Corneal Epithelial-Stromal Interactions During Regeneration and Fibrosis

摘要 角膜伤口愈合是一个复杂的过程，涉及角膜上皮细胞增殖、肌成纤维细胞 细胞外基质(ECM)的生成和沉积。在角膜创伤模型中，角膜内皮细胞的破坏 分离上皮和间质的鲍曼层经常被观察到，这种破坏可能导致 不同的病理(纤维化)状态，我们认为这可以通过细胞间信号转导 上皮细胞和间质细胞部分通过旁分泌因子。转化生长因子-β是一种多效性细胞因子，存在于三种 异构体(转化生长因子-β1、-β2和-β3)，通过维持角膜的信号通路发挥生物学效应 正直和伤口愈合。先前，我们发现转化生长因子-β1通过以下途径参与角膜纤维化伤口的愈合 而转化生长因子-β-3在角膜损伤后应用可逆转和 分别在体外和体内抑制肝纤维化反应。尽管转化生长因子-β的功能存在差异 亚型，抑制纤维化的分子机制仍然知之甚少。 在其他旁分泌因子中，细胞外小泡(EVS)被认为是细胞-细胞之间的介质。 沟通。EVS可以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(如蛋白质或脂类)，并通过随后的生理作用抵消它们进入受体细胞的货物 改变。我们已经证明，角膜上皮细胞来源的EVS可以触发肌成纤维细胞的分化和 创造一个细胞外基质微环境，促进肌成纤维细胞的持久性，这是角膜疤痕形成的关键； 然而，驱动这一现象的生物活性货物仍不清楚。对于疾病相关性，圆锥角膜(KCN)是 感兴趣，因为它会导致角膜基质疤痕，即使没有任何急性创伤或已知的潜在原因 病因学。在病理学上，KCN与我们的伤口愈合模型相似，因为KCN角膜：1)形成缝隙 在鲍曼氏层中，允许上皮和间质之间直接接触；2)在 上皮细胞和间质；以及3)形成肌成纤维细胞分化，导致断裂下的瘢痕形成 在鲍曼的层面上。转化生长因子-β的S在肌纤维细胞分化和细胞外基质重塑中的作用提示参与了 KCN的发病机制，无论是在致病修复中还是在继发性修复中，都会导致KCN结构的改变。 在这项提议中，我们假设将转化生长因子-β3装载到电动汽车上并应用于角膜伤口将推动 通过抑制纤维化反应和防止疤痕形成而愈合而不形成疤痕； 但转化生长因子-β-1-EVS可促进角膜创面的瘢痕形成。通过这些实验，我们还将 确定KCN上皮细胞来源的EVS中的生物活性物质，这是导致KCN角膜瘢痕形成和 疾病的发展。与公共卫生的相关性--总的来说，这项提案将提供关键的机制 深入了解角膜EV生物学及其相应的货物，以及识别具有生物活性的EV货物 可用于预测未来KCN患者的角膜瘢痕形成。

SUMMARY Corneal wound healing is a complex process involving corneal epithelial cell proliferation, myofibroblast generation, and extracellular matrix (ECM) deposition. In corneal wound models, the disruption of the Bowman's layer that separates the epithelium from stroma is often observed, and this disruption may lead to a different pathological (fibrotic) state, which we propose can be mediated by the intercellular signaling between epithelial and stromal cells in part via paracrine factors. TGF-β is a pleiotropic cytokine that exists in three isoforms (TGF-β1, -β2, and -β3), which exert biological effects through signaling pathways to maintain corneal integrity and wound healing. Previously, we found that TGF-β1 is involved in corneal fibrotic wound healing by stimulating myofibroblast differentiation; whereas, TGF-β3 application after corneal wounding reversed and diminished the fibrotic response in vitro and in vivo, respectively. Despite the functional differences in TGF-β isoforms, the molecular mechanisms in dampening fibrosis remain poorly understood. Amid other paracrine factors, extracellular vesicles (EVs) are recognized as mediators for cell-cell communication. EVs can selectively engulf a part of their parental cell and become enriched in a repertoire of bioactive cargo (e.g., proteins or lipids), and offset their cargo into recipient cells by ensuing physiological changes. We have shown that corneal epithelial cell-derived EVs can trigger myofibroblast differentiation and generate an ECM microenvironment that promotes myofibroblast persistence, which is key for corneal scarring; however, the bioactive cargo driving this remains unclear. Of disease relevance, keratoconus (KCN) is of interest because it leads to corneal stromal scarring even without any acute trauma or known underlying etiology. Pathologically, KCN is similar to our wound-healing models in that the KCN corneas: 1) develop gaps in Bowman's layer that allow direct contact between epithelium and stroma; 2) have EVs present between epithelial cells and stroma; and 3) develop myofibroblast differentiation that leads to scarring under the breaks in Bowman's layer. TGF-β's role in myofibroblast differentiation and ECM remodeling suggests involvement in KCN's pathogenesis, either in a causative or secondary repair role, leading to structural changes in KCN. In this proposal, we hypothesize that loading TGF-β3 onto EVs and applying to corneal wounds will drive healing without scarring by dampening the fibrotic response and preventing the onset of scar formation; however, TGF-β1-EVs will enhance the scarring in corneal wounds. Through these experiments we will also determine bioactive cargo from KCN-epithelial cell derived EVs that is contributing to KCN corneal scarring and disease progression. Relevance to Public Health—Collectively, this proposal will provide key mechanistic insights into corneal EV biology and their corresponding cargo, as well as identify the bioactive EV cargo that could be used to predict corneal scarring in KCN patients in the future.