Role of Inositol requiring enzyme 1 in regulating angiogenesis for diabetic wound repair.

 描述（由申请人提供）：糖尿病患者的难治性伤口通常导致截肢。内皮祖细胞（EPC）归巢至创伤部位后，通过血管生成积极参与创伤修复。然而，EPC功能在糖尿病中受损，其机制尚不清楚。我们的初步研究表明，一个重要的ER传感器ER传感器，肌醇需要酶1（IRE 1）的活性降低，以及一个有效的血管生成因子血管生成素-1（ANGPT 1）在人和2型糖尿病小鼠的EPC中的表达降低，导致EPC功能障碍。最重要的是，我们的初步研究表明，IRE 1 α抑制已知对血管生成有害的microRNA（miR）簇的子集。我们推测，这种抑制是由于IRE 1 α直接剪接到前体miR（pre-miR）的活性，导致功能性miR的耗尽。特别有趣的是，我们的初步数据表明，IRE 1 α缺陷显著增加了糖尿病小鼠EPCs中miR-200家族的表达，导致其靶基因之一ANGPT 1下调。然而，IRE 1如何调节糖尿病中的EPC功能和伤口修复尚不清楚。因此，本研究的目的是确定IRE 1调节血管生成和伤口修复的机制。基于我们广泛的初步研究和已发表的工作，该假设是IRE 1 α的缺陷导致糖尿病中pre-miRNA降解不足，导致内皮祖细胞（EPC）血管生成受损和伤口愈合延迟。我们的假设将在三个特定的目标进行测试：1）确定IRE 1 α在体外维持糖尿病EPC功能中的重要作用的分子机制; 2）确定IRE 1 α靶向的pre-miR如何在体外调节EPC功能; 3）确定IRE 1 α如何在体内改善糖尿病伤口愈合。我们的方法包括利用腺病毒介导的基因操作、全基因组RNA分析、IRE 1 α floxed小鼠和新产生的内皮特异性IRE 1 α敲除小鼠的体外和体内研究。此外，将从2型糖尿病患者和健康受试者中获得人EPC，以确定IRE 1 α通路和miR的水平。这项研究意义重大，因为它将在翻译水平上揭示ER应激反应在糖尿病血管生成受损和伤口愈合中的作用。对IRE 1 α介导的应激miR调节的研究将为研究难治性伤口发病机制的分子机制开辟新的范式，这将使未来能够开发这种影响数百万美国人的毁灭性情况的治疗方法。

 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.