Gene Therapy Using a VEGF165/Ang-1 Plasmid Construct: Evaluation of Vascular Density in Chronic Hindlimb Ischemia in Rats

Abstract

Introduction . The prevalence of chronic lower limb ischemia among individuals over 50 years of age does not exceed 8 % . However, in the presence of risk factors such as smoking, diabetes mellitus, or hypertension, the risk of its development increases to 30 % . This study evaluates the efficacy of therapeutic angiogenesis using a combined circular DNA-based construct. Objective . To determine, in an in vivo experiment, the features of the dynamics of blood vessel density changes in the ischemic muscles of the rat hind limb after local application of a plasmid construct containing gene segments encoding vascular endothelial growth factor 165 and angiopoietin-1 (pcDNA_VEGF165/Ang-1). Materials and Methods . Three observation groups were formed: «CHI» (chronic hindlimb ischemia without treatment; n=12), «CHI + PSS» (administration of pyrogen-free physiological saline, 200 μL; n=12), and the experimental «CHI + VEGF165/Ang-1» group (administration of pcDNA_VEGF165/Ang-1 at a dose of 100 μg/animal; n=24). The solutions were administered once intramuscularly into the animal’s right hindlimb. An immunohistochemical examination of the skeletal muscle tissue of the thigh and lower leg was performed using monoclonal antibodies against blood vessel endothelial cells (CD31) and against smooth muscle α-actin (α-SMA). Results . The use of the combined VEGF165/Ang-1 plasmid led to a statistically significant stimulation of angiogenesis and arteriogenesis, which made it possible to completely restore vascular density in the ischemic limb to the level of a healthy limb. In contrast to the persistent ischemic decrease in the number of microvessels in the control groups («CHI» and «CHI + PSS»), gene therapy ensured a steady increase in the vascular network. By day 28, the density of CD31-positive capillaries in the thigh and lower leg increased by 55.3  % and 33.3  %, respectively, and the density of α-SMA-positive arterioles increased by 42.2  % and 44.5  %, respectively, reaching normal values. Conclusion . The use of a domestic plasmid construct containing the genes of the angiogenesis-activating proteins VEGF165 and Ang-1 (pcDNA_VEGF165/Ang-1) in chronic hindlimb ischemia in rats accelerates capillarogenesis and arteriogenesis in ischemic lower leg muscles starting from day 14, and in the thigh muscles from day 28 after administration, as evidenced by a significant increase in the density values of CD31-positive capillaries and α-SMA-positive arterioles.