Portal hypertension is a syndrome accounting for significant morbidity and mortality in patients with liver cirrhosis. However, the molecular events which contribute to hepatic vasoconstriction and ensuing portal hypertension remain poorly understood. In this regard the nitric oxide (NO) system has elicited significant attention owing to its vital role in vascular homeostasis. The Long-Term Goal of our program is to understand the biological control mechanisms of the endothelial nitric oxide synthase (eNOS) enzyme in liver endothelial cells (LEC) and the pathobiologic role of this system in development of portal hypertension. In support of this goal, our Preliminary Studies have 1) established the vital role of eNOS in hepatic vascular regulation, 2) identified a deficiency of shear stress dependent NO production in LEC as a pathogenic mechanism in the development of portal hypertension, 3) determined that dynamin-2, a protein with distinct vesicle trafficking and signal transduction properties, binds directly with eNOS and promotes NOS function, and 4) identified caveolin-1, as a key contributory protein in development of deficient NOS activation in portal hypertension. These studies have led us to formulate the well-supported Central Hypothesis of this Proposal, that dynamin and caveolin play reciprocal and vital roles in the molecular regulation of eNOS and thereby contribute to deficient NO production in portal hypertension. In our Specific Aims we will: 1) determine the interaction domains between dynamin and eNOS using deletion mutagenesis and then dissect the mechanisms by which dynamin-vesicle trafficking and -NOS binding determine NOS localization and function, 2) explore the mechanism by which dynamin binding activates eNOS by delineating the effect of binding on specific biochemical components of the NOS catalytic activation process, and 3) assess how dynamin and caveolin act in concert to modulate the shear stress activation o about eNOS and the role of these protein interactions in portal hypertension. To address these Aims, we will utilize a variety of feasible and state-of-the-art approaches in primary LEC, novel cell lines, and validated models of portal hypertension including adenoviral based gene delivery, caveolae isolation, and quantitative measurement of NO from single cells. In sum, this hypothesis-driven proposal will delineate novel regulatory mechanisms of eNOS derived NO as they relate to hepatic vascular biology and thereby enhance our understanding of the pathogenic mechanisms underlying portal hypertension.
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