Molecular mechanisms of cold storage-induced damage to the corneal endothelium

冷藏引起角膜内皮损伤的分子机制

• 批准号: 10741168
• 负责人: SANGLY P SRINIVAS
• 金额: $ 43.22万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-30 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10741168
• 关键词: Actomyosin; Acute; Adverse effects; Affect; Antioxidants; Cadaver; Cardiolipins; Cause of Death; Cell Death; Cell Death Induction; Cell Density; Cell membrane; Cells; Chelating Agents; Clinical Management; Cornea; Corneal Diseases; Corneal Endothelium; Cryopreservation; Custom; Cytoplasm; Cytoskeleton; Deferoxamine; Descemet' s membrane; Disease; Endothelial Cells; Endothelium; Exhibits; Failure; Family suidae; Functional disorder; Genetic Diseases; Gifts; Glutathione; Goals; Human; Iron Chelation; Island; Keratoplasty; Lipid Peroxidation; Lipids; Liquid substance; Measurement; Measures; Membrane Potentials; Microtubule Disassembly Inhibition; Microtubule-Associated Proteins; Microtubules; Mitochondria; Molecular; Operative Surgical Procedures; Organ Transplantation; Oxidation-Reduction; Oxidative Stress; Oxidative Stress Induction; Permeability; Pharmaceutical Preparations; Phosphorylation; Population; Potassium Channel; Procedures; Protein Kinase; Pump; Reactive Oxygen Species; Recovery; Scanning; Signal Transduction; Small Interfering RNA; Solid; Stress; Surface; Temperature; Testing; Tight Junctions; Transplantation; Vision; cell motility; clinically significant; cold stress; cost; experimental study; falls; inhibitor; knock-down; macromolecule; mitochondrial dysfunction; mitochondrial membrane; monolayer; oxidation; p38 Mitogen Activated Protein Kinase; peroxidation; pharmacologic; preservation; response; standard of care; success; uptake; Actomyosin; Acute; Adverse effects; Affect; Antioxidants; Cadaver; Cardiolipins; Cause of Death; Cell Death; Cell Death Induction; Cell Density; Cell membrane; Cells; Chelating Agents; Clinical Management; Cornea; Corneal Diseases; Corneal Endothelium; Cryopreservation; Custom; Cytoplasm; Cytoskeleton; Deferoxamine; Descemet' s membrane; Disease; Endothelial Cells; Endothelium; Exhibits; Failure; Family suidae; Functional disorder; Genetic Diseases; Gifts; Glutathione; Goals; Human; Iron Chelation; Island; Keratoplasty; Lipid Peroxidation; Lipids; Liquid substance; Measurement; Measures; Membrane Potentials; Microtubule Disassembly Inhibition; Microtubule-Associated Proteins; Microtubules; Mitochondria; Molecular; Operative Surgical Procedures; Organ Transplantation; Oxidation-Reduction; Oxidative Stress; Oxidative Stress Induction; Permeability; Pharmaceutical Preparations; Phosphorylation; Population; Potassium Channel; Procedures; Protein Kinase; Pump; Reactive Oxygen Species; Recovery; Scanning; Signal Transduction; Small Interfering RNA; Solid; Stress; Surface; Temperature; Testing; Tight Junctions; Transplantation; Vision; cell motility; clinically significant; cold stress; cost; experimental study; falls; inhibitor; knock-down; macromolecule; mitochondrial dysfunction; mitochondrial membrane; monolayer; oxidation; p38 Mitogen Activated Protein Kinase; peroxidation; pharmacologic; preservation; response; standard of care; success; uptake

Molecular mechanisms of cold storage-induced damage to the corneal endothelium The endothelium at the posterior surface of the cornea is a non-regenerative monolayer. It maintains corneal transparency through its barrier and fluid pump functions. Thus, a rapid decline in endothelial cell density (ECD), observed during genetic disorders or in response to intraocular surgery, disturbs corneal transparency. Moreover, since there are no drugs to halt endothelial cell loss (ECL) during these disorders, corneal transplantation is the standard of care when ECD < 600-800 cells/mm2. In 2021, > 49,000 transplants were carried out in the USA at the cost of ~ $40,000/transplant. In addition to the gift of sight, the procedure saved an estimated lifetime value of $5.8 Billion. Although corneal transplants are more successful than other solid organ transplants, delayed recovery of corneal transparency and a rapid decline in ECD after the surgery are two significant challenges yet to be fully understood. The decrease in the ECD after surgery is in addition to ECL that occurs during cold storage (CS). However, the molecular mechanisms for ECL during CS or acute ECL after surgery are unknown. Our recent studies have shown that CS adversely impacts the endothelium. Specifically, when freshly isolated porcine corneas were held in prolonged CS (> 3 days at 4 C), endothelium exhibited microtubule (MT) disassembly, destruction of its peri-junctional actomyosin ring (PAMR), lipid peroxidation, and cell death. These effects were inhibited by including antioxidants or iron chelators (deferoxamine) in the CS medium. Thus, we hypothesize that CS induces oxidative stress in the endothelium, leading to (a) a breakdown of its tight junctions via the destruction of its PAMR and (b) the formation of islands of denuded Descemet’s membrane (iDDMs) because of cell death. To regain corneal transparency, the extreme loss in barrier function must be corrected by the repopulation of iDDMs and normalization of the tight junctions. The main goal of this project is to test all our hypotheses based on two specific aims. First, in Aim 1, we will determine how CS induces mitochondrial dysfunction in the endothelium, leading to oxidative stress. We will also assess if the mode of cell death also involves ferroptosis. Subsequently, in Aim 2, we will determine how oxidative stress destroys the PAMR, thereby causing a breakdown of tight junctions. In addition, we will test the assumption that the repopulation of iDDMs is slowed by oxidative stress prevailing in the endothelium, leading to a delay in the rapid recovery of corneal transparency after CS. Overall, this project will dissect the molecular mechanisms that adversely affect the endothelium during CS. We expect our findings to enable pharmacological approaches by which we can limit ECL during CS and acutely after surgery for enhanced corneal transplantation.

冷储存引起的角膜内皮损伤的分子机制 角膜后表面的内皮是非再生单层。它保持角膜 通过其屏障和流体泵功能的透明度。这是内皮细胞密度（ECD）的迅速下降， 在遗传疾病或对眼内手术的响应中观察到的，扰乱了角膜透明度。 此外，由于在这些疾病期间没有药物可以阻止内皮细胞损失（ECL） 当ECD <600-800细胞/mm2时，移植是护理标准。在2021年，> 49,000次移植 在美国进行，费用约为40,000美元/移植。除了视线之外，该程序还保存了 估计终身价值为58亿美元。 尽管角膜移植比其他固体器官移植更成功，但角膜的恢复延迟 手术后的透明度和ECD迅速下降是两个重大挑战 理解。手术后的ECD减少是在冷藏期间发生的ECL（CS）。 但是，尚不清楚CS或急性ECL期间ECL的分子机制。 我们最近的研究表明，CS对内皮产生不利影响。具体而言，新鲜隔离时 猪角膜在长时间的CS中（在4°C时> 3天），内皮暴露的微管（MT）持有 拆卸，破坏其骨周围的肌球蛋白环（PAMR），脂质过氧化和细胞死亡。这些 通过在CS培养基中包括抗氧化剂或铁螯合剂（脱氧明）来抑制效果。那，我们 假设CS会影响内皮中的氧化应激，导致（a）其紧密连接的分解 通过破坏其PAMR和（b）裸露的Descemet膜（IDDMS）的形成 由于细胞死亡。为了保持角膜透明度，必须纠正屏障功能的极端损失 IDDM的重生和紧密连接的归一化。该项目的主要目标是测试我们的所有 基于两个具体目标的假设。首先，在AIM 1中，我们将确定CS如何诱导线粒体 内皮功能障碍，导致氧化应激。我们还将评估细胞死亡的模式 涉及铁铁作用。随后，在AIM 2中，我们将确定氧化应激如何破坏PAMR，从而 导致紧密连接的崩溃。此外，我们将测试以下假设：IDDM的重生为 在内皮中流行的氧化应激减慢，导致角膜快速恢复 CS后透明。总体而言，该项目将剖析对影响的分子机制 CS期间的内皮。我们希望我们的发现能够实现我们可以限制的药物方法 CS期间的ECL和手术后急性，以增强角膜移植。