Effects of genetically modified adipose stromal cells implantation in rabbits with experimental peripheral vascular disease

Abstract

Purpose: Adipose Stromal Cells (ASCs) have been shown to display reparative and regenerative potential in diverse tissues, including skeletal muscle, mainly due to the paracrine effect of secreted angiogenic factors. On the other hand, we have demonstrated that gene transfection with a plasmid encoding human Vascular Endothelial Growth Factor (pVEGF165) promotes angio-arteriogenesis and affords protection against ischemic muscle lesions in rabbits with hind limb ischemia. We therefore hipothesised that in this animal model ASCs genetically modified to overexpress VEGF165 would exert greater anti-ischemic effect than non-modified ASCs. Methods: Rabbit ASCs were isolated, identified, cultured and transfected with pVEGF165. Rabbits with extirpation of the superficial femoral artery and ligature of the deep femoral artery of the left hind limb were randomized to receive 10 local intramuscular injections of 10 million (total amount) allogeneic modified ASCs (ASC-VEGF group, n=10), non-modified ASCs (ASC group, n=10) or PBS (placebo group, n=10). Prior to surgery, and at 1 and 30 days after treatment, Peak Systolic flow Velocity (PSV) was measured by Doppler echo in both hind limbs. At 30 days post-treatment vascular density of both hind limbs was assessed by angiography, and after euthanasia (sodium thiopental overdose followed by potassium chloride) tissue samples from the adductor, gastrocnemius and quadriceps muscles were harvested for assessment of ischemic muscle lesions. Investigators involved in data collection and analysis were blinded to the animals' study groups. Results: PSV increased in the ASC-VEGF group (pre-treatment: 11.1±3.3, post-treatment: 23.4±19 cm/sec; p<0.05, mean ± SD, 2-way ANOVA-Bonferroni), but not in the ASC (from 11±4.7 to 16±10.7 cm/sec, p=NS) or placebo (from 10.1± to 17±10.9 cm/sec p=NS) groups. In the angiography, only the ACS-VEGF group showed greater vascular density than the placebo group (5.6±1.1 vs. 4.5±0.6 vessels/cm2; p<0.04). Ischemic vascular lesions were present in 20% of the harvested samples in ASC-VEGF group, 33% in ASC group, and 73% in placebo group (p<0.05 vs. ASC-VEGF and ASC, Fisher's exact test). Conclusions: In rabbits with experimental peripheral vascular disease, the local injection of ASCs overexpressing human VEGF165 improves vascularisation and hemodinamics of the affected limb more than non-modified ASCs. However, both treatments afford protection against ischemic muscle lesions to a similar extent.