Therapeutic angiogenesis involves the transfer of angiogenic growth factors to ischemic tissues to promote the growth of new blood vessels. It shows promise for the treatment of both peripheral limb ischemia andl myocardial ischemia. Current protocols including all of the clinical trials in progress use plasmid or adenoviral vectors with strong unregulated viral promoters to deliver and express vascular endothelial growth factor: (VEGF) or fibroblast growth factor genes. Powerful promoters are used because of the transient lifetime of the: vectors and the requirement for high-level short-term expression. Disadvantages include an absence of directional cues for vessel growth and lack of control over the local dose and time of exposure to the growth factors. Recent evidence suggests that this may lead to disorganized unstable vessels that are leaky and prone to form hemangiomas. We hypothesize that the signals for stimulating the growth of collateral vessels should emanate from the ischemic tissue and the duration of the stimulus should be related to the persistence of ischemia. This is the case during pathology-related angiogenesis in the adult where ischemic regions provide the directional and temporal cues for the growth of new vessels. We have developed a novel gene switch that provides such cues for pro-angiogenic growth factor genes delivered to ischemic muscle. The switch includes promoter elements that are activated by hypoxia and represser elements that conditionally silence gene expression under conditions of normal perfusion. We have confirmed the function of this switch in adenoviral vectors after transfer to ischemic skeletal and cardiac tissues. Studies are proposed here to characterize angiogenesis in models of PAOD and CAD in which the VEGF gene is delivered under the direction of the switch in semi-permanent AAV vectors. We propose to characterize the angiogenic pathway and determine the properties, function, and stability of the collateral vessels in these models. The spatial cues for collateral vessel growth, stability, and recruitment of endothelial progenitor cells will be analyzed in Matrigel plugs and intact muscle. These studies will provide a basis for evaluating the efficacy of adenoviral versus regulated vectors and address some of the outstanding mechanistic questions involved in therapeutic angiogenesis. ? ?
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