Molecular Regulation of Corneal Wound Healing

项目摘要 该实验室的长期目标是研究角膜伤口愈合的分子和细胞生物学。 在目前的资助期间，实验室的研究大大提高了我们对 糖尿病性神经营养性角膜病变（DNK）是角膜发病的主要原因。我们 证明角膜上皮细胞通过释放可溶性因子进行交流， TGFβ3、IL-1 RA、Sema 3C（上皮细胞）、CNTF（树突状细胞，DC）和VIP（神经元）， 伤人糖尿病破坏细胞间相互作用，导致DNK。最近，细胞外囊泡， 特别是外来体，被发现是细胞间通讯的额外机制。 外泌体由大多数细胞类型分泌，并封装一组选择性的蛋白质、遗传物质（用于 例如，mRNA、miRNA、lncRNA、DNA）、代谢物和脂质，并将它们的货物水平地转移到 受体细胞此外，这些天然纳米尺寸的膜囊泡是极好的递送载体， 药物和治疗。我们发现，培养的小鼠角膜上皮细胞（CEC）和骨 骨髓来源的树突状细胞（BMDC）释放外泌体，外泌体可以容易地被纯化并被 在体外和/或在体内通过感觉神经相互作用。此外，我们发现， 外泌体的内容物根据病理/生理学而变化;外泌体来源于愈合，而不是来自 静止CEC加速糖尿病小鼠角膜中减弱的伤口愈合; 从BMDC分离的DCs可以在功能上替代DCs在支持角膜伤口闭合中的作用。我们 已发表的和初步的研究导致了这样的假设，即外泌体传递关键信号， 在伤口愈合过程中角膜中的事件，并且可以被修改以提高治疗潜力， 治疗DNK。这一应用包括三个目标。目的1：探讨其分子机制 CEC外泌体（其已经从静止和愈合的CEC中分离并在正常和高浓度条件下培养）。 葡萄糖）对于DNK是治疗有效的或致病的。目的2：探讨BMDC的作用 从正常和糖尿病型DC中纯化的外泌体（已从骨髓来源的DC中分离） 1和2型糖尿病小鼠在介导角膜伤口愈合和巨噬细胞活化中的作用。目标3：确定 改变Exo含量是否可以改善DNK以及1型和2型中受损的皮肤伤口愈合 小鼠完成拟议的研究将证明外泌体是细胞功能的重要介质 或功能障碍，这取决于外泌体的性质，鉴定包封的分子，如蛋白质， 负责外泌体再生能力的特异性microRNA，并修饰纯化的外泌体以增强 它们用于治疗DNK的治疗潜力，其中存在未满足的临床需求。

PROJECT ABSTRACT This laboratory's long-term goal is to research the molecular and cellular biology of corneal wound healing. During the current grant period, studies from the lab have significantly improved our understanding of the pathogenesis of diabetic neurotrophic keratopathy (DNK), a major cause of the morbidity of the cornea. We demonstrated that cells in the corneal epithelium communicate through the release of soluble factors, such as TGFβ3, IL-1RA, Sema3C (epithelial cells), CNTF (dendritic cells, DC), and VIP (neuron), in response to wounding. Diabetes mellitus disrupts cell-cell interaction, resulting in DNK. Recently, extracellular vesicles, particularly exosomes, were discovered as an additional mechanism for intercellular communication. Exosomes are secreted by most cell types and encapsulate a selective set of proteins, genetic material (for example, mRNAs, miRNAs, lncRNAs, DNA), metabolites, and lipids, and horizontally transfer their cargo to recipient cells. Moreover, these natural nanosized membrane vesicles are excellent delivery vehicles for drugs and therapeutics. We discovered that both cultured mouse corneal epithelial cells (CECs) and bone marrow-derived dendritic cells (BMDCs) release exosomes, which can be readily purified and taken up by each other and by sensory nerves in vitro and/or in vivo. Additionally, we found that the miRNA and protein contents of exosomes vary depending on patho/physiology; that exosomes derived from healing, but not from quiescent CECs, accelerate attenuated wound healing in diabetic mouse corneas; and that exosomes isolated from BMDCs can functionally replace the role of DCs in supporting corneal wound closure. Our published and preliminary studies led to the hypothesis that exosomes communicate critical signaling events in the cornea during wound healing and may be modified to boost therapeutic potential for treating DNK. This application includes three Aims. Aim 1: To explore the molecular mechanism by which CEC exosomes (which have been isolated from quiescent and healing CECs and cultured in normal and high glucose) are therapeutically effective or pathogenic for DNK. Aim 2: To investigate the role of BMDC exosomes (which have been isolated from bone marrow derived DCs) purified from normal and diabetic type 1 and 2 diabetic mice in mediating corneal wound healing and macrophage activation. Aim 3: To determine whether modifying Exo contents can ameliorate DNK as well as impaired skin wound healing in type 1 and 2 mice. Completion of the proposed study will demonstrate exosomes are a significant mediator of cell function or dysfunction depending on the nature of exosomes, identify encapsulated molecules such as proteins and specific microRNAs responsible for exosome regenerative power, and modify purified exosomes to boost their therapeutic potential for treating DNK where there is an unmet clinical need.