Increased resistance to sinusoidal blood flow is an important component of early as well as advanced portal hypertension. This results from an imbalance in intrahepatic vasoconstrictor and vasodilator molecules, the latter including nitric oxide (NO). Our laboratory has been on the cutting edge of advances in understanding the molecular basis for the imbalance in NO. Seminal among our discoveries is that there is a (dramatic and remarkable) reduction in endothelial NO synthase (eNOS) dependent NO release by sinusoidal endothelial cells (SECs). Our focus has been on understanding the mechanism underlying this defect. Preliminary data presented in the current application has identified highly novel post-translational defects in eNOS in SECs after liver injury, including reduced phosphorylation of eNOS caused by reduced levels of a novel protein known as G-protein coupled receptor (GPCR) kinase interactor-1 (GIT1) that regulates eNOS phosphorylation, activity and NO production. In the basal state, GIT1 is the most potent stimulator of eNOS we have identified to date. Extensive new preliminary data provide a foundation for the hypothesis that GIT1 acts GIT1 acts as a scaffolding partner for a macromolecular complex including eNOS and other proteins that together regulate eNOS function in a spatiotemporal fashion. Importantly, the reduction in GIT1 expression after liver injury, have led us to hypothesize that post-translational defects in eNOS after liver injury are critical in the sinusoidal endothelialopathy that accompanies liver injury, and further, that GIT1 is a central component. The overarching goals of this new project are twofold. First, with a future objective being to translate our work to humans with liver disease, we wish to validate the importance of GIT1 in vivo. Secondly, we wish to uncover fundamental basic mechanisms that regulate eNOS function. Therefore, our specific aims are as follows: We will (1) define the functional importance of GIT1 in vivo in normal and injured liver, (2) characterize the molecular interaction between GIT1 and eNOS, and determine how GIT1 tyrosine phosphorylation affects this interaction and eNOS function, and (3) determine how the GIT1 binding partner, ??PIX (an integral component of a putative multiprotein signaling module) contributes to regulating eNOS function. The proposed experiments will uncover novel mechanistic aspects of eNOS structure and function and as such have fundamental therapeutic implications not only for patients with liver disease and portal hypertension, but also for those with other vascular disorders.
Liver damage leads to elevated resistance to blood flow through the liver. This leads to intestinal bleeding and severe clinical problems (such as bleeding and fluid build up), which are typical in patients with liver damage. This proposal aims to understand the cause of this elevated resistance to blood flow in the liver, and to develop new therapies to treat this problem.
