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.

高达 70% 的 2 型糖尿病患者受到无法治愈的角膜损伤的影响，这是一个重要原因 该人群的视力丧失。尽管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，但这种手术并不容易实现 全世界。通过检查细胞中参与伤口愈合反应的各种蛋白质的变化 水平，我希望了解为什么糖尿病患者的角膜无法有效愈合。当否则 健康的角膜受到损伤，伤口旁边的细胞会经历许多协调所需的变化 迁移和伤口闭合。其中一项变化涉及嘌呤能受体 P2X7 的激活，该受体存在于 细胞表面。当激活时，P2X7 会产生特定模式的钙信号传导事件，这些信号从 通过激活离子通道 Pannexin-1 实现细胞与细胞之间的连接。这些传播的信号事件代表细胞 细胞通讯，最终导致细胞骨架蛋白的重新排列和协调伤口 关闭。我们之前的研究已经发现 2 型糖尿病前期 P2X7 的异常定位和激活 模型。我们有初步证据表明，受伤的糖尿病细胞的信号传导谱缺乏 特征性 P2X7 信号响应。使用 P2X7 的特定激动剂并观察 糖尿病细胞的反应大大减弱。该提案的目标是揭示细胞间如何 P2X7 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成束并最终影响细胞运动。我的 初步数据已识别出一组我们推测为控制者或领导者细胞的细胞，因为它们发起 相邻小区中的通信事件和传播的信号事件大大减少 缺席。目标 1 将使用机器学习方法来研究这些领导细胞在两个细胞中的存在 糖尿病细胞培养和角膜模型。目标 1 还将解决 Pannexin-1 在生成 独特的前导细胞信号传导谱。此外，P2X7/Pannexin-1 信号传导的下游影响将是 使用 3D 电子显微镜评估受伤糖尿病患者和对照组的肌动蛋白排列 角膜。在目标 2 中，P2X7 和 Pannexin-1 蛋白和 mRNA 在角膜细胞内的定位 将检查样品。这将产生有关糖尿病患者之间表达的总体趋势的数据 和对照组，以及单个样本内的表达差异可以解释功能 前导细胞和上皮层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（伤口前后）的共定位对于伤口修复是必要的。 这些目标将共同推动我们对监管的理解取得重大进展。 P2X7/Pannexin-1 信号级联，该级联的 mRNA、蛋白质和功能水平的改变 糖尿病，以及这些畸变对肌动蛋白细胞骨架的下游影响。