Occlusive vascular disease of the heart, brain and peripheral limbs is the primary cause of morbidity and mortality in the US. Angiogenesis and growth (remodeling) of collateral vessels are major adaptations that limit end-organ damage. Yet, compared to angiogenesis, much less is known about the mechanisms directing collateral growth. And no studies have determined how or when COLs develop. Collateral density and remodeling vary widely among species and humans, suggesting a genetic basis. Yet nothing is known about the source of this variation. Our preliminary studies show that COLs develop during the late embryonic-to-early postnatal period. We also find that compared to C57BL6 (B6) mice, COL formation in BALB/c (BC) mice is impaired --most dramatically in the cerebral circulation where BC essentially lack COLs. These findings create an excellent opportunity to identify factors specifying COL formation. Although multiple genes are likely involved, our preliminary work shows that BC are deficient in VEGF-A and are polymorphic at/near its locus. This, plus data from mutant mice overexpressing VEGF, suggest the novel hypothesis that VEGF signaling is crucial in COL formation. This is the first factor to be identified that regulates COL density in normal tissues. Many signals postulated to regulate COL growth in adult ischemic disease are known to be important in angiogenesis, in particular VEGF. However, VEGF's role is controversial because of difficulties in previous studies in localizing manipulation of VEGF. Also, no studies have identified the source (s) of VEGF in COL growth. To address these problems, we have developed new methods permitting 1) non-invasive imaging of COL perfusion and 2) local manipulation of VEGF signaling at the site of COL formation. Our preliminary results in the mouse hindlimb model suggest VEGF is a critical determinant of COL remodeling in ischemia. This proposal will investigate the overall hypothesis that COLs form perinatally, that VEGF signaling is central to this process, and that VEGF is also a key determinant of COL enlargement in adult ischemic disease. Aim I will determine how and when collaterals develop. Nothing is known about this fundamental process. We will also test the hypothesis that VEGF is critical in specifying collateral formation. We will characterize perinatal COL development and test the hypothesis that VEGF signaling is a key determinant and identify VEGF-R1 and -R2 involvement. Aim II will use array and genomic mapping to identify polymorphisms and VEGF-dependent and VEGF-independent candidate genes directing COL formation. Aim III will test the hypothesis that VEGF is important in collateral remodeling in adult ischemic disease, using the mouse hindlimb model, and will seek to identify the responsible mechanisms. We will use VEGF mutants, alter VEGF and its receptors, and identify the source of VEGF during COL growth. We hope our work will lead to therapies to stimulate formation of new COLs and augment growth of pre-existing COLs in occlusive vascular diseases. PUBLIC HEALTH RELEVANCE: Ischemic disease of the heart, brain and peripheral limbs, which is the most common cause of morbidity and death in western countries, is increasing worldwide, and many affected individuals are not candidates for percutaneous or bypass revascularization and are deemed "no option" (or face amputation) because of multiple vessel disease, diffuse disease or adverse preconditions. Therapies to augment angiogenesis (increase in capillary density) and arteriogenesis (enlargement of collaterals) have tremendous potential to treat ischemic disease. While several "second generation" angiogenesis trials are underway, development of therapies directed at arteriogenesis, which involves different mechanisms that are much less well understood, cannot come without a better understanding of the genetic and physiological mechanisms that specify collateral formation in normal tissue and that regulate their enlargement in ischemic disease - the specific aims of this proposal.