DESCRIPTION (provided by applicant): Refractory wounds in diabetic patients often result in amputation. Endothelial progenitors cells (EPC) actively participate in wound repair through angiogenesis after homing to the wounding site. However, EPC functions are impaired in diabetes with mechanisms poorly understood. Our pilot studies demonstrate a reduced activity of an important ER sensor ER sensor, Inositol requiring enzyme 1 (IRE1), and a decreased expression of a potent angiogenic factor angiopoietin-1 (ANGPT1) in EPCs from human and mice with type 2 diabetes, contribute to EPC dysfunction. Most importantly, our preliminary studies show that IRE1α suppresses a subset of microRNA (miR) clusters known to be detrimental to angiogenesis. We speculate that this suppression is due to IRE1α's direct splicing activity to precursor miRs (pre-miRs), resulting in depletion of functional miRs. Of particular interest, our preliminary data demonstrate that IRE1α deficiency significantly increased miR-200 family in EPCs from diabetic mice, leading to down-regulation of one of their target genes - ANGPT1. Yet how IRE1 regulates EPC function and wound repair in diabetes is unknown. Therefore, the objective in this proposal is to determine the mechanisms of IRE1 regulation of angiogenesis and wound repair. The hypothesis, built upon our extensive preliminary studies and published work, is that deficiency in IRE1α, leads to insufficient degradation of pre-miRNA in diabetes, resulting in impaired endothelial progenitor cell (EPC) angiogenesis and delayed wound healing. Our hypothesis will be tested in three specific aims: 1) Determine the molecular mechanisms underlying the essential role of IRE1α in maintaining EPC function in diabetes in vitro; 2) Determine how IRE1α-targeted pre-miR modulates EPC function in vitro; 3) Determine how IRE1α improves wound healing in diabetes in vivo. Our approaches encompass in vitro and in vivo studies utilizing adenovirus-mediated gene manipulations, whole genome RNA profiling, IRE1α floxed mice and newly generated endothelium-specific IRE1α knockout mice. Furthermore, human EPCs will be obtained from type 2 diabetic patients and healthy subjects in order to determine the levels of IRE1α pathway and miRs. The proposed study is significant, because it will uncover a previously unrecognized role of the ER stress response in impaired angiogenesis and wound healing in diabetes at the translational level. The investigation of IRE1α-mediated regulation of stress miRs will open a new paradigm for the study of the molecular mechanisms responsible for pathogenesis of refractory wounds, which will enable future development of therapeutics for this devastating situation affecting millions of Americans.