Critical limb ischemia is estimated to develop in 500 to 1000 individuals per million per year. Despite maximal pharmacological therapy and major advances in both percutaneous and surgical techniques, the disease frequently follows an inexorable down-hill course. There is an important group of patients, who are not candidate to conventional revascularization therapies. Thus, the need for alternative strategies is compelling.

Therapeutic angiogenesis is a novel concept consisting in the use of angiogenic growth factors to expedite and/or augment collateral artery development in ischemic tissues. Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, has been shown to promote therapeutic angiogenesis in animal models of hindlimb and myocardial ischemia.

Ischemic skeletal muscle is advantageous for taking up and expressing foreign genes transferred as naked plasmid DNA. In this regard, gene therapy operates as a sustained delivery system. The present study was designed to test the hypothesis that intramuscular administration of naked plasmid DNA encoding the 121-amino acid isoform of VEGF could augment collateral development and tissue perfusion in a dog model of hindlimb ischemia.

The human VEGF₁₂₁ gene was inserted into the expression vector pAEC-D2, under control of the human citomegalovirus promoter. The resultant plasmid, named pVEGF₁₂₁, was purified to transfect mammal CHO-K1 cells. These cells expressed biologically active VEGF₁₂₁ in the culture supernatant. Human microvascular endothelial cells grown in the presence of VEGF₁₂₁-containing supernatants became elongated, forming thin cords of interconnecting cells. These data demonstrate that VEGF₁₂₁ is able to mediate dramatic cell reorganization, which would be necessary in vivo for endothelial cell sprouting and tube formation.

pVEGF₁₂₁ was assayed for in vivo angiogenesis in a dog hindlimb ischemia model. Animals were subjected to a surgical procedure in order to induce unilateral ischemia. An interval of 10 days was permitted for spontaneous development of collateral vessels. Then, animals received intramuscular injections of pVEGF₁₂₁ directly in the ischemic muscles. Anatomic and physiological effects of VEGF₁₂₁ gene transfer were evaluated by angiography, calf blood pressure ratio and vasomotor reserve analyses.

Thirty days after gene transfer (day 40), angiographically recognizable collateral vessels were increased in pVEGF₁₂₁-treated animals compared with controls. At day 40, calf blood pressure ratio was significantly higher for pVEGF₁₂₁ versus controls (P<0.01). Moreover, when compared with day 10 (baseline), a significant increase was found at day 40 in pVEGF₁₂₁ group (P<0.01), but not in the control group. These results indicate an improvement in blood flow at large and medium diameter vessels.

Vasodilator responses were assessed by intravenous infusion of 2 mg nitroglycerine. They were classified as total, partial, or no response. At baseline, no animal showed response. At day 25, 50% of control animals partially responded, while 67% of pVEGF₁₂₁-treated animals were partial or total responders. By day 40, responder animals remained 50% for the control group. In contrast, 100% of pVEGF₁₂₁-treated animals showed partial or total responses. These data suggest amelioration in blood availability at the microcirculation level.

An important set of hematological variables were measured in order to evaluate possible side effects of pVEGF₁₂₁ on animal homeostasis. The statistical analysis showed no significant differences between groups for all hematological variables at all measurement points. Moreover, all variables remained within or close to the physiological ranges. This finding suggests a safety feature of our procedure, in that no side effects could be associated to the administration of pVEGF₁₂₁ plasmid.

The present research provides evidences that intramuscular injection of naked plasmid DNA encoding VEGF₁₂₁ is an effective method for gene transfer in a dog model of hindlimb ischemia. The expression of VEGF₁₂₁ results in increased collateral vessel development and tissue perfusion, with no adverse side effects. Our results suggest the feasibility of pVEGF₁₂₁ plasmid to promote therapeutic angiogenesis in patients with critical limb ischemia. Such a strategy may be suitable in patients who are not candidate to conventional revascularization procedures.