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

糖尿病角膜上皮伤口愈合延迟：损伤后蛋白质反应减少和细胞间通讯不协调

• 批准号: 10387681
• 负责人: Kristen Segars
• 金额: $ 5.18万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10387681
• 关键词: 3-Dimensional; Actins; Address; Affect; Agonist; Anterior; Biological Assay; Blindness; Bundling; Calcium Signaling; Cell Communication; Cell Culture Techniques; Cell surface; Cells; Characteristics; Communication; Complication; Computer Analysis; Control Groups; Cornea; Corneal Injury; Cytoskeletal Proteins; Cytoskeleton; Data; Defect; Development; Diabetes Mellitus; Diabetic mouse; Diffuse; Electron Microscopy; Epithelial; Etiology; Event; Eye; Functional disorder; Generations; Goals; Golgi Apparatus; Human; Impaired wound healing; Injury; Ion Channel; Keratoplasty; Knowledge; Lead; Ligation; Machine Learning; Messenger RNA; Modeling; Mus; Non-Insulin-Dependent Diabetes Mellitus; Pain; Patients; Pattern; Persons; Phenotype; Population; Prediabetes syndrome; Probability; Procedures; Process; Proteins; Purinoceptor; Recurrence; Regulation; Rest; Risk; Role; Sampling; Severity of illness; Signal Pathway; Signal Transduction; Signaling Protein; Surface; System; Time; Translations; Travel; Type 2 diabetic; Ulcer; Vision; cell motility; corneal epithelial wound healing; corneal epithelium; diabetic; diabetic patient; diabetic ulcer; experience; healing; injured; intercellular communication; knock-down; migration; mouse model; non-diabetic; programs; response; sensor; stem cells; symptomatic improvement; trafficking; trend; wound; wound closure; wound healing

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.

不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 视力丧失的几率虽然有治疗方法可以改善不良症状- 治愈角膜伤口，唯一的永久解决方案是角膜移植，这是一种不容易获得的手术 国际吧通过检测细胞内参与伤口愈合反应的各种蛋白质的变化， 在这个层面上，我希望了解糖尿病患者的角膜为什么不能有效愈合。当一个原本 当健康的角膜受伤时，伤口旁边的细胞会经历一些必要的变化， 迁移和伤口闭合。其中一个变化涉及嘌呤能受体P2 X7的激活，P2 X7发现于 细胞表面。当激活时，P2 X7产生一种特定的钙信号传导事件模式， 通过激活离子通道Pannexin-1在细胞间传递。这些传播的信号事件代表细胞- 细胞通讯，并最终导致细胞骨架蛋白的重新排列和协调伤口 结束我们以前的研究已经确定了2型糖尿病前期P2 X7的异常定位和激活， 模型我们有初步的证据表明，受伤的糖尿病细胞的信号转导模式缺乏 特征性P2 X7信号传导反应。使用P2 X7的特异性激动剂证实了这一点，并观察到 大大降低了糖尿病细胞的反应。这项提案的目的是揭示细胞-细胞 P2 X7信号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和最终细胞运动。我 初步的数据已经确定了一组细胞，我们推测它们是控制者或领导者细胞，因为它们启动 在相邻小区中的通信事件，以及传播的信令事件在其 缺席Aim 1将使用机器学习方法来研究这些领导细胞在两种细胞中的存在。 糖尿病细胞培养和角膜模型。Aim 1还将解决Pannexin-1在产生一种免疫应答中的作用。 独特的前导细胞信号特征此外，P2 X7/Pannexin-1信号传导的下游影响将是 通过使用3D电子显微镜研究创伤糖尿病患者和对照组的肌动蛋白排列来评估 角膜目的2：研究P2 X7和Pannexin-1蛋白及其mRNA在角膜上皮细胞中的定位， 样品将被检查。这将产生关于糖尿病患者之间表达的一般趋势的数据。 和对照组，以及单个样本中表达的差异，这可能解释了功能性 前导细胞和上皮片的其余部分之间的差异。此外，目标2将讨论 P2 X7和Pannexin-1蛋白的共定位（在创伤之前和之后）对于创伤修复是必需的。 这些目标将使我们对监管的理解取得重大进展， P2 X7/Pannexin-1信号级联，在mRNA，蛋白质和功能水平上的改变，该级联在 糖尿病，以及这些畸变对肌动蛋白细胞骨架的下游影响。