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. 2012 Nov 20;21(2):445–455. doi: 10.1038/mt.2012.234

Adipose-derived Stromal Cells Overexpressing Vascular Endothelial Growth Factor Accelerate Mouse Excisional Wound Healing

Allison Nauta 1,2, Catharina Seidel 1,3, Lorenzo Deveza 4,5, Daniel Montoro 1, Monica Grova 1,6, Sae Hee Ko 1,7, Jeong Hyun 1, Geoffrey C Gurtner 1, Michael T Longaker 1,*, Fan Yang 4,5,*
PMCID: PMC3594010  PMID: 23164936

Abstract

Angiogenesis is essential to wound repair, and vascular endothelial growth factor (VEGF) is a potent factor to stimulate angiogenesis. Here, we examine the potential of VEGF-overexpressing adipose-derived stromal cells (ASCs) for accelerating wound healing using nonviral, biodegradable polymeric vectors. Mouse ASCs were transfected with DNA plasmid encoding VEGF or green fluorescent protein (GFP) using biodegradable poly (β-amino) esters (PBAE). Cells transfected using Lipofectamine 2000, a commercially available transfection reagent, were included as controls. ASCs transfected using PBAEs showed enhanced transfection efficiency and 12–15-fold higher VEGF production compared with cells transfected using Lipofectamine 2000 (*P < 0.05). When transplanted into a mouse wild-type excisional wound model, VEGF-overexpressing ASCs led to significantly accelerated wound healing, with full wound closure observed at 8 days compared to 10–12 days in groups treated with ASCs alone or saline control (*P < 0.05). Histology and polarized microscopy showed increased collagen deposition and more mature collagen fibers in the dermis of wound beds treated using PBAE/VEGF-modified ASCs than ASCs alone. Our results demonstrate the efficacy of using nonviral-engineered ASCs to accelerate wound healing, which may provide an alternative therapy for treating many diseases in which wound healing is impaired.

Introduction

Wound healing is a complex, dynamic process, and efficient wound healing is essential to protect the body against debris and foreign pathogens.1 Controlled angiogenesis occurs during the proliferative phase of repair and results in new blood vessel formation and vascular hyperpermeability, facilitating oxygen and nutrient delivery. These events are critical to tissue replacement. In many settings—such as chronic disease, infection, and radiation exposure—wound healing can be significantly impaired, resulting in chronic ulcers. Wound management is a major health concern, as over 23 million people in the USA suffer from diabetes, and ~25% of these patients develop non-healing ulcers.2

Growth factors capable of stimulating blood vessel growth are promising targets to accelerate wound closure. The application of exogenous vasoactive polypeptides has shown limited success due to the tendency to lose bioactivity with topical application.3,4 As an alternative, plasmid-encoded target genes have been delivered directly to the wound with or without the use of viral vectors,3,4 but this approach is flawed. For example, vascular endothelial growth factor (VEGF) is an endogenous angiogenic protein; however, topical application of its recombinant form produces only slight accelerations in repair and often results in incompetent or “leaky” vasculature.3,5,6 Furthermore, broad applications of growth factors for wound repair is limited due to their poor biostability, short half-life in vivo, and risk of overdose.7

A strategy to deliver growth factors in situ via a safe and efficient manner would be highly desirable for accelerating wound repair. Most current methods involve viral vectors to achieve gene delivery in order to overexpress therapeutic factors in situ.8,9 Although these approaches produce impressive results,4,10 their promise for broad clinical application is limited due to risks of immunogenicity, insertional mutagenesis, and tumorigenesis.

Cell-based therapy may improve wound healing directly by contributing to the regeneration of new tissue, or indirectly by secreting paracrine factors to promote tissue repair. Stem cells are attractive candidates to promote wound healing due to their ability to self-renew, migrate toward injury sites, and secrete paracrine signals to attract other cell types to the wound bed.7,11 Adipose-derived stromal cells (ASCs) are a promising cell source for wound repair due to their relative abundance, isolation ease, and multipotentency. Furthermore, ASCs secrete angiogenic growth factors12 and may function as pericytes to the endothelium of newly developing blood vessels.13,14 ASCs are known to migrate toward hypoxic environments such as the wound bed15 and release paracrine signals.16 Several studies support that bone marrow-derived marrow stromal cells (MSCs) and ASCs accelerate wound closure when applied to full thickness excisional wounds.17 However, it is difficult to compare results across these wound-healing studies due to differences in mouse strains, wound-healing model employed, treatment dose, and delivery modality.18 It is generally believed that in vivo ASC survival is limited upon engraftment,19 and ASCs' paracrine signals are insufficient to produce a significant acceleration in healing. To amplify paracrine signal production in situ, stem cell and gene-based therapy can be combined to genetically engineer stem cells to overexpress desired therapeutic factors in situ.20 However, current methods rely on viral vectors to achieve efficient gene delivery, and safety concerns remain a challenge with this approach.

Nonviral methods of gene transfer—such as lipofection with commercially available Lipofectamine 2000—are associated with their own limitations, such as low efficiency, short duration of gene expression, acute toxicity, and inflammatory responses in vivo.21 Cytotoxicity may be a result of the formation of reactive oxygen species,22 cell membrane destabilization,23 or the activation of mitochondrial cell death pathways that trigger apoptosis.24 Therefore, current nonviral methods are not appropriate for broad clinical application.

To address these concerns, we previously reported that biodegradable, polymeric nanoparticles can be used to deliver DNA encoding VEGF into human bone marrow-derived MSCs, and transplantation of such VEGF-overexpressing MSCs led to significantly enhanced angiogenesis and limb salvage in a mouse hindlimb ischemia model.25 The aim of this study was to exploit the potential of VEGF-overexpressing ASCs26 for wound healing using biodegradable polymeric nanoparticles. Specifically, mouse ASCs modified with polymer/VEGF nanoparticles were characterized in vitro, and their efficacy in accelerating wound healing was examined in a mouse excisional wound-healing model.

Results

In vitro transfection optimization

Lipofectamine 2000 is a commercially available reagent used as a positive control for in vitro optimization studies. Dose–response experiments using a cell proliferation assay and VEGF ELISA were performed to confirm the pDNA-loading dose recommended by the manufacturer's protocol (1 µg) was appropriate for ASCs to be used in subsequent experiments. Viability assay confirmed that at higher pDNA-loading doses above 1 µg, cell proliferation decreased (Supplementary Figure S1a). Furthermore, VEGF ELISA confirmed a progressive decrease in VEGF protein production with DNA doses over 1 µg (Supplementary Figure S1b).

Green fluorescent protein (GFP) DNA was used as a reporter to validate the transfection efficiency in ASCs. Fluorescence microscopy confirmed GFP expression in cells transfected using polymer/GFP nanoparticles (Figure 1a, left). Fluorescence-activated cell sorting analysis performed 24 hours following transfection showed increasing transfection efficiency with DNA dose increased to 4 µg. At the optimal dose of 4 µg, efficiency of transfection varied from 6 to 10% with poly (β-amino) esters (PBAE) transfection, compared to 3–4% with Lipofectamine 2000 transfection (*P < 0.05) (Figure 1a, middle and right).

Figure 1.

Figure 1

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Optimizing gene delivery to ASCs using biodegradable polymeric vectors. (a) Transfection efficiency was visualized using fluorescence microscopy (left) and quantified with FACS analysis for GFP production (middle and right). FACS analysis generally supported a 4 µg optimal treatment dose, with efficiencies ranging from 6 to 10%. A one-way ANOVA was used to test for differences among groups, showing statistically significant differences across the eight groups, F (7, 16) = 101.6, *P < 0.05. Tukey post-hoc comparisons showed that cells treated with the 4 µg treatment dose consistently demonstrated twofold higher transfection efficiency than cells transfected with Lipofectamine 2000. (Mean difference = -3.5, 99.9% CI of difference = -5.356 to -1.644, ***P < 0.001). All experiments were performed in triplicates and were repeated nine times to confirm findings. (b) VEGF ELISA demonstrated increased VEGF protein production from PBAE/VEGF transfected cells on day 2 post-transfection. One-way ANOVA showed statistically significant differences overall between the eight groups, F (7, 16) = 31.23, *P < 0.05. At the optimal dose (4 µg), PBAE transfection produced 12–15 times higher VEGF protein than Lipofectamine 2000, as determined by Tukey post-hoc comparison (mean difference = -6,807, 99.9% CI of difference = -10,656 to 2,957, ***P < 0.001). With higher DNA load, VEGF concentration decreased, as demonstrated by 4 versus 6 µg loading doses (mean difference = 3,143, 99% CI of difference = 129.7 to 6,156, **P < 0.01). (c) Cell viability was slightly lower with transfection with any reagent compared to the untreated group (**P < 0.01, as applied to every treatment group compared with untreated), but viability after transfection with PBAE at any dose was not statistically significantly different from viability after transfection with Lipofectamine 2000, as evaluated by one-way ANOVA and Tukey post-hoc comparison. All values are expressed as mean ± SEM (n = 3). ANOVA, analysis of variance; CI, confidence interval; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; PBAE, poly (β-amino) esters; VEGF, vascular endothelial growth factor.

When transfected using polymer/VEGF nanoparticles, ASCs produced increasing amount of VEGF protein as VEGF pDNA doses increased up to 4 µg. The optimum dose (4 µg) consistently produced a high level of VEGF secretion (*P < 0.01) in transfected ASCs, which ranged from 12- to 15-fold higher compared with ASCs transfected using Lipofectamine 2000 (Figure 1b). Further increases in DNA doses over 4 µg led to a decrease in VEGF production by ASCs (*P < 0.05). Cells transfected with polymeric vectors showed comparable viability to those transfected with Lipofectamine 2000. (Figure 1c).

PBAE/VEGF-transfected ASCs enhanced endothelial cell tube formation in vitro

The effects of paracrine signals from ASCs on endothelial cells in vitro were then examined using a tubulogenesis assay. Human umbilical vein endothelial cells (HUVECs) were cultured in the conditioned medium harvested from untransfected ASCs or ASCs transfected to overexpress VEGF using Lipofectamine 2000 or PBAE, and the ability of HUVEC to form tubular structures was examined. Our results showed that conditioned medium from PBAE/VEGF-transfected ASCs induced a marked increase in HUVEC tubule formation (~10.6 × 103 pixels total tubule length) compared to HUVEC treated with medium from controls (Lipo/VEGF: ~8.23 × 103 pixels total tubule length; untreated ASCs: 6.33 × 103 pixels total tubule length) (Figure 2). Conditioned medium from untreated ASCs only led to sporadic clusters of cell tubules without connectivity, whereas conditioned medium from PBAE/VEGF-transfected ASCs demonstrated interconnected networks. The tubules in the Lipofectamine 2000 group were to be intermediate between the groups, with sporadic interconnections and some areas devoid of tubular formation. The results of this assay suggest that paracrine signals from ASCs transfected with PBAE/VEGF nanoparticles may promote angiogenesis via the facilitation of endothelial cell tube formation.

Figure 2.

Figure 2

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In vitro matrigel tubulogenesis assay. HUVECs treated with conditioned media from PBAE/VEGF-transfected ASCs showed significantly increased tubulogenesis compared to cells treated with conditioned media collected from untreated ASCs. Differences were statistically significant, as evaluated by one-way ANOVA and Tukey post-hoc test. ANOVA: F (2, 20) = 16.70; ***P < 0.001; Tukey (untreated versus PBAE): mean difference: -4,226, ***P < 0.001, 99.9% CI of difference: -7,361 to -1,090; Tukey (Lipo versus PBAE): mean difference: -2,321,*P < 0.05, 95% CI of difference: -4,171 to -471.0. Values are expressed as mean ± SEM (n = 10 high-powered fields per well, three wells per group). ANOVA, analysis of variance; ASC, adipose-derived stromal cell; CI, confidence interval; HUVEC, human umbilical vein endothelial cell; PBAE, poly (β-amino) esters; VEGF, vascular endothelial growth factor.

PBAE/VEGF-transfected ASCs accelerated excisional wound closure

The efficacy of VEGF-overexpressing ASCs on wound healing was evaluated in a mouse excisional wound-healing model. Our results showed that PBAE/VEGF-transfected ASCs accelerated wound healing and achieved complete wound healing by 8 days. In contrast, groups treated using untransfected ASCs and vehicle only (phosphate-buffered saline (PBS)) did not achieve complete wound healing until days 10–12 (Figure 3a,b).

Figure 3.

Figure 3

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Wound healing in a mouse excisional wound model. (a) Percent of open wound was evaluated every 2 days post-wounding. PBAE/VEGF-modified ASCs produced significantly accelerated wound healing compared to groups treated with ASCs or PBS control. Values expressed as mean ± SEM (n = 20 wounds total per group, 10 mice per group). Statistics were performed on each day using one-way ANOVA and showed statistically significant differences between groups from days 2–10. For days 2, 8, and 10, all three combinations of groups (PBS and ASC, PBS and PBAE, and ASC and PBAE) were statistically significant with *P < 0.05 or better, as evaluated by Tukey post-hoc test. (b) Representative images show accelerated wounds closure in group treated with PBAE/VEGF-transfected ASCs, with full epithelialization observed by day 8. ANOVA, analysis of variance; ASC, adipose-derived stromal cell; PBAE, poly (β-amino) esters; PBS, phosphate-buffered saline; VEGF, vascular endothelial growth factor.

In the early phase of repair (day 0 through 4), groups treated with PBAE/VEGF-transfected ASCs showed no apparent improvement in wound size compared to groups treated with untransfected ASCs or vehicle alone. In fact, wounds treated with ASCs, either VEGF enhanced or untreated, showed a slight increase in wound size by day 2 (Figure 3a), which may be due to the recruitment of inflammatory cells to the wound bed. Many reports in the literature suggest that bone marrow-derived stromal cells possess immunomodulatory properties and may suppress inflammatory cells such as natural killer cells and T lymphocytes.27,28,29 However, several studies specific to wound healing also reported that MSCs implanted into the wound bed act via paracrine mechanisms to recruit inflammatory cells, such as macrophages, which are essential to effective wound healing.30,31 To confirm that the slight increase in wound size on day 2 may be due to edema as a result of inflammatory cell activation and recruitment, we quantified the number of leukocytes in the skin surrounding the wound on day 2 with May-Grünwald Giemsa staining (Figure 4a,b). Results showed that PBAE/VEGF-transfected cells implanted in wounds caused increased inflammatory cell recruitment with significantly increased number of leukocytes compared to wounds injected with untreated ASCs (6.12 × 10-5 versus 2.76 × 10-5 leukocytes per pixel area; *P < 0.01). As expected, F4/80 staining of skin sections from day 2 confirmed that macrophages are present in high numbers in wounds treated with PBAE/VEGF-transfected ASCs (Figure 4c). Macrophage recruitment typically reaches its peak in wound healing at day 2; however, this effect is more pronounced in our study by treating wounds with PBAE/VEGF-transfected ASCs on the day of wounding. Macrophages respond to increased VEGF production due to their cell surface VEGF receptor Flt-1, which induces chemotaxis and migration to the wound bed.32 Once in the wound environment, macrophages then release tumor necrosis factor-α and other growth factors that may induce VEGF release by resident fibroblasts and keratinocytes, effectively further increasing VEGF production.33,34,35 Therefore, the inflammatory stimulus (evidenced by increased edema and wound size on day 2), combined with enhanced VEGF angiogenic factor expression, is likely responsible for the accelerated healing observed on days 6–8.

Figure 4.

Figure 4

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Evaluation of inflammation. (a) May-Grünwald Giemsa quantification of leukocytes from wounds harvested on day 2 show increased leukocyte recruitment in the dermis on either side of wounds in the PBAE/VEGF-transfected ASC group compared to both the untreated ASC and PBS control groups (**P < 0.05). (n = 3 wounds per group, six regions of interest per group). Differences between ASC- and PBS-treated controls were not statistically significant, as evaluated by one-way ANOVA and Tukey post-hoc test. (b) From left to right, representative images showing hematoxylin and eosin stains (×20) of the wound edges showing dermal cellularity, May-Grünwald Giemsa staining for leukocytes and the wound borders (×20), and higher magnification of May-Grünwald Giemsa-stained tissue, demonstrating marked leukocytosis in tissue treated with PBAE/VEGF-transfected ASCs. Yellow arrow points to leukocytes. Bars: 20 µm. (c) Immunofluorescence of F4/80 stained tissue sections on day 2 show a high density of macrophages at the periphery of wounds treated with PBAE/VEGF-transfected ASCs. Red arrows points to macrophages. Bars: 20 µm. ANOVA, analysis of variance; ASC, adipose-derived stromal cell; DAPI, 4′,6-diamidino-2-phenylindole; PBAE, poly (β-amino) esters; PBS, phosphate-buffered saline; VEGF, vascular endothelial growth factor.

Interestingly, wounds treated with ASCs alone showed a delay in closure on day 2 compared to those treated with PBS vehicle; however, leukocyte quantification showed only a slight increase in inflammatory cell recruitment, which was not statistically significant. This result suggests that ASC implantation alone initially hinders wound healing due to disruption in physiological events, perhaps due to either inhibition of key cellular players or stimulation of apoptotic factors with resultant cell death. Despite this delay shown on day 2, wound-healing kinetics in the ASC treatment group accelerate thereafter to match vehicle-treated controls and even surpass closure rates (though differences at days 10 and 12 were not statistically significant). Overall, enhanced wound repair kinetics was observed in the PBAE/VEGF treatment group between days 4 and 8 compared with controls. Wound healing is a highly evolved and efficient biological process; therefore, any acceleration in healing of 2–4 days is thought to be meaningful.

To evaluate potential effects of transfection process itself on wound heading, ASCs transfected with GFP, a nonfunctional gene, were also transplanted into the excisional wound-healing model and examined as a control. Our results showed no difference in wound-healing kinetics in wounds treated with ASCs and wounds treated with ASCs transfected using PBAE/GFP nanoparticles, confirming that the transfection process itself did not influence the wound-healing process (Supplementary Figure S2a). Lipofectamine 2000, a commercially available transfection reagent, was used as a control in our excisional wound-healing model. Wounds were treated with 1 × 106 ASCs transfected with VEGF at the optimized loading dose for Lipofectamine 2000 (1 µg). Data shown in Supplementary Figure S2b confirm that healing was not significantly accelerated using this commercially available reagent.

PBAE/VEGF-transfected ASCs led to increased VEGF protein production in vivo

VEGF ELISA was used to evaluate VEGF production in skin tissue harvested on day 1 after transfection and treatment. Results of human VEGF ELISA performed on skin tissue lysates showed high levels of VEGF protein in excisional wounds treated with PBAE/VEGF-transfected ASCs, demonstrating that these cells produce high levels of VEGF protein during the first 24 hours after injection into excisional wounds (~836 pg/ml) (Figure 5a). Mouse VEGF ELISA performed on the same tissue lysates showed that both PBAE/VEGF-transfected ASCs and unaltered ASCs increased mouse VEGF protein in the wound bed over the level shown in PBS-treated controls (~332 and ~185 pg/ml versus 55.4 pg/ml). Even more notable was the increase in mouse VEGF production in the PBAE/VEGF treatment group compared with ASC-treated controls; transfected cells not only produced higher levels of human VEGF protein attributed to genetic manipulation, but also produced a higher level of mouse VEGF protein (Figure 5b). As discussed previously, this result could be due to either increased production by the implanted ASCs themselves or by paracrine interactions. These genetically modified cells recruit other cells to the wound bed—such as macrophages—that contribute to VEGF protein levels in the inflammatory milieu. In physiological wound healing, VEGF levels reach a peak during the proliferative phase (days 3–7).35 Treatment with PBAE/VEGF-transfected ASCs likely accelerated the peak in VEGF concentration, thereby accelerating angiogenesis and wound repair.

Figure 5.

Figure 5

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VEGF ELISAs of skin tissue lysates. (a) Human VEGF ELISA of skin tissue lysates from day 1 show high levels of human VEGF protein production in wounds treated with PBAE/VEGF-transfected ASCs, versus negligible levels in ASC- and PBS-treated controls (***P < 0.001 for comparisons of PBAE/VEGF versus ASC and PBAE/VEGF versus PBS; ANOVA: F (2, 15) = 198.8. Tukey mean difference: 818.8 and 827.0; 99.9% CI of difference = 600.8 to 1,037 and 609.0 to 1,045, respectively). (b) Mouse VEGF ELISA showed elevated levels of mouse VEGF in tissue treated with PBAE/VEGF-transfected ASCs compared to ASC- and PBS-treated controls (**P < 0.01 and ***P < 0.001, respectively. ANOVA: F (2, 15) = 26.43. Tukey mean difference: 146 and 275, 99% CI of difference = 16.31 to 275.7 and 145.9 to 405.3). Differences between ASC- and PBS-treated controls were not statistically significant. For each group, n = 2 wounds evaluated. Each sample was run in triplicate. All values are expressed as mean ± SEM. Statistical analysis was evaluated using one-way ANOVA and Tukey post-hoc test. ANOVA, analysis of variance; ASC, adipose-derived stromal cell; CI, confidence interval; PBAE, poly (β-amino) esters; PBS, phosphate-buffered saline; VEGF, vascular endothelial growth factor.

Increased VEGF protein production in vivo led to increased angiogenesis with normal blood vessel formation

Immunofluorescence with anti-CD31 staining of day 14 tissue was performed to quantify blood vessel formation both in and surrounding the wound bed. Results from histological analysis showed statistically significant increases in endothelial cell staining in the wound environment of skin treated with PBAE/VEGF-transfected ASCs compared with untreated ASC and vehicle-treated controls (~13.9% compared with ~3.52 and ~3.23%, respectively) (Figure 6a). This finding is not surprising given the evidence that PBAE/VEGF transfection of HUVECs increased expression of cell surface receptors KDR/Flk-1 and Tie-2, as well as adhesion molecule VECAM-1.36

Figure 6.

Figure 6

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Evaluation of blood vessel formation. (a) CD31 labeling of endothelial cells (red) and fluorescence microscopy at ×20 shows increased staining on day 14 in the wound and surrounding tissue in the PBAE/VEGF ASC-treated group compared with ASC and PBS controls (**P < 0.01 and *P < 0.05, respectively; n = 3 wounds evaluated per group; ANOVA: F (2, 8) = 10.72; Tukey mean difference: -10.40 and 10.69; 95% CI of difference: -17.4 to -3.40 and 2.86 to 18.5, respectively). All values are expressed as mean ± SEM. Quantification (left) and representative images (right). White arrows point to endothelial cells. (b) Hematoxylin and eosin (H&E) and Verhoeff-Van Gieson (V-VG) stains were used to evaluate quality of blood vessels. From left to right, H&E and V-VG stains of dermal scar show blood vessels forming a continuous networks parallel to the skins' surface. H&E and V-VG of skin flanking dermal scar demonstrates mature blood vessels coursing parallel to the skin's surface. Black arrows point to blood vessels. Bars: 20 µm. ANOVA, analysis of variance; ASC, adipose-derived stromal cell; CI, confidence interval; DAPI, 4′,6-diamidino-2-phenylindole; PBAE, poly (β-amino) esters; PBS, phosphate-buffered saline; VEGF, vascular endothelial growth factor.

To evaluate the quality of blood vessel formation, Verhoeff-Van Gieson stains were performed on VEGF/PBAE- and PBS-treated wounds harvested at day 14. Comparison of both scar and the unwounded edge of the dermis in PBAE/VEGF-treated and PBS-treated controls using hematoxylin and eosin (H&E) and Verhoeff-Van Gieson stains showed similar orientation of blood vessels in the reticular dermis, running continuous and parallel to the skin surface (Figure 6b). As expected, blood vessels in scar tissue appeared to be less mature than those at the edge of the wound in both groups. Although CD31 staining showed an increase in blood vessel formation in the PBAE/VEGF-transfected ASC group, this proliferation did not subjectively appear to affect the quality of blood vessels formed in replacement tissue.

PBAE/VEGF-transfected ASCs led to prolonged cell survival in vivo

Bioluminescence imaging was used to evaluate cell viability from the day of cell transplantation through full wound closure. The largest decrease in luminescence was observed within the first 24 hours post-transplantation, with no statistical significance between the transfected ASC group (log (average radiance) ~5.50 p/second/cm2/sr) and the untransfected ASC control (log (average radiance) ~5.24 p/second/cm2/sr). Between days 6 and 10, however, PBAE/VEGF-transfected ASCs showed statistically higher luminescence (~5.64–5.18 p/second/cm2/sr) in comparison to untransfected ASCs (~5.17–4.10 p/second/cm2/sr), indicating higher cell viability and/or proliferation (Figure 7a,b). These findings are not surprising given that PBAE/VEGF transfection of HUVECs was recently shown to increase endothelial cell antiapoptotic factors PIK3 and Akt-1.36 By day 12 postimplantation, the level of luminescence in both groups had decreased to the level of baseline, indicating low number of transplanted cells after complete wound closure. (Figure 7a).

Figure 7.

Figure 7

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Cell survival post-transplantation in vivo. (a) Bioluminescence imaging showed comparable cell engraftment 24 hours post-transplantation (right), with prolonged cell survival detected in PBAE/VEGF-transfected ASCs between days 6 and 10 (*P < 0.05; left). Statistical analysis was performed using Student's t-test between the two ASC groups; PBS injection was included as negative control and is shown in this figure only to illustrate background luminescence. Values expressed as mean ± SEM (n = 6). (b) Representative images from bioluminescence imaging studies on day 6 post-wounding showed higher level of cell viability in groups treated with PBAE/VEGF ASCs than ASCs alone. (c) Immunofluorescence shows increased cellularity in groups treated with PBAE/VEGF/ASCs on day 4 post-wounding. Transplanted cells (labeled green: anti-luciferase) frequently colocalized in proximity to blood vessels (labeled red: anti-CD31). ASC, adipose-derived stromal cell; DAPI, 4′,6-diamidino-2-phenylindole; PBAE, poly (β-amino) esters; PBS, phosphate-buffered saline; VEGF, vascular endothelial growth factor.

Immunofluorescence staining showed transplanted ASCs located in close proximity to blood vessels in the wound bed, suggesting the acceleration in wound closure produced by PBAE/VEGF-transfected ASCs is likely due to paracrine effects (Figure 7c). These findings were similar between transfected and untransfected ASCs. Accelerated in vivo wound closure shown in Figure 3 is likely due to enhanced VEGF protein production by these cells, stimulating the migration, proliferation, and organization of local endothelial cells.

PBAE/VEGF-transfected ASCs produced more mature collagen in scar tissue

Histological staining at day 14 showed increased collagen deposition in groups treated with PBAE/VEGF-transfected ASCs or untransfected ASCs alone compared with PBS-treated control (Figure 8a). H&E staining showed increased dermal cellularity in the group treated with PBAE/VEGF-transfected ASCs. The increase in cellularity is likely due to infiltration of fibroblasts and myofibroblasts that contribute to tissue turnover and reorganization, though further investigation would be necessary to definitively define this population.

Figure 8.

Figure 8

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Histology of wound beds day 14 post-treatment. (a) Hematoxylin and eosin and Masson's trichrome staining shows increased cellularity and collagen deposition in the dermis treated with both ASC-transplanted groups than PBS-treated control. (b) PBAE/VEGF/ASC-treated group shows most abundant mature collagen fiber (red-orange bifringent, by Picrosirius red staining), whereas PBS-treated group showed highest level of immature collagen fiber (green-yellow bifringent, by polarization microscopy). ASC, adipose-derived stromal cell; PBAE, poly (β-amino) esters; VEGF, vascular endothelial growth factor.

Picrosirius red staining at day 14 shows that the dermal scar in ASC and PBAE/VEGF/ASC-treated groups contain a greater percentage of mature, thick, and round collagen fibers (red-orange) versus less mature, thin, and flat collagen fibers (yellow-green). The opposite was true of PBS-treated controls (Figure 8b). This observation suggests that ASCs not only increase the rate of healing at its initiation but also led to enhanced proliferation and remodeling that is sustained throughout the course of healing. For example, treatment with VEGF-overexpressing ASCs causes an increase in blood vessel formation initially, and these new blood vessels persist and may allow the delivery of increased oxygen and nutrients to the healing wound well beyond the time point of wound closure. Enhanced angiogenesis begets enhanced wound remodeling, at least at this early stage of collagen turnover.

Discussion

Wound healing is a process that is highly evolved and tightly regulated. Many disease conditions are associated with impaired wound healing, and therapy-accelerating wound repair would be beneficial to these patients. Many paracrine signals have been identified as key regulators in the overlapping phases of inflammation, proliferation, and remodeling. Among these targets is VEGF, which is one of the most potent and active factors promoting angiogenesis. Upregulation of genes such as VEGF through gene therapy may facilitate more rapid wound closure through enhanced delivery of oxygen and nutrient-rich blood to the wound bed.

Viral-based gene delivery, due to its high transduction efficiency, has allowed researchers to establish cell sources and genes that are favorable targets for wound healing. A few publications have demonstrated statistically significant accelerations in repair with direct recombinant VEGF therapy and viral-mediated gene delivery.3,4,5 However, these methods often led to inconsistent and variable results. Viral vectors produce impressive results with ex vivo pVEGF transfer, one report showing VEGF supernatant protein concentration of 3.78 ng/ml on day 3.37 Despite the high efficiency and efficacy of viral-mediated gene transfer, viral vector use is limited due to safety concerns. As gene therapy moves closer to its translational applications in the clinic, it is important to develop safe gene delivery approaches with high potential for clinical trial and FDA approval. Current commercially available nonviral-based gene delivery reagents suffer from low transfection efficiency and high toxicity.38

To solve for these limitations, we have engineered mouse ASCs to transiently overexpress VEGF using biodegradable polymeric nanoparticles. Our previous studies established an optimal ratio of polymer weight to pDNA weight of 30:1.39 Therefore, increasing pDNA-loading dose in optimizing studies was accompanied by a proportional increase in polymer weight. Results of initial in vitro studies defined an optimal dose that maximizes transfection efficiency and enhancement of VEGF protein expression while minimizing toxicity to transfected cells. PBAE demonstrated much higher transfection efficiency in ASCs compared with Lipofectamine 2000, a commercially available transfection reagent. These results are consistent with previous studies in which human ASCs were transfected with PBAE/VEGF using the optimized polymer weight to pDNA weight ratio of 30:1. These experiments produced PBAE/VEGF transfection efficiency approximately three times the efficiency of Lipofectamine 2000 transfection.39 Subsequent experiments transfecting human bone marrow-derived MSCs with PBAE/VEGF demonstrated one to threefold higher VEGF protein production compared with Lipofectamine/VEGF-transfected controls. These cells increased angiogenesis and improved ischemic limb salvage in vivo.25 Similarly, the current study shows that PBAE/VEGF nanoparticle-transfected mouse ASCs transplanted into a mouse excisional wound-healing model accelerated wound closure by ~2 days. Wound healing in healthy mammals is a highly evolved process; therefore, the results produced by this study are considered meaningful and significant.

Previous studies have shown the promise of ASCs for angiogenesis and wound healing due to their ability to directly contribute to blood vessel formation.13,18 In this study, ASCs were exploited to contribute to wound healing mostly via paracrine pathway, with very little evidence of direct endovascular contribution. Therefore, ASCs can be considered delivery vehicles for both their own well-established endogenous cytokine and growth factor arsenal (including VEGF, hepatocyte growth factor, and fibroblast growth factor-2),18 as well as other growth factors transiently overexpressed via gene delivery. In this case, VEGF overexpression resulted in statistically significant ASC angiogenic potential, both in vitro and in vivo. In addition, cell survival in vivo was higher with VEGF-overexpressing cells than the untransfected form, suggesting that increasing VEGF expression may improve ASC cell survival in the wound bed and prolong beneficial effects.

Angiogenesis occurs throughout the wound-healing process, and VEGF has been shown to be essential during the proliferative and remodeling phases. In this study, we chose to transplant VEGF-overexpressing ASCs to the wound bed at the time of wounding to evaluate whether single treatment would be sufficient to induce therapeutic efficacy. Compared with approaches in which multiple cell transplantation treatments are required, single transplantation therapy would be much cheaper and easier for broad clinical translation. Our results show that even with one single cell transplantation, significantly accelerated wound healing, increased normal-appearing blood vessel formation, and more mature collagen fiber formation in the wound site.

Wound healing may be further accelerated if several growth factors are co-delivered to produce synergistic effects.40 VEGF administered alone improves wound healing slightly but sometimes produces “leaky” vasculature.3,5,6 Combination of VEGF and Ang1 has been shown to augment angiogenesis with competent vessel formation in place of these “leaky” structures.41,42 Therefore, future studies using polymeric nanoparticles to overexpress multiple synergistic factors in stem cells may further enhance their efficacy in wound healing.

The results from this study demonstrate the efficacy of using nonviral-engineered ASCs to accelerate wound healing in healthy wild-type animals. Given that impaired wound healing is often accompanied by diseases conditions, it would be clinically relevant to examine the efficacy of such nonviral-engineered stem cells in disease models such as diabetes. Diabetes has rapidly become a major global health issue. Therefore, developing a safe and effective therapy to treat chronic ulcers in diabetic patients with impaired angiogenesis would hold broad and significant impacts. Much like humans with type II diabetes, leptin receptor-deficient db/db mice have been previously shown to exhibit significantly delayed healing in excisional wounds compared with their wild-type counterparts.43 ASCs harvested from inguinal fat pads of db/db mice also showed decreased VEGF expression compared with those harvested from wild-type mice.44 Future studies will examine the potential of such nonviral-engineered stem cells for restoring VEGF expression of ASCs harvested from diabetic mice, and their efficacy in accelerating wound healing in diabetic mouse models.

Materials and Methods

Isolation and culture of ASCs. ASCs were collected by harvesting the inguinal fat pads from 5 to 6 weeks old luciferase-positive/GFP-positive transgenic male mice (Joseph Wu, MD, PhD laboratory, Stanford University) and FVB wild-type male mice (purchased from Jackson Laboratories, Bar Harbor, ME). Following serial betadine washes, the fat pads were finely minced in cold, sterile PBS, followed by digestion in 0.075% Collagenase II (Sigma-Aldrich, St Louis, MO) at 37 °C for 30 minutes in a shaking water bath. Digestion was neutralized with Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin (Sigma-Aldrich). Neutralized cells were centrifuged at 4 °C and 1,000 rpm for 5 minutes, and floating adipocytes and media were removed before pelleted ASCs from the stomal-vascular portion were resuspended in culture media, passed through a 100 µm Facon nylon cell strainer (Becton Dickinson, Bedford, MA) and plated on a 10 cm dish. Cells were incubated at 37 °C and 5% atmospheric CO2. Twenty-four hours after transfection, the cells were washed in PBS to remove debris and nonadherent cells, and fresh medium was added to the cells. Medium was changed every 2 days, and adherent spindle-shaped cells were grown to subconfluence, passaged using standard methods of trypsinization, and propagated through passage 2.

Transfection conditions. ASCs were transfected with either the reporter gene enhanced GFP (pEGFP) (ELIM Biopharm, Hayward, CA) or VEGF (pVEGF) (Aldevron, Fargo, ND). ASCs were seeded at 65,000 cells per well in clear 24-well plates and incubated over night at 37 °C. Cells were then transfected utilizing end-modified PBAE polymers (C32-122), as previously reported.39 Briefly, polyplexes were formed by mixing pDNA and PBAE polymers at a weight-to-weight ratio of 30:1 in sodium acetate. Current PBAE synthesis is still under development; therefore, a high polydispersity index exists, with molecular weights ranging from 4–18 kDa.45 Polyplexes were added to the cell culture after 10 minutes of complexation. Cells were then incubated with polyplexes for 4 hours, at which time cell medium was replaced with 10% serum containing Dulbecco's modified Eagle's medium. Transfection efficiency was optimized by varying pDNA-loading dose (1, 2, 3, 4, 5, and 6 µg) and observing GFP expression, where Lipofectamine 2000 (Invitrogen) served as a positive control and nontransfected cells served as a negative control. Lipofectamine 2000 was used to transfect the cells according to the manufacturer's protocol using 2.5 µg Lipofectamine and pDNA-loading dose of 1 µg. This optimum dose, consistent with methods reported in the literature,46 was confirmed by performing the transfection at increasing pDNA amounts (1, 2, 3, 4, 5, and 6 µg), keeping the ratio of Lipofectamine and pDNA weight consistent. Conditioned medium collected on day 2 was evaluated for VEGF protein production by ELISA, and Cell Titer 96 Aqueous One Solution assay kit (Promega, Madison, WI) was performed on transfected cells at day 2 to determine metabolic activity at increasing pDNA doses. ASCs were transfected with pVEGF using Lipofectamine 2000 under the optimized conditions (1 µg pDNA) for subsequent in vivo experiments.

Flow cytometric analysis of ASC transfection efficiency. Twenty-four hours after transfection of wild-type FVB ASCs with pEGFP, transfection efficiency was evaluated by fluorescence microscopy using the Leica DMI4000 B microscope (Leica Microsystems, Buffalo, IL) and flow cytometry on the Scanford analyzer using CellQuest Pro software. Data were analyzed using FlowJo software (Treestar, Ashland, OR).

Cell viability/proliferation assay. Cell viability or proliferation following transfection was evaluated at 48 hours post-transfection using the Cell Titer 96 Aqueous One Solution assay kit (Promega) to measure metabolic activity. Measurements of cells transfected with polymers + pEGFP and lipofectamine + pEGFP were converted to percentage viability in comparison to untreated control cells.

VEGF ELISA on conditioned medium. Conditioned medium was collected from transfected and untreated control cells at 2 and 4 days post-transfection. Quantikine human VEGF ELISA kits (R&D Systems, Minneapolis, MN) were used according to the manufacturer's protocol. Briefly, VEGF standards (1–1,000 pg/ml) and samples were pipetted into wells coated with antibody specific for human VEGF. The plates were washed with ELISA buffer, and an enzyme-linked polyclonal antibody specific for VEGF was added to the wells. After a second wash, a substrate solution was added, producing a color signal. The absorbance of samples and standards was measured spectrophotometrically at 450 nm with wavelength correction of 570 nm using a microplate reader. VEGF concentrations were calculated (in pg/ml) with the standard curve via four parameter logistic (4-PL) curve-fit model using GraphPad Prism (GraphPad Software, La Jolla, CA).

In vitro angiogenesis assay. HUVECs (Lonza, Allendale, NJ) were maintained in culture in the supplier's recommended complete medium (EGM-2) at 37 °C, 5% CO2; 10,000 HUVECs were plated in 200 ml of conditioned media on chamber slides (Sigma-Aldrich) coated with Growth Factor Reduced Matrigel (EMD Millipore, Billerica, MA). Cells were incubated for 24 hours. Tubulogenesis, the alignment of endothelial cells and generation of a patent lumen, was evaluated using a Zeiss Axioplan 2 light-fluorescent microscope (Carl Zeiss Microscopy, Thronwood, NY). Adobe Photoshop software (Adobe Systems, San Jose, CA) was used to measure the total length of tubules in pixels. A tubule was defined as a structure exhibiting length four times its width.

Mouse excisional wound-healing model. All experiments were performed in accordance with the Stanford University Animal Care and Use Committee Guidelines and approved APLAC protocols. FVB female mice, age 10–12 weeks, were housed one per cage in a 12-hour light/dark cycle and provided ad libitum with standard food and water. After depilation, two 6 mm full thickness wounds extending through the panniculus carnosus were made at the same level on the dorsum of mice as previously described.47 A silicone 12 mm diameter washer (Invitrogen) was placed around the perimeter of the wound and secured with cyanoacrylate glue and interrupted Ethilon 6-0 sutures (eSutures.com, Mokena, IL). Mice were randomized to three groups receiving different treatments including: PBS (negative control), untreated ASC (positive control), PBAE/VEGF-transfected ASCs (n = 10 animals per group, 20 wounds per group). To verify that the PBAE transfection process did not alter wound-healing process, ASCs transfected using PBAE/GFP and Lipofectamine 2000/VEGF were also examined as controls (n = 3 animals, six wounds). Wounds treated with transfected and untreated ASCs received 1 × 106 cells suspended in 80 µl of PBS, injected subdermally at the four quadrants of the wound using a tuberculin syringe. Negative controls received an equivalent volume of sterile PBS injected in four quadrants. Wounds were dressed with Tegaderm sterile dressing (3M Healthcare, St Paul, MN), which was changed every other day until wound closure. Digital photographs were taken at the time of surgery and every other day until closure, defined as the time at which the wound bed was completely re-epithelialized and filled with new tissue. Wound area was quantified using ImageJ software (NIH, Bethesda, MD) and expressed as a ratio of wound circumference to silicone stent circumference.

VEGF ELISA on tissue lysates. Wounded tissue was harvested on day 1 after transfection, wounding, and implantation by bisecting a ring of skin tissue measuring 12 mm in diameter. This tissue was then ground by mortar and pestle technique in 100 µl of solution containing RIPA buffer (Sigma-Aldrich) and protease inhibitor (Sigma-Aldrich). The tissue was allowed to lyse at 4 °C, after which the solution was centrifuged and the supernatant was collected for ELISA. Quantikine human and mouse VEGF ELISA kits (R&D Systems) were used according to the manufacturer's protocol, as previously described for in vitro experiments.

Bioluminescent Imaging. ASC survival was monitored post-injection with bioluminescence imaging technology. Bioluminescence imaging was performed on the day of surgery, day 1, day 2, and every other day thereafter through 14 days. Animals were anesthetized by 2–3% inhaled isoflurane and injected intraperitoneally with the reporter probe D-Luciferin at 100 mg/kg body weight. IVIS Spectrum system (Xenogen, Alameda, CA) was used to image the animals while under 2% inhaled isoflurane anesthesia. Each animal was scanned until the peak signal was reached, recording radiance of regions of interest in photons. Radiance was quantified in photons per second per centimeter squared per steridian.

Immunofluorescence. Tissue samples harvested on days 2 and 4 post-wounding and implantation were fixed overnight in 4% paraformaldehyde, washed with PBS, and embedded in optimal cutting temperature for cryosectioning. Sections of the wound bed were collected at 8 µm thickness. Macrophages were detected at the periphery of day 2 skin excisional wounds with Anti-F4/80 (Abcam, Cambridge, MA). Transplanted GFP/luciferase-positive cells were detected in day 4 wounds with Anti-Firefly Luciferase (Abcam) using Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA), and endothelial cells with anti-CD31 (Biolegend, San Diego, CA) using Vector Laboratories' M.O.M. kit and appropriate fluorescent secondary antibodies (Alexa Fluor 488, 594; Invitrogen). Sections were imaged with confocal microscopy.

Histology. Tissue samples harvested on days 2 and 14 post-wounding and implantation were fixed overnight in 4% paraformaldehyde, washed in D5W, dehydrated with sequential alcohol (30, 50, 70, 95, and 100%), xylene, and paraffin washes, and embedded in paraffin for sectioning. Day 2 sections of the wound bed were collected at 8 µm thickness and stained using a May-Grünwald Giemsa staining protocol. In brief, after deparaffinization, sections were washed with May-Grünwald reagent (Sigma-Aldrich), followed by 1% acetic acid. Sections were then treated with Giemsa reagent (Sigma-Aldrich), then rinsed in water. Sections were mounted with permount. Day 14 sections of the wound bed were collected at 8 µmol/l thickness and stained using H&E, Verhoeff-Van Gieson (Sigma-Aldrich), Masson's trichrome, and Sirius Red protocols (IHC World, Woodstock, MD). Scar was evaluated using light microscopy for H&E, Masson's trichrome, and Picrosirius Red (×20) and polarization microscopy for Picrosirius red staining. Leukocyte numbers were quantified in day 2 histology using Adobe Photoshop software (Adobe Systems). Each May-Grünwald Giemsa-stained section was visualized under light microscopy at ×40 magnification (Leica Microscope, Leica DM 4000B; Leica Microsystems) and photographed using the Leica DFC 500 camera (Leica, Allendale, NJ). Photographs were taken of the dermis flanking each wound (two regions of interest per section), and three wounds were evaluated per group (N = 6 regions of interest per group). The number of leukocytes per field of view were counted and expressed as a ratio of number of cells per pixel area of dermis. Anti-CD31 quantification was performed on day 14 sections using Adobe Photoshop software (Adobe Systems). Each section was visualized under fluorescence microscopy at ×20 magnification (Leica Microscope, Leica DM 4000B) and photographed using the Leica DFC 500 camera (Leica). Photographs were taken at the center of the dermal scar and one field of view on either side of the scar, for a total of three images per wound. Quantity of red staining, indicating anti-CD31 antibody staining, was quantified per area of dermis in pixels, and the three measurements for each wound were averaged. Four wounds were evaluated per group and averaged to determine percent of anti-CD31 staining per treatment group.

Statistical analysis. All data were expressed as mean ± SEM. Student's t-test and one-way analysis of variance for multiple comparisons with Tukey post-hoc test were used to determine statistical significance, which was defined by a *P value <0.05.

SUPPLEMENTARY MATERIAL Figure S1. Optimizing gene delivery to ASCs using Lipofectamine 2000. Figure S2. In vivo wound-healing experiments with pGFP and Lipofectamine 2000.

Acknowledgments

We would like to acknowledge the Oak Foundation and the Hagey Laboratory for Pediatric Regenerative Medicine for financial support. F.Y. and L.D. thank American Heart Association (10SDG2600001) and Stanford Medical Scholars Research Program for funding. The authors would like to acknowledge Timothy C Doyle, D.Phil. for his assistance and support in the use of the SCi3 Small Animal Imaging Facility, a Stanford shared resource facility used the generation of the data reported in this study. The authors declared no conflict of interest.

Supplementary Material

Figure S1.

Optimizing gene delivery to ASCs using Lipofectamine 2000.

Click here for additional data file. (92.4KB, tiff)
Figure S2.

In vivo wound-healing experiments with pGFP and Lipofectamine 2000.

Click here for additional data file. (123.1KB, tiff)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Optimizing gene delivery to ASCs using Lipofectamine 2000.

Click here for additional data file. (92.4KB, tiff)
Figure S2.

In vivo wound-healing experiments with pGFP and Lipofectamine 2000.

Click here for additional data file. (123.1KB, tiff)

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