Molecular Regulation of Corneal Wound Healing

描述（由申请人提供）：实验室的长期目标是获得有关角膜伤口愈合的分子和细胞生物学的基本信息。本资助期内的研究表明，表皮生长因子受体（EGFR）是创伤后激活的关键受体酪氨酸激酶，创伤诱导的EGFR及其下游信号通路（特别是ERK和PI3K）的激活在介导角膜上皮创伤愈合中发挥核心作用。虽然在正常角膜中上皮伤口通常以及时的方式愈合而没有任何并发症，但是在糖尿病患者的角膜中上皮伤口愈合显著延迟。上皮缺损的延迟愈合（称为糖尿病性角膜病）可导致威胁视力的并发症，例如持续性上皮缺损、复发性糜烂和微生物性角膜炎。使用链脲佐菌素（SZT）处理的大鼠作为1型糖尿病模型和人类患病角膜的初步研究揭示，EGFR信号传导，特别是PI3K通路，在糖尿病大鼠角膜和人类糖尿病角膜的上皮中受损，但在正常和饮食控制的角膜中不受损。我们已发表的和初步的研究导致了这样的假设，即上皮中的高血糖症废除了严格调节的EGFR信号传导，并且增强减弱的EGFR信号传导，沿着减少氧化应激，可以保持上皮完整性并促进糖尿病角膜中的上皮伤口愈合。提出了三个具体目标来检验这一假设。1)表征SZT糖尿病大鼠角膜中的EGFR信号通路和氧化损伤。将表征SZT诱导的和人糖尿病角膜的上皮中EGFR信号传导的改变，并将其与在这些患病角膜中观察到的上皮异常相关联。2)确定高血糖症如何影响表皮生长因子受体信号传导和角膜上皮完整性和功能。将使用培养的猪角膜和原代人角膜上皮细胞。将评估模拟高血糖症的高葡萄糖对活性氧生成、EGFR信号传导以及上皮完整性和功能（包括屏障特性和伤口愈合）的影响。3)确定如何操纵EGFR信号通路可能用于加速糖尿病角膜上皮伤口愈合。EGFR配体将与抗氧化剂组合，并且将阐明它们对促进上皮伤口愈合和屏障功能的协同作用。这些研究的结果应该阐明糖尿病性角膜病变的发病机制，并导致目前缺乏的延迟糖尿病性角膜上皮伤口愈合的有效治疗。

DESCRIPTION (provided by applicant): The long-term goal of the laboratory has been to obtain basic information about the molecular and cellular biology of corneal wound healing. The studies in the current grant period demonstrated that epidermal growth factor receptor (EGFR) is the key receptor tyrosine kinase activated upon wounding, and the wound-induced activation of EGFR and its downstream signaling pathways, particularly ERK and PI3K, play a central role in mediating corneal epithelial wound healing. While in normal cornea an epithelial wound is usually healed in a timely fashion without any complications, epithelial wound healing is considerably delayed in the corneas of diabetic patients. The delayed healing of the epithelial defect (termed diabetic keratopathy) may result in sight threatening complications, such as persistent epithelial defect, recurrent erosion, and microbial keratitis. Preliminary studies using streptozotocin (SZT)-treated rats as a type 1 diabetes model and human diseased corneas revealed that EGFR signaling, in particular PI3K pathways, is compromised in the epithelia of diabetic rat corneas and of human diabetic, but not in normal and diet-controlled corneas. Our published and preliminary studies lead to the hypothesis that the tightly regulated EGFR signaling is abrogated by hyperglycemia in the epithelium and that strengthening of the weakened EGFR signaling, along with reducing oxidative stress, may preserve epithelial integrity and promote epithelial wound healing in the diabetic corneas. Three specific aims are proposed to test this hypothesis. 1) To characterize EGFR signaling pathways and oxidative damage in SZT diabetic rat corneas. Alterations of EGFR signaling in the epithelia of SZT-induced and human diabetic corneas will be characterized and correlated to the epithelial abnormalities observed in these diseased corneas. 2) To determine how hyperglycemia affects EGFR signaling and epithelial integrity and function in the cornea. Cultured porcine corneas and primary human corneal epithelial cells will be used. The effects of high glucose that mimics hyperglycemia on the generation of reactive oxygen species, on EGFR signaling, and on epithelial integrity and function including barrier properties and wound healing will be assessed. 3) To determine how manipulation of EGFR-signaling pathways might be used to accelerate epithelial wound healing in diabetic corneas. EGFR ligands will be combined with antioxidant and their synergistic effects on promoting epithelial wound healing and barrier function will be elucidated. The results of these proposed studies should shed light on the mechanisms underlying pathogenesis of diabetic keratopathy and lead to the identification of effective treatments for the delayed diabetic corneal epithelial wound healing which currently are lacking.