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

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.

冷藏致角膜内皮损伤的分子机制 角膜后表面的内皮是一层不可再生的单层。它能维持角膜 通过其屏障和流体泵功能实现透明。因此，内皮细胞密度(ECD)迅速下降， 在遗传性疾病期间或在眼内手术后观察到的，干扰角膜透明度。 此外，由于在这些疾病期间没有阻止内皮细胞丢失(ECL)的药物，角膜 当ECD为600-800个/mm2时，移植是治疗的标准。2021年，&gt；进行了4.9万例移植 在美国进行，每例移植的费用约为4万美元。除了视力的天赋，这个手术还挽救了一个 估计终身价值为58亿美元。 虽然角膜移植比其他实体器官移植更成功，但角膜恢复延迟 透明度和术后ECD的迅速下降是两个尚未完全完成的重大挑战 明白了。手术后ECD的下降是在冷藏(CS)期间发生的ECL的补充。 然而，CS期间ECL或术后急性ECL的分子机制尚不清楚。 我们最近的研究表明，CS对内皮细胞有不利影响。具体地说，当新分离的 猪角膜在4C恒温条件下放置3天，内皮细胞呈微管状。 解体，破坏其连接周围的肌动球蛋白环(PAMR)，脂质过氧化和细胞死亡。这些 在CS培养液中加入抗氧化剂或铁络合剂(去铁胺)可抑制上述作用。因此，我们 假设CS诱导内皮细胞的氧化应激，导致(A)其紧密连接的破坏 通过破坏其PAMR和(B)形成剥落的Descemet膜岛(IDDMS) 因为细胞死亡。为了恢复角膜的透明性，屏障功能的极度丧失必须通过 IDEM的重新填充和紧密连接的正常化。这个项目的主要目标是测试我们所有的 假设基于两个特定的目标。首先，在目标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模式是否也 涉及上睑下垂。随后，在目标2中，我们将确定氧化应激如何破坏PAMR，从而 导致紧密连接的破裂。此外，我们还将检验这样一种假设，即iDEM的重新人口是 由于内皮细胞中普遍存在的氧化应激而减慢，导致角膜快速恢复的延迟 CS之后的透明度。总体而言，这个项目将剖析对人类健康产生负面影响的分子机制。 CS过程中的内皮功能。我们希望我们的发现能够使我们能够通过药理学方法限制 增强角膜移植术中及术后急性期ECL。