We propose to examine changes in coronary and peripheral microvascular reactivity and identify the respective mechanisms of change, which occurs as a consequence of extracorporeal circulation/cardiopulmonary bypass (CPB) and cardioplegia. This will be accomplished by a unique approach to the study of the coronary, mesenteric and skeletal muscle microcirculations using both in vivo and in vitro techniques, such that auto regulatory and metabolic influences are minimized or eliminated. Standard cellular and molecular approaches will be used to determine what specific changes occur in vasomotor pathways. Porcine models will be employed to define the effects of CPB and cardioplegia on vascular signal transduction, while isolated micro vessels from rats will be used to examine the effects of specific pathologic processes on vascular reactivity. Since CPB and cardioplegia inherently involve numerous simultaneous pathologic processes, whenever possible, the effects of specific pathologic processes will be inhibited and the consequent change in vascular function assessed. In vitro experiments on intact isolated micro vessels will also be performed to identify the changes in signal transduction responsible for altered tone operating through conventional and novel isoforms of protein kinase C (PKC), mitogen activated protein (MAP) kinases and myosin light chain (MLC) phosphorylation pathways in vascular smooth muscle. The following specific aims will be addressed: 1) to identify the mechanisms responsible for altered agonist-induced and myogenic microvascular contraction after cardioplegia in the porcine model. 2) To examine the mechanisms causing reduced microvascular smooth muscle myogenic and agonist induced contraction of peripheral (skeletal muscle and mesenteric) micro vessels after CPB. 3) To examine the effects of hydrogen peroxide and other enhancers of oxidative stress and inhibitors of oxidant stress on PKC-mediated contraction in isolated peripheral (skeletal and mesenteric) rat vessels. In addition, we will study the effects of prior exposure of vessels to alpha-1 adrenergic (phenylephrine), thromboxane A2 (U46619), and vasopressin. 4) The relationship of vasomotor regulation of peripheral micro vessels to direct cytokine exposure will be examined in vitro. Specifically, peripheral micro vessels will be exposure to platelet-derived growth factor, vascular endothelial growth factor, tumor necrosis factor-alpha interleukin-6, and interleukin-8 alone and in combination and in the presence of increased oxidative stress. These studies should provide a better understanding of the cause, potential prevention and treatment of systemic hypotension and mal-perfusion and may improve the outcome of patients (reduced morbidity and shorter length of hospitalization) undergoing cardiac surgery in which CPB support is utilized. Finally, given that alterations in vasomotor regulation and other pathological conditions such as increased vascular permeability often share common mechanisms, the findings of the experiments described in this proposal may have universal implications.
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