Arginase II, A Novel Target in Atherosclerosis

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DESCRIPTION (provided by applicant): An early and critical event in the development of endothelial dysfunction in atherosclerosis is the interaction of the oxidized low density lipoprotein (Ox-LDL) with the lectin-like oxidized low density lipoprotein receptor-1 (LOX-1). Our work to date has shown that activation of arginase-2 (Arg2) is a key step in Ox-LDL-mediated atherogenesis, likely leading to competitive depletion of the substrate L-arginine for eNOS, leading to decreased NO bioavailability. Our preliminary studies with human aortic endothelial cells (HAEC) suggest that Ox-LDL exposure releases a pre-existing pool of Arg2 into the cytosol from mitochondria. This, in turn, diminishes the concentration of cytosolic L-arginine leading to eNOS uncoupling. Ox-LDL-evoked changes in cytosolic Arg2 activity are muted by Rho-kinase inhibition, and they do not occur at all in LOX-1 null endothelial cells. In addition, inhibition or siRNA knockdown of mitochondrial processing peptidase (MPP) reduce the cytosolic abundance and activity of Arg-2 following Ox-LDL stimulation. Furthermore, blocking MPP attenuates OxLDL-mediated changes in EC reactive oxygen species (ROS) and NO. We therefore hypothesize that an Ox-LDL-LOX1-Rho kinase signaling axis triggers an increase in EC cytosolic Arg2 activity via MPP-mediated decompartmentalization of Arg2 from the mitochondria to cytosol through the following sequence of events: Mitochondrial MPP is activated, and removes the mitochondrial targeting sequence (MTS) from the N-terminus of Arg2; Arg2 then moves to the cytosol where increased Arg2 activity decreases the concentration of L- arginine; this impairs the bioavailability of NO by depleting substrate for eNOS, and also increases ROS (eNOS uncoupling). These events result in EC dysfunction and contribute to atherogenesis. In the first aim, we will test the hypothesis that MPP-mediated cleavage of Arg2 at the MTS site is responsible for the activation of Arg2 in vitro, and test whether Arg2 abundance in the cytosol that is induced by Ox-LDL is due to increased MPP activity and retrograde transport out of mitochondria. Studies in the second aim will define changes in the localization of potential partners in the LOX-1 signaling complex with OxLDL stimulation, and will test the hypothesis that mechanotransduction mediates OxLDL activation of Arg2 through LOX-1 and the following signaling intermediaries: MT1-MMP, p27kip1, RhoA, ROCK, and mDia1. In the third aim we will examine Arg2 activition and subsequent depletion of L-Arginine substrate as mechanisms of eNOS uncoupling. In the fourth aim, an atherogenic diet in genetically hypercholesterolemic (ApoE-/-) mice will be used to evaluate the consequences of inhibiting Arg2 with a small molecule inhibitor, or genetic deletion (ApoE- /- Arg2-/-, double KO). Primary outcome variables for this last aim will include endothelial dysfunction, vascular stiffness, thickening of the aortic intima and media, and the atherosclerotic plaque burden. Taken together, these studies will allow us to better understand the role of Arg2 in the pathobiology of atherosclerosis, and determine whether Arg2 represents a novel target for the effective treatment of this disease process.