The goal of this proposal is to delineate the signals involved in coronary collateral growth or non-budding angiogenesis. The general thesis is that collateral growth is critically dependent on the time-dependent expression of specific growth factors and their receptors. The PI hypothesizes that: At the onset of ischemia: a. angiogenic factors regulated by hypoxia (insulin-like growth factor-2 [IGF-2] and vascular endothelial growth factor [VEGF] and the cellular redox state (transforming growth factor beta-1 [TGF-beta 1] and basic fibroblast growth factor [beta FGF]) are expressed. b. receptors for these angiogenic factors will be expressed (IGF-II receptor, flk-1 receptor for VEGF, TGF receptor Types I and II, FGF-receptor-1). During angiogenesis, and establishment of sufficient perfusion to ameliorate ischemia in the occluded territory: a. angiogenic factors regulated by shear stress (platelet derived growth factor-B [PDGF-B] and its receptors (PDGF-alpha and -beta receptor), and endothelial nitric oxide synthase [eNOS]) will be expressed. b. angiogenic factors and their receptors regulated by hypoxia and redox state will have their expression return toward baseline. After achievement of normalized perfusion and coronary vasodilator reserve to the occluded territory: a. proteins and genes that are markers of endothelial and vascular smooth muscle cell quiescence (gax, gas-1) will be expressed. b. growth factors and their receptors associated with angiogenesis and/or cellular proliferation will have their expression return to baseline. Coronary angiogenesis will be induced in chronically-instrumented dogs by 2 minute repetitive coronary artery occlusions: 8 occlusions/day, 7 days/week. Angiogenesis will be evaluated at several different times: 1. Early response: 2, 24, and 48 hours after initiation of the occlusions; 2. Rapid growth phase: 7, 10, and 14 days after initiation of the occlusions; and 3. Maintenance phase: 18 and 21 days after initiation of the occlusions when collateral conductance enables flow to reach levels comparable that in the normal zone; and 4. Final growth phase: 28 and 35 days after initiation of occlusions when collateral conductance allows for vasodilator reserve and growth ceases. Sham control groups will be studied in addition to the animals receiving the repetitive ischemic stimuli. Collateral conductance will be evaluated by measurements of myocardial function in the occluded territory, collateral flow (radioactive microspheres), and diminution of reactive hyperemic responses following the occlusions. Mitogens associated with angiogenesis will be assayed from myocardial interstitial dialysate of the normal and ischemic vascular regions (cell proliferation, tube formation, Western analysis). Northern analysis, and reverse transcriptase polymerase chain reaction will be used to evaluate expression of the specific transcripts in the myocardium and vasculature. This approach which integrates a range of techniques from the molecular to physiological levels will facilitate a complete understanding of the signals associated with coronary angiogenesis and the impact of hypertension on these responses.
