Biomimetic Hydrogels with VEGF Induce Angiogenic Processes in

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Biomacromolecules 2011, 12, 242–246

Biomimetic Hydrogels with VEGF Induce Angiogenic Processes in Both hUVEC and hMEC Alex M. Porter,† Carolyn M. Klinge,‡ and Andrea S. Gobin*,†,§ Physiology and Biophysics, Biochemistry and Molecular Biology, and Bioengineering, University of Louisville, Louisville, Kentucky 40292, United States Received October 14, 2010; Revised Manuscript Received November 15, 2010

Angiogenesis is the process by which new blood vessels arise from the pre-existing vasculature. Human endothelial cells are known to be involved in three key cellular processes during angiogenesis: increased cell proliferation, degradation of the extracellular matrix during cell migration, and the survival of apoptosis. The above processes depend upon the presence of growth factors, such as vascular endothelial growth factor isoform 165 (VEGF165) that is released from the extracellular matrix as it is being degraded or secreted from activated endothelial cells. Thus, the goal of the current study is to develop a system with a backbone of polyethylene glycol (PEG) and grafted angiogenic signals to compare the initial angiogenic response of human umbilical vein endothelial cells (hUVEC) or human microvascular endothelial cells (hMEC). Adhesion ligands (PEG-RGDS) for cell attachment and PEG-modified VEGF165 (PEG-VEGF165) are grafted into the hydrogels to encourage the angiogenic response. Our data suggest that our biomimetic system is equally effective in stimulating proliferation, migration, and survival of apoptosis in hMEC as compared to the response to hUVEC.

Introduction Current research in wound healing and tissue engineering has recently placed emphasis on understanding in vitro angiogenic processes involving endothelial cells in biomimetic materials, such as polyethylene glycol (PEG)-based hydrogels, Matrigels, and hyaluronic acid-based hydrogels.1-4 For these biomimetic systems to be successful, they need to recapitulate some of the elaborate biological recognition and signaling functions of the extracellular milieu. In each of these systems, various aspects of the system have been manipulated to mimic the natural environment, such as growth factor presentation, degradability, or adhesivity. However, all systems have only been assessed with only one endothelial cell type, more often the umbilical vein endothelial cell. It has been shown in previous works that the endothelial cell type can have various angiogenic responses.5-7 Specifically, Cavallaro et al. observed that microvascular cells reorganized actin microfilaments, decreased thrombospondin1, and induced urokinase-type plasminogen activator (uPA) in response to FGF, while aortic cells had little or no response to FGF or VEGF.3 Angiogenesis involves a myriad of cytokines.8,9 Many of the well-studied cytokines, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and angiopoietin-1, act on the endothelial cells during different times in the process of angiogenesis.2,10-12 For instance, VEGF165 (isoform 165) has been shown to be involved in the initiation of angiogenesis, causing vasodilation and subsequent leaking of capillaries, extracellular matrix degradation, and cell migration.12,13 After migrating into the newly degraded ECM, the endothelial cells engage in cell-cell junction formation, thus, creating new lumens. Increases in cell proliferation are also observed, allowing for extension of the budding lumens to further invade * To whom correspondence should be addressed. E-mail: andrea.gobin@ louisville.edu. † Physiology and Biophysics. ‡ Biochemistry and Molecular Biology. § Bioengineering.

the ECM and eventually form a functioning vessel network.14,15 The cells avoid oxidation-induced apoptosis through cellular signaling processes that are inhibited by VEGF receptor signaling.16,17 These signaling processes have been shown to be vital in wound healing and tumor formation.18-20 PEG-based hydrogels are an effective system for studying angiogenesis: PEG hydrogels are biocompatible with the vascular cells21-25 and are resistant to protein adsorption, hence, allowing the ability to tailor in specific biochemical compounds to elicit specific cellular responses.6,22,26 Our system makes use of the 6000 Da PEG diacrylate (PEGDA6K), which is photopolymerized to form a sustaining network of polymer. Adhesion peptide sequences, such as RGDS derived from fibronectin, and proangiogenic cytokines, such as VEGF165, are covalently incorporated into the system via PEG linker chains. Grafting in such biochemical cues enables us to assess bioactivity of the signals without the reduction in response due to equilibrium diffusion or steric hindrance.22 Recently, it has been demonstrated that the covalently incorporated VEGF in PEG hydrogels can increase cell-cell junction and direct migration of human UVEC (hUVEC) on and in PEG hydrogels.6 However, the response of all endothelial cells was not examined to determine if similar responses would be seen with endothelial cells actually involved in angiogenesis. Our present studies have focused on hUVEC and human microvascular endothelial cells (hMEC). We demonstrate the similarities and differences between each of these cell types in their response to VEGF grafted into a PEG biomimetic system.

Materials and Methods Chemical Supplies. All chemicals are supplied from Fisher Scientific (Pittsburgh, PA) unless otherwise stated. Formulation of Hydrogel Components. Biomimetic hydrogels are comprised of PEG diacrylate (PEGDA), acryloyl-PEG-VEGF165, and acryloyl-PEG-RGDS adhesion ligand. The PEGDA was synthesized by combining dry PEG (6000 Da), 0.4 mmol/mL acryloyl chloride, and 0.2 mmol/mL triethyl amine in anhydrous dichloromethane

10.1021/bm101220b  2011 American Chemical Society Published on Web 12/03/2010

Hydrogels with VEGF Induce Angiogenic Processes overnight under argon. The PEGDA was then purified via phase separation using 2 M K2CO3. The organic phase containing the PEGDA was dried with MgSO4 and filtered. PEGDA was then precipitated with diethyl ether, filtered, and vacuum-dried overnight. The biochemical components of the hydrogel were synthesized by combining VEGF165 (Promokine; Heidelberg, Germany) or RGDS peptide with acrylate -PEG-N-hydroxysuccinimide (PEG-NHS; molecular weight 3400 Da; Laysan-Bio; Arab, AL) at a 5:1 or 1:1 molar ratio, respectively, at room temperature for 2 h. After the reaction was complete, the components were dialyzed (MWCO 3400 Da) overnight at room temperature to remove unreacted reactants. The components were then frozen and lyophilized. Hydrogel Formation. Hydrogels were prepared by a photopolymerization reaction. To form the hydrogels, PEGDA (100 mg/mL) and PEG-RGDS (3.7 µM/mL) was dissolved in HEPES buffer (pH 7.4; 10 mM). To assess the effects of PEG-VEGF, 50 ng/mL PEG-VEGF is also added to the solution. The prepolymer solution is then filter sterilized through a 0.22 µm pore syringe filter. A total of 10 µL of acetophenone photoinitiator (600 mg/mL in N-vinyl pyrollidinone) was then added per mL of hydrogel solution to initiate polymerization. Prepolymer solution is then placed into an appropriate mold and exposed to UV light (365 nm, 10 mW/cm2) for 1 min, which converts it into a solid. Cell Maintenance. Human umbilical vein endothelial cells (hUVEC) and human microvascular endothelial cells (hMEC) were purchased from Cascade Biologics (Portland, OR) and cultured in endothelial cell growth medium 200 and 131, respectively. Endothelial growth medium (EGM) contained a cocktail of penicillin (10000 IU/mL), streptomycin (10000 µg/mL), and glutamine (29.2 mg/mL) and was supplemented with 2% v/v fetal bovine serum (FBS), hydrocortisone (1 µg/mL), human epidermal growth factor (10 ng/mL), basic fibroblast growth factor (3 ng/mL), and heparin (10 µg/mL). Endothelial cells were cultured at 37 °C, 5% CO2 incubator. Cells from passages 2-6 were used in reported studies. Proliferative Response of Endothelial Cells to Hydrogels with Grafted VEGF. Hydrogel solution (180 µL/well) was pipetted into the wells of sterile 48-well plates and was polymerized. Hydrogels were then allowed to swell in cell medium for 1 h. hUVEC and hMEC cell suspensions were then added to their respective wells at an initial confluence of 25% (10000 cells/cm2) and 10% (4000 cells/cm2), respectively. The endothelial cells were placed in an incubator for 4 h to ensure adequate cell adhesion. After which, the EGM was removed, and replaced with supplement-free medium with 2% FBS and antibiotics. Supplement-free medium was used as basal experimental medium because of the fact the supplement additive contains bFGF at 1 ng/ mL, which may skew the data. Experimental groups were exposed to 50 ng/mL VEGF165 suspended in the medium (VEGF in Media), 50 ng/mL PEGylated VEGF165 suspended in the medium (PEG-VEGF in Media), or 50 ng/mL PEGylated VEGF165, which was grafted within the hydrogel matrix (VEGF in Hydrogel), and control groups contained no VEGF (VEGF-free). Studies were conducted in triplicate. Cells were then incubated for 24 or 72 h, after which, they were trypsinized and counted via hemocytometer. Migration of Endothelial Cells on Hydrogels with Grafted VEGF. Hydrogels with RGDS and with or without VEGF were prepared in glass molds with the dimensions 20 × 70 × 0.5 mm and then fitted into two-chambered compartment slides (Lab-Tek; Rochester, NY). Polydimethylsiloxane (PDMS) fences were placed into the chambers atop each hydrogel, approximately 1/3 the length down of the hydrogels, and cells were seeded to near confluence behind the fence. Cells were allowed to attach for 4 h and cell medium was changed to medium containing 2% FBS with 0.5 µg/mL mitomycin C to study migration without the influence of proliferation. Mitomycin is a carcinostatic agent, which inhibits DNA synthesis by blocking the cell cycle at the G2 phase. The same experimental groups as described above were used for the migration studies. Grids (20 × 20 µm) were aligned on the underside of the chamber slide with the PDMS fences.

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At t ) 0, the PDMS fences were removed and the cells were allowed to migrate randomly for 48 h in a 37 °C incubator. After which the cells were fixed with 4% formaldehyde and their nuclei stained with DAPI. Images were taken with a CCD camera (Q Imaging, 32-0129A452, Surrey, British Columbia) on a Nikon epifluorescent microscope (TE2000-U, Melville, NY; excitation: 358 nm, emission: 461 nm) at various locations as identified by the underlying grid. Data obtained was used to determine the average distance the cells migrated for each group. Endothelial Cell Apoptosis Survival on Hydrogels with Grafted VEGF. Cells were seeded on hydrogels in 48-well plates to near confluence and allowed to attach for ∼4 h. For assessment of intrinsic apoptosis survival, cells were serum-starved for 24 h in a 37 °C incubator and then rescued from apoptosis via VEGF (50 ng/mL) for a subsequent 24 h incubation at 37 °C. Positive control groups, which were cells that had apoptosis induced but not rescued, were fixed with 4% formaldehyde after the 24 h starvation and stored in PBS thereafter. For assessment of extrinsic apoptosis, cells were exposed to 50 ng/mL TNF-R related apoptosis inducing ligand (TRAIL; Biosource; Camarillo, CA) in 2% FBS only containing medium for 6 h at 37 °C. After exposure to TRAIL, cells were then rescued with VEGF either in the media (VEGF in media; 50 ng/mL) or grafted in the hydrogels (VEGF in hydrogel; 50 ng/mL) for a subsequent 24 h incubation. In either intrinsic or extrinsic apoptosis induction, 24 h after VEGF rescue, endothelial cells were fixed with 4% formaldehyde and assessed with the DeadEnd fluorometric TUNEL system (Promega; Madison, WI). To assess apoptosis survival in each of the groups, the system measures nuclear DNA fragmentation, an important biochemical hallmark of apoptosis in many cell types. Results are described as percentage compared to cells not rescued by VEGF. The number of cells counted per image was found to be not statistically different; hence, variation was not introduced in the data due to probable decreased cell adhesion. Statistical Analysis. Results are represented as averages with standard deviations. All studies were performed in at least triplicate and statistically analyzed via Kruskal-Wallis ANOVA with Tukey/ Dunn post hoc analysis. Results with p < 0.05 were considered significantly different.

Results Endothelial Cell Proliferation on Biomimetic Hydrogels. hUVEC and hMEC have different proliferative responses. Hence, both cell types are assessed from a confluency which VEGF treatments in preliminary studies responded most robustly (data not shown). Cell counts for groups exposed to VEGF in the cell medium or grafted within the hydrogel were compared to counts for a VEGF-free group. The proliferation response of hUVECs on the biomimetic hydrogels is seen in Figure 1A. Though the initial cell adhesion to the hydrogel surface is lower than the seeding density, it was observed that this was not statistically different from hUVEC adhesion to tissue culture polystyrene. The endothelial cells exposed to VEGF in the medium or grafted within the hydrogel had significant percent increase in growth as compared to the VEGF-free control group at 24 h (VEGF in media ) 48.85% ( 4.28, VEGF in hydrogel ) 49.60% ( 5.13). This increase was maintained at 72 h. hMEC reacted to VEGF stimulation when presented in the hydrogel system in a similar manner as hUVEC (VEGF in media ) 42.47% ( 4.30, VEGF in hydrogel ) 42.06% ( 2.63; Figure 1B). These results suggest that the modification and covalent incorporation of VEGF into the hydrogel do not affect its bioactivity in regard to cell proliferation. However, hUVEC showed a slight increase in sensitivity to VEGF as compared to hMEC at the same time points and VEGF concentration.

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Figure 1. Endothelial cell mitogenic response on biomimetic hydrogels: Cells were treated with VEGF (50 ng/mL) for either 24 or 72 h, at which times cells were counted. (A) hUVECs on hydrogels. (B) hMECs on hydrogels. Legend definitions: VEGF Free, no VEGF present; VEGF in Media, VEGF added to the media; VEGF in Hydrogel, VEGF is grafted into the hydrogel; *p < 0.0001, #p < 0.001.

Figure 2. Endothelial cell mitogenic response on biomimetic hydrogels: Endothelial cells were treated with 50 ng/mL VEGF either in the media or grafted within the hydrogel for 48 h, after which they were imaged and quantified: VEGF-Free, no VEGF present; VEGF in Media, VEGF was added at 50 ng/mL to the medium; VEGF in Hydrogel, 50 ng/mL VEGF was covalently grafted into the hydrogel; *p < 0.0001; $p < 0.05.

Endothelial Cell Migration on Biomimetic Hydrogels. hUVEC migration on hydrogels was analyzed by comparing zero time point images to images at 48 h. Figure 2 shows hUVEC average distance transversed. Groups exposed to VEGF in the medium migrated an average 22 ( 10 µm, which were not statistically different from the overall migrated distance of cells seeded on hydrogels with grafted VEGF (29 ( 16 µm). Neither the modification of VEGF or grafting into the hydrogels reduced the motogenic activity of the VEGF, however, they did show a significant increase (p ) 0.008) when compared to the average migrated distance of cells not exposed to VEGF (11 ( 9 µm). Figure 2 also shows hMEC migration on engineered hydrogels. Average migration for the grafted VEGF group was not statistically different (27 ( 14 µm) from the average migration distance of the VEGF group (28 ( 16 µm). Each of the average migration distances of the VEGF-stimulated groups were significantly increased over when VEGF was not present (p ) 0.0014). The data suggest the covalent incorporation of VEGF into the hydrogel system does not hinder its ability to bind the VEGF receptor and initiate endothelial cell migration.

Endothelial Cell Apoptosis Survival. Figure 3 illustrates hUVEC extrinsic apoptosis survival after VEGF administration. After the 6-h incubation with 50 ng/mL TRAIL in the experimental media, the cells were rescued with 50 ng/mL VEGF either grafted in the hydrogels or suspended in the media. Figure 3 also illustrates hUVEC intrinsic apoptosis survival after VEGF administration. After a 24-h serum starvation, VEGF was administered at 50 ng/mL for a subsequent 24-h incubation to induce cell rescue. In both cases, the data shows VEGF was able to successfully rescue the cells to 100% survival after apoptosis was allowed to ensue. The survival data for the rescued groups were significantly improved over the apoptosisinduced groups, both extrinsic and intrinsic, which were not rescued (p < 0.0001). There was no statistical difference between the data of the VEGF-rescued groups and the control group in which no apoptosis was induced. hMEC were also exposed to apoptotic inducing conditions. hMEC were subjected to 50 ng/mL TRAIL for a 6 h incubation, with a subsequent 50 ng/mL VEGF rescue incubation for 24 h for extrinsic induction. For intrinsic induction, hMEC were serum-starved for 24 h, after which time VEGF was adminis-

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Figure 3. Endothelial cell apoptosis survival on biomimetic hydrogels: Extrinsic apoptosis was induced via 50 ng/mL TRIAL for 6 h, followed by 24 h VEGF rescue. Intrinsic apoptosis was induced via serum deprivation for 24 h, followed by 24 h VEGF rescue: VEGF-Free, no VEGF present; VEGF in Media, VEGF was added at 50 ng/mL to the medium; VEGF in Hydrogel, means 50 ng/mL VEGF was covalently grafted into the hydrogel; *p < 0.001.

tered for a subsequent 24 h incubation to induce cell survival. The data showed that the VEGF rescue groups successfully accomplished survival from apoptotic stimuli as compared to the apoptosis-induced group without VEGF rescue (Figure 3). Though the rescue for the grafted VEGF group only reached 95% survival, this was not significantly different as compared to the survival attained by the media-suspended VEGF group (both unmodified and PEG-VEGF, 92 and 100%, respectively). The data shows the hydrogel-grafted VEGF at 50 ng/mL was equally effective in initiating the cell survival cascade as compared to the noninduced group. The results were significantly increased (p < 0.0001) as compared to the apoptosisinduced group without VEGF rescue.

Discussion The goal of the present study is to develop a biomimetic system capable of encouraging angiogenesis in endothelial cells in vitro. To accomplish this, we have identified VEGF as an important growth factor in the angiogenic process, and chosen it to be one of the key components to modulate within our model. We first covalently modified VEGF with a PEG linker chain and then tethered the modified VEGF molecules into the hydrogel network via photopolymerization at concentrations that have been shown to be effective in eliciting stimulatory responses in endothelial cells. Others have reported similar studies,27-29 though it differs in that we have chosen to incorporate studies instrumental to proving each of the key biological processes instrumental to fully understand angiogenesis: (1) endothelial cell proliferation; (2) endothelial cell migration; and (3) survival of apoptosis by endothelial cells.18,30,31 Previously reported hydrogel systems have addressed the first two processes,6,32 but no group to date has reported the effects that covalently modifying and tethering VEGF has on all three of the angiogenic processes with one system. In addition, the work presented highlights difference in the source cells examined and how these differences can be detrimental in tissueengineered designs.

It is shown that the covalent modification of VEGF and subsequent tethering of the resulting moiety into the PEG hydrogel does not alter its ability to interact with the extracellular domain of the VEGF receptor on the membrane of endothelial cells. This is first demonstrated by comparison of modified VEGF to VEGF alone in media. None of the essential cellular processes involved in angiogenesis, proliferation, migration, or apoptosis survival were significantly reduced. For that reason, we have demonstrated that adding PEG chains to the growth factor does not affect binding to the cell surface VEGF receptor or reduce its bioactivity. Additionally, when grafted into the hydrogel, activity was still comparable to that as when added directly to the media. This indicates that grafting the growth factor into the hydrogel does not produce steric hindrance, reducing the ability of the factor to interact with the cell receptor. Studies were conducted in parallel between hUVEC and hMEC cells. Very little literature is present on development of angiogenic models with capillary source cells, whereas there is an abundance of information with hUVEC. Moreover, comparisons of the two cell sources are limited. It has been shown by Cavallaro et. al that microvascular cells reorganized actin microfilaments, decreased thrombospondin-1, and induced urokinase-type plasminogen activator (uPA) in response to FGF, while aortic cells had little or no response to FGF or VEGF.3 We found that the potency of VEGF on hMEC are reduced to that seen on hUVEC. Though VEGF elicits a strong growth response of both cell types, hMECs do not grow as effectively as the hUVECs. This is not only important in the angiogenic process, but also in the expansion of the cells for tissue engineering applications. In addition, similar responses are seen when investigating the migratory responses. Both cell types show a significant increase in random migration when exposed to VEGF either in the media or in the hydrogel. However, the hUVECs showed a slight increase in distance traveled on hydrogels with grafted VEGF than those where the VEGF was in the media. This increase is not due to the proliferative response of the cells, as in these studies proliferation was

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inhibited. Hence, the hUVEC are capable of migrating further when the microenvironment presents the growth factor. No differences were seen with the hMEC when comparing between VEGF in the media or in the hydrogel. Lastly, VEGF has abilities to rescue cells from apoptosis. hUVEC being a little more robust are capable to 100% survival, whereas hMEC could only reach just above 90%. Though there is a small difference between the cell types, there is no significant difference. Biomimetic hydrogels containing VEGF are able to instruct endothelial cells behavior adherent to the surface, encouraging proliferation, enhancing migration, and maintaining viability. However, the response seen can vary dependent on the source of the endothelial cells.

Conclusion This study on the ability of PEGDA-based hydrogels provides new insights into the uses of biomimetic systems involved in tissue engineering. By providing results that clearly display the ability of hydrogels to study all of the processes of angiogenesis, we propose that our observations are indicative of the potential of hydrogel-based systems to study complex biological processes in vitro. Future studies are to include degradative components to the system and assess cellular behavior in three dimensions to more realistically mimic the native microenvironment. Acknowledgment. We thank the American Heart Association (0635023N) and the National Science Foundation (0814194) for the support of this work. In addition, we are very thankful for the hard work and support Jeremy Phillips and Dr. Youling Yuan provided in the duration of the studies.

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