MONOCYTE OXIDATION OF LIPOPROTEIN LIPIDS

单核细胞是肿瘤病理特征的积极参与者， 动脉粥样硬化斑块氧化脂质和最近氧化的 脂蛋白已经在动脉粥样硬化病变中被鉴定， 假设作为深层脂质的病理介质参与 积累和细胞损伤。我们的实验室致力于定义 人单核细胞低密度氧化的机制和途径 脂蛋白（LDL）脂质，因为我们第一次报告说，单核细胞是 能够氧化LDL。我们已经报道了单核细胞介导的LDL 氧化需要单核细胞活化，单核细胞产生超氧化物 阴离子（O2-），单核细胞15-脂氧合酶活性和细胞内 来自两种钙源的钙水平，细胞内膜储存 和细胞外钙的流入。我们最近还表明， LDL脂质的单核细胞氧化依赖于蛋白激酶C（PKC） 活动在本申请中，我们提出进行实验， 将进一步阐明人单核细胞 LDL脂质的氧化。首先，我们建议严格评估 15-LO在这一过程中发挥的作用。由于我们最近的研究表明， 单核细胞氧化LDL上的主要氧化脂肪酸是13-HPODE， 13-HPODE是15-LO作用于亚油酸的产物，我们建议 首先探索15-LO是否显著有助于催化 在单核细胞氧化的LDL上形成13-HPODE。我们还将研究 从人动脉粥样硬化中提取的13-HPODE和13-HODE是否 损伤由15-LO酶活性形成。这些研究将揭示 15-LO如何参与LDL脂质的单核细胞氧化， 直接作为在LDL上形成氧化脂质的酶，或 间接作为诱导的次级信使级联的一部分， 单核细胞活化在目标2中，我们建议确定 亚油酸被氧化成HPODE（无论13- HPODE形成），并阐明了产生HPODE- 胆固醇酯和HPODE-磷脂酯，我们最近 显示了在单核细胞氧化的LDL上发现的HPODE的化学形式。 最后，在目标3中，我们将评估可能的信号转导途径， 它们参与这种脂质氧化过程， 单核细胞是诱导它们氧化LDL脂质所必需的。在这 目的探讨蛋白激酶C同工酶、钙离子通道蛋白 和磷脂酶A2，我们将研究它们对两个关键的 这个过程的元素，O2-的生产和15-的活化， LO活动。从这些研究中获得的知识将有助于 对我们理解单核细胞 氧化LDL脂质，并可能促进更好地理解 病理过程导致这些氧化脂肪的存在， 酸在血管疾病中的作用

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.