In myocardial ischemia-reperfusion (I/RP), the production of reactive oxygen species (ROS), such as superoxide radicals (023, from vascular endothelial cells (ECs) during RP is thought to play a critical role in tissue injury. The injury is attributed partially to peroxynitrite (ONOO-), a product of the reaction between nitric oxide (NO) and O2-, and to the small GTPase Racl that activates the O2--producing NAD(P)H oxidase. In vitro studies have used hypoxia/reoxygenation (H/RO) in static ECs to simulate I/RP, ignoring possible flow effects on the cellular response. It is known, however, that the onset of laminar shear stress triggers NO and 02- generation, ONOO-formation and redox-sensitive gene expression. Thus, this study aims to investigate the role of endogenous ROS on EC dysfunction following the exposure of static hypoxic ECs to the onset of steady laminar (or oscillatory) shear stress concurrently with oxygen readmission. It is hypothesized that at RP, ECs will produce ROS of different levels/time profiles resulting in different extents of dysfunction compared to RO. Production rates of NO and 02- will be measured during RP vs. RO. EC dysfunction will be quantified by assaying for: (a) lipid peroxidation and apoptosis/activation of pro-apoptotic signaling molecules (such as the transcription factor NF-rB), (b) expression of leukocyte adhesion molecules and the associated EC-neutrophil adhesive interactions. While measuring each marker of dysfunction, inhibitors of key ROS sources (including adenoviral expression of a dominant negative form of Racl) and ROS scavengers (eg. a ONOO- decomposition catalyst) will help us identify the ROS responsible for the EC injury. If the injury is inhibited by both NO and 02- inhibitors, then ONOO-is implicated and its relative changes (both intracellularly and extracellularly) will be monitored during RP with or without the presence of inhibitors/scavengers. Extracellular ONOO- was shown to be an index of loss of myocardial function, so the ONOO' concentrations that the ECs are exposed to will be estimated using a mathematical transport/reaction model of ROS in the extracellular space. In sum, the new RP model will advance our knowledge on EC survival under conditions where both changes in oxygen tension and fluid flow occur.
