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。本课题研究表明表皮生长因子受体（epidermal growth factor receptor， EGFR）是酪氨酸激酶在损伤时激活的关键受体，而表皮生长因子受体及其下游信号通路，尤其是ERK和PI3K，在介导角膜上皮损伤愈合中起着核心作用。在正常角膜中，上皮性创面通常及时愈合而无任何并发症，而糖尿病患者角膜上皮性创面愈合明显延迟。上皮缺损的延迟愈合（称为糖尿病性角膜病变）可能导致威胁视力的并发症，如持续性上皮缺损、复发性糜烂和微生物性角膜炎。用链脲佐菌素（SZT）治疗的大鼠作为1型糖尿病模型和人类病变角膜的初步研究表明，糖尿病大鼠和人类糖尿病的角膜上皮中EGFR信号通路，特别是PI3K通路受到损害，而在正常和饮食控制的角膜中则没有。我们已发表的和初步的研究提出了这样的假设，即上皮内受到严格调控的EGFR信号被高血糖所取消，而增强被削弱的EGFR信号，以及减少氧化应激，可能会保持上皮的完整性，促进糖尿病角膜上皮伤口愈合。为了验证这一假设，提出了三个具体目标。1)研究SZT糖尿病大鼠角膜EGFR信号通路及氧化损伤。szt诱导和人糖尿病角膜上皮中EGFR信号的改变将被表征，并与这些病变角膜中观察到的上皮异常相关。2)确定高血糖如何影响EGFR信号和角膜上皮的完整性和功能。将使用培养的猪角膜和原代人角膜上皮细胞。将评估模拟高血糖的高葡萄糖对活性氧生成、EGFR信号传导、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配体将与抗氧化剂结合，并在促进上皮伤口愈合和屏障功能方面发挥协同作用。这些研究的结果将揭示糖尿病角膜病变的潜在发病机制，并导致目前缺乏的糖尿病角膜上皮伤口延迟愈合的有效治疗方法的确定。

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.