Research Project II - Vesicant-Induced Corneal Injury

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.