PROJECT SUMMARY Refractory wounds in diabetic patients often result in amputation. Bone marrow derived endothelial progenitor cells (EPCs) actively participate in wound repair through angiogenesis after homing to the wounding site. However, progenitor cell functions are impaired in diabetes with mechanisms poorly understood. Reactive carbonyl species (RCS) are the intermediates and by-products generated during energy metabolism. Our pilot studies demonstrate one of the most potent RCS and the major precursor of the advanced glycation endproducts (AGE), methylglyoxal (MGO), exerted immediate inhibitory effects on progenitor cell functions in vitro. The glyoxalase I (GLO1), the key enzyme detoxifying MGO, was deficient in diabetic EPCs. These observations unveil an important message: Theses RCS actually play a major role in compromising progenitor cell function in diabetes, and this is due to the deficient glyoxalase defense system. The Major Goal of this project is to understand the molecular mechanisms of disrupted angiogenesis induced by RCS and to identify therapeutic targets for diabetic wound repair. Our recent report has demonstrated that an endoplasm reticulum response sensor, Inositol-Requiring Enzyme 1α (IRE1α), is essential to progenitor cell-mediated angiogenesis during wound repair. The endothelial-specific deletion of IRE1α leads to aberrant wound angiogenesis in vivo. However, how IRE1α functionality in EPCs is damaged in diabetes is not clear yet. Our pilot data strongly suggest that MGO directly diminishes IRE1α’s ribonuclease (RNase) function, and that IRE1α activation in EPCs is severely inhibited by MGO but rescued by GLO1 over-expression. We further found out that chronic wounds in diabetic animals started to heal upon receiving GLO1 gene transfer in vivo. Based on these findings, we propose Central Hypothesis that accumulated MGO in diabetes compromises progenitor cell function via interfering with IRE1α function, resulting in disrupted angiogenesis and delayed wound healing. To test the hypothesis, we propose Three Specific Aims: 1) Elucidate mechanisms by which MGO causes EPC dysfunction and IRE1α deficiency in diabetes in vitro; 2) Determine the molecular basis for MGO-induced IRE1α deficiency in vitro; 3) Determine the therapeutic effects of lowering MGO in diabetic wound healing in vivo. Our proposed studies will use newly developed Liquid chromatography–mass spectrometry (LC-MS) protocol to quantify free MGO accumulation in human plasma and diabetic foot ulcer tissues, representing the first effort to acquire the dynamic changes of free MGO generation in the microenvironment. We will employ both gain-of-function and loss-of-function technologies for gene manipulations, IRE1α gene engineered animals, and a newly established chronic diabetic wound animal model with cell therapies. Our project will allow us to uncover novel molecular mechanisms of impaired angiogenesis and wound healing in diabetes in which RCS-induced prog...