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 1) VEGF overexpression in skeletal muscle at therapeutic doses induces angiogenesis by intussusception and not by sprouting; 2) Notch-1 regulates the initial stages of intussusception by a different pattern of activation compared to sprouting; 3) EphrinB2/EphB4 signaling is a crucial determinant of the switch between normal and aberrant angiogenesis by different VEGF doses.

Specific aims. In the current funding period we propose to extend these results to investigate whether: 1) PDGF-BB co-expression induces normal angiogenesis by switching from intussusception to sprouting; 2) PDGF-BB normalizes aberrant VEGF-induced angiogenesis by preventing excessive Notch activation; and 3) stimulation of EphB4 signaling can prevent aberrant angiogenesis by high levels of VEGF alone.

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.

Expected value of the proposed project. The experiments proposed are expected to provide fundamental knowledge on the basic mechanisms by which pericyte recruitment regulates the dose-dependent effects of VEGF and the induction of normal or aberrant angiogenesis, identifying novel and possibly more specific molecular targets to modulate the effects of VEGF gene delivery for the treatment of ischemic diseases. One such target (EphrinB2/EphB4 signaling) will be tested for its therapeutic potential in a clinically relevant gene therapy approach.