INCREASE OF REACTIVE OXYGEN SPECIES (ROS) IN ENDOTHELIAL-CELLS BY SHEAR-FLOW AND INVOLVEMENT OF ROS IN SHEAR-INDUCED C-FOS EXPRESSION

Intracellular reactive oxygen species (ROS) may participate in cellula r responses to various stimuli including hemodynamic forces and act as signal transduction messengers. Human umbilical vein endothelial cell s (ECs) were subjected to laminar shear flow with shear stress of 15, 25, or 40 dynes/cm(2) in a parallel plate flow chamber to demonstrate the potential role of ROS in shear-induced cellular response. The use of 2',7'-dichlorofluorescin diacetate (DCFH-DA) to measure ROS levels in ECs indicated that shear flow for 15 minutes resulted in a 0.5- to 1.5-fold increase in intracellular ROS. The levels remained elevated u nder shear flow conditions for 2 hours when compared to unsheared cont rols. The shear-induced elevation of ROS was blocked by either antioxi dant N-acetyl-cysteine (NAG) or catalase. An iron chelator, deferoxami ne mesylate, also significantly reduced the ROS elevation. A similar i nhibitory effect was seen With a hydroxyl radical ((OH)-O-.) scavenger , 1,3-dimethyl-2-thiourea (DMTU), suggesting that hydrogen peroxide (H 2O2), (OH)-O-., and possibly other ROS molecules in ECs were modulated by shear flow. Concomitantly, a 1.3-fold increase of decomposition of exogenously added H2O2 was observed in extracts from ECs sheared for 60 minutes. This antioxidant activity, abolished by a catalase inhibit or (3-amino-1,2,4-triazole), was primarily due to the catalase. The ef fect of ROS on intracellular events was examined in c-fos gene express ion which was previously shown to be shear inducible. Decreasing ROS l evels by antioxidant (NAG or catalase) significantly reduced the induc tion of c-fos expression in sheared ECs. We demonstrate for the first time that shear force can modulate intracellular ROS levels and antiox idant activity in ECs. Furthermore, the ROS generation is involved in mediating shear induced c-fos expression. Our study illustrates the im portance of ROS in the response and adaptation of ECs to shear flow. ( C) 1998 Wiley-Liss, Inc.