Background. Ischemic cardiovascular disease is the most common cause of death in the western world and, despite advances in medical and surgical therapy, the morbidity and mortality remain very high. Therapeutic angiogenesis aims to induce the formation of new blood vessels to improve the perfusion of ischemic tissue in patients with end-stage coronary artery or peripheral arterial disease that are not amenable to other treatment options. Vascular Endothelial Growth Factor-A (VEGF) is the most potent and specific angiogenic factor and it has been tested in several clinical trials with a variety of delivery methods. However, preclinical studies clearly demonstrated that high doses of VEGF can also induce aberrant vascular structures which resemble cavernous hemangiomas and grow progressively. The results of placebo-controlled clinical trials have been disappointing and yielded mostly negative results. The main conundrum of VEGF delivery-based strategies for therapeutic angiogenesis is its apparently limited therapeutic window in vivo, such that low doses are safe, but mostly inefficient, and higher doses become rapidly unsafe. Rationale. Our previous results show that the transition between normal and aberrant angiogenesis takes place in an all-or-none fashion across a discrete threshold level of VEGF expression. However, we found that this threshold is not an intrinsic property of VEGF dose alone, but rather depends on the balance between angiogenic stimulation by VEGF and vascular maturation by PDGF-BB-mediated pericyte recruitment. Our results in the previous funding period show that coordinated co-expression of PDGF-BB and pericyte recruitment effectively improves both safety and efficacy of VEGF gene delivery, by inducing robust normal and homogeneous angiogenesis despite heterogeneous and high VEGF levels. Specific aims. In the current funding period we propose to extend these results along two lines: 1) to apply these biological concepts in a clinically applicable gene therapy strategy for therapeutic angiogenesis, and 2) to investigate the mechanism underlying the modulation of VEGF effects by PDGF-BB co-expressio. We will dissect the role of pericytes and specific pericyte-mediated signaling pathways in the normalization of VEGF-induced angiogenesis by PDGF-BB (Aim 1). Further, we will investigate whether dose-dependent co-expression prevents angioma growth and leads instead to normal capillary networks by regulating the signaling pathways that control the endothelial cell fate decision to become a sprouting tip cell or a circumferentially growing stalk cell (Aim 2). We will then test the feasibility of AV and AAV-based coordinated co-expression of VEGF and PDGF-BB as gene therapy strategies to achieve safe and efficacious angiogenesis (Aim 3). Experimental design. Monoclonal populations of retrovirally-transduced myoblast, which stably secrete different amounts of VEGF or VEGF+PDGF, will be used to achieve specific expression levels in skeletal muscle. We will then co-express specific molecules to block or activate specific pathways involved in the pericyte-endothelium and endothelium-endothelium cross talk. Finally we will test the applicability of VEGF+PDGF co-delivery to a clinically relevant delivery system using a bicistronic AV or AAV vector. Expected value of the proposed project. The proposed research is expected to provide the basic preclinical testing of a novel therapeutic angiogenesis strategy based on coordinated targeting of both vascular induction and maturation. Furthermore, these experiments are expected to dissect the mechanisms by which PDGF-BB signaling regulates endothelial cells and blood vessel growth and to identify alternative molecular targets, which might provide more specific therapeutic effects to modulate the dose-dependent effects of VEGF gene delivery.