Research Project II - Vesicant-Induced Corneal Injury

研究项目2.发泡剂引起的角膜损伤 项目负责人：马里恩·K·戈登博士。 共同调查者：劳里·B·约瑟夫，博士 项目摘要/摘要 包括硫芥末(SM)和氮芥末(NM)在内的芥末发泡剂会导致一系列的衰弱 对人类眼睛的伤害，可能导致长期的病理和失明。在以前的研究中，我们 证明芥末损伤的部分原因是角膜上皮与基质的分离； 此外，这是包括基质金属蛋白酶(MMP9)在内的蛋白水解酶激活的结果。 内肽酶，ADAM17，降解必要的上皮细胞锚定蛋白。在最后一次拨款期间 期间，我们证明了MMPs的抑制剂多西环素有效地钝化了两者的毒性。 NM和SM在培养的角膜中，以及在兔SM蒸汽角膜损伤模型中。根据我们的发现，有两个 IND前会议与FDA举行，眼科药物产品土霉素目前正在 迈向超前发展。EMMPRIN(细胞外)诱导MMP9和ADAM17的表达 基质金属蛋白酶诱导因子(CD147)，一种由上皮细胞合成的跨膜糖蛋白。 EMMPRIN的配体是亲环素A，它是细胞在损伤或氧化应激时释放出来的。 在接触芥末后，我们发现EMMPRIN在角膜中显著上调，这表明 治疗干预的潜在靶点。免疫抑制剂环孢素A(CsA)已被 显示在纳摩尔浓度下抑制亲环素A的活性。我们发现Restasis®，一种CSA 乳剂能有效地阻止角膜暴露于NM后的上皮脱落。此外，它还 减少角膜组织中MMP9的表达。这些令人兴奋的发现表明Restasis®有潜力成为 被开发为对抗芥子的目测对策。基于这些发现，我们假设 芥末诱导的角膜氧化应激导致角膜上皮细胞释放亲环素A 这会上调EMMPRIN；这会导致导致上皮-间质分离的蛋白酶增加； EMMPRIN还调节临时基质蛋白(如SPARC、Hevin、纤维连接蛋白)的表达。作为一名 结果，伤口愈合延迟，持续的上皮损伤最终导致慢性 疾病。为了验证这一假设，我们计划：(1)分析芥末发泡剂对EMMPRIN的影响 (2)评估芥末发泡剂对临时基质沉积的影响 角膜中的分子；以及(3)确定阻断亲环素A是否诱导EMMPRIN激活 抑制芥末引起的角膜损伤。我们的研究结果将阐明调解机制 芥末发泡剂诱导的角膜损伤将导致新的有效治疗方法的确定 减轻毒性。

Research Project 2. Vesicant-induced Cornea Injury Project Lead: Marion K. Gordon, Ph.D. Co-Investigator: Laurie B. Joseph, Ph.D. Project Summary/Abstract Mustard vesicants including sulfur mustard (SM) and nitrogen mustard (NM) cause a range of debilitating injures to human eyes that can lead to long term pathologies and blindness. In previous studies we demonstrated that mustard injury is due in part to separation of the corneal epithelium from the stroma; moreover, this is a consequence of activation of proteases including matrix metalloproteinase (MMP)-9 and the endopeptidase, ADAM17, which degrade essential epithelial cell anchoring proteins. During the last grant period, we demonstrated that doxycycline, an inhibitor of MMPs, was effective in blunting the toxicity of both NM and SM in cultured corneas, and in a rabbit SM vapor model of corneal injury. Based on our findings, two pre-IND meetings were held with the FDA, and an ocular drug product, oxytetracycline, is currently moving towards advanced development. Expression of MMP9 and ADAM17 is induced by EMMPRIN (Extracellular Matrix Metalloproteinase Inducer, CD147), a transmembranous glycoprotein synthesized by epithelial cells. The ligand for EMMPRIN is cyclophilin A, which is released from cells in response to injury or oxidative stress. Following mustard exposure, we found that EMMPRIN is markedly upregulated in the cornea, suggesting a potential target for therapeutic intervention. The immunosuppressive agent cyclosporine A (CsA) has been shown to inhibit cyclophilin A activity at nanomolar concentrations. We discovered that Restasis®, a CsA emulsion, is effective in blocking epithelial detachment after exposure of the cornea to NM. Moreover, it reduced MMP9 expression in the cornea. These exciting findings suggest that Restasis® has potential to be developed as an ocular countermeasure against mustards. Based on these findings, we hypothesize that mustard induced oxidative stress in the cornea causes the release of cyclophilin A from corneal epithelial cells which upregulates EMMPRIN; this results in increases in proteases which cause epithelial-stromal separation; EMMPRIN also dysregulates expression of provisional matrix proteins (e.g., SPARC, hevin, fibronectin). As a consequence, there is delayed wound healing and persistent epithelial injury which ultimately leads to chronic disease. To test this hypothesis, plans are to: (1) Analyze the effects of mustard vesicants on EMMPRIN expression in the cornea; (2) Assess the effects of mustard vesicants on deposition of provisional matrix molecules in the cornea; and (3) Determine whether blocking cyclophilin A-induced activation EMMPRIN suppresses mustard-induced corneal damage. Results of our studies will elucidate mechanisms mediating mustard vesicant-induced corneal injury which will lead to the identification of new efficacious therapeutics for mitigating toxicity.