Molecular Mechanisms of corneal wound repair

根据世界卫生组织的数据，2型糖尿病是导致 增加了伤口愈合不当的风险，现在被认为是导致 可预防的失明。在损伤时，伤口边缘的相邻上皮细胞必须沟通并 协调他们的反应，向前迈进。我们在这个提案中的目标是检查这些相互作用 P2X7和pAnnexin1之间的相互作用对体内平衡和伤口修复至关重要。我们有证据表明 PAnnexin1介导了P2X7诱导的细胞通讯和细胞运动。这两种蛋白质对离子至关重要。 在炎症和疼痛中发挥作用的动员、ATP运输和信号复合体。其他 研究人员表明，P2X7在2型糖尿病的发病机制中发挥了作用，包括 微血管并发症、血视网膜屏障受损和神经病理性疼痛(Solini和Novak(2019))。 我们和我们的合作者已经在角膜上皮细胞中证明：1.P2X7和pAnnexin1 定位随损伤而改变，并在对照角膜前缘附近增强；2.抑制 P2X7减少细胞间的通讯，改变运动成分，改变酪氨酸 粘着斑蛋白和接头蛋白的磷酸化；4.P2X7和pAnnexin1在体外相互作用 有初步数据显示在对照小鼠中使用邻近性来证明P2X7和pAnnexin1的相互作用 5.我们有初步数据表明，未受伤的人的pAnnexin-1的差异很小 对照和糖尿病角膜上皮；但对损伤的反应不同；以及6.抑制 Pannexin1在对照角膜和体外抑制细胞迁移。这让我们假设，P2X7- 角膜上皮细胞中pAnnexin1的相互作用是细胞间有效沟通和信号传递所必需的 对损伤作出反应，是调节细胞运动所需的细胞骨架和力所必需的。这个 我们将解决的具体目标是：确定嘌呤受体和 Pannexin1在角膜伤口愈合过程中被调节；确定是否抑制和/或激活这2 蛋白质改变肌动蛋白细胞骨架的组织和细胞的运动性，并决定伤口是否愈合 而运动性是由通过pAnnexin-P2X7相互作用施加的力来调节的，如果它受到 衬底硬度。 我们的数据表明，治疗延迟的角膜上皮细胞的可能性将会愈合， 以及2型糖尿病患者复发的角膜侵蚀。

According to the World Health Organization, type 2 diabetes is one of the leading pathologies that increases the risk of improper wound healing and is now considered to be one of the leading causes of preventable blindness. Upon injury, adjacent epithelial cells at the wound edge must communicate and coordinate their response to move forward. Our goal in this proposal is to examine if the interactions between P2X7 and pannexin1 are critical for homeostasis and wound repair. We have evidence that pannexin1 mediates P2X7-induced communication and cell motility. These 2 proteins are critical for ion mobilization, ATP transport and signaling complexes that play roles in inflammation and pain. Other investigators have shown that P2X7 has a role in the pathogenesis of type 2 diabetes including microvascular complications, impaired blood retinal barrier and neuropathic pain (Solini and Novak (2019). We and our collaborators have demonstrated in corneal epithelium that: 1. P2X7 and pannexin1 localization changes with injury and is enhanced near the leading edge of control corneas; 2. Inhibition of P2X7 diminishes communication between cells, alters components of motility and alters tyrosine phosphorylation of focal adhesion and adaptor proteins; 4. P2X7 and pannexin1 interact in vitro and we have preliminary data demonstrating the interaction of P2X7 and pannexin1 in control mice using proximity ligation assays; 5. We have preliminary data that there is minimal difference in pannexin1 in unwounded control and diabetic corneal epithelium; however the response to injury differs; and 6. Inhibition of pannexin1 impedes cell migration in control corneas and in vitro. These led us to hypothesize that P2X7- pannexin1 interaction in corneal epithelium is required for effective cell-cell communication and signaling in response to injury and is needed to regulate the cytoskeleton and forces required for cell motility. The specific aims that we will address are: determine how changes in association of purinoreceptors and pannexin1 are regulated during corneal wound healing; determine if inhibition and/or activation of these 2 proteins changes the organization of the actin cytoskeleton and cell motility and determine if wound healing and motility is mediated by a force exerted through the pannexin-P2X7 interaction and if it is affected by substrate stiffness. Our data suggest the potential for therapeutic approaches to treat delayed corneal epithelial would healing, and recurrent corneal erosions in type 2 diabetes.