Regulation of Limbal Niche in Normal and Diabetic Cornea by Extracellular Vesicles

细胞外囊泡对正常和糖尿病角膜角膜缘生态位的调节

• 批准号: 10570179
• 负责人: Mehrnoosh Saghizadeh Ghiam
• 金额: $ 53.48万
• 依托单位: CEDARS-SINAI MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10570179
• 关键词: Adhesions; Blindness; Budgets; Cell Culture Techniques; Cell Maintenance; Cell Proliferation; Cell Survival; Cell physiology; Cells; Circulation; Coculture Techniques; Complement; Complex; Conditioned Culture Media; Confocal Microscopy; Cornea; Corneal Diseases; Corneal Injury; DNA; Defect; Diabetes Mellitus; Diameter; Disease; Endosomes; Epithelial Cells; Epithelium; Extracellular Space; Functional disorder; Goals; Health Professional; Healthcare Systems; Homeostasis; Human; In Vitro; Infiltration; Investigation; Keratopathy; Label; Maintenance; Maps; Mediating; Messenger RNA; MicroRNAs; Molecular; Molecular Target; Multivesicular Body; Organ Culture Techniques; Pathologic; Patients; Phenotype; Physiological; Play; Process; Proliferating; Proteins; Proteome; Proteomics; Public Health; Quality of life; Recurrence; Regulation; Reporting; Research; Role; Signal Transduction; Small RNA; Stromal Cells; System; Techniques; Therapeutic; Transmission Electron Microscopy; Vesicle; Vision; cell type; corneal epithelial wound healing; corneal regeneration; diabetic; diabetic wound healing; differential expression; epithelial stem cell; exosome; extracellular vesicles; healing; intercellular communication; limbal; mRNA Expression; microvesicles; migration; nanosized; next generation sequencing; protein expression; stem cell biomarkers; stem cell function; stem cell niche; stem cell proliferation; stem cells; therapeutic target; vesicular release; wound healing

PROJECT SUMMARY/ABSTRACT Corneal diseases have a significant impact on the quality of life of patients and also constitute a major problem for health care system. Corneal diseases such as limbal stem cell deficiency (LSCD) and diabetic keratopathy, associated with limbal epithelial stem cell (LESC) dysfunction/loss, may lead to epithelial defects, conjunctivalization, recurrent epithelial erosions, abnormal wound repair and altered vision. Therefore, mechanisms of LESC function in normal and diseased corneal homeostasis and wound healing process could be key to understanding various corneal diseases. The important part of the stem cell niche functioning is interaction between stem cells and their neighboring stromal cells. LESC are in close contact with the cells of the underlying limbal stroma and the vasculature surrounding limbal crypts. It is well established that there is a crosstalk between stromal cells, or keratocytes, and limbal epithelial cells (LECs) through their secreted factors, which are essential for the stem cell maintenance. Any damage to LESC or limbal stromal niche due to the external insults or diseases such as diabetes may lead to pathological state of altered vision and, in severe cases of LESC loss, results in limbal stem cell deficiency and blindness. Therapeutic potentials of extracellular vesicles (EVs) released from various cell type into extracellular space have been widely reported. EVs are nano-sized vesicles containing mRNA, miRNA, DNA, and protein cargo and mediating physiological intercellular crosstalk. They play important roles in intercellular communication and in stem cell maintenance and activation. We propose to investigate limbal EV contribution to corneal regeneration and wound healing, and examine possible differences in EV cargos released from normal and diabetic limbal stromal cells (LSCs) and LECs. We show that normal LSC-derived EVs (LSC-EVs) significantly promote epithelial healing in wounded organ-cultured human corneas. In addition, we found that there is a difference in cargos of EVs derived from normal and diabetic LSC. We hypothesize that limbal resident cell-derived EVs contribute to limbal epithelial and stromal homeostasis. We predict that the unique complement of EV cargos mediates corneal regeneration and wound healing in normal and diseased conditions. The goal of this proposal is to investigate the major mechanisms of action of both human LSC- and LEC-EVs, which are taken up by neighboring cells, in stem cell maintenance or survival and wound repair in cell cultures and in normal and diabetic human organ-cultured corneas. Techniques to be used include, EV isolation, miRNA isolation, next generation sequencing, proteomics, transmission electron and confocal microscopy, and trans-well co-culture system. Relevance to Public Health: investigation of molecular changes in diabetic corneas is essential for understanding of underlying causes and identification of therapeutic targets for diseased cornea and wound healing process in different layers of the cornea.

项目摘要/摘要 角膜疾病对患者的生活质量有重大影响，也构成了一个主要问题 用于医疗保健系统。角膜疾病，例如长缘干细胞缺乏症（LSCD）和糖尿病性角膜病， 与边缘上皮干细胞（LESC）功能障碍/损失相关，可能导致上皮缺陷， 结膜化，复发性上皮侵蚀，异常伤口修复和视力改变。所以， LESC功能在正常和患病的角膜稳态和伤口愈合过程中的机制可以 是理解各种角膜疾病的关键。干细胞生态位功能的重要部分是 干细胞及其邻近基质细胞之间的相互作用。 LESC与细胞密切接触 缘缘隐窝周围的层层基质和脉管系统。已经很确定有一个 基质细胞或角膜细胞和缘缘上皮细胞（LEC）之间的串扰。 因素，这对于干细胞维护至关重要。由于 外部侮辱或疾病（例如糖尿病）可能导致视力改变的病理状态，并且在严重的情况下 LESC损失的病例，导致干细胞缺乏症和失明。细胞外的治疗潜力 从各种细胞类型释放到细胞外空间的囊泡（EV）已被广泛报道。电动汽车是 含有mRNA，miRNA，DNA和蛋白质货物的纳米大小囊泡，并介导生理 细胞间串扰。它们在细胞间通信和干细胞维护中起重要作用 和激活。我们建议研究角膜膜对角膜再生和伤口愈合的贡献， 并检查从正常和糖尿病层间基质细胞（LSC）释放出的EV货物的可能差异（LSC） 和lecs。我们表明，正常LSC衍生的EV（LSC-EVS）显着促进上皮愈合 受伤的器官培养的人角膜。此外，我们发现电动汽车的cargos存在差异 源自正常和糖尿病LSC。我们假设边缘驻留的居民细胞衍生的电动汽车有助于 边缘上皮和基质稳态。我们预测EV Cargos的独特补充会介导 在正常和患病的情况下，角膜再生和伤口愈合。该提议的目的是 研究人类LSC和LEC-EV的主要作用机理，这些机制由 相邻细胞，在干细胞维持或生存以及细胞培养物以及正常和 糖尿病人体器官培养角膜。要使用的技术包括，EV隔离，miRNA隔离，接下来 生成测序，蛋白质组学，透射电子和共聚焦显微镜以及反孔培养 系统。与公共卫生有关：糖尿病角膜分子变化的调查对于 了解潜在原因和鉴定患病的角膜和伤口的治疗靶标 角膜不同层中的康复过程。