Gene Therapy by Adenovirus-Mediated Vascular Endothelial Growth Factor and Angiopoietin-1 Promotes Perfusion of Muscle Flaps

Abstract

An experimental study was conducted to investigate the potential use of intravascular gene therapy with adenovirus-mediated (Ad) vascular endothelial growth factor (VEGF) or angiopoietin-1 (Ang-1) for the enhancement of muscle flap perfusion and to evaluate the effect of therapy on microcirculatory hemodynamics and microvascular permeability in vivo by using a cremaster muscle flap model in the rat. The cremaster tube flap was left intact after isolation of the pudo-epigastric pedicle. A total of 90 male Sprague-Dawley rats were divided into five groups of 18 each, according to the type of intraarterial treatment. Control flaps received phosphate-buffered saline. Group 2 (the control gene encoding green fluorescent protein, Ad-GFP) served as the adenovirus control. In Groups 3, 4, and 5, flaps were pretreated with Ad-VEGF, Ad-Ang-1, and Ad-Ang-1 + Ad-VEGF, respectively. Flaps were preserved in a subcutaneous pocket in the hindlimb for evaluation of functional capillary density and microvascular permeability indices at 3, 7, and 14 days by intravital microscopy system. At day 7 and 14, Ad-VEGF, Ad-Ang-1, and combined treatment groups showed significantly higher numbers of capillary densities when compared with control and Ad-GFP groups (p < 0.05). At day 14, Ad-VEGF was the superior treatment group compared with Ad-Ang-1 and Ad-VEGF + Ad-Ang-1 (p < 0.05). Overall, there was a linear increase in the number of functional capillaries in all treatment groups (p < 0.05). At day 3 after Ad-Ang-1 therapy, a significantly lower permeability index was found when compared with Ad-VEGF + Ad-Ang-1 and Ad-VEGF alone treatment (p < 0.05). At day 7, the Ad-VEGF group had the highest score of permeability index compared with control, combined, and Ad-Ang-1 groups (p < 0.05). Histologic evaluation of muscle flaps demonstrated mild focal inflammation. There was evidence of mild vasculitis in all flaps except control muscles. Intravascular angiogenic therapy with Ad-VEGF or Ad-Ang-1 was technically feasible, as demonstrated by expression of the control gene, GFP, along the vascular tree. All treatment groups increased perfusion of the muscle flap over a period of 14 days, indicating a long-lasting effect of gene therapy. Ang-1 alone or in combination with VEGF was as effective as VEGF alone in augmenting muscle perfusion with more stable vessels 1 week after gene therapy.

Angiopoietin-1 Promotes Cardiac and Skeletal Myocyte Survival Through Integrins

“…Ang1 overexpression in the heart resulted in smaller infarct zones and preserved cardiac function following myocardial infarctions. 29 -31 Improved myoblast survival and reduced tissue necrosis with hindlimb ischemia 96 and enhanced survival of muscle 59,60 and skin 97 flaps have also been reported. These studies showed increases in perfusion and/or capillary density, which were thought to explain the protective effects of Ang1 on the muscle and other tissues.…”

“…mediate skeletal muscle survival 59,60 during insults such as limb ischemia from peripheral vascular disease. We propose that this new angiopoietin function acts in concert with effects on the vasculature to protect the heart and peripheral tissues under adverse conditions.…”

Improved survival of rat ischemic cutaneous and musculocutaneous flaps after VEGF gene transfer

“…5), confirming a property already described in literature. 6,13,14,22,39,40 Immunostaining the same samples with an antibody specific for a-Smooth Muscle Actin, emphasized the neoangiogenetic process taking place in AAV-VEGF-treated tissues, by demonstrating the massive presence of arterial vessels (Fig. 6), particularly in samples from group 9 (vector inoculation 14 days before surgery).…”

The Effects of Transpositional Muscle Flaps Transfected with Vascular Endothelial Growth Factor Gene in the Treatment of Experimental Osteomyelitis

“…Studies have shown that adenovirus vector inserted with VEGF or liposome-mediated VEGF gene are effectively transferred and expressed via direct intramuscular injection or arterial access. [29,30] However, in addition to gene delivery techniques, the simplest method of gene delivery is the direct injection of naked plasmid DNA into tissues. Advantages of this method are local delivery to the target organ and the ability to act on non-dividing cells.…”