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: 10663786
• 负责人: Kristen Segars
• 金额: $ 5.27万
• 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/10663786
• 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; Epithelium; Etiology; Event; Eye; Functional disorder; Generations; Goals; Golgi Apparatus; Human; Impaired wound healing; Injury; Ion Channel; Keratoplasty; Knowledge; 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到高达70%的2型糖尿病患者，这是一个重要的原因