Delayed wound healing in diabetic corneal epithelia: reduction in protein response after injury and uncoordinated cell-cell communication

无法愈合的角膜损伤影响高达70%的2型糖尿病患者，这是一个重要的原因 在这群人中失明的可能性。虽然有治疗方法可以改善不良的症状- 治愈角膜伤口，唯一的永久解决方案是角膜移植，这是一种不易获得的程序 全世界。通过检测参与伤口愈合反应的各种蛋白质在细胞内的变化 在这个层面上，我希望了解为何糖尿病患者的角膜不能有效愈合。当不是这样的时候 健康的角膜受到损伤，伤口旁的细胞会经历一些必要的变化，以协调 移位和伤口闭合。其中一个变化涉及嘌呤能受体P2X7的激活，在 细胞表面。当被激活时，P2X7产生一种特定的钙信号事件模式，从 通过激活离子通道PAnnexin-1实现细胞间的相互作用。这些传播的信令事件代表细胞- 细胞通讯，并最终导致细胞骨架蛋白重排和协调创伤 结案了。我们先前的研究已经发现，在2型糖尿病前期患者中，P2X7的异常定位和激活 模特们。我们有初步证据表明，受损的糖尿病细胞的信号特征缺乏 典型的P2X7信号反应。使用P2X7的特定激动剂证实了这一点，并观察到 糖尿病细胞的反应大大减弱。这项提议的目标是揭示细胞-细胞如何 P2X7信令级联中的通信事件受到调节，这种调节是如何在 糖尿病系统，以及这种调节变化如何影响肌动蛋白捆绑并最终影响细胞运动。我的 初步数据已经确定了一组细胞，我们推测这些细胞在启动时是控制者或引导者细胞 相邻小区中的通信事件和传播的信令事件在其 不在了。Aim 1将使用机器学习方法来调查这两个区域中是否存在这些领头细胞 糖尿病细胞培养和角膜模型。目标1还将讨论PAnnexin-1在血管生成中的作用 唯一的领导者细胞信号配置文件。此外，P2X7/PAnnexin-1信号的下游影响将是 应用三维电子显微镜研究创伤糖尿病患者和对照组肌动蛋白的排列 眼角膜。在目标2中，P2X7和PAnnexin-1蛋白和mRNA在角膜细胞中的定位 将对样品进行检查。这将产生关于糖尿病患者之间表达的两个一般趋势的数据 和对照组，以及单个样本内表达的差异，这可能解释功能 前导细胞和上皮片的其余部分之间的差异。此外，目标2将解决是否 P2X7和PAnnexin-1蛋白的共同定位(创伤前和创伤后)对于伤口修复是必要的。 这些目标加在一起将在我们对监管的理解方面取得重大进展 P2X7/PAnnexin-1信号转导通路及其在mRNA、蛋白质和功能水平上的变化 糖尿病，以及这些异常对肌动蛋白细胞骨架的下游影响。

Non-healing corneal injuries affect up to 70% of patients with type 2 diabetes, representing a significant cause of vision loss in this population. Although there are treatments available to improve the symptoms of poorly- healing corneal wounds, the only permanent solution is a corneal transplant, a procedure not readily available worldwide. By examining changes in various proteins involved in the wound healing response at the cellular level, I hope to understand why the corneas of diabetic patients fail to heal effectively. When an otherwise healthy cornea is injured, cells next to the wound experience a number of changes necessary for coordinated migration and wound closure. One change involves the activation of the purinergic receptor, P2X7, found on the cell surface. When active, P2X7 generates a specific pattern of calcium signaling events that travel from cell to cell through activation of the ion channel Pannexin-1. These propagated signaling events represent cell- cell communication, and ultimately lead to re-arrangement of cytoskeletal proteins and coordinated wound closure. Our previous studies have identified aberrant localization and activation of P2X7 in pre-type 2 diabetic models. We have preliminary evidence that the signaling profile of wounded diabetic cells lacks the characteristic P2X7 signaling response. This was confirmed using specific agonists to P2X7, and observing a greatly diminished response in diabetic cells. The goal of this proposal is to uncover how the cell-cell communication events in the P2X7 signaling cascade are regulated, how this regulation is thrown off in diabetic systems, and how this change in regulation affects actin bundling and ultimately cell motility. My preliminary data has identified a set of cells that we speculate are controllers or leader cells, as they initiate communication events in neighboring cells, and propagated signaling events are greatly reduced in their absence. Aim 1 will use a machine learning approach to investigate the presence of these leader cells in both diabetic cell culture and corneal models. Aim 1 will also address the role of Pannexin-1 in the generation of a unique leader cell signaling profile. Furthermore, the downstream impact of P2X7/Pannexin-1 signaling will be assessed by using 3D electron microscopy to study actin arrangement in wounded diabetic and control corneas. In Aim 2, the localization of P2X7 and Pannexin-1 protein and mRNA within the cells of corneal samples will be examined. This will yield data regarding both general trends in expression between diabetic and control groups, and differences in expression within a single sample that may explain the functional difference between leader cells and the rest of the epithelial sheet. In addition, Aim 2 will address whether the co-localization of P2X7 and Pannexin-1 proteins (before and after a wound) is necessary for wound repair. Together these Aims will produce significant advances in our understanding of the regulation of the P2X7/Pannexin-1 signaling cascade, alterations at the mRNA, protein, and functional level of this cascade in diabetes, and downstream effects of these aberrations on the actin cytoskeleton.