(from abstract). Corneal infections with herpes simplex virus (HSV) are estimated to affect 400,000 individuals in the U.S. and over 20,000 new cases present annually. This application focuses on the mechanism(s) by which HSV infections induce corneal edema, the most common complication of herpetic keratitis. We propose that HSV-induced edema results from inhibition of corneal endothelial cell Na+K+ATPase by products of an inducible nitric oxide synthase (NOS). NOS2 is induced and regulated by cytokines produced during HSV infections and products of this enzyme, nitric oxide (NO) and its metabolites, can alter the function of proteins including Na+K+ATPase.
Aim 1 is to measure corneal endothelial cell Na+K+ATPase during development and recovery from HSV-induced edema in a rabbit model. Pump function will be measured by rubidium uptake and enzyme activity by phosphate release from ATP. ATPase subunit mRNA will be quantitated by RT-PCR using mimics and standard curve methodology. Western blots of endothelial cell extracts from edematous corneas will be used to detect nitrotyrosine residues in the ATPase, definitive evidence of the action of NOS2 products.
Aim 2 will determine which cell types in the anterior segment express NOS2 during disease. NOS2 assays will measure the Ca+-independent conversion of L-anginine to L-citrulline. RT-PCR will be used to estimate changes in steady state NOS messages from all three NOS isoforms. In situ hybridization will be used to locate and tentatively identify cells expressing NOS2.
Aim 3 is to assess the effects of NO production on endothelial cell function. The effects of exogenously generated NO upon endothelial cell function in vivo will be evaluated by measuring corneal thickness, endothelial cell number, ouabain binding, and barrier function. NO generators and inducers of NOS2 will be used to determine the effects of NO on Na+K+ATPase activity in cultured cells. The nitration of proteins in corneal cells will be examined using immunochemical methods and electron spin resonance.
Aim 4 will determine the effects of regulators on Na+K+ATPase and NOS2 activity in corneal cells in vivo and in vitro. The results of these studies will determine whether NO generated by NOS2 is responsible for HSV-induced corneal edema and whether NOS isoforms or NO-regulated functions contribute to the pathogenesis of this disease. The long-term goal is to understand the pathogenesis of edema in order to develop effective and specific alternatives to corticosteroids for the therapy of HSV-induced edema and stromal disease.
| O'Brien, W J; Heimann, T; Tsao, L S et al. (2001) Regulation of nitric oxide synthase 2 in rabbit corneal cells. Invest Ophthalmol Vis Sci 42:713-9 |
