CONSTITUTIVE EXPRESSION OF PHVEGF(165) AFTER INTRAMUSCULAR GENE-TRANSFER PROMOTES COLLATERAL VESSEL DEVELOPMENT IN PATIENTS WITH CRITICAL LIMB ISCHEMIA

Background - Preclinical studies have indicated that angiogenic growth factors can stimulate the development of collateral arteries, a conce pt called ''therapeutic angiogenesis.'' The objectives of this phase 1 clinical trial were (1) to document the safety and feasibility of int ramuscular gene transfer by use of naked plasmid DNA encoding an endot helial cell mitogen and (2) to analyze potential therapeutic benefits in patients with critical limb ischemia. Methods and Results - Gene tr ansfer was performed in 10 limbs of 9 patients with nonhealing ischemi c ulcers (n = 7/10) and/or rest pain (n = 10/10) due to peripheral art erial disease. A total dose of 4000 mu g of naked plasmid DNA encoding the 165-amino-acid isoform of human vascular endothelial growth facto r (phVEGF(165)) was injected directly into the muscles of the ischemic limb. Gene expression was documented by a transient increase in serum levels of VEGF monitored by ELISA. The ankle-brachial index improved significantly (0.33 +/- 0.05 to 0.48 +/- 0.03, P = .02); newly visible collateral blood vessels were directly documented by contrast angiogr aphy in 7 limbs; and magnetic resonance angiography showed qualitative evidence of improved distal flow in 8 limbs. Ischemic ulcers healed o r markedly improved in 4 of 7 limbs, including successful limb salvage in 3 patients recommended for below-knee amputation. Tissue specimens obtained from an amputee 10 weeks after gene therapy showed foci of p roliferating endothelial cells by immunohistochemistry. PCR and Southe rn blot analyses indicated persistence of small amounts of plasmid DNA , Complications were limited to transient lower-extremity edema in 6 p atients, consistent with VEGF enhancement of vascular permeability. Co nclusions - These findings may be cautiously interpreted to indicate t hat intramuscular injection of naked plasmid DNA achieves constitutive overexpression of VEGF sufficient to induce therapeutic angiogenesis in selected patients with critical limb ischemia.