Alleviating Reactive Carbonyl Species-Induced Progenitor Cell Dysfunction in Diabetic Wound Healing

项目总结 糖尿病患者的难治性伤口经常导致截肢。骨髓源性内皮祖细胞 细胞(EPC)在归巢到创伤部位后，通过血管生成积极参与创伤修复。 然而，糖尿病患者的祖细胞功能受损，其机制尚不清楚。反应性 羰基物种(RCS)是能量代谢过程中产生的中间产物和副产物。我们的飞行员 研究表明，最有效的RCS之一，也是晚期糖基化终末产物的主要前体 甲基乙醛(MGO)对体外培养的造血祖细胞功能有即刻抑制作用。这个 糖醛酸酶I(GLO1)是MGO解毒的关键酶，在糖尿病内皮祖细胞中缺乏。这些观察结果 揭示一个重要的信息：这些RC实际上在损害造血祖细胞功能方面发挥着重要作用 糖尿病，这是由于乙二酸酶防御系统的缺陷。这个项目的主要目标是 了解RCS诱导血管生成障碍的分子机制并确定治疗方法 糖尿病伤口修复的靶点。我们最近的报告表明，内质网反应 肌醇需要酶1α(IRE1α)是一种感受器，在前体细胞介导的血管生成中起着至关重要的作用。 伤口修复。IRE1α内皮细胞特异性缺失可导致体内创面血管生成异常。然而， 糖尿病时内皮祖细胞的IRE1α功能如何受损尚不清楚。我们的试点数据强烈表明 氧化镁直接抑制IRE1α的S核糖核酸酶功能，使EPC中IRE1α激活严重 被MGO抑制，但被GLO1过表达拯救。我们进一步发现糖尿病患者的慢性创面 动物在体内接受GLO1基因转移后开始愈合。根据这些研究结果，我们建议中环 糖尿病患者体内累积氧化镁通过干扰IRE1α影响祖细胞功能的假说 功能，导致血管生成受阻，延迟伤口愈合。为了检验这一假设，我们建议 三个具体目标：1)阐明氧化镁导致内皮祖细胞功能障碍和IRE1α缺陷的机制 2)体外确定氧化镁诱导的IRE1α缺陷的分子基础；3)确定 降镁在糖尿病创面愈合中的体内治疗作用我们建议的研究将使用新的 建立了液质联用(LC-MS)方法来定量体内游离氧化镁的累积 人血浆和糖尿病足溃疡组织，代表了首次努力获得的动态变化的游离 在微环境中生成氧化镁。我们将同时采用功能增益和功能损耗技术 对于基因操作，IRE1α基因工程动物和新建立的慢性糖尿病伤口动物 接受细胞疗法的模特。我们的项目将使我们能够发现受损的新的分子机制 糖尿病患者的血管生成和创面愈合，其中RCS诱导的祖细胞功能障碍正在发挥作用 举足轻重的角色。该项目的发现将为开发新的治疗方法提供有价值的信息。 糖尿病创面愈合通过增强RCS清道夫GLO1或ER应激反应传感器IRE1α。

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 progenitor cell dysfunction is playing a pivotal role. Findings from this project will provide valuable information for novel therapeutics development for diabetic wound healing by augmenting RCS scavenger GLO1 or ER stress response sensor IRE1α.