Inhibition Of VEGF–Induced Endothelial Cell Poliferation And Differentiation By Steroidal And Non–steroidal Cox Inhibitors With Variable Cox–1/Cox–2 Selectivity

Abstract

Abstract: : Purpose: Endothelial cell proliferation and differentiation are critical components of angiogenesis. Inhibition of VEGF–induced bovine retinal microvascular endothelial cell (BRMEC) proliferation and tube formation (differentiation) in vitro was tested with the following series of steroidal and non–steroidal cyclooxygenase (COX) inhibitors with varied COX–1/COX–2 selectivity: the deaminated derivative of nepafenac, celocoxib, rofecoxib, diclofenac salt, ketorolac tromethamine salt, NS398, SC560 and dexamethasone. The PKC–beta inhibitor, LY333531, and the active metabolite of anecortave acetate were used as known active controls. Methods: BRMEC were grown on 96–well plates coated with fibronectin/hyaluronic acid. Growth medium was added for 2d followed by serum free medium overnight. Test medium containing 25 ng/ml VEGF, 0.1%DMSO and COX inhibitor at concentrations ranging from 0.01 to 10 mM was added for 24 hr. BRMEC proliferation was assayed using a modified MTT assay. For tube formation, BRMEC were grown on 6–well plates, within a Vitrogen 100 growth matrix sandwich and incubated in growth medium for 2d. The medium was replaced with 0.5% FBS plus 25 ng/ml VEGF with and without COX inhibitor and 0.1% DMSO. The mean tube length of each experimental group was digitally analyzed after 48hr in the experimental medium. Results: All test compounds except dexamethasone demonstrated inhibition of VEGF–induced BRMEC proliferation ranging from 42%–100%. The deaminated derivative of nepafenac, the active metabolite of anecortave acetate and celecoxib appeared to be the most potent. All test compounds showed inhibition of tube formation ranging from 28%–100%, with the deaminated derivative of nepafenac, celecoxib, rofecoxib, diclofenac and ketorolac being the most effective. Conclusions: Rofecoxib and celecoxib are COX–2 selective NSAIDs, while the nepafenac derivative and diclofenac are non–selective. Yet, the abilities of these compounds to inhibit endothelial cell proliferation and differentiation are comparable. Thus, it appears that COX selectivity is not a determining factor in the angiostatic activity demonstrated by these compounds.

Keywords: retinal neovascularization