524: Effect of hypoxia and hyperthermia on eNOS protein concentration in cultured ovine umbilical vein endothelial cells

Abstract

CULTURED OVINE UMBILICAL VEIN ENDOTHELIAL CELLS JUAN A ARROYO, RUSSELL V. ANTHONY, BRADLEY T. ZIEBELL, HENRY L. GALAN, University of Colorado Health Sciences Center, Obstetrics and Gynecology, Aurora, Colorado, University of Colorado Health Sciences Center, Pedriatics, Aurora, Colorado OBJECTIVE: Nitric Oxide (NO) and endothelial nitric oxide synthase (eNOS), important regulators of placental blood flow, are reduced in a hyperthermic-induced (HT) ovine model of fetal growth restriction (FGR) with fetal hypoxia (HX) and hypertension. Our objective was to differentiate the effect of hypoxia and HT on NO and eNOS production in ovine umbilical vein endothelial cells (OUVEC). STUDY DESIGN: At 130 days gestational age (dGA), OUVEC were isolated with a collagenase B solution and cultured in 2% gelatin coated flasks for up to three passages. CD31 and Factor VIII staining confirmed endothelial cell type. Endothelial cells were treated with HX (2% O2) or HT (40°C) for 24, 48 and 72 hours (H). At each time point, OUVEC eNOS protein and NO from culture media were assessed by Western blot and a NOx assay kit, respectively. RESULTS: Compared to controls, hypoxia-exposed OUVEC showed increased eNOS protein concentration at 48H (2-fold, p 0.0005), but decreased at 72H (1.6-fold, p 0.03) while OUVEC media NOx concentration was only increased at 48H (figure) with otherwise no changes. In HT exposed OUVEC, eNOS protein was increased at 72H (2-fold, p 0.05) with no changes observed for NOx at any time point. CONCLUSION: Hypoxia and hyperthermia independently regulate endothelial eNOS protein production in OUVEC. Hypoxia induced upregulation of this protein is temporary as a concomitant increase in NO production is only seen at 48H, which may reflect ongoing cell death or a decrease in the eNOS substrate L-arginine. (Supported by NIH grant R01 HL071990-01A1).