VEGF-Encoding, Gene-Activated Collagen-Based Matrices Promote Blood Vessel Formation and Improved Wound Repair

Abstract

Disruption in vascularization during wound repair can severely impair healing. Proangiogenic growth factor therapies have shown great healing potential; however, controlling growth factor activity and cellular behavior over desired healing time scales remains challenging. In this study, we evaluated collagen-mimetic peptide (CMP) tethers for their capacity to control growth factor gene transfer and growth factor activity using our recently developed gene-activated hyaluronic acid-collagen matrix (GAHCM). GAHCM was comprised of DNA/polyethyleneimine (PEI) polyplexes that were retained on hyaluronic acid (HA)-collagen hydrogels using CMPs. We hypothesized that using CMP-collagen tethers to control vascular endothelial growth factor-A (VEGF-A) gene delivery in fibroblasts would provide a powerful strategy to modulate the proangiogenic behaviors of endothelial cells (ECs) for blood vessel formation, resulting in enhanced wound repair. In co-culture experiments, we observed that CMP-modified GAHCM induced tunable gene delivery in fibroblasts as predicted, and correspondingly, VEGF-A produced by the fibroblasts led to increased growth and persistent migration of ECs for at least 7 days, as compared to non-CMP-modified GAHCM. Moreover, when ECs were exposed to fibroblast-containing VEGF-GAHCM with higher levels of CMP modification (50% CMP-PEI, or 50 CP), high CD31 expression was stimulated, resulting in the formation of an interconnected EC network with a significantly higher network volume and a larger diameter network structure than controls. Application of VEGF-GAHCM with 50 CP in murine splinted excisional wounds facilitated prolonged prohealing and proangiogenic responses resulting in increased blood vessel formation, improved granulation tissue formation, faster re-epithelialization, and overall enhanced repair. These findings suggest the benefits of CMP-collagen tethers as useful tools to control gene transfer and growth factor activity for improved treatment of wounds.

Keywords: VEGF-A; angiogenesis; collagen-mimetic peptide (CMP); gene therapy; wound healing.

Figure 1.

Endothelial cell net growth stimulated by the condition media collected from fibroblasts cultured in VEGF-GAHCM at days 1, 3, 5, and 7. The net growth (%) was normalized to the endothelial cell growth without the condition media treatment. Each data point represents the mean ± standard deviation for n=4. The statistically significant differences state +P<0.05 compared to HCM and *P<0.05 compared to PEI.

Figure 2.

Endothelial cell invasion in fibroblasts cultured in HCM hydrogel, rVEGF+HCM hydrogel, VEGF-GAHCM with PEI, 20 CP, or 50 CP at days 3, 5, and 7. The schematics in the graph are fibroblast location in VEGF-GAHCM at days 3, 5, and 7. The red dotted line is the minimum threshold used to detect signal of calcein-AM pre-stained endothelial cells on the z-stack images of full thickness of samples. Each data point represents the mean ± standard deviation for n=7.

Figure 3.

Endothelial cellular networks on fibroblast cultured in HCM hydrogel, rVEGF+HCM hydrogel, VEGF-GAHCM with PEI, 20 CP, or 50 CP at days 2,4,6, and 10. The representative 3D plot of z-stack images for calcein-AM pre-stained endothelial cells (Green). Scale bar is 200 μm.

Figure 4.

Image quantification for endothelial cellular networks on fibroblast cultured in HCM hydrogel, rVEGF+HCM hydrogel, VEGF-GAHCM with PEI, 20 CP, or 50 CP at days 2,4,6, and 10. (A) The total volume of capillary network. (B) The average diameter of capillary network. Each data point represents the mean ± standard deviation for n=5. +P<0.05, ++P<0.001 compared to HCM, #P<0.05, ##P<0.001 compared to rVEGF, *P<0.05, **P<0.001 compared to PEI

Figure 5.

CD31 immunostaining on the endothelial cells network formed on fibroblast cultured in HCM hydrogel, rVEGF+HCM hydrogel, VEGF-GAHCM with PEI, 20 CP, or 50 CP at day 6. (A) The representative confocal microscope images of CD31 expressed endothelial cells within the capillary network. CD31 (Green) and F-actin (Red). Scale bar is 50 μm. (B) Image quantification for CD31 expression on the endothelial cells within the capillary network. Each data point represents the mean ± standard deviation for n=4. +P<0.05 compared to HCM, #P<0.05 compared to rVEGF, and *P<0.05, compared to PEI

Figure 6.

In vivo wound healing evaluation using histological analysis of mouse splinted excisional wounds treated with saline, HCM hydrogel, rVEGF+HCM hydrogel, and VEGF-GAHCM with PEI, 20 CP, and 50 CP after 7 and 14 days. The tiled images of H&E stained mouse skin wound tissue section at 7 or 14 days of treatments and the zoom-in image of black-dotted box in the tiled images. s = native skin and w = wound. Black arrow indicates the scab. Scale bar is 1.5 mm and scale bar for zoom-in image is 250 μm.

Figure 7.

In vivo wound healing evaluation using image analysis of histological stained mouse splinted excisional wounds treated with saline, HCM hydrogel, rVEGF+HCM hydrogel, and VEGF-GAHCM with PEI, 20 CP, and 50 CP. (A) Granulation tissue area quantification after 7 and 14 days of treatment. Each data point represents the granulation tissue area for each mouse per group. (B) Epidermal thickness measurements after 14 days of treatment. Each data point represents the average of epidermal thickness in the wound per field of image for three images per each mouse. The horizontal line represents the mean ± standard deviation for total 6 mouse per group. The statistically significant differences state ^%P<0.05 for samples relative to Native, ^&P<0.05 for samples relative to Saline, ⁺P<0.05 for samples relative to HCM, and ^#P<0.05 for samples relative to rVEGF

Figure 8.

Blood vessel formation analysis of mouse wounds treated with saline, HCM hydrogel, rVEGF+HCM hydrogel, and VEGF-GAHCM with PEI, 20 CP, and 50 CP after 7 and 14 days. (A) The representative confocal microscope images of CD31/α-SMA Immunostained mouse skin wound tissue section after 7 or 14 days of treatment (CD31 for red, α-SMA for green, and nuclei for blue). Scale bar is 40 μm. The white dotted circles indicate blood vessels on the images. (B) Total blood vessels count per field of image. (C) The number of mature blood vessels within the total blood vessels counts per field of image. Each data point represents the average of number of blood vessels in the wound per field of image for five images per each mouse. The horizontal line represents the mean ± standard deviation for total 6 mouse per group. The statistically significant differences state ^&P<0.05 for samples relative to Saline, ⁺P<0.05 for samples relative to HCM, and ^#P<0.05 for samples relative to rVEGF.