One-year Results of pCMV-VEGF165 Gene Transfer in Patients with Critical Lower Limb Ischemia Due to Peripheral Atherosclerosis and Diabetes Mellitus

Abstract

Introduction - atherosclerotic peripheral arterial disease is common in patients with diabetes mellitus (DM) and leads to a significant increase in amputation rates. Induction of angiogenesis may be a promising technique in improving limb salvage rates and quality of life in patients with critical limb ischemia (CLI) and DM. Methods - The study was divided into experimental and clinical parts. Experiment was performed using a model of surgical skin wound in Wistar rats with alloxan-induced DM. Experimental animals were treated with 60mcg (N=8) or 200mcg (N=8) of pCMV-VEGF165. Animals in control group were treated with water for injections. At 10 days the animals were withdrawn from the experiment with following morphological and immunohistochemical assessment of the wound and surrounding soft tissues. Clinical study included 140 patients with CLI and DM. Patients were divided into 4 groups: group I – arterial revascularization and conservative therapy (N=45), group II –conservative treatment (N=40), group III – arterial revascularization, conservative therapy, and pCMV-VEGF165 gene transfer (N=30), group IV – conservative treatment and pCMV-VEGF165 gene transfer (N=25). Limb salvage rates, lethal outcomes, degree of ischemia, pain-free walking distance, ankle-brachial index (ABI), transcutaneous oxygen tension (tcp02), blood velocities, and neurological symptom score (NSS) were assessed within 1 year after treatment. Results - experimental part: epithelization and granulation rates were higher in diabetic animals treated with pCMV-VEGF165 (p=0.038). Histological and immunohistochemical studies have revealed a better multi-caliber vessel growth in rats which received gene therapy. Clinical part: after one year amputation rates and lethal outcomes were lower in patients in group III (16.7% of amputations as compared to group I, p=0.041; 13.3% of lethal outcomes). When assessing the results of treatment among the patients who received conservative treatment, amputation rates and lethal outcomes were lower in subjects treated by gene transfer (36% of amputations, p = 0.0496; 16% of lethal outcomes). Postprocedural patency rates at 6 months were comparable between the patients in groups I and III (66.7% and 70%, respectively), while the number of amputations was significantly lower in subjects treated by pCMV-VEGF165 gene transfer (33.3% and 86.7% of amputations in groups III and I, respectively, p=0.013). When comparing the difference in pain-free walking distance, better results were obtained in patients in group IV as compared to group II (p=0.032). When comparing the perfusion parameters and neurological score between the patients in groups II and IV, ABI was not affected by gene therapy, while the subjects in group IV had improved tcpO2 values, blood velocity, and NSS score (p=0.028, p=0.047, p=0.044, respectively). Conclusion - 1. pCMV-VEF165 gene transfer leads to improved wound healing rates and increased vascularization of granulation tissue in diabetic experimental animals (p=0.038). 2. Gene therapy improves perfusion and increases limb salvage rates in diabetic patients with CLI when used in combination with arterial revascularization or conservative therapy within 1 year after treatment.