Project Summary Impaired vascular repair/angiogenesis is a major clinical problem in aged patients that often leads to ischemic peripheral artery and cardiovascular diseases. Recent studies have focused on strategies to improve aged cells? angiogenesis capability and subsequent tissue repair. MicroRNAs (miRNAs) have emerged as crucial regulators of vascular function. We propose that altered expression of miRNAs in hind limbs during aging contributes to the age-dependent decline in vascular function. Among the numerous miRNAs, miR-34a has shown promise as a biomarker for organ aging. It correlates with impaired function of bone marrow-derived mononuclear cells from patients with cardiovascular disease. Remarkably, the mechanism underlying miRNAs? regulation of angiogenesis, especially in aging, is not completely understood. Our central hypothesis is that the miR-34a retards angiogenesis in aging by directly inhibiting the GCH1/BH4 pathway resulting in excessive oxidative stress, which accelerates senescence, activates inflammasome, and suppresses mitophagy. Remarkably, little information exists on miRNAs regulation of angiogenesis, especially under aging. This hypothesis was formulated after a careful analysis of published work in the field and the generation of some key preliminary data in our laboratory. We plan to test our central hypothesis and accomplish our objective by pursuing three specific aims. In Aim 1, we will establish the critical role of miR-34a in regulation of angiogenesis in aging. Our working hypothesis is that miR-34a inhibits angiogenesis in aging via impairing endothelial progenitor cell (EPC) functions. In Aim 2, we will delineate the effect of the GCH 1/BH4 pathway on miR-34a induced EPC dysfunction in aging. We hypothesize that that miR-34a retards EPC functions in aging by directly inhibiting the GCH1/BH4 pathway, which results in excessive oxidative stress that decreases telomerase activity and accelerates senescence via suppressing Silent Information Regulator 1 (SIRT1). In Aim 3, we will determine how miR-34a affects mitophagy and inflammasome activation in aging after hind limb ischemia. Deficiency of GCH1/BH4 results in elevated reactive oxygen species (ROS) level, which may activate inflammasome, and thus inhibit mitophagy and accelerate the aging process. We hypothesize that miR-34a inhibits mitophagy in aging via Nlrp3 (NOD-like receptor family, pyrin domain containing 3) inflammasome activation, which can be triggered by accumulation of mitochondrial superoxide. The major significance of this study is that it will determine how miRNAs regulate angiogenic functions during aging. Once such knowledge is gained, it is possible that new modalities may be developed to therapeutically rescue impaired angiogenesis in the growing number of aging patients today.