Corneal Epithelial-Stromal Interactions During Regeneration and Fibrosis

总结 角膜创伤愈合是一个复杂的过程，涉及角膜上皮细胞增殖、肌成纤维细胞增殖、成纤维细胞增殖等。 生成和细胞外基质（ECM）沉积。在角膜创伤模型中， 经常观察到将上皮与间质分离的鲍曼层，并且这种破坏可能导致 不同的病理（纤维化）状态，我们提出可以通过细胞间信号传导介导， 上皮细胞和基质细胞部分通过旁分泌因子。TGF-β是一种多效性细胞因子，存在于三种细胞中， 同种型（TGF-β1，-β2和-β3），通过信号通路发挥生物学作用，以维持角膜 完整性和伤口愈合。以前，我们发现TGF-β1通过以下途径参与角膜纤维化伤口愈合： 刺激肌成纤维细胞分化;而角膜创伤后应用TGF-β3逆转， 分别在体外和体内减少纤维化反应。尽管TGF-β在功能上存在差异， 对于同种型，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，细胞外囊泡（EV）被认为是细胞-细胞粘附的介质。 通信EV可以选择性地吞噬其亲本细胞的一部分，并在一个库中富集， 生物活性货物（例如，蛋白质或脂质），并通过随后的生理学过程将其货物抵消到受体细胞中。 变化我们已经表明，角膜上皮细胞来源的EV可以触发肌成纤维细胞分化， 产生促进肌成纤维细胞持久性的ECM微环境，这是角膜瘢痕形成的关键; 然而，驱动这一趋势的生物活性货物仍不清楚。与疾病相关的圆锥角膜（KCN）是 因为它导致角膜基质瘢痕，即使没有任何急性创伤或已知的潜在的 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）形成间隙 在允许上皮和基质之间直接接触Bowman层中; 2）在上皮和基质之间存在EV 上皮细胞和间质;和3）发展成肌纤维细胞分化，导致断裂处下的瘢痕形成 在鲍曼的层。TGF-β在肌成纤维细胞分化和ECM重塑中的作用表明TGF-β参与了 KCN的发病机制，无论是在致病或继发性修复作用，导致KCN的结构变化。 在这个提议中，我们假设将TGF-β3负载到EV上并应用于角膜伤口将驱动 通过抑制纤维化反应和防止瘢痕形成的发生而愈合而不形成瘢痕; 但TGF-β1-EV可促进角膜创面瘢痕形成。通过这些实验，我们还将 确定来自KCN上皮细胞衍生的EV的生物活性货物，其有助于KCN角膜瘢痕形成， 疾病进展。与公共卫生的相关性-总体而言，本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，以及确定生物活性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.