Monocytes are active participants int he pathologic features of the atherosclerotic plaque. Oxidized lipids and recently oxidized lipoproteins have been identified in atherosclerotic lesions and are hypothesized to participate as pathologic mediators of the profound lipid accumulation and in cell injury. Our laboratory has focused on defining the mechanisms and pathways of human monocyte oxidation of low density lipoprotein (LDL) lipids since we first reported that monocytes were capable of oxidizing LDL. We have reported that monocyte-mediated LDL oxidation requires monocyte activation, monocyte production of superoxide anion (O2-), monocyte 15-lipoxygenase activity and rises in intracellular calcium levels from two calcium sources, intracellular membrane stores and influx of extracellular calcium. We have also recently shown that monocyte oxidation of LDL lipids is dependent on protein kinase C (PKC) activity. In this application, we propose to perform experiments that will further elucidate the mechanisms involved in human monocyte oxidation of LDL lipids. First, we propose to rigorously assess the role that 15-LO plays in this process. Since our recent studies show that the predominant oxidized fatty acid on monocyte-oxidized LDL is 13-HPODE and 13-HPODE is the product of 15-LO acting on linoleic acid, we propose to first explore whether 15-LO significantly contributes to the catalytic formation of 13-HPODE on monocyte-oxidized LDL. We also will examine whether the 13-HPODE and 13-HODE extracted from human atherosclerotic lesions is formed by 15-LO enzymatic activity. These studies will reveal how 15-LO is participating in monocyte oxidation of LDL lipids, either directly as the enzyme that forms the oxidized lipids on LDL or indirectly as part of the secondary messenger cascade induced upon monocyte activation.
In Aim 2 we propose to determine the source of the linoleic acid that becomes oxidized to HPODE (regardless of how the 13- HPODE is formed) and elucidate the process for generating HPODE- cholesteryl esters and HPODE-phospholipid esters, which we have recently shown are the chemical forms of HPODE found on monocyte oxidized LDL. Finally in Aim 3, we will assess likely signal transduction pathways for their participation in this lipid oxidation process since activation of monocytes is essential for inducing them to oxidize LDL lipids. In this Aim we will primarily focus on further studies on PKC isoenzymes, calcium and phospholipases A2 and we will investigate their regulation of two key elements of this process, the production of O2- and the activation of 15- LO activity. Knowledge acquired from these studies will contribute substantially to our understanding of the mechanisms of monocyte oxidation of LDL lipids and likely promote a better comprehension of the pathological processes leading to the presence of these oxidized fatty acids in vascular disease.
Showing the most recent 10 out of 27 publications
