Pulsatile flow increases the expression of eNOS, ET-1, and prostacyclin in a novel in vitro coculture model of the retinal vasculature.
OBJECTIVE
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 (PGI2), 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 mmHg; shear stress, 0.5 dyne/cm2) or high flow (flow rate, 25 mL/min; pulse pressure, 56 mmHg; shear stress, 23 dynes/cm2) 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 phosphorylated-eNOSSer1179 (pp-eNOS) were determined by Western blot analysis. Nitrate, PGI2, 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, PGI2 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.
Walshe
Tony E
TE
Vascular Health Research Centre, Faculty of Science and Health, Dublin City University, Dublin, Ireland.
Ferguson
Gail
G
Connell
Paul
P
O'Brien
Colm
C
Cahill
Paul A
PA
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D018920:Coculture Techniques; D051546:Cyclooxygenase 2; D004591:Electrophoresis, Polyacrylamide Gel; D019332:Endothelin-1 / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D011464:Epoprostenol / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D007527:Isoenzymes / Q000378:metabolism; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D020286:Pericytes / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D011673:Pulsatile Flow / Q000502:physiology*; D012171:Retinal Vessels / Q000166:cytology / Q000502:physiology*
D019332:Endothelin-1; D007527:Isoenzymes; D011464:Epoprostenol; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases