Pulsatile flow increases the expression of eNOS, ET-1, and prostacyclin in a novel in vitro coculture model of the retinal vasculature. Invest Ophthalmol Vis Sci 46: 375–382

Abstract

PURPOSE. By the development of a novel retinal microvascular endothelial and pericyte cell coculture system, this study determined the effects of pulsatile flow on the activation of the endothelial cell markers nitric oxide (NO), prostacyclin (PGI 2 ), and endothelin (ET)-1. METHODS. Monocultured bovine retinal endothelial cells (BRECs) and cocultured BRECs with bovine retinal pericytes (BRPs) were exposed to low flow (flow rate, 0.3 mL/min; pulse pressure, 6 mm Hg; shear stress, 0.5 dyne/cm 2 ) or high flow (flow rate, 25 mL/min; pulse pressure, 56 mm Hg; shear stress, 23 dynes/cm 2 ) for 24 hours, by using a novel perfused transcapillary culture system. The cells were characterized by immunohistochemistry and electron and confocal microscopy. Endothelial nitric oxide synthase (eNOS) and phosphorylatedeNOS Ser1179 (pp-eNOS) were determined by Western blot analysis. Nitrate, PGI 2 , and ET-1 levels were quantified in the medium perfusate by using fluorometric and enzyme-linked immunosorbent assays, respectively. Activation of cyclooxygenase (COX)-2 in BRECs was determined by measuring COX-2 promoter activity with a luciferase reporter assay. RESULTS. The presence of BRPs and BRECs was confirmed by Western blot, immunocytochemistry, and scanning electron microscopy. Phosphorylated eNOS (pp-eNOS) protein levels in BRECs were significantly increased from low to high flow in both mono-and cocultures, concomitant with a significant increase in nitrate levels in the conditioned medium after exposure to pulsatile flow. In parallel cultures, PGI 2 levels were also significantly enhanced concomitant with an increase in the transactivation of a COX-2 promoter BREC after exposure to pulsatile flow. ET-1 levels were also increased in both mono-and cocultured cells. CONCLUSIONS. In this study a novel, functioning, in vitro model of retinal microvascular endothelial and pericyte cells that respond to changes in pulsatile flow was established. (Invest Ophthalmol Vis Sci. 2005;46:375-382