Mechanisms of corneal stromal activation during regeneration and fibrosis by exosomes

摘要 众所周知，角膜瘢痕形成(或纤维化)会导致中到重度伤口的视力障碍，而且没有 单一有效的预防或治疗方法，需要新的工具来治疗和减少 留下疤痕的风险是紧迫的。细胞间的通讯是角膜的重要组成部分，它包括 动态平衡、再生和纤维化。我们已经证明了角膜的伤口愈合机制 上皮细胞由其分泌体组成，细胞外小泡(EVS)是关键载体。在.期间 EV的产生，EV选择性地吞噬其亲代细胞的一部分，并在一系列 生物活性货物(例如，蛋白质、脂类和RNA)。我们最近报道了人角膜成纤维细胞(Hcf) 与转化生长因子-β3不同的是，转化生长因子-β1可增加纤维化标记物的表达，从而形成纤维化基质 (抗纤维化)。此外，我们的数据表明，来源于人角膜上皮细胞的EVS(HCE-EV)可以 Hcf诱导分化，含转化生长因子-β1或-β3的HcE-EV的蛋白质组可能是其驱动因素 可能与角膜瘢痕形成的机制有关。虽然，尽管 HCE-EVS的创面愈合和角膜基质激活能力及其生物活性的研究 这种效应的性质和分子机制尚不清楚。在这项提案中，我们假设 EV亚群HcE(转化生长因子-β1KD/-β3KD)-EVS将触发角膜肌成纤维细胞分化，产生 激活的间质微环境，支持纤维化愈合。我们提出以下目标来测试这一点 假设：目的1)调节和表征HCE-EV亚群；目的2)定义HcE-EV的基质激活能力 体外2D和3D模型中的HCE-EV亚群；以及目的3)确定HCE-EV亚群如何影响角膜 在活体内形成疤痕。与公共卫生的相关性-总的来说，这项建议将为HCE提供重要的见解- EV生物学和角膜成纤维细胞-靶细胞相互作用可用于开发新型EV 预防和治疗角膜瘢痕形成的治疗方法。

SUMMARY Corneal scarring (or fibrosis) is known to cause visual impairment in moderate to severe wounds, and with no single effective therapy for either the prevention or treatment, the need for new tools to treat and reduce the risk of scarring is urgent. Cell-cell communication is a vital component for the cornea that encompasses homeostasis, regeneration, and fibrosis. We have shown that the wound-healing mechanisms of corneal epithelial cells are comprised in their secretome, with extracellular vesicles (EVs) being a key vector. During EV production, the EVs selectively engulf a part of their parental cell and become enriched in a repertoire of bioactive cargo (e.g., proteins, lipids, and RNAs). We recently reported that human corneal fibroblasts (hCF) treated by TGF-β1 increased expression of fibrotic markers that established a fibrotic matrix, unlike TGF-β3 (anti-fibrotic). Also, our data suggests that EVs derived from human corneal epithelial cells (hCE-EV) can trigger hCF differentiation, and the proteome of hCE-EV containing TGF-β1 or -β3 could be a driving mechanism that could contribute to the mechanistic action of corneal scar formation. Although, despite the wound-healing and corneal stromal activation capacity of hCE-EVs, our understanding of their bioactive properties and molecular mechanisms for such effects remains unclear. In this proposal, we hypothesize that the EV subsets, hCE (TGF-β1KD/-β3KD)-EVs, will trigger corneal myofibroblast differentiation to generate an activated stromal microenvironment that supports fibrotic healing. We propose the following Aims to test this hypothesis: Aim 1) Modulate and characterize hCE-EV subsets; Aim 2) Define stromal activation capacity of hCE-EV subsets in 2D and 3D models in vitro; and Aim 3) Determine how hCE-EV subsets effect corneal scarring in vivo. Relevance to Public Health—Collectively, this proposal will provide important insight for hCE- EV biology and corneal fibroblast-target cell interaction, which can be leveraged to develop novel EV-based therapies for the prevention and treatment of corneal scarring.