Efficacy and mechanism of adenovirus-mediated VEGF-165 gene therapy for augmentation of skin flap viability

Abstract

Skin ischemic necrosis due to vasospasm and/or insufficient vascularity is the most common complication in the distal portion of the skin flap in reconstructive surgery. This project was designed to test our hypothesis that preoperative subdermal injection of adenoviral vectors encoding genes for vascular endothelial growth factor-165 (Ad.VEGF-165) or endothelial nitric oxide (NO) synthase (Ad.eNOS) effectively augments skin viability in skin flap surgery and that the mechanism of Ad.VEGF-165 gene therapy involves an increase in synthesis/release of the angiogenic and vasodilator factor NO. PBS (0.5 ml) or PBS containing Ad.VEGF-165, Ad.eNOS, or adenovirus (Ad.Null) was injected subdermally into the distal half of a mapped rat dorsal skin flap (4 × 10 cm) 7 days preoperatively, and skin flap viability was assessed 7 days postoperatively. Local subdermal gene therapy with 2 × 107–2 × 1010 plaque-forming units of VEGF-165 increased skin flap viability compared with PBS- or Ad.Null-injected control ( P < 0.05). Subdermal Ad.VEGF-165 and Ad.eNOS gene therapies were equally effective in increasing skin flap viability at 5 × 108 plaque-forming units. Subdermal Ad.VEGF-165 therapy was associated with upregulation of eNOS protein expression, Ca2+-dependent NOS activity, synthesis/release of NO, and increase in capillary density and blood flow in the distal portion of the skin flap. Injection of the NOS inhibitor Nω-nitro-l-arginine (15 mg/kg im), but not the cyclooxygenase inhibitor indomethacin (5 mg/kg im), 45 min preoperatively completely abolished the increase in skin flap blood flow and viability induced by Ad.VEGF-165 injected subdermally into the mapped skin flap 7 days preoperatively. We have demonstrated for the first time that 1) Ad.VEGF-165 and Ad.eNOS mapped skin flap injected subdermally into the mapped skin flap 7 days preoperatively are equally effective in augmenting viability in the rat dorsal skin flap compared with control, 2) the mechanism of subdermal Ad.VEGF-165 gene therapy in augmenting skin flap viability involves an increase in NO synthesis/release downstream of upregulation of eNOS protein expression and Ca2+-dependent NOS activity, and 3) the vasodilating effect of NO may predominantly mediate subdermal Ad.VEGF gene therapy in augmenting skin flap blood flow and viability.

Identification of the Fra-1 transcription factor in healing skin flaps transplants: A potential role as a negative regulator of VEGF release from keratinocytes

“…It is generally believed that insufficient vascularization of transplanted skin flap tissue contributes to necrosis and loss of the affected tissue (Myers and Cherry, 1968;Kerrigan, 1983;McFarlane et al, 1965). Based on this belief the management of necrotic tissue loss has mainly been focused on the exploitation of pro-angiogenic factors such as VEGF (Scalise et al, 2004;Michlits et al, 2007;Huang et al, 2006) or basic fibroblast growth factor (bFGF) (Fujihara et al, 2005). The most recent two reports from our laboratory suggested an additional regulatory process in the control of skin flap survival or loss: the wound inflammatory response.…”

“…Those attributes may define VEGF as a mediator to provide potential tissue protection in clinical situations of disturbed skin flap integration. VEGF has been shown to have beneficial effects in the maintenance of disturbed skin flaps when applied either as recombinant protein (Scalise et al, 2004), by using diverse expression vector systems (Michlits et al, 2007;Huang et al, 2006) or VEGFtransduced stem (Zheng et al, 2008) and endothelial progenitor cells (Yi et al, 2006).…”

Hemoglobin vesicles improve wound healing and tissue survival in critically ischemic skin in mice

“…Besides stimulating new vessel formation, it attenuates endothelial cell apoptosis (1,15). eNOS is therefore a pivotal player in wound healing (3,14) and contributes to maintain or improve flap tissue survival (11,13). eNOS expression is oxygen dependent (12,16,23), stimulated by fluid shear stress (15,24), and inhibited by proinflammatory cytokines (1,23,27).…”

Effects of Natural and Recombinant Hirudin on VEGF Expression and Random Skin Flap Survival in a Venous Congested Rat Model

“…Finally, VEGF induces endothelial nitric oxide synthase expression in endothelial cells and thereby increases nitric oxide synthesis and release, leading to the dilation of blood vessels. 15 Our quantitative RT-PCR results showed that VEGF mRNA levels in the control group subsequently decreased on day 5 and on day 7 from the level recorded on postoperative day 3. These results are consistent with previous reports by Han et al…”