Proof that therapeutic angiogenesis could be successfully extended to human subjects was first demonstrated using gene transfer of naked DNA encoding for VEGF (phVEGF) for the treatment of critical limb ischemia (9). Using a dose-escalating design, treatment was initiated with 100 µg of phVEGF. Three patients presenting with rest pain (but no gangrene) and treated with 1,000 µg of phVEGF were subsequently shown at 1-year follow-up to have improved blood flow to the ischemic limb as well as no rest pain. With the increase in dose of phVEGF to 2,000 µg, angiographic and histologic evidence of new blood vessel formation became apparent (9). More recently, the use of intramuscular gene transfer, employed initially as a means of treating patients in whom vascular disease in the ischemic limb was too extensive to permit an intra-arterial approach, achieved marked improvement in collateral vessel development in patients with critical limb ischemia (10). Objective findings of bioactivity in this preliminary report included improvement in the ankle-brachial index, angiographic evidence of newly visible collateral blood vessels, and demonstration by magnetic resonance angiography of improved lower extremity blood flow. Ischemic ulcers healed or markedly improved in 4 of 7 limbs, including successful limb salvage in 3 patients recommended for below-knee amputation.

Successful application of both gene transfer and recombinant protein administration for the treatment of myocardial ischemia in human subjects was reported in 1998. Gene transfer involved direct intramyocardial injection of phVEGF as sole therapy for myocardial ischemia refractory to conventional therapy (11). Among 28 consecutive patients treated with this strategy to date, anginal episodes requiring sublingual nitroglycerin were reduced from nearly 60 per week to less than 3 per week. Objective evidence of improved perfusion was documented by a near doubling of treadmill exercise time and improved myocardial blood flow on stress and resting nuclear perfusion scans. The improvement in perfusion observed at rest is consistent with resolution of hibernating myocardium, a finding that has been recently confirmed using catheter-based electromechanical mapping (Vale, P., and Losordo, D., unpublished data). Recombinant protein administration using FGF-1 has also been reported to augment myocardial revascularization and improve functional status in patients undergoing concurrent coronary artery bypass surgery (12). In vitro studies have suggested certain mechanisms that may have contributed to the apparent benefit and safety of phVEGF gene transfer in these early trials. While ECs were previously viewed solely as the target for VEGF, it is now clear that ECs subjected to hypoxia can synthesize VEGF as well (13). This autocrine feature of VEGF creates the opportunity for amplifying the effects of even a small amount of exogenous VEGF, as EC proliferation in the ischemic territory creates additional potential cellular sources of VEGF synthesis and secretion. Moreover, the recent observation that VEGF may upregulate its own receptor (VEGFR-2, or KDR)(14) establishes a second basis for autocrine and paracrine amplification. VEGF has also been shown to inhibit EC apoptosis by activating the serine-threonine protein kinase Akt through a process requiring integrin ligation (15). This finding suggests a mechanism other than mitogenesis by which a net increase in EC viability may be accomplished. Given the limited 2-to 4-fold increase shown for VEGF on cellular proliferation, it is possible that the contribution of enhanced EC survival under conditions of severe ischemia is …