Combination of Injectable Multiple Growth
Factor–Releasing Scaffolds and Cell Therapy as an
Advanced Modality to Enhance Tissue Neovascularization
Jaimy Saif, Theresa M. Schwarz, David Y.S. Chau, James Henstock, Paramjit Sami, Simon F. Leicht,
Patrick C. Hermann, Sonia Alcala, Francisca Mulero, Kevin M. Shakesheff,
Christopher Heeschen, Alexandra Aicher
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Objective—Vasculogenic progenitor cell therapy for ischemic diseases bears great potential but still requires further
optimization for justifying its clinical application. Here, we investigated the effects of in vivo tissue engineering by
combining vasculogenic progenitors with injectable scaffolds releasing controlled amounts of proangiogenic growth
factors.
Methods and Results—We produced biodegradable, injectable polylactic coglycolic acid– based scaffolds releasing single
factors or combinations of vascular endothelial growth factor, hepatocyte growth factor, and angiopoietin-1. Dual and
triple combinations of scaffold-released growth factors were superior to single release. In murine hindlimb ischemia
models, scaffolds releasing dual (vascular endothelial growth factor and hepatocyte growth factor) or triple
combinations improved effects of cord blood– derived vasculogenic progenitors. Increased migration, homing, and
incorporation of vasculogenic progenitors into the vasculature augmented capillary density, translating into improved
blood perfusion. Most importantly, scaffold-released triple combinations including the vessel stabilizer angiopoietin-1
enhanced the number of perivascular smooth muscle actin⫹ vascular smooth muscle cells, indicating more efficient
vessel stabilization.
Conclusion—Vasculogenic progenitor cell therapy is significantly enhanced by in vivo tissue engineering providing a
proangiogenic and provasculogenic growth factor-enriched microenvironment. Therefore, combined use of scaffoldreleased growth factors and cell therapy improves neovascularization in ischemic diseases and may translate into more
pronounced clinical effects. (Arterioscler Thromb Vasc Biol. 2010;30:1897-1904.)
Key Words: angiogenesis 䡲 ischemia 䡲 progenitor cells 䡲 tissue engineering
I
studies could be demonstrated.3,4 Because the outcome of
patients is still poor, optimization protocols for enhanced cell
therapy have emerged.5,6 In this context, injection of biodegradable scaffolds in vivo with controlled release of proangiogenic growth factors7 facilitated in vivo tissue engineering
and compensated for the disadvantage of in vitro– engineered
3-dimensional tissues lacking appropriate vascularization.
Here, we investigated the combined effects of vasculogenic
cell therapy with scaffolds delivering proangiogenic growth
factors in a protracted manner. Biocompatible polylactic
coglycolic acid (PLGA) microparticles were used as injectable scaffolds.8 Their gradual degradation into water-soluble,
nontoxic products can be controlled by choosing polymers
with release profiles ranging from 1 week to several months.
Because dual and multiple growth factor-based therapeutic
schemic diseases such as peripheral artery disease and
coronary artery disease are devastating diseases of the
cardiovascular system caused by lack of sufficient blood
supply for skeletal or cardiac muscles. To date, these diseases
are on the rise because of an increasing elderly population and
the prevalence of obesity and diabetes mellitus. Moreover,
conventional therapies, including surgical treatments, are not
yet sufficient to promote adequate recovery of the blood flow
in ischemic areas.1
Initially, growth factor-based approaches without additional cell application were widely used to enhance neovascularization, but the rapid protein degradation in vivo hinders
a sustainable success.2 More recently, cell-based therapies
have attracted great interest, as improved neovascularization
in both experimental hindlimb ischemia models and clinical
Received on: December 17, 2009; final version accepted on: July 14, 2010.
From the School of Science and Technology, Nottingham Trent University, Nottingham, United Kingdom (J.S., J.H., P.S., A.A.); Experimental
Medicine, Ludwig-Maximilians-University, Munich, Germany (T.M.S., S.F.L.); Division of Drug Delivery and Tissue Engineering, School of Pharmacy,
Centre for Biomolecular Sciences, University of Nottingham, Nottingham, United Kingdom (D.Y.S.C., K.M.S.); Stem Cells and Cancer Clinical Research
Group, (P.C.H., S.A., C.H.); Molecular Imaging Core Unit, Biotechnology Programme, Spanish National Cancer Research Centre, Madrid, Spain (F.M.).
Jaimy Saif and Theresa Schwarz contributed equally to this work. Dr Heeschen and Dr Aicher contributed equally to this work.
Correspondence to Alexandra Aicher, MD, School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS,
United Kingdom. E-mail [email protected]
© 2010 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
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DOI: 10.1161/ATVBAHA.110.207928
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Arterioscler Thromb Vasc Biol
October 2010
approaches have been reported to be superior to single–
growth factor therapy,9 –11 we used a mixture of dual and
triple combinations of proangiogenic growth factors. The
combination of hepatocyte growth factor (HGF) and vascular
endothelial growth factor (VEGF) has resulted in a more
robust proangiogenic response.12 We therefore also designed
scaffolds incorporating VEGF and HGF as basic proangiogenic growth factors along with additional vessel-stabilizing
angiopoietin-1 (Ang-1) with and without concomitant infusion of cord blood– derived vascular progenitors.13
Methods
For more information, see supplemental material, available online at
http://atvb.ahajournals.org.
Animals
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Animal procedures performed for this study were all approved by the
Institutional Animal Care and Use Committee of the Spanish
National Cancer Research Centre and the Administrative Panel on
Laboratory Animal Care (Government of Upper Bavaria, Germany).
Scaffold Preparation
Microparticles containing individual growth factors were produced
using a double-emulsion technique as previously described.8
Isolation of Human Cord Blood–Derived
Vasculogenic Progenitor Cells
Isolation of ECFC was performed by density gradient centrifugation
with Ficoll as previously described.13
Chicken Chorioallantoic Membrane Assay
Chorioallantoic membrane assays were performed on 4-day-old
eggs. After a window was cut, the scaffolds with or without growth
factors were placed into the egg, which was sealed and incubated for
6 days at 37°C.
Matrigel Plug In Vivo Assay
VEGF, HGF, and Ang-1 incorporated into PLGA scaffolds were
mixed with growth factor-reduced Matrigel (BD Biosciences) and
subcutaneously implanted into nude mice. After 1 week,
fluorochrome-conjugated lectin was administered intravenously as
described previously.14
Hindlimb Ischemia Model
Unilateral hindlimb ischemia was induced as described previously.15,16
Histological Analysis of Ischemic Muscles
Capillaries were scored in frozen sections of the adductor muscles
by staining for CD31-allophycocyanin (APC) (BD Biosciences).
Conductant vessels were stained by smooth muscle actin (SMA)Cy3 or SMA–fluorescein isothiocyanate (Sigma-Aldrich). Human
ECFC were detected by human-specific APC-conjugated human
leukocyte antigen (HLA)-ABC (Biolegend).
Results
Scaffold-Derived Proangiogenic Growth Factors
Enhance Neovascularization in Matrigel Plugs
First, we produced 50 to 100 ␮m microparticles containing
VEGF, HGF, and Ang-1, which were designed to release
their incorporated proangiogenic growth factors for up to 2
weeks. Scanning electron microscopy indicated that the
microparticles had a spherical shape with smooth surface
(Figure 1A). We then tested the effectiveness of single,
double, and triple sets of scaffold-released growth factors in
vivo in Matrigel plugs. Local delivery of the single growth
factors in Matrigel plugs resulted in an increased vessel
growth, as shown by intravenous lectin injection visualizing
the vascular network (Figure 1B and 1C; Supplemental
Figure VA). All growth factor-containing scaffolds induced a
significantly (P⬍0.05) increased vessel growth compared
with growth factor-free scaffolds (blank). The efficacy of the
dual combination of scaffold-released VEGF and HGF on the
vascular network formation was superior to VEGF alone, and
the triple combination of VEGF, HGF, and Ang-1 was
superior to the double combination of VEGF and HGF
(Figure 1B and 1C). Therefore, we decided to proceed using
the dual and triple combination of scaffold-released growth
factors for our further experiments in an ischemic setting.
Scaffolds Releasing the Dual Combination of
VEGF and HGF in Combination With
Vasculogenic Progenitors Enhance
Neovascularization in Hindlimb Ischemia Models
We intramuscularly injected scaffolds releasing VEGF and
HGF in a model of unilateral hindlimb ischemia. Here, we
combined growth factor therapy with a cell therapeutic
approach using cord blood– derived ECFC (Supplemental
Figure I). Using this combinational approach, we could
demonstrate as a proof-of-concept that the scaffold-mediated
release of VEGF and HGF increased the blood flow in the
ischemic hindlimb (Figure 2A). The additional presence of
vasculogenic progenitor cells administered either intravenously or intramuscularly further enhanced perfusion. In
particular, intramuscularly injected vasculogenic progenitor
cells, which on their own exhibited only a small increase in
perfusion,6 profited most from the additional scaffolddelivered growth factors. These data could be confirmed by
measuring the microvessel density in the ischemic adductor
muscles (Figure 2B and 2C). To evaluate the migration and
subsequent homing of intravenously administered ECFC to
the ischemic site, we identified human ECFC by staining with
human-specific HLA-ABC antibodies (Figure 2D; Supplemental Figure IVB and IVC). We found significantly
(P⬍0.05) increased numbers of human HLA⫹ cells in the
ischemic muscles of mice that had received VEGF and
HGF-secreting scaffolds compared with blank scaffolds (Figure 2D and 2E). Likewise, the number of human ECFC that
had incorporated into the vasculature was also more pronounced (Figure 2D and 2F). These data imply that in vivo
tissue engineering using a combination of injectable VEGF
and HGF-releasing scaffolds and additional vasculogenic cell
therapy improves local vessel growth, homing, and incorporation of vasculogenic progenitor cells, resulting in restored
blood flow.
Scaffold-Derived Ang-1 Further Improves
Neovascularization Induced by VEGF and HGF
The effects of different factors and their combinations were
further investigated in a chick chorioallantoic membrane
assay. Blank or growth factor-containing scaffolds were
covered with filter paper and placed into a fertilized egg.
After 6 days, scaffolds containing Ang-1 alone significantly
(P⬍0.05) increased neovascularization compared with blank
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Figure 1. Scaffolds releasing single, dual, and triple
sets of proangiogenic growth factors increase the
number of vessels growing into Matrigel plugs in
vivo. A, Representative scanning electron micrographs of PLGA microparticles. B and C, Quantification (B) and representative pictures (C) of lectin⫹
vessels after local administration of growth factorcontaining scaffolds in Matrigel plugs by in vivo
injection of lectin (*P⬍0.05 versus blank; n⫽4 to 6).
scaffolds (Figure 3A and 3B). Most importantly, Ang-1
further significantly (P⬍0.05) enhanced neovascularization
when combined with VEGF and HGF.
Scaffolds Releasing Ang-1 Reduce VEGF-Mediated
Vascular Leakage
In addition to its proangiogenic action, Ang-1 is also known
to stabilize newly formed vessels.17 Using an ear tissue
leakage assay,18 we show that VEGF alone drastically decreases vascular integrity, as evidenced by enhanced extravasation of intravenously injected Evans blue, whereas Ang-1
alone showed a pronounced reduction of Evans blue leakage
(Figure 4A and 4B; Supplemental Figure VB). Interestingly,
the combination of HGF and VEGF already reduced the
leakage caused by VEGF alone. These positive effects were
further enhanced by the coadministration of Ang-1. The triple
combination of VEGF, HGF, and Ang-1 induced formation
of significantly more vessels compared with the dual combination (Figures 1B, 1C, 3A, and 3B), which are significantly
(P⬍0.05) less leaky than those induced by the dual combination of VEGF and HGF.
Scaffolds Releasing the Triple Combination of
VEGF, HGF, and Ang-1 in Combination With
Vasculogenic Progenitors Efficiently Enhance
Neovascularization in Hindlimb Ischemia Models
Finally, we set out to test whether the triple combination of
growth factors including the vessel stabilizing factor Ang-1
might further improve the effects of vasculogenic progenitor
cells. Here, we focused on intravenous ECFC injection,
because we obtained more pronounced treatment results
using this route of administration. We observed a robust
increase in perfusion using the triple growth factor combination and intravenous ECFC injection, as shown by laser
Doppler measurements (Figure 5A). In addition, we performed near-infrared in vivo imaging of indocyanine green
distribution using an IVIS-200 noninvasive optical imaging
device; this imaging further underlined the effect of the triple
growth factor combination along with ECFC compared with
blank controls (Figure 5B). The triple scaffold combination
plus ECFC reduced the presence of toe necrosis and efficiently restored the blood flow (Figure 5B). Histologically,
these effects correlated strongly with increased numbers of
SMA conductant vessels in the adductor muscles (Figure 5C
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Figure 2. Scaffolds releasing VEGF and HGF together with ECFC enhance neovascularization in hindlimb ischemia models. A, Relative
blood flow (ischemic/nonischemic hindlimb) evaluated 14 days after surgery (nⱖ4). VEGF- and HGF-releasing scaffolds were injected
intramuscularly. ECFC were delivered either intravenously or intramuscularly. B, Capillary density measured as CD31⫹ cells per myocyte in the ischemic adductor muscles. Representative pictures of the histological analysis with CD31 (red) and nuclei (blue) are shown.
C, Quantification of the capillary density. D, Left, Identification of intravenously injected human ECFC by HLA-ABC (red) and their incorporation into lectin⫹ vessels (green). Arrows denote incorporated HLA⫹lectin⫹ cells (yellow). Right, Higher magnification of incorporated
human HLA⫹lectin⫹ cells (yellow). E, Quantification of the homing of HLA⫹ cells into ischemic muscles (nⱖ8). F, Quantification of incorporated HLA⫹lectin⫹ cells per vessel (nⱖ8).
and 5D). Of note, only the triple combination of scaffoldreleased growth factors, not the dual combination, increased
numbers of SMA⫹ conductant vessels (Figure 5C). Interestingly, pericytes were not derived from infused ECFC (Sup-
plemental Figure II). These data suggest that a combination of
the 3 growth factors VEGF, HGF, and Ang-1 robustly
improved neovascularization by strongly increasing the number of vascular smooth muscle cells, including pericytes.
Saif et al
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Figure 3. Effect of scaffolds releasing Ang-1 alone or in combination with VEGF and HGF to enhance neovascularization in
chorioallantoic membrane assays. A, Scaffolds releasing the
indicated growth factors were placed into fertilized eggs covered with filter paper and assessed after 6 days. B, Quantification of the chorioallantoic membrane assay (*P⬍0.05 versus
blank; nⱖ6).
Discussion
In the present study, we show that the effects of vasculogenic
cell therapy can be additively enhanced by the release of
proangiogenic growth factors delivered by injectable
microparticle-based scaffolds in a murine model of hindlimb
ischemia. Vasculogenic cell therapy using cord blood– derived ECFC was most efficiently enhanced in the presence of
scaffolds releasing a triple growth factor combination of
VEGF, HGF, and Ang-1. Using this combination, we observed a robust increase in perfusion of the ischemic hindlimb. We assessed the functional improvements by noninvasive in vivo analyses using laser Doppler-derived blood flow
measurements and optical imaging. These in vivo measurements were corroborated by corresponding increases in homing and incorporation of intravenously administered cells,
microvessel density, and the number of SMA⫹ vascular
smooth muscle cells representing perivascular mural cells
essential for vessel stabilization. Moreover, the triple combination containing Ang-1 reduced vascular permeability.
Clinical needs for vascular regeneration in the treatment of
ischemic peripheral artery disease and myocardial infarction
are still unmet. Although the results of experimental and first
clinical studies investigating cardiovascular cell therapy were
Vascular Progenitor In Vivo Tissue Engineering
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Figure 4. Vascular leakage into ear skin in the presence of
VEGF, HGF, and Ang-1 in vivo. A, One week after local application of scaffolds releasing VEGF, HGF, Ang-1, or combinations
thereof into the dorsal ear base, Evans blue was intravenously
injected after local application of mustard oil to the right ear. In
vivo fluorescence of Evans blue was assessed using an IVIS200 noninvasive optical imaging device. Quantification of Evans
blue dye extravasated into ear skin in vivo is depicted (*P⬍0.05
versus blank; n⫽4). B, Representative images of the right ear of
mice (ratio of the retained fluorescence in the right ear versus
the left ear is given in parentheses).
promising, the overall level of the therapeutic effects clearly
indicates that additional strategies will be necessary to enhance the proangiogenic effects of cell therapy.19 This could
be achieved in a variety of ways, but combinational approaches seem to be most promising. To date, some investigators have focused on combining different cell sources to
produce more pronounced effects with respect to tissue
neovascularization.20,21 In these experimental settings, vasculogenic progenitor cells were coadministered along with cell
types replacing the function of pericytes, which are essential
for producing mature and stable vascular networks. However,
the implementation of different cell types sometimes requires
invasive procedures and renders the therapeutic approach
even more personalized, limiting its broader clinical application. Another strategy is to combine cell therapeutics with
additional growth factors. Interestingly, a very recently published study using VEGF and HGF gene therapy combined
with mesenchymal stem cells showed beneficial effects on
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Figure 5. Scaffolds releasing VEGF, HGF, and Ang-1 together with ECFC enhance neovascularization in hindlimb ischemia models. A,
Relative blood flow (ischemic/nonischemic hindlimb) evaluated 14 days after surgery (nⱖ4). Scaffolds were injected intramuscularly.
ECFC were delivered intravenously. B, Top, Disappearance of toe necrosis in mice receiving triple growth factor scaffolds and ECFC
compared with controls. Bottom, In vivo fluorescence imaging was performed after intravenous injection of indocyanine green to
assess vascular distribution. Red color indicates high perfusion, yellow low perfusion. C, Quantification of SMA⫹ conductant vessels in
the adductor muscles. D, Representative confocal images showing SMA⫹ vascular smooth muscle cells, including pericytes (red), and
nuclei (4⬘,6-diamidino-2-phenylindole [DAPI]; blue).
cardiomyocyte survival and function in myocardial infarction
models.22 However, gene therapy approaches can bear safety
risks that may limit their clinical application.
Here, we use a novel dual approach strategy involving
ECFC and multiple scaffold-released growth factors for
vascular tissue regeneration. Growth factor-releasing scaffolds providing the matrix for coadministered ECFC represent a new in vivo tissue engineering approach. Injectable
scaffolds are an emerging concept for tissue engineering
purposes and have initially been used for bone and cartilage
regeneration.23 Incorporated growth factors stimulate local
vascularization of the host tissue and support and attract
simultaneously injected stem and progenitor cells. In the past,
less well-characterized matrices, such as collagen, have been
used to support local cell injection and restoration of vascular
networks.24 However, these matrices do not provide controlled release of defined growth factors, making them less
suitable for clinical applications.
In this report, we used PLGA microparticles as scaffolds,
because they represent a defined drug delivery system that is
injectable, biodegradable, and approved by the US Food and
Drug Administration, making them attractive candidates for
clinical vascular tissue engineering. Microparticles have the
advantage that they can be specifically designed for longterm release over months. The sustained release of erythropoietin using biodegradable gelatin hydrogel microspheres
has very recently been reported to persistently improve
hindlimb ischemia.25 However, hydrogels do not offer protection during release in in vivo environments. Moreover,
PLGA microparticles allow a variety of factors to be incorporated, including hydrophobic substances that might not be
suitable for hydrogels.
In our experimental setup, the scaffold mixture was injected into the ischemic muscle, followed by vasculogenic
progenitor cell therapy. In contrast to Bible et al,8,26 we did
not attach our vasculogenic progenitors to the surface of the
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microparticles, because we administered our cell therapy by
intravenous and intramuscular routes for direct comparison of
the optimal route of delivery. Because of the relatively large
size of the microparticles ranging from 50 to 100 ␮m,
intravascular delivery may potentially lead to microembolism
and should be avoided. Moreover, as a further improvement
compared with Bible et al,8,26 we used scaffolds releasing up
to 3 growth factors.
Optimal results were obtained using injectable scaffolds
releasing a multifactorial growth factor combination that
included the 2 proangiogenic growth factors and chemoattractants VEGF and HGF, as well as Ang-1 with additional
vessel stabilizing capacities. Although VEGF is the classic
factor to induce vascularization, it has been shown to have
dichotomous roles and is a known mediator of inflammatory responses and microvascular permeability leading to
edema.12,27 Recently, inhibition of vascular smooth muscle
cells, including pericytes, has been reported as another
unexpected effect of VEGF.28 Interestingly, these data imply
that VEGF might even retard, not promote, tumor progression.29 Therefore, we decided to use additional proangiogenic
growth factors, namely Ang-1 and HGF. Vascularization is
regulated mainly by the interplay between VEGF and the
angiopoietin system, in which Ang-1 acts as an essential
factor for vessel maturation.17 Ang-1 has also been reported
to have a chemotactic effect on bone marrow– derived hematopoietic stem cells expressing the Ang-1 receptor Tie2, thereby
inducing recruitment of hematopoietic stem cells.30 Mobilized hematopoietic stem cells may then undergo Ang-1-induced
endothelial differentiation as previously reported,31,32 further
facilitating neovascularization. However, enhanced endothelial commitment seems to be restricted to undifferentiated
hematopoietic stem cells, because endothelially committed
ECFC did not respond to Ang-1 pretreatment (Supplemental
Figure III).
In addition, we used HGF, a potent endothelial mitogen
that has been shown to enhance vascularization without
inducing vascular permeability.33 Moreover, HGF has been
shown to mediate the Ang-1-induced pericyte recruitment
and seems to lack proinflammatory effects.34,35 Therefore, in
our experiments, the triple combination of scaffold-released
VEGF, HGF, and Ang-1 resulted in a robust enhancement of
vascularization that was superior to any of the administered
factors alone. The increased muscular capillary density and
the enhanced number of arterioles/arteries (conductant vessels) clearly demonstrate the increased capacity of our coadministered scaffold-released growth factors and vasculogenic
progenitor therapy to promote therapeutic neovascularization.
The increased number of SMA⫹ smooth muscle vascular
cells and pericytes in the presence of the triple scaffoldreleased growth factor combination indicated sufficient vessel stabilization and maturation.
For our studies, we used cord blood– derived ECFC, which
bear greater potential for cell-based clinical therapies compared with dysfunctional autologous patient-derived progenitors,36 provided they are transplanted in an HLA-matched
manner.37
Finally, the beneficial effect of our combinational approach
of scaffold-based growth factor release including in vivo cell
Vascular Progenitor In Vivo Tissue Engineering
1903
therapy is strongly supported by a recent report.38 In this
study, release of VEGF from an alginate matrix strongly
supported coadministered vasculogenic progenitor cells,
whereas intramuscular injection of the cells together with a
bolus injection of VEGF was only minimally effective.38
In conclusion, our results demonstrate that vasculogenic
progenitor cell therapy can be very efficiently enhanced by a
suitable controlled-release microenvironment of growth factors and chemoattractants. Therefore, we propose this
scaffold-based multifactorial growth factor approach as a
novel optimization strategy to promote neovascularization in
ischemic peripheral artery disease.
Sources of Funding
This work was supported by a Healthcare and Bioscience iNet
Higher Education Collaboration Fund (HECF) grant funded by East
Midlands Development Agency (EMDA) (to A.A.), the Fresenius
Foundation (A23), and a Europeans Research Council (ERC) Advanced Investigator Grant (to C.H.).
Disclosures
K.M.S. is affiliated with Regentec Ltd (Nottingham, UK) as Chief
Scientific Director. Aside from the aforementioned, the authors have
no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the
subject matter or materials discussed in the manuscript. This includes
employment, consultancies, honoraria, stock ownership or options,
expert testimony, grants or patents received or pending, or royalties.
In addition, this work constitutes part of the doctoral thesis of
Theresa Schwarz at the Ludwig-Maximilians-University Munich.
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Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
Combination of Injectable Multiple Growth Factor−Releasing Scaffolds and Cell Therapy
as an Advanced Modality to Enhance Tissue Neovascularization
Jaimy Saif, Theresa M. Schwarz, David Y.S. Chau, James Henstock, Paramjit Sami, Simon F.
Leicht, Patrick C. Hermann, Sonia Alcala, Francisca Mulero, Kevin M. Shakesheff, Christopher
Heeschen and Alexandra Aicher
Arterioscler Thromb Vasc Biol. 2010;30:1897-1904; originally published online August 5, 2010;
doi: 10.1161/ATVBAHA.110.207928
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
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Supplement Material
Material & Methods
Animals
Female NMRI nude mice from Janvier (Elevage Janvier, Le Genest Saint Isle, France) at 6 to
10 weeks were used. For all invasive procedures, mice were anesthetized with isoflurane or
ketamine (100 mg/kg intraperitoneally) and xylazine (10 mg/kg intraperitoneally). For postoperative analgesia carprofen (5 mg/kg subcutaneously) was administered. Animal procedures
performed for this study were all approved by the Institutional Animal Care and Use
Committee of the CNIO and the Administrative Panel on Laboratory Animal Care
(Government of Upper Bavaria, Germany).
Scaffold preparation
Microparticles containing individual growth factors were produced using a double-emulsion
technique as previously described
1, 2
. The optimised method for the production of protein-
incorporated 50-100 µm-sized particles is described as follows. PLGA copolymer (1 g of
50:50 D,L-lactide:glycolide; DLG 2A (2-week release polymer), Lakeshore Biomaterials,
Alabama, USA, per growth factor) was dissolved in 5 ml dichloromethane (DCM) in a glass
vial at 37°C for 2 hours. Growth factors (VEGF, HGF and angiopoietin-1; 1 mg / each) were
obtained from Peprotech and separately dissolved in 100 µl PBS + 1% heat-inactivated foetal
calf serum (FCS). Growth factors in solution were added to polymer-DCM solution and
vortexed for 1 minute to create a primary emulsion. Blank particles were produced by adding
100 µl PBS + 1% heat-inactivated FCS to polymer solution. The microparticles were then
formed by the addition of 7 ml 0.3% (w/v) polyvinyl alcohol (PVA) in dH2O carefully to the
surface of the polymer/DCM solution and vortexed immediately for 2 minutes to produce the
double emulsion of the polymer/DCM/cytokine in the aqueous PVA solution. This emulsion
was immediately poured into a hardening bath comprising 200 ml 0.3% PVA/ dH2O in a glass
beaker which was magnetically stirred at ~100 RPM. The particle hardening/solvent
evaporation was performed in a fume hood overnight. The particles were recovered by
vacuum filtration using Whatman #1 filter paper and were washed with 2 x 200 ml dH2O.
Once filtered, the particles were removed to glass vials and freeze-dried for 48 hours,
separated and sieved using a Retsch AS200 sieve shaker (amplitude: 1.40, 40 s interval time
and 10 min total running time) and then weighed and stored appropriately. Mean particle
diameters and surface morphology were examined by scanning electron microscopy (SEM).
Particles were stored at 4°C and pre-treated with antibiotic / antimycotic solution (Sigma)
prior to use. The incorporation efficiency of the microparticles was determined using
QuantiPro BCA assay kit (Sigma) according to the manufacturer’s instructions. Lysozyme
(chicken egg white, 1 mg; Sigma) as a model protein test substance was incorporated into the
microparticles and the incorporation efficiency was found to be 28.97 ± 2.66 %. Per mouse, 8
mg of each type of scaffold releasing a total of 2.5 µg of the growth factors, respectively,
were intramuscularly injected.
Isolation of human cord blood-derived vasculogenic progenitor cells
After informed consent, full term deliveries served as sources for umbilical cord blood
(Department of Obstetrics, University of Munich). Briefly, mononuclear cells were cultured
with endothelial basal medium-2 (Lonza, Basel, Switzerland) with 10% FCS and EGM-2
SingleQuots in 6-well tissue culture plates pre-coated with type 1 rat tail collagen (BD
Biosciences). After 24 hours, non-adherent cells were discarded. Medium was changed daily
for 7 days and then every other day until colonies were arising. Cells from colonies were
picked, expanded in T75 flasks, and used for further experiments till passage 4.
Chicken chorioallantoic membrane (CAM) assay
CAM assays were performed on 4 days old eggs (stored horizontally in incubator at 37°C).
The exact position of the embryo and blood vessels was located and 5-6 ml of albumin was
withdrawn using a 19 G blunt needle and 10 ml syringe. Using a sterile saw, a small window
of approx. 1 by 1.5 cm size was cut and the shell window slowly peeled off using tweezers.
The blood vessels and the tiny pulsating embryo are then visible through the window. The
scaffold (8 mg) with or without growth factors was placed near a blood vessel away from the
embryo and covered by a 0.5 x 0.5 cm2 filter paper. The window was sealed with clear tape
and the egg horizontally incubated for 6 days at 37°C.
Matrigel plug in vivo assay
VEGF, HGF, and Ang-1 incorporated into PLGA scaffolds (8 mg of scaffold / each) were
mixed with 0.2 ml of ‘growth factor-reduced’ Matrigel (BD Biosciences) and hardened in a
mold at 37 °C for 30 min. After skin incision, Matrigel plugs were implanted subcutaneously
into nude mice in vivo. After one week, 100 µl FITC-conjugated griffonia simplicifolia lectin
I (isolectin B4), Texas Red-labeled tomato lectin (both from Vector Laboratories, Burlingame,
CA, USA; 1 mg/ml), or Alexa Fluor 647-labeled griffonia simplicifolia lectin (Invitrogen)
were intravenously injected 30 min prior to sacrifice as described previously 3. Matrigel plugs
were then excised, embedded in OCT Compound tissue freezing medium, and blood vessel
infiltration of the Matrigel plugs was assessed on cryosections counterstained for DAPI. In
some cases, cyrosections were additionally stained for lectin (1:100 in 10 mM HEPES buffer
+ 0.15 M NaCl + 0.1 mM Ca2+ (CaCl2) for 60 min at 37 °C) to improve the intensity of the
signal. The vascularized area (µm2) was densitometrically analyzed using the volocity
software (Improvision, Coventry, United Kingdom).
Vascular leakage assay
One week after local application of scaffolds releasing VEGF, HGF, Ang-1 or combinations
thereof into the dorsal ear base, Evans blue (Reactifs RAL, Martillac, France; 30 mg/kg; 50 µl)
was intravenously injected 15 min after local application of 5 % mustard oil (Pestanal™,
Sigma-Aldrich, Dorset, UK; diluted in peanut oil) to the right ear. In vivo fluorescence of
Evans blue was assessed using an IVIS-200 non-invasive optical imaging device. The ratio of
the retained fluorescence in the right ear versus the left ear was determined.
Hind limb ischemia model
To induce unilateral hind limb ischemia, the proximal portion of the right femoral artery and
vein including the superficial and the deep branch were occluded using an electrical
coagulator as described previously
4, 5
. The overlying skin was closed using surgical staples.
After 24 hours, scaffolds were intramuscularly injected into the ischemic adductor muscles.
Two hours later, 0.5 x 106 ECFCs were either intramuscularly or intravenously delivered.
Limb perfusion was assessed with an O2C laser Doppler flowmetry probe (LEA
Medizintechnik, Giessen, Germany) after two weeks. Calculated perfusion is expressed as the
ratio of ischemic to non-ischemic hind limb perfusion. Additionally, we performed intravital
near infrared (NIR) fluorescence imaging after intravenously injecting 100 µl of indocyanine
green (ICG; Sigma-Aldrich, Dorset, United Kingdom: 400 µM), a NIR fluorophore 6. Images
were obtained using an In Vivo Imaging System (IVIS)-200 (Caliper Life Sciences,
Hopkinton, MA, USA) and analyzed using the Living ImageTM 3.2 software.
Histological analysis of ischemic muscles
Mice were sacrificed for histological analysis two weeks after induction of hindlimb ischemia.
Capillaries were scored in 50 µm frozen sections of the adductor muscles by staining for
CD31 (APC-labeled; BD Biosciences; 1:100). Conductant vessels were stained by SMA-Cy3
or SMA-FITC (Sigma-Aldrich; 1:300). Moreover, human ECFC were detected by human
APC-conjugated HLA-ABC (Biolegend, Uithoorn, The Netherlands; 1:100). For all stainings,
1 % milk powder in PBS was used for blocking (60 min at room temperature). For most
stainings, sections were incubated with the antibodies for 2 hours, apart from staining
involving CD31, which was incubated overnight. DAPI (4'-6-Diamidino-2-phenylindole) was
used to counterstain nuclei. Images were obtained by confocal microscopy (Leica TCS-SP2)
using Leica Confocal Software.
Flow cytometry analysis
ECFC were trypsinized, blocked using Fc block (Miltenyi, Bergisch Gladbach, Germany),
and stained with CD34-APC (BD Biosciences, Erembodegem, Belgium; 1:10), CD45-FITC
(BD Biosciences; 1:10), c-met (HGF receptor; 1:10)-FITC (R&D Abingdon, United
Kingdom), tie-2-APC (R&D; 1:10), or biotinylated VEGF receptor 2 (Reliatech, Manchester,
United Kingdom; 1:10) followed by streptavidin-APC (BD Biosciences; 1:50) and compared
to their corresponding isotype controls. All antibodies were applied for 30 min at 4°C. Cells
were washed twice with PBS and analyzed on a FACS Calibur (BD Biosciences).
Corresponding isotype controls were used to set the gates.
Statistical Analysis
Results are expressed as mean ± SEM. Overall comparison of the treatment groups was
performed with the Kruskal-Wallis test followed by post-hoc pairwise comparison using the
Mann-Whitney test. P values < 0.05 were considered statistically significant. Analyses were
performed with SPSS 15.0 (SPSS Inc.).
Results
Expression of surface markers on cord blood-derived ECFC
The flow cytometry profile of the utilized ECFC showed strong expression of the endothelial
marker CD31 and the progenitor marker CD34 without noticeable expression of the
hematopoietic pan-leukocyte marker CD45, confirming their endothelial progenitor
phenotype. In addition, VEGF receptor 2, the major receptor mediating the pro-angiogenic
effects of VEGF, and tie-2, the receptor for Ang-1, were strongly expressed, whereas c-met,
the receptor for HGF, was expressed at a lower level.
Figure I. Expression of surface markers on cord blood-derived ECFC as assessed by flow
cytometry (n ≥ 3).
ECFC do not differentiate into SMA+ cells
Newly formed vessels after injecting ECFC into nude mice that underwent unilateral hindlimb
ischemia are chimeric vessels composed of human ECFC (HLA in green) and smooth muscle
cells / pericytes (SMA in red) derived from the murine host, because we did not observe any
co-staining of HLA and SMA.
HLA
SMA
DAPI
HLA
SMA
DAPI
50 µm
HLA
SMA
DAPI
50 µm
50 µm
Figure II. Expression of HLA (green) indicative for human ECFC and SMA (red)
representing smooth muscle cells and pericytes in ischemic hindlimbs.
Ang-1 does not further enhance endothelial commitment of ECFC
To test the effect of Ang-1 pre-treatment on the endothelial commitment of ECFC, we
examined the expression of the progenitor / endothelial marker CD34 in the presence of Ang1 (Figure III. A). However, ECFC are already strongly committed to the endothelial lineage
(Figure I). We performed cytometry analyses for the expression of CD34 in ECFC but did
not detect any changes in the expression of Ang-1. In addition, we ran in vitro Matrigel
TM
assays to induce tube formation using ECFC in the presence of Ang-1 (Figure III B). On
MatrigelTM, ECFC further differentiate and form capillary-like structures. However, Ang-1
did not affect the tube formation capacity of ECFC quantified as cumulative branch length.
Figure III. Effect of Ang-1 on endothelial commitment in ECFC in vitro. (A) Expression of
CD34 on ECFC in the presence of 500 ng/ml or 1000 ng/ml Ang-1 for 48 h. (B) Cumulative
length of branches produced by ECFC (500,000 ECFC per 24-well) in the presence or
absence of 500 ng/ml or 1000 ng/ml Ang-1 for 48 h (n=5).
Ang-1 enhances migration but not proliferation in ECFC
BrdU incorporation was measured to assess proliferation in ECFC (Figure IV.A). As shown
in the representative pictures, Ang-1 stimulation did not induce proliferation, while
stimulation with basic fibroblast growth factor (bFGF) used as a positive control clearly
augmented proliferation. Our results are consistent with a study of Davis et al. 7 that could not
elicit proliferative responses on mature human endothelial cells in the presence of Ang-1.
However, opposite results on Ang-1-induced proliferation on mature endothelial cells have
been reported
8, 9
. Moreover, we investigated the invasive migratory capacity of ECFC
through Matrigel-coated Boyden chamber inserts towards Ang-1 used as chemoattractant in
the lower well. Here, we demonstrate that Ang-1 induced migration of ECFC (Figure IV. B).
These data are in line with previous results about Ang-1-induced migration in mature
endothelial cells 8-10.
Figure IV. Effect of Ang-1 on proliferation and migration of ECFC. (A) Percentage of cells
incorporating BrdU (purple) in the presence of Ang-1 or bFGF (used as positive control).
Cells were starved for 3 hours in DMEM + 2 % FCS, then EBM-2 (without growth factors) +
10 % FCS with or without Ang-1 or bFGF was added for 48 h. Representative flow cytometry
pictures are shown.
(B) Representative pictures of migrating ECFC (given in orange) through 8 µm pores
covered with Matrigel (BD Matrigel invasion chamber, BD Biosciences) are shown. ECFC
were starved for 3 hours in DMEM + 2 % FCS (starving medium), then 500,000 ECFC were
seeded in starving medium into the Boyden chamber inserts and allowed to migrate for 12
hours towards DMEM + 10 % FCS with or without Ang-1 as chemoattractant. Cells were
then fixed and stained with DAPI. Color-coded z-stack projections were acquired on a Leica
TCS SP5 confocal microscope to assess the number of cells migrating through the membrane.
Color coding by the Leica Confocal Software assigned an orange color to the most distant
cells that migrated most, whereas less distant cells were depicted in blue or green. (C)
Quantification of the number of migrated cells / mm2 is given (n=8).
Ang-1 enhanced vessel growth and reduced vascular leakage in MatrigelTM plugs
containing ECFC and MSC
The co-implantation of endothelial progenitor cells together with MSC serving as perivascular
cells has been described to produce stable vascular networks in MatrigelTM plugs
11
. We
examined whether this dual cell combination could also profit from scaffolds releasing Ang-1.
Indeed, scaffold-released Ang-1 improved vascularization and reduced vessel permeability
(Figure V. A and B).
Figure V. Effect of scaffold-released Ang-1 on vessel growth and stability in MatrigelTM
plugs containing ECFC and MSC. (A) Vascularization was assessed by lectin staining. The
vascularized area per total area was analyzed by Image J software (n=9). (B) Assessment of
vascular permeability measured by Evans blue extravasation in the presence of Ang-1. Four
weeks after intradermal application of MatrigelTM containing human ECFC and MSC (1 x 106 :
0.25 x 106) in the presence of scaffolds releasing Ang-1 or no growth factors (blank), Evans
blue (30 mg/kg; 50 µl) was intravenously injected, and in vivo fluorescence of Evans blue was
assessed. Quantification of the extravasation of Evans blue dye into the intradermally
administered MatrigelTM is given. In vivo fluorescence over the intradermally injected
Matrigel plug is determined using an IVIS-200 device (n=3).
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