Abstract
Abstract: :
Purpose:Topical nepafenac uniquely inhibits pathologic ocular angiogenesis in a variety of animal models, as compared to other marketed NSAIDs. Following topical ocular administration, Nepafenac is rapidly metabolized to its active metabolite, amfenac, a potent COX–1/–2 inhibitor. Vascular endothelial cell growth factor (VEGF) –induced endothelial cell proliferation and differentiation are critical components of angiogenesis. In vitro, cyclooxygenase (COX) inhibitors, such as Amfenac and Celecoxib, are NSAIDs that have been shown to reduce the VEGF–induced proliferation and tube formation of human retinal microvascular endothelial cells (HRMEC). In this study, we have examined the effect of Amfenac and Celecoxib on the phosphorylation state of the VEGF downstream signaling intermediate ERK, in VEGF–treated HRMEC. Methods: HRMEC were grown to 70–80% confluency, serum starved overnight and treated with VEGF for 30 minutes in the presence of 0.1ºM Amfenac or 1.0ºM Celecoxib. Some cells were maintained in serum–free medium and served as a control group. These drug concentrations were chosen on the basis of the least concentration required for maximal inhibition of HRMEC proliferation determined in previous studies. ERK phosphorylation was determined by Western blot analysis using a phospho–specific ERK antibody. Band intensities were determined by densitiometric analysis. Results:Amfenac treatment produced an 88% decrease (p < 0.005) in VEGF–induced phosphorylation of ERK whereas Celecoxib at 10–fold higher concentration had no apparent effect. Conclusions:Amfenac may inhibit VEGF–induced HRMEC proliferation by blocking the phosphorylation of ERK. This may occur through a COX–2–independent mechanism, since the COX–2–selective inhibitor Celecoxib produced no effect on ERK phosphorylation. This novel finding may account, in part, for the unique antiangiogenic efficacy provided by topical nepafenac in vivo.
Keywords: neovascularization • retina • signal transduction