Laminar shear stress is an important stimulus for vasodilation. In many species the mechanism involves endothelial release of nitric oxide. In humans with coronary artery disease (CAD), we have determined that endothelial derived hyperpolarization factor (EDHF) formed by cytochrome P450 mediates flow-induced dilation, possibly to compensate for loss of nitric oxide (NO). Shear stress has also been shown to induce changes in the vascular redox state. Flow-dependent release of superoxide, which is dismutated to H2O2., is observed in conduit arteries and endothelial cells in culture. Production of reactive oxygen species (ROS) has traditionally been considered a pathological response that leads to impaired vasomotor function. However, ROS may plan an important role in the physiological regulation of vessel function. Recent data suggests that H2O2 is an EDHF. Since cytochrome P450, a key enzymatic pathway in the formation of EDHF, also generates ROS, we hypothesize in Aim 1 that H2O2 mediates flow-induced dilation in human coronary arterioles. The signaling pathway transducing shear stress to NO release has been characterized and involves activation of tyrosine kinases linked to integrins and focal adhesions, heterotrimeric G-proteins, and phospholipases. Whether these cell processes are also involved in shear- mediated release of ROS is not known. Furthermore the endothelial oxidant enzyme systems responsible for generating ROS are incompletely understood. These critical features of shear-induced redox changes will be examined.
In Aim 2, we shall determine which key enzyme systems including cytochrome P450, nitric oxide synthase, and NADPH oxidase are responsible for shear-induced generation of ROS.
In Aim 3 we shall determine the intracellular signaling pathways producing ROS, focusing on the role of tyrosine kinases, phospholipases, and G-proteins. The effects of inhibiting endothelial function on ROS production will be examined. These questions will be examined in isolated human coronary arterioles prepared for in vitro measurement of flow-induced dilation, smooth muscle hyperpolarization, and fluorescence as well as ESR detection of ROS. Our model system is well-suited for studying ROS-mediated flow- induced vasomotor responses since these vessels demonstrate flow- mediated vasodilation (FMD), release superoxide in response to flow, and do not produce vasoactive levels of NO in response to shear. NO may interfere with ROS activity by converting superoxide to peroxynitrite, producing direct vasomotor responses, and by inhibiting cytochrome P450. The proposed experiments are designed to provide novel mechanistic insight into pathophysiological regulation of the human coronary microcirculation. The results should improve our understanding of the shear-mediated events leading to release of ROS.
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