Angiogenic Therapy for Critical Lower Limb Ischemia

Background. In several preclinical and clinical studies, VEGF gene therapy has proven successful in the treatment of critical limb ischemia (CLI). CLI is estimated to develop in 500-1000 individuals/million/year. It is a severe disease associated with a high risk of amputation and mortality. Therapeutic angiogenesis is a novel concept consisting of the use of proangiogenic growth factors to promote collateral artery development in ischemic tissues. To test a potential clinical application of this method, the authors prepared a nonviral expression vector encoding VEGF(165) protein. Objectives. The purpose of this study was to construct a functional expression vector encoding isoform VEGF(165) which can be used for therapeutic angiogenesis in no-option patients with critical limb ischemia. Material and Methods. Total RNA was extracted from vein and RT-PCR was used to prepare a cDNA sequence for cloning into the plasmid vector. The naked pcDNA3/VEGF(165) plasmid was administered alone (group I) or combined with mononuclear cells (group II) directly into the skeletal muscle of the ischemic lower limb. The vector's ability of expression in mammalian cells, clinical outcome, serum level of VEGF protein, and endothelial cell proliferation in muscle tissue were evaluated. Results. pcDNA3/VEGF(165) was expressed in transfected CHOPro5 cells. Due to the gene transfer, 9 of 24 patients did not require amputation. A higher serum VEGF concentration was observed than in healthy controls and the level of cytokine increased on day 14 and decreased on day 90 after plasmid administration. Histological and immunohistochemical analysis of the muscles derived from amputated limbs revealed the presence of VEGF protein and signs of new blood vessel formation. Conclusions. This therapy is safe, but a single intramuscular gene transfer of VEGF(165) is insufficient to promote angiogenesis efficiently. Better results were observed when combined therapy was used (Adv Clin Exp Med 2010, 19, 3, 347-359).