A novel peroxidase in vascular endothelium and the development of atherosclerosis

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DESCRIPTION (provided by applicant): Evidence implicates a role for heme-containing peroxidase in atherogenesis due in part to oxidation of low density lipoprotein (LDL). While generally attributed to the phagocyte-specific enzyme myeloperoxidase (MPO), it is unclear whether a peroxidase other than MPO in vascular wall plays a role in atherogenesis. We have now identified and have begun to characterize a new heme-containing peroxidase that is present in heart, vascular smooth muscle and endothelial cells. This Vascular PerOxidase, termed VPO, is highly homologous to MPO, but also contains multiple domains that are likely to participate in protein-protein interactions. We have partially purified VPO from mouse heart and overexpressed cells, and in preliminary studies, some important enzymatic properties are similar to those of MPO. Our earlier investigations focused on novel NADPH- oxidases (Noxes) including Nox1, Nox4 and Nox5, which are major sources of reactive oxygen species in and adjacent to the vessel wall. Nox1 and perhaps other Noxes (but not the phagocyte NADPH-oxidase) are implicated in animal models of Angiotensin II-induced of atherosclerosis and hypertension. VPO, when co- expressed in cells with novel vascular Noxes, utilizes hydrogen peroxide produced by these Noxes to catalyze peroxidative reactions. Like MPO, VPO also generates HOCl from chloride, an important oxidant for the development of atherosclerosis. We hypothesize that VPO can carry out peroxidative reactions in the vascular system (e.g. LDL) that may be involved in the development of atherosclerotic disease. Aim #1 will purify VPO from mammalian cells stably overexpressing VPO and will characterize its basic substrate specificity and enzymatic properties. Aim #2 will investigate the oxidative modification of lipoproteins and lipids by VPO. The ability of purified VPO to oxidize LDL lipoprotein and lipid will be emphasized. Aim #3 will explore the subcellular localization of VPO and compare this with that of the vascular Noxes. In addition, the ability of vascular Noxes to support VPO-dependent oxidation will also be evaluated. Aim #4 will investigate the localization of VPO in intact vasculature and atherosclerotic lesions. These studies are an essential prelude to (patho)physiological studies (e.g., in knockout mice) of the role of VPO in cardiovascular disease. Understanding the role of this new peroxidase in the development of atherosclerosis may provide new diagnostic and/or therapeutic interventions targeted to VPO. PUBLIC HEALTH RELEVANCE: Our research aim is to understand why atherosclerosis, also known as the "hardening of blood vessels," occurs in cardiovascular system. It is clear that chemical change of low density lipoprotein (LDL), which contains "bad cholesterol," is one of the major reasons causing the blood vessels to become harden. Recent findings show that peroxidases are related to cardiovascular diseases including atherosclerosis. Peroxidases are enzymes that catalyze hydrogen peroxide reduction and play important roles in physiology, immunology and pathology such as killing of bacteria, modulation of cell signal transduction pathway and tissue damage. My proposal focuses on relationship between mechanism of cardiovascular disease causing by peroxidases and reactive oxygen species (in general, toxic agents). In the proposal, a novel peroxidase gene has be identified and cloned, called vascular peroxidase (VPO), which mainly exists in cardiovascular system. We will study VPO enzymatic features and in what conditions it changes LDL. First, we will study the properties of this enzyme, including substrate specificity and optimal chemical conditions. Then, we will decide at which part of cell the enzyme is located and explore what causes the increase of the enzyme's activity. We will also evaluate the features of chemical change of LDL and find out the possible ways to block the change. Finally, we will explore whether VPO and other related enzymes exists in intact vasculature and atherosclerotic lesions. Thus, we can elucidate whether the new enzyme is a new risk factor for atherosclerosis. If it is, then we can develop a new diagnostic tool to test the level of this enzyme and its activity in blood, allowing people to find "hardening of blood vessels" earlier. Furthermore, we anticipate this research will lead to the development of a new drug that inhibits the enzyme's activity and blocks the change of LDL.