Corneal Epithelial-Stromal Interactions During Regeneration and Fibrosis

再生和纤维化过程中角膜上皮-基质相互作用

• 批准号: 10521703
• 负责人: Joseph B. Ciolino
• 金额: $ 52.47万
• 依托单位: SCHEPENS EYE RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10521703
• 关键词: 3-Dimensional; Acute; Address; Automobile Driving; Basement membrane; Biological; Biological Markers; Biological Process; Biology; Biophysics; Blindness; Cell Communication; Cell Line; Cell Lineage; Cell Proliferation; Cell physiology; Cells; Cellular Structures; Cicatrix; Complex; Complication; Cornea; Corneal Diseases; Corneal Injury; Corneal Stroma; Deposition; Disease; Disease Progression; Elements; Epithelial; Epithelial Cell Proliferation; Epithelial Cells; Epithelial-Stromal Communication; Etiology; Extracellular Matrix; Fibroblasts; Fibrosis; Future; Generations; Genetic Markers; Growth Factor; Homeostasis; Human; In Vitro; Infection; Infiltration; Inflammation; Inflammatory; Injury; Keratoconus; Lead; Lipids; Mediating; Mediator of activation protein; Messenger RNA; Modeling; Molecular; Myofibroblast; Natural regeneration; Oryctolagus cuniculus; Pathogenesis; Pathologic; Pathway interactions; Patients; Penetrating Wounds; Physiological; Pilot Projects; Process; Property; Protein Isoforms; Proteins; Proteomics; Public Health; Quality of life; RNA; Research; Role; Signal Pathway; Signal Transduction; Stromal Cells; Surgical Models; Testing; Therapeutic; Therapeutic Effect; Tissues; Transforming Growth Factor beta; Trauma; Validation; Visual impairment; Wound models; biophysical properties; cell motility; cell stroma; clinically relevant; corneal epithelial wound healing; corneal epithelium; corneal regeneration; corneal scar; cytokine; experimental study; extracellular vesicles; healing; in vivo; insight; interest; paracrine; phenotypic biomarker; prevent; repaired; response; tool; transforming growth factor beta3; vesicular release; wound healing

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.

概括 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 生成和细胞外基质（ECM）沉积。在角膜伤口模型中， 经常观察到将上皮与基质分开的鲍曼层，这种破坏可能会导致 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 同种型（TGF-β1、-β2 和 -β3），通过信号通路发挥生物效应以维持角膜 完整性和伤口愈合。此前，我们发现TGF-β1通过以下方式参与角膜纤维化伤口愈合： 刺激肌成纤维细胞分化；然而，角膜损伤后应用TGF-β3可逆转并 分别减少体外和体内的纤维化反应。尽管 TGF-β 存在功能差异 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，细胞外囊泡 (EV) 被认为是细胞间的介质 沟通。 EV 可以选择性地吞噬其亲代细胞的一部分，并丰富了 生物活性货物（例如蛋白质或脂质），并通过随后的生理作用将其货物抵消到受体细胞中 变化。我们已经证明角膜上皮细胞来源的 EV 可以触发肌成纤维细胞分化和 产生促进肌成纤维细胞持久存在的 ECM 微环境，这是角膜疤痕形成的关键； 然而，驱动这一现象的生物活性物质仍不清楚。圆锥角膜 (KCN) 与疾病相关 感兴趣，因为即使没有任何急性创伤或已知的潜在因素，它也会导致角膜基质疤痕 病因学。从病理学上讲，KCN 与我们的伤口愈合模型相似，KCN 角膜：1）产生间隙 在鲍曼层中，允许上皮和基质之间直接接触； 2) 之间有电动汽车 上皮细胞和基质； 3) 肌成纤维细胞分化，导致骨折处形成疤痕 在鲍曼层。 TGF-β 在肌成纤维细胞分化和 ECM 重塑中的作用表明参与 KCN的发病机制，无论是起致病作用还是继发修复作用，导致KCN的结构变化。 在该提案中，我们假设将 TGF-β3 装载到 EV 上并应用于角膜伤口将推动 通过抑制纤维化反应并防止疤痕形成，实现无疤痕愈合； 然而，TGF-β1-EVs 会加剧角膜伤口的疤痕形成。通过这些实验我们还将 确定 KCN 上皮细胞来源的 EV 中导致 KCN 角膜疤痕形成的生物活性物质，以及 疾病进展。与公共卫生的相关性——总的来说，该提案将提供关键机制 深入了解角膜 EV 生物学及其相应的货物，并确定具有生物活性的 EV 货物 未来可用于预测 KCN 患者的角膜疤痕。