Angiogenesis therapy has been described as a "biological bypass", the idea being that through administration of agents capable of inducing collateralization, a more natural type of "bypass" can be achieved. Indeed it has been observed that ischemic muscles secrete angiogenic factors in response to hypoxia and that to some extent natural angiogenesis does occur in animal models of CLI and in humans [5, 6]. One of the angiogenic factors noted in many ischemic conditions, including cardiac ischemia, stroke, and CLI is vascular endothelial growth factor (VEGF) [7–9]. In 1994, Isner's group sought to enhance ischemia-associated angiogenesis using single bolus intra-arterial administration of VEGF-165 in a rabbit model of CLI. Rabbits with resected femoral arteries demonstrated augmentation of perfusion, increased capillary density, and overall better function as compared to control rabbits . Subsequent experiments sought to optimize therapeutic effect using different dosing schedules. Daily VEGF-165 administration for 10 days subsequent to ligation and resection of the external iliac artery and femoral artery, respectively was performed . Not only was dose-dependent increase in collateralization observed, but rabbits receiving the highest dose of VEGF-165 (1 mg) had no incidence of calf muscle atrophy and distal limb necrosis, whereas this was present in 85.7% of control rabbits. A similar study in the rabbit model of CLI demonstrated superior benefit in terms of limb function in animals receiving VEGF by osmotic pump for 28-days in comparison to nitroglycerin or saline treatment . A variety of studies have been performed with VEGF protein which confirmed the proangiogenic, as well as angiogenesis-independent muscle reparative properties in the limb ischemia model [13–15]. These positive preclinical data unfortunately were not successfully reproduced in the clinic. Trials using VEGF protein , or DNA, did not show significant benefit at reducing leg amputations in a double blind setting [15, 17]. One of the concerns with VEGF therapy is its vasodilatory effects, as well as induction of blood vessels that are relatively immature and "leaky".
Another approach involved use of the cytokine fibroblast growth factor-1 (FGF-1). Given that FGF-1 is considered "upstream" of VEGF, it is believed to stimulate numerous angiogenic processes so as to result in creation of more mature vessels . Indeed, it is believed that one of the differences between tumor neo-vasculature, which is characteristically leaky, and neo-vasculature associated with physiological angiogenesis, is the ratio of VEGF to FGF . Specifically, it was demonstrated both in animal models  and in clinical trials  that muscular ischemia is associated with up-regulation of FGF gene transcription, suggesting that this is part of the endogenous pro-angiogenic response to ischemia. The critical role of FGF in endogenous angiogenesis was conclusively demonstrated in FGF knockout mice, which displayed inhibited ability to heal post-wounding . Although FGF-1 gene therapy has clinically been used in CLI patients with some improvement in ABI and perfusion, statistically powered randomized clinical trials have not been conducted to date .
It is known that additive and/or synergistic effects are observed in terms of neoangiogenesis when several angiogenic factors are combined. For example, Cao et al demonstrated synergy between administration of PDGF-BB and FGF-2 in terms of increasing blood vessel formation and function in the femoral artery ligation model in rats and rabbits . Similarly, in cancer angiogenesis, it is known that several tumor-derived angiogenic factors synergize for acceleration of neovascularization . Accordingly, investigators have attempted to activate upstream mediators of several angiogenic signals through transfection of genes encoding transcription factors such as HIF-1 alpha . In fact, this approach has been demonstrated to be superior to VEGF gene administration in terms of new capillary sprouting. In a Phase I dose-escalating trial, transfection of HIF-1 alpha into CLI patients demonstrated tolerability with some indication of efficacy .
In conclusion, while administration of angiogenic factors to patients with CLI does induce some benefit in early trials, data from randomized trials to date do not support widespread use. The transfection of upstream transcription factors such as HIF-1 alpha is a promising approach since it mimics natural angiogenesis in that a plurality of growth factors are induced following transfection [26, 28]. However clinical results are too premature to draw firm conclusions. Additionally, the transfection of foreign genes may possess unintended consequences in the long run due to the uncontrolled nature of the transfected gene insert.