain. Mixed clinical results of PRP addition to lipofilling could be explained by its concentration depended effect on ASC fenotype, proliferation and secrotome as demonstrated in vitro. Future lipofilling/ASC/SVF based therapies have the potential of regenerating damaged tissue.
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Increases in the number of circulating endothelial cells (CECs) and progenitors (CEPs) have been reported in various pathological conditions including cancer. Preclinical studies have shown that CEC and CEP kinetics correlate well with several standard laboratory angiogenesis assays, which cannot be used in humans. At the clinical level, evidence is emerging that CEC kinetics and viability might correlate with clinical outcomes in cancer patients who undergo anti- angiogenic treatment. Therefore, CEC and CEP measurement has potential as a surrogate marker for monitoring anti-angiogenic treatment and drug activity, and could help to determine the optimal biological dose of anti-angiogenic drugs, which are being used with increasing frequency in medical oncology.
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The hematopoietic system of vertebrates can be completely reconstituted with hematopoietic stem cells derived from the bone marrow, fetal liver, or cord blood, or even from peripheral-blood-derived cells. A cellular marker to identify those cells is the proteoglycan CD34, although we have shown that the earliest identifiable hematopoietic stem cell is a CD34− fibroblast-like cell which can differentiate into CD34+ hematopoietic precursors. Peripheral blood mononuclear cells were isolated from the heparinized blood of a dog and incubated in tissue culture in the presence of interleukin 6. After 10-14 days, an adherent layer of fibroblast-like cells had developed and cells were immortalized using the SV-40 large T antigen. Cells were cloned and subcloned by measures of limiting dilution, and various fibroblast-like clones were established. These fibroblast-like cells either do not express the CD34 antigen or express CD34 on a low level, although transcribing CD34. The CD34−/low cells express osteocalcin as a mesenchymal cell marker. The fibroblast-like cells eventually differentiate spontaneously in vitro into CD34+ precursors and show colony formation. Prior to autologous stem cell transplantation, one clone of choice (IIIG7) was transfected with a retroviral construct containing the green-fluorescence protein (GFP). The recipient dog was totally irradiated with 300 cGy and received a stem cell transplant with GFP-containing, immortalized, fibroblast-like monoclonal autologous stem cells (0.5 × 108/kg dog). No additional growth factors were applied. The peripheral blood counts recovered after 23 days (WBC >500; platelets >10,000). A peripheral blood smear showed some dim but definite, although timely, limited expression of the GFP protein in nucleated peripheral blood cells just five weeks after transplantation. A bone marrow biopsy showed GFP-positive cells in the marrow cavity predominantly as “bone-lining cells.”
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MSCs reside within their niche and pathologic conditions such as hypoxia and inflammation can lead to mobilization and migration of Mesenchymal Stem Cells (MSCs). Xenograft animal models using immundeficient mouse demonstrated that MSCs migrated to and distributed throughout the tumors and were found to engraft into tumor stroma and vasculature. In contrast, MSCs primarily incorporated within tumor-capsula and did not invade the tumor using immuncompetent tumor allograft models. Here we hypothesize that MSCs migrate primarily towards an inflammatory milieu independent of the underlying biological process causing the inflammation. Murine MSCs (mASCs) were isolated from subcutaneous fat tissues and transduced at passage 0 with lentiviral vector encoding green fluorescent protein (GFP) and luciferase reporter. Breast cancer was established in BALB/c mice by subcutaneous injection of 4T1 cells into the left mammary fat pad. E. coli were injected subcutaneously in the right 4th mammary fat pad. After 24 h luciferase labeled mASCs were administered intraperitoneal (i.p.) and monitored with IVIS Bioluminescence camera for 72 hours. Control group received either tumor implantation or E. coli injection. MSCs significantly migrated towards tumor when compared to control mice without tumor or inflammatory process. However, mASCs injected in 4T1 bearing mice with E. coli only migrated towards the bacterial inflammatory focus. Our results substantiate the notation the MSCs response predominantly to the inflammatory milieu created by bacteria or tumor rather than specifically to the tumor. Thus, it is suggested that the migration of MSCs in immunodeficient mice depends on cancer secreted cytokines due to the lack of the inflammatory response by the immune system. Therefore, in vivo studies investigating the role of MSCs in tumor angiogenesis have shown controversy results and should be interpreted with caution in terms of tumor secreted cytokine dependent stem cell migration.
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Neutrophils are the all-terrain vehicle of the innate immune system because of their ability to gain entry into tissues and organs, and thus, play an essential role in host defense. Exactly how this marvel of nature works is still incompletely understood. In the past 2-3 years, new players and processes have been identified in the endothelial-leukocyte adhesion cascade. Novel signaling pathways have been discovered in both the endothelium and the neutrophils that regulate various steps in the recruitment process. This review focuses on these emerging pathways and the mechanisms that regulate neutrophil recruitment across endothelium.
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Carcinoma-associated fibroblasts (CAF) have recently been implicated in important aspects of epithelial solid tumor biology, such as neoplastic progression, tumor growth, angiogenesis, and metastasis. However, neither the source of CAFs nor the differences between CAFs and fibroblasts from nonneoplastic tissue have been well defined. In this study, we show that human bone marrow-derived mesenchymal stem cells (hMSCs) exposed to tumor-conditioned medium (TCM) over a prolonged period of time assume a CAF-like myofibroblastic phenotype. More importantly, these cells exhibit functional properties of CAFs, including sustained expression of stromal-derived factor-1 (SDF-1) and the ability to promote tumor cell growth both in vitro and in an in vivo coimplantation model, and expression of myofibroblast markers, including alpha-smooth muscle actin and fibroblast surface protein. hMSCs induced to differentiate to a myofibroblast-like phenotype using 5-azacytidine do not promote tumor cell growth as efficiently as hMSCs cultured in TCM nor do they show increased SDF-1 expression. Furthermore, gene expression profiling revealed similarities between TCM-exposed hMSCs and CAFs. Taken together, these data suggest that hMSCs are a source of CAFs and can be used in the modeling of tumor-stroma interactions. To our knowledge, this is the first report showing that hMSCs become activated and resemble carcinoma-associated myofibroblasts on prolonged exposure to conditioned medium from MDAMB231 human breast cancer cells.
Protein kinase B (PKB) has emerged as the focal point for many signal transduction pathways, regulating multiple cellular processes such as glucose metabolism, transcription, apoptosis, cell proliferation, angiogenesis, and cell motility. In addition to acting as a kinase toward many substrates involved in these processes, PKB forms complexes with other proteins that are not substrates, but rather act as modulators of PKB activity and function. In this review, we discuss the implications of these data in understanding the multitude of functions predicted for PKB in cells.
After the developing embryo has formed a primary vascular plexus by a process termed vasculogenesis, further blood vessels are generated by both sprouting and non-sprouting angiogenesis, which are progressively pruned and remodelled into a functional adult circulatory system. Recent results, particularly from the study of mice lacking some of the signalling systems involved, have greatly improved our understanding of the molecular basis underlying these events, and may suggest new approaches for treating conditions such as cancer that depend on angiogenesis.
Abramoff, M.D., Magelhaes, P.J., Ram, S.J. "Image Processing with ImageJ". Biophotonics International, volume 11, issue 7, pp. 36-42, 2004.