1. Art and Diseño

TISSUE-SPECIFIC STEM CELLS
Mesenchymal Stem Cells Regulate Angiogenesis According to Their
Mechanical Environment
GRIT KASPER,a,b NIELS DANKERT,a JENS TUISCHER,a MORITZ HOEFT,a TIMO GABER,c JULIANE D. GLAESER,a,d
DESIREE ZANDER,a MIRIAM TSCHIRSCHMANN,e MARK THOMPSON,a GEORG MATZIOLIS,a GEORG N. DUDAa,b
a
Musculoskeletal Research Center Berlin, bBerlin-Brandenburg Center for Regenerative Therapies, and cDepartment
of Rheumatology and Clinical Immunology, Charite´–Universita¨tsmedizin, Berlin, Germany; dDepartment of Biology,
Chemistry, and Pharmacy, Free University Berlin, Berlin, Germany; eMedical Biotechnology Department, University
for Technologies, Berlin, Germany
Key Words. Angiogenesis • Endothelial cells • Mesenchymal stem cells • Mechanical loading • Regeneration • Vascularization
ABSTRACT
In fracture and bone defect healing, MSCs largely drive
tissue regeneration. MSCs have been shown to promote
angiogenesis both in vivo and in vitro. Angiogenesis is a
prerequisite to large tissue reconstitution. The present
study investigated how mechanical loading of MSCs influences their proangiogenic capacity. The results show a
significant enhancement of angiogenesis by conditioned
media from mechanically stimulated compared with unstimulated MSCs in two-dimensional tube formation and
three-dimensional spheroid sprouting assays. In particular, proliferation but not migration or adhesion of endothelial cells was elevated. Promotion of angiogenesis was
dependent upon fibroblast growth factor receptor 1
(FGFR1) signaling. Moreover, stimulation of tube forma-
tion was inhibited by vascular endothelial growth factor
receptor (VEGFR) tyrosine kinase blocking. Screening
for the expression levels of different soluble regulators of
angiogenesis revealed an enrichment of matrix metalloprotease 2, transforming growth factor ␤1, and basic
fibroblast growth factor but not of vascular endothelial
growth factor in response to mechanical stimulation. In
conclusion, mechanical loading of MSCs seems to result in
a paracrine stimulation of angiogenesis, most likely by the
regulation of a network of several angiogenic molecules.
The underlying mechanism appears to be dependent on
the FGFR and VEGFR signaling cascades and might be
mediated by an additional cross-talk with other pathways.
STEM CELLS 2007;25:903–910
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION
At present, the prevention of tissue-engineered implant failure
due to the lack of blood vessels and resulting hypoxia is still a
fundamental challenge in regenerative medicine. To address
these problems, approaches based on cell delivery systems harboring proangiogenic properties seem to be preferable to pharmacological stimulation, since the agents delivered, such as
recombinant vascular endothelial growth factor (VEGF) or basic
fibroblast growth factor (bFGF), are rapidly cleared from the
target site and may lead to unwanted side effects, such as
vascular leakage and hypotension [1, 2].
MSCs have proangiogenic properties, are relatively easy to
harvest, and harbor a great expansion potential [3]. These cells
are able to differentiate not only into mesenchymal cells, such as
osteoblasts and chondrocytes, but also into nonmesenchymal
cells, such as endothelial cells (ECs) and neural cells [3–5].
These properties make MSCs an attractive cell source for a wide
variety of tissue engineering strategies. Indeed, these cells are
used in clinical applications as single cell-type constructs and
show promise in combination with ECs in multicell-type in vitro
prevascularized constructs for bone regeneration [6, 7].
It is well established that MSCs are able to influence EC
behavior and vice versa. For example, the presence of ECs
appears to promote osteogenic differentiation of MSCs [8 –10].
In addition, MSCs are capable of neoangiogenesis [11] induction in an in vivo model [12]. A recent study consolidates these
observations in vitro by demonstrating that MSCs promote EC
migration and tube formation [13]. Furthermore, MSCs seeded
on three-dimensional (3D) constructs seem to support endothelial cell growth [14]. VEGF is one factor that could potentially
mediate the cross-talk between MSCs and ECs. MSCs were
shown to secrete VEGF, the expression of which was elevated
during osteogenesis [15]. In addition, MSCs have been shown to
generate sufficient VEGF to support the survival and differentiation of ECs [16]. Apart from VEGF, other molecules expressed by MSCs, including transforming growth factor-␤
(TGF-␤) and matrix metalloproteases (MMPs) (e.g., MMP-2
and MMP-14), could contribute to the complex interaction of
MSCs and ECs [17, 18]. In addition, mechanical boundary
conditions are known to alter the gene expression pattern and
consequently the functional behavior of MSCs. For example,
osteogenic differentiation and proliferation of MSCs appear to
be stimulated by mechanical loading [19 –22].
In conclusion, there is clear evidence for a complex interplay between MSCs and ECs, and thereby MSCs seem to be
Correspondence: Grit Kasper, Ph.D., Musculoskeletal Research Center Berlin, Charite´–Universita¨tsmedizin Berlin, Augustenburger Platz,
113353 Berlin, Germany. Telephone: 49-30-450615116; Fax: 49-30-450559969; e-mail: [email protected] Received July 14, 2006;
accepted for publication December 28, 2006; first published online in STEM CELLS EXPRESS January 11, 2007. ©AlphaMed Press
1066-5099/2007/$30.00/0 doi: 10.1634/stemcells.2006-0432
STEM CELLS 2007;25:903–910 www.StemCells.com
MSCs Regulate Angiogenesis
904
able to promote angiogenesis. However, the influence of mechanical loading on this interaction remains unknown. In the
present study, we report data demonstrating that mechanical
stimulation of MSCs increases their paracrine proangiogenic
properties by the induction of molecules other than VEGF. The
results presented are relevant for the generation of in vitrovascularized tissue-engineered constructs, as well as for the
stimulation of physiological regeneration processes.
MATERIALS
AND
METHODS
Cell Culture and Characterization of MSCs
Bone marrow was obtained from patients undergoing hip surgery.
All donors gave informed consent. The median age of male and
female donors was 50 years (range, 33– 84 years). MSCs were
isolated within 4 hours by centrifugation with Histopaque-1077
(Sigma-Aldrich, Steinheim, Germany, http://www.sigmaaldrich.
com) density separation and subsequent adherence to tissue culture
plastic. MSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Karlsruhe, Germany, http://www.
invitrogen.com) supplemented with 10% fetal calf serum (FCS)
(Biochrom AG, Berlin, http://www.biochrom.de) and 100 U/ml
penicillin ⫹ 100 ␮g/ml streptomycin. Cells were passaged at 70%–
80% confluence and seeded at a density of 2.5 ⫻ 103 cells per cm2.
Only cells from passages 3–7 were used for experiments. For flow
cytometry, 1 ⫻ 105 cells were trypsinized and washed with phosphate-buffered saline/bovine serum albumin before a 15-minute
incubation with fluorescence-conjugated antibodies on ice. After
washing, propidium iodide was added, and cells were analyzed
using FACSCalibur (BD Biosciences, Heidelberg, Germany, http://
www.bdbiosciences.com). Twenty thousand events were acquired
and analyzed using the FCSExpress 2 software (De Novo Software,
Thornhill, ON, Canada, http://www.denovosoftware.com). Antibodies used were as follows: mouse (␣-human CD73): phycoerythrin (PE) (BD Biosciences), mouse (␣-human CD44): fluorescein isothiocyanate (FITC) (BD Pharmingen, Heidelberg, Germany,
http://www.bdbiosciences.com/pharmingen),mouse(␣-humanCD105):
FITC (Serotec, Du¨sseldorf, Germany, http://www.serotec.com),
mouse (␣-human CD106):PE (Pharmingen), mouse (␣-human
CD34):PE (BD Biosciences), mouse (␣-human CD45):FITC (BD
Biosciences), and mouse (␣-human CD90):FITC (BD Biosciences).
As negative controls, cells were stained with nonspecific molecules
of the same isotype as the antibodies used. In addition, MSC
capacity to differentiate into the osteogenic and adipogenic lineage
by addition of the appropriate media [3] was confirmed. SV40immortalized human dermal microvascular endothelial cells
(HMEC-1) were used [23]. This cell line was kindly provided by
Prof. G. Scho¨nfelder (Charite´, Universita¨tsmedizin, Berlin) and was
cultured in MCB-131 (Invitrogen) supplemented with 1 ␮g/ml
hydrocortisone, 2 mM L-glutamine, 5% FCS (Biochrom), and 100
U/ml penicillin ⫹ 100 ␮g/ml streptomycin.
Generation of Conditioned Media and
Characterization of the Bioreactor
The bioreactor system used has been described previously [24].
Briefly, MSCs were trypsinized, and 2 ⫻ 106 cells in 350 ␮l of
culture medium were mixed with 300 ␮l of fibrinogen per medium
(1:2) mixture and 50 ␮l of thrombin S/medium mixture (1:2)
(Tissucol; Baxter, Munich, Germany, http://www.baxter.com). The
construct mixture was placed between two spongiosa chips (4 mm
in height and 15 mm in diameter) and allowed to solidify for 30
minutes at 37°C. The solid sandwich was placed into the bioreactor,
and 25 ml of MSC culture medium was added containing 0.6 ml of
Trasylol (Bayer, Leverkusen, Germany, http://www.bayer.com)
and, if indicated, 6.7 ␮M MMP-2 inhibitor (Calbiochem, Darmstadt, Germany, http://www.merckbiosciences.com). Oxygen partial
pressure was measured by Licox MCB Revooxide Oxygen Probe
(Integra Neuroscience, Ratingen, Germany, http://www.integra-ls.
com). Cell viability in the constructs was determined by CellTiter
96 AQueous test (Promega, Mannheim, Germany, http://www.
promega.com). Levels of cell proliferation were determined based
on 5-bromo-2⬘-deoxyuridine incorporation using the cell proliferation enzyme-linked immunosorbent assay (ELISA) kit (Roche,
Manheim, Germany, http://www.roche.com). Results showed that
in contrast to positive controls (MSCs on cell culture plastic), MSCs
within the construct do not proliferate either with or without mechanical loading. This was confirmed visually by hematoxylin staining that revealed single cells in the construct. Mechanical loading
was carried out for 72 hours at 1 Hz and 10 kPa (approximately 30%
strain). Afterward, conditioned medium (CM) was harvested and
centrifuged at 500g. The supernatant was stored in working aliquots
at ⫺20°C and analyzed within 3 weeks. All measurements were
made in three independent experiments.
RNA Isolation, cDNA Synthesis, and Quantitative
Reverse Transcription-Polymerase Chain Reaction
Total RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden,
Germany, http://www1.qiagen.com) according to the manufacturer’s instructions. RNA quality was assessed by determination of the
18S/28S rRNA ratio using the Agilent Bioanalyzer (Agilent Technologies, Waldbronn, Germany, http://www.agilent.com). Subsequently, cDNA was obtained by reverse transcription of total RNA
using the TaqMan reverse transcription reagents (Applied BioSystems, Foster City, NJ, http://www.appliedbiosystems.com) according to the manufacturer’s instructions. The quantification of HIF1␣, VEGF, and ␤-actin transcripts were assessed by quantitative
reverse transcription-polymerase chain reaction (qRT-PCR) using
the LightCycler FastStart DNA Master SYBR Green I Kit and the
Roche LightCycler instrument and software (Roche, Mannheim,
Germany). VEGF and HIF-1␣ transcript expression were normalized versus the housekeeping gene ␤-actin. The following primers
used in the real-time PCR assay were purchased from TIB Molbiol
(Berlin, http://www.tib-molbiol.com): ␤-actin forward primer, 5⬘gAC Agg ATg CAg AAg gAg ATC ACT-3⬘; ␤-actin reverse primer,
5⬘-TgA TCC ACA TCT gCT ggA Agg T-3⬘; HIF-1␣ forward primer,
5⬘-CCA TTA gAA AgC AgT TCC gC-3⬘; HIF-1␣ reverse primer,
5⬘-Tgg gTA ggA gAT ggA gAT gC-3⬘; VEGF forward primer,
5⬘-TCC ATg gAT gTC TAT CAg Cg-3⬘; VEGF reverse primer,
5⬘-gCT CAT CTC TCC TAT gTg CT-3⬘. The accuracy of the
qRT-PCR assay as determined by the amplification efficiency (E)
was assessed by measurement of dilution series of a randomized
cDNA mix (␤-actin, E ⫽ 1.95 ⫾ 0.05; HIF-1␣, E ⫽ 1.98 ⫾ 0.03;
VEGF, E ⫽ 1.94 ⫾ 0.04). Transcripts from four MSC donors were
analyzed.
Angiogenesis Assays
For tube formation assays, 24-well plates were coated with 50 ␮l of
Matrigel (10 mg/ml; Invitrogen) and allowed to solidify for 30
minutes at 37°C. Afterward, 4 ⫻ 104 HMEC-1 cells were seeded in
100 ␮l of endothelial cell medium per well, and 500 ␮l of CM was
added. Sprouting assays were conducted according to Korff et al.
[25]. Briefly, spheroid formation was performed by suspending 1 ⫻
103 HMEC-1 cells in culture medium containing 0.25% (wt/vol)
methylcellulose and seeded into nonadherent round-bottom 96-well
plates. After cultivation for 24 hours, spheroids were embedded into
collagen gels (Vitrogen; Nutacon BV, Leimuiden, The Netherlands,
http://www.nutacon.nl), where three parts collagen were mixed with
one part medium containing the spheroids. The gel was allowed to
polymerize (30 minutes at 37°C) and overlaid with 100 ␮l of CM.
Both assays were incubated for 17 hours before results were visualized by light microscopy. Images were digitalized at a magnification of ⫻10 and a resolution of 1,600 ⫻ 1,200 pixels. Quantification was performed by means of NIH ImageJ software package
(http://rsb.info.nih.gov/nih-image/). The length of the two-dimensional (2D) capillary network was analyzed in five independent
fields per well. Cumulative sprout length was summed from sprouts
over the threshold length of 55 pixels. Four spheroids per sample
were evaluated. Experiments were repeated at least three times with
duplicates. Equal cell numbers were confirmed by a CellTiter 96
AQueous test (Promega) at the end of the assays. Inhibitors used in
the assays were as follows: a monoclonal ␣-human VEGF antibody
(25 ␮g/ml; R&D Systems, Wiesbaden, Germany, http://www.
rndsystems.com), a vascular endothelial growth factor receptor
Kasper, Dankert, Tuischer et al.
905
(VEGFR) tyrosine kinase inhibitor (20 ␮M; Calbiochem), a MMP-2
inhibitor (6.7 ␮M; Calbiochem), a monoclonal ␣-human TGF-␤1
antibody (1 ␮g/ml; Calbiochem), and the fibroblast growth factor
(FGF) receptor tyrosine kinase inhibitor SU5402 (80 ␮M; Calbiochem).
Proliferation Assay
HMEC-1 cells (4 ⫻ 103) were seeded in HMEC-1 culture medium
into 96-well plates. The following day, cells were washed twice
with DMEM without FCS, and CM was added. After 3 days of
incubation, a CellTiter 96 AQueous test (MTS test; Promega) was
performed according to the manufacturer’s instructions. Six independent experiments using different MSC donors were conducted
using five wells per experiment.
Transwell Migration Assay
Migration was measured by a modified Boyden chamber assay [26]
using polycarbonate filters (8-␮m pore size; Nunc, Wiesbaden,
Germany, http://www.nuncbrand.com) coated with Matrigel (1 mg/
ml; Invitrogen). CM (500 ␮l) from stimulated or unstimulated
MSCs was placed in the lower chamber. HMEC-1 cells (2 ⫻ 104)
were seeded onto the filters in 500 ␮l of CM from unloaded
samples. After 1 hour of incubation at 37°C, cells were fixed by
paraformaldehyde. Nonmigrated cells were removed by scraping,
and remaining migrated cells were stained by Hoechst. Cell numbers of five microscopic fields per filter were analyzed using the
NIH ImageJ software package (http://rsb.info.nih.gov/nih-image/).
Six independent experiments using different MSC donors with
duplicates in each were conducted.
Adhesion Assay
After trypsinization, 2 ⫻ 104 HMEC-1 cells were resuspended in
100 ␮l of CM of MSC and seeded into 96-well plates. After 20
minutes of incubation, nonadherent cells were removed by inverting
the plates. Remaining adherent cells were quantified by CellTiter 96
AQueous test (MTS test; Promega) according to the manufacturer’s
instructions. Five independent experiments using different MSC
donors were conducted using five wells per experiment.
Zymogram and ELISA
Gelatin zymography was performed by the Novex system (Invitrogen) according to the manufacturer’s protocol. To detect MMP-2,
CM from six MSC donors was tested at a dilution of 1:30. All
ELISAs were obtained from R&D Systems and performed in triplicate according to the manufacturer’s instructions. In VEGF and
TGF-␤1 ELISAs, original CM was used. For bFGF ELISA, the CM
was concentrated 30 times. CM with and without MMP-2 inhibitor
application via conditioning was tested from three MSC donors.
Statistical Analysis
The SPSS 12.0 software package (SPSS, Inc., Chicago, http://www.
spss.com) was used for statistical evaluation. Data from functional
assays were analyzed by nonparametric testing and are displayed in
box plots showing medians as bars and interquartile ranges as
boxes. Effects of CM from mechanically loaded versus unloaded
MSCs from the same donors were analyzed by means of the
Wilcoxon test. For analysis of the effect of inhibitor supplementation on the ratio of angiogenesis stimulation by CM from loaded to
unloaded MSCs versus this ratio in control samples without inhibitors, the Mann-Whitney U test was used. Results from expression
analyses were analyzed by Student’s t tests. All tests were two-sided
and at a significance level of p ⬍ .05. In Figs. 3– 6, statistical
significance is indicated by asterisks.
RESULTS
Characterization of the Bioreactor System for
Mechanical Stimulation
The MSCs used were characterized by their marker protein
expression, as well as their potential to differentiate (Fig. 1; data
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Figure 1. Flow cytometry analysis of cell surface markers in MSCs.
The cells were negative for CD34 and CD45 and stained positive for
CD13, CD44, CD73, CD90, CD105, and CD106. Shown are cell counts
(y-axis) and relative fluorescence intensities (x-axis). Isotype controls
are displayed in gray, and samples incubated with specific antibodies are
indicated in black. Abbreviations: FITC, fluorescein isothiocyanate; PE,
phycoerythrin.
not shown). The bioreactor system presented [24] was used for
the generation of CM from mechanical loaded MSCs. For a
characterization of the system, oxygen partial pressure was
measured throughout the construct (Fig. 2B). The partial pressure for the setting used was not lower than 75% of that of
saturated cell culture media, which corresponds to approximately 15 kPa. Thus, compared with physiological oxygen
pressure, the conditions in the bioreactor can be considered as
nonhypoxic. Furthermore, it was demonstrated that the viability
of MSCs could be maintained over the time of conditioning of
media and that cell numbers were similar in mechanically stimulated and unstimulated cell constructs (data not shown).
Influence of CM from Mechanically Stimulated
MSCs on Angiogenesis
To address the question of how mechanical stimulation of MSCs
might influence their angiogenesis-promoting capacity, the CM
was tested in in vitro angiogenesis assays. Beforehand, expression levels of hypoxia-induced HIF-1␣ and VEGF were determined and found to be unchanged in response to mechanical
stimulation of MSCs (Fig. 2C). CM of unstimulated MSCs
906
MSCs Regulate Angiogenesis
loaded MSCs, inhibitory agents were used (Fig. 4). In 2D tube
formation assays, the inhibitor of the VEGFR tyrosine kinase, as
well as the inhibitor of the fibroblast growth factor receptor
(FGFR), led to a significantly reduced enhancement of angiogenesis by CM from mechanically loaded versus unloaded
MSCs (mediancontrol ⫽ 263%, medianVEGFR inhibitor ⫽ 94%,
medianFGFR inhibitor ⫽ 123%; pVEGFR inhibitor ⫽ 0.011,
pFGFR inhibitor ⫽ 0.020). The supplementation of an inhibitory
antibody against TGF-␤1 resulted in a trend of suppression of
angiogenesis stimulation, which was not statistically significant
(median␣-TGF-␤1 ⫽ 149%; p ⫽ .088). In agreement with the 2D
results, the application of FGFR inhibitor in 3D sprouting assays
resulted in a diminished angiogenesis promotion (mediancontrol
⫽ 174%, medianFGFR inhibitor ⫽ 93%; p ⫽ .045). Furthermore,
the supplementation of CM with a MMP-2 inhibitor showed a
tendency to inhibit the described effect without reaching significance (medianMMP-2 inhibitor ⫽ 121%; p ⫽ .055). Since the level
of induction of angiogenesis varied between donors (Fig. 3),
results for MMP-2 and TGF-␤1 inhibition might reach statistical
significance in a larger donor cohort.
Influence of CM from Mechanically Stimulated
MSCs on EC Function
Figure 2. Characterization of oxygen supply in the bioreactor system.
(A): The bioreactor consists of a closed cylindrical polyethylene chamber communicating via four sterile filters with the surrounding gas.
Mechanical stimulation of cell loaded constructs is mediated by a
pneumatically driven displacement of the upper silicone membrane. The
lower membrane is connected to a pressure transducer, enabling pressure regulation. (B): Construct used for mechanical loading. Oxygen
saturation within constructs is displayed relative to saturated cell culture
media. (C): mRNA levels of HIF-1␣ and VEGF as determined by
quantitative reverse transcription-polymerase chain reaction from MSCs
relative to ␤-actin. The ratio of expression levels in mechanically loaded
relative to unloaded controls is presented. Abbreviation: VEGF, vascular endothelial growth factor.
showed no significant effect on angiogenesis in the 2D or 3D
assays in comparison to control media from constructs without
cells (Fig. 3). In contrast, CM from mechanically stimulated
MSCs enhanced angiogenesis compared with CM from unstimulated cells in both assays (2D: medianstimulated ⫽ 142 ⫻
105 pixels, medianunstimulated ⫽ 49 ⫻ 105 pixels; p ⫽ .028; 3D:
medianstimulated ⫽ 96 ⫻ 104 pixels, medianunstimulated ⫽ 40 ⫻
104 pixels; p ⫽ .028). This effect was not detectable in assays
using CM from mechanically loaded control constructs without
cells. Absolute levels of sprouting and tube formation were
rather diverse between CM from different MSC donors. However, due to the small sample size, no correlation to donor
characteristics, such as sex or age, could be made.
Involvement of bFGF and VEGFR
Signaling Cascade
To investigate the relevance of different signaling cascades for
the paracrine stimulation of angiogenesis by mechanically
Since angiogenesis involves several functional activities of ECs,
the proliferation, migration, and adhesion of HMEC-1 cells was
investigated in appropriate in vitro models. CM from untreated
MSCs showed a stimulatory effect on HMEC-1 migration compared with media from constructs without cells (medianMSCs ⫽
32 cells per field, medianno cells ⫽ 17 cells per field; p ⫽ .043;
Fig. 5). Proliferation and adhesion of HMEC-1 cells were similar in CM from MSCs and from control constructs without
cells. However, mechanical loading of MSCs led to a significant
enhancement of proliferation of HMEC-1 cells (medianstimulated
⫽ 0.754 optical density [OD]490 nm, medianunstimulated ⫽ 0.439
[OD]490 nm, p ⫽ .028; Fig. 5A), whereas CM from mechanically
loaded control constructs without cells showed no effect (p ⫽
.917). Migration and adhesion of HMEC-1 cells was not affected by CM from mechanically stimulated MSCs (pmigration ⫽
0.528, padhesion ⫽ 0.500; Fig. 5B, 5C).
Levels of Angiogenic Factors in CM of MSCs
To reveal factors that are potentially involved in the enhancement of angiogenesis by mechanically stimulated MSCs, levels
of the angiogenesis regulators MMP-2, VEGF, TGF-␤1, and
bFGF were investigated in CM of stimulated and unstimulated
MSCs. MMP-2 was upregulated by mechanical loading, as
shown by zymography (Fig. 6A; G. Kasper, J.D. Glaeser, S.
Giessler, A. Ode, J. Tuischer, G. Matziolis, C. Perka, G.N.
Duda, manuscript in preparation). The presence of MSCs led to
a significant accumulation of VEGF in CM compared with CM
from control constructs without cells (meanno cells ⫽ 2 ng/ml,
meanMSCs ⫽ 158 ng/ml; p ⬍ .001). However, no consistent
change of VEGF levels in response to mechanical loading was
observed (Fig. 6B). TGF-␤1 was not enriched in cell culture
supernatant from untreated MSCs (meanno cells ⫽ 120 pg/ml,
meanMSCs ⫽ 121 pg/ml). However, after mechanical stimulation, TGF-␤1 levels were significantly increased (Fig. 6C).
When investigating bFGF concentrations in CM of MSCs, a
wide variation in absolute expression levels of this growth factor
was observed between MSC donors (Fig. 6D). bFGF showed a
trend of accumulation in the CM of untreated MSCs that did not
reach statistical significance (meanno cells ⫽ 0.01 pg/ml,
meanMSCs ⫽ 1.04 pg/ml). However, protein levels were increased by mechanical loading of MSCs. Since MMPs are able
to release growth factors [27], the elevated levels of TGF-␤1
and bFGF could be due to the observed enhanced MMP-2
Kasper, Dankert, Tuischer et al.
907
Figure 3. Conditioned medium of mechanically stimulated MSCs enhanced in vitro
angiogenesis. Representative photographs
and analysis of the cumulative length of the
tubular network from two-dimensional tube
formation (A) and sprouts per spheroid from
three-dimensional sprouting (B). Shown are
six MSC donors, as well as the average of
these donors and of the corresponding negative controls (constructs without cells). Original magnification of photographs, ⫻10.
Slashed circles define extreme values. Abbreviation: w/o, without.
Figure 4. Potential involvement of fibroblast growth factor receptor and the VEGFR
signaling cascades in angiogenesis stimulation. Displayed are ratios of the cumulative
length resulting from conditioned medium of
loaded to unloaded MSCs from tubular structures in two-dimensional tube formation
assays (A) and sprouts per spheroid in threedimensional sprouting assays (B). Abbreviations: MMP, matrix metalloprotease; ␣TGF-␤, transforming growth factor-␤ blocking antibody; ␣-VEGF, vascular endothelial
growth factor blocking antibody; VEGFR,
vascular endothelial growth factor receptor.
activity. However, the application of an MMP-2 inhibitor
showed no effect on the increased TGF-␤1 or bFGF levels,
indicating that this protease is not involved in an increased
release of these growth factors (Fig. 6C, 6D).
The effects described were not seen in CM from control
constructs without cells. Thus, mechanical loading leads to a
specific accumulation of MMP-2, TGF-␤1, and bFGF in the
microenvironment of MSCs.
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DISCUSSION
In the present study, the influence of mechanical loading on the
paracrine stimulation of angiogenesis by MSCs was investigated. CM from untreated MSCs was not capable of stimulating
angiogenesis. However, enhanced tube formation in endothelial
cells cultivated with CM from unstimulated MSCs has recently
908
MSCs Regulate Angiogenesis
Figure 5. Proliferation of endothelial cells
was influenced by conditioned medium
(CM) from mechanically stimulated MSCs.
Shown are OD490 nm values corresponding to
HMEC-1 cell numbers (A), migrated
HMEC-1 cells per microscopic field (B), and
OD490 nm values corresponding to numbers
of adhered HMEC-1 cells (C). All assays
were conducted with CM from mechanically
stimulated and unstimulated MSCs and negative control constructs. Abbreviation: OD,
optical density.
Figure 6. MMP-2, TGF-␤1, and bFGF were enriched by mechanical loading. (A): A representative zymogram using MSCs from one donor is
displayed. Shown are VEGF (B), TGF-␤1 (C), and bFGF (D) concentrations as determined by enzyme-linked immunosorbent assay in conditioned
medium of mechanically loaded and unloaded MSCs from three different donors in the presence and absence of an MMP-2 inhibitor. Abbreviations:
bFGF, basic fibroblast growth factor; MMP, matrix metalloprotease; TGF, transforming growth factor; VEGF, vascular endothelial growth factor.
been reported [13]. This discrepancy might be due to the different experimental settings used (e.g., a 3D system in fibrin to
approach physiological conditions versus a 2D cultivation of
MSCs on tissue culture plastic). The data from the present study
suggest that unstimulated MSCs lack the ability to promote
angiogenesis. Instead, the cells seem to gain this capability in
response to changes in their mechanical boundary conditions.
This regulatory mechanism might be of high physiological
relevance, since angiogenesis is an essential process for tissue
regeneration [28] but on the other hand needs to be tightly
controlled spatially and temporally to prevent tumor formation
[29].
It has been shown that ECs and osteoprogenitor cells interact by gap junctions [9]. The results presented here demonstrate
that paracrine mechanisms for a cross-talk between MSCs and
ECs, independent of direct cell-cell contacts, appear to exist in
response to mechanical loading of MSCs. The transcription
factor HIF-1␣ and its downstream target VEGF could represent
Kasper, Dankert, Tuischer et al.
candidate mediators for the translation of mechanical signals
into a proangiogenic response, since these angiogenesis regulators were shown to be upregulated because of mechanical stress
[30, 31]. However, under the conditions in this study, neither
HIF-1␣ nor VEGF expression was enhanced after mechanical
stimulation of MSCs. Since these factors are also induced by
hypoxia [30 –32], it is important to note that the mechanical
loading setting described was demonstrated to run under nonhypoxic conditions by direct oxygen measurement. Furthermore, the upregulation of MMP-2 indicates a nonhypoxic environment, since a report of Annabi et al. [33] showed that
hypoxia downregulates MMP-2 expression in MSCs. The hypothesis that VEGF is not the mediating factor of the stimulatory effect of mechanical loading on angiogenesis shown in this
study is further supported by the inability of a VEGF inhibitory
antibody to repress the proangiogenic response. However, our
data indicate that although the promotion of tube formation
occurs independently of VEGF, there is still a dependence on
the activity of the VEGFR pathway. This suggests that the effect
might be mediated by cross-talk to another pathway. Additional
candidates for soluble factors mediating the observed effect are
the angiogenesis regulators MMP-2, TGF-␤1, and bFGF, which
were shown to be upregulated in response to mechanical loading. Since the proproliferative molecule VEGF [34, 35] was not
enhanced in response to mechanical stimulation, the reported
effect of angiogenesis promotion by mechanically loaded MSCs
is not a likely result of this factor. However, microvascular cells
have the potential to respond not only to VEGF but also to
TGF-␤1 and bFGF, since they were shown to express the
corresponding cell surface receptors [36, 37]. Indeed, we could
demonstrate that FGFR signaling, which is known to have the
potential to stimulate survival, proliferation, migration, and differentiation of endothelial cells [34, 35, 38], is involved in
angiogenesis stimulation by CM from mechanically loaded
MSCs. MMP-2 is postulated to be essential for the initiation of
angiogenesis [39]. Our results suggest that MMP-2 is not involved in the enrichment of CM by TGF-␤1 or bFGF but might
contribute to angiogenesis by other mechanisms, such as the
removal of mechanical barriers by extracellular matrix degradation or the generation of regulatory breakdown products from
the extracellular matrix [40]. In fact, 3D sprouting assays hint at
a potential involvement of MMP-2 in mediating the observed
effect. TGF-␤1 seems to play a dual role in angiogenesis. Low
concentrations (ⱕ0.5 ng/ml) stimulate tube formation, whereas
higher concentrations (1–5 ng/ml) are inhibitory [41– 43]. Similar effects are seen for EC proliferation [44]. ELISA results
from this study point to TGF-␤1 concentrations lower than 0.2
ng/ml in CM from mechanically stimulated MSCs. Therefore,
the proproliferative and tube formation-enhancing effect on ECs
observed in this study could be mediated by TGF-␤1. Indeed,
inhibition of TGF-␤1 showed a tendency to repress the enhance-
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ACKNOWLEDGMENTS
This study was partially supported by the Bundesministerium
fuer Bildung und Forschung excellence cluster Berlin-Brandenburg Center for Regenerative Therapies and the AO Foundation,
Switzerland. We are grateful to Prof. U. Dirnagl (Neurology,
Charite´, Universita¨tsmedizin, Berlin, Germany) for the use of
the oxygen probe. We thank Dr. A. Rump (Molecular Genetics,
TU-Dresden) for critical reading of the manuscript and M. Princ
for excellent technical assistance.
DISCLOSURE
OF POTENTIAL
OF INTEREST
CONFLICTS
The authors indicate no potential conflicts of interest.
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