Lipid glyceryl ester homeostasis in macrophages and perturbation by environmental

巨噬细胞中的脂质甘油酯稳态和环境扰动

基本信息

批准号：
8232778
负责人：
MATTHEW K ROSS
金额：
$ 42.55万
依托单位：
MISSISSIPPI STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-01 至 2017-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8232778
关键词：
2-arachidonylglycerol Accounting Agonist Anabolism Animal Model Atherosclerosis Blood Blood Vessels CNR1 gene CNR2 gene Cannabinoids Carboxylic Ester Hydrolases Cardiovascular Diseases Catabolism Cell Line Cells Cholesterol Cholesterol Esters Cholesterol Homeostasis Chronic Clinical Combined Modality Therapy Complement Cues Culture Media Development Dinoprostone Disease Endocannabinoids Enzymes Esters Exposure to Foam Cells Funding Genes Health Homeostasis Human Hydrolysis Inflammatory Insecticides Intestines Knockout Mice Lead Ligands Lipids Liver Macrophage Activation Metabolism Monoacylglycerol Lipases Morbidity - disease rate Mus Organophosphates Oxygen Pathway interactions Pesticides Phospholipase Physiological Prostaglandin-Endoperoxide Synthase Prostaglandins Receptor Activation Research Role Serine Hydrolase Signal Pathway Stress System Testing Tissues Toxic Environmental Substances Toxin Wild Type Mouse Work anandamide atheroprotective cannabinoid receptor carboxylesterase cell type cholesterol transporters fatty acid amide hydrolase inhibitor/antagonist lipid metabolism macrophage monocyte mortality oxidized low density lipoprotein receptor scavenger receptor toxicant

项目摘要

DESCRIPTION (provided by applicant): Atherosclerosis leads to cardiovascular disease, thus causing significant amounts of morbidity and mortality. In animal models, cannabinoid (CB)1 receptor antagonism can reduce atherosclerosis, whereas activation of CB2 receptors by agonists are atheroprotective. These findings suggest that the endocannabinoid system (ECS) has an important role in vascular homeostasis and that its perturbation may lead to clinical disease. The ECS is composed of several components, including CB receptors, ligands (2-arachidonoylglycerol, 2AG; anandamide, AEA), endocannabinoid biosynthetic enzymes, transporters, and hydrolytic enzymes that degrade 2AG and AEA thereby terminating their actions. In addition to this complexity, 2AG can be oxygenated by cyclooxygenases to yield prostaglandin glyceryl esters (PG-Gs), which can elicit pro-inflammatory activities. Although monoacylglycerol lipase (MAGL) and fatty-acid amide hydrolase (FAAH) are known to catalyze the hydrolysis of 2AG and AEA, respectively, we recently showed that carboxylesterases (CES) 1 and 2 are also efficient at hydrolyzing 2AG and PG-Gs, but not AEA. Moreover, CES1 accounted for 40-50% and 80-95% of the 2AG and PG-G hydrolysis activity in THP1 macrophages, respectively. An enzyme (Mr 31-32kDa) of un- known function was also detected by serine hydrolase activity profiling and may be responsible for the remaining glyceryl ester hydrolysis activity. Pesticides, such as organophosphates (OPs), are environmental toxicants that can be detoxified by CES; however, CES are irreversibly inhibited by the bioactive metabolites of OPs during attempted catalytic turnover. CES1 has been shown to liberate free cholesterol from neutral lipid droplets in macrophages and in the last funding cycle we showed that this function can be blocked by CES1 inhibitors. Additionally, we recently showed that human THP1 macrophage foam cells synthesize and release 2AG and PG-Gs into the culture medium and levels of these bioactive lipids are augmented by bioactive metabolites of OP pesticides. Thus, endocannabinoids may have physiological roles in the vasculature that modulate development of atherosclerosis. Since macrophages in the vessel wall express CB1 and CB2, and 2AG biosynthesis is augmented when macrophages are stimulated by external cues, it is unclear how these opposing signaling pathways are integrated to influence atherosclerosis development. Therefore, the local concentration of 2AG in the vessel wall may be an important determinant of foam cell formation and atherosclerosis. We hypothesize that the endocannabinoid tone of vessel wall macrophages is significantly perturbed by chronic exposure to oxLDL and bioactive metabolites of OP insecticides, and that elevated levels of 2AG and its COX derived metabolite, PGE2-G, modulate cholesterol metabolism in macrophages. We will test our hypothesis with the following 3 aims: SA 1. Compare the metabolism of 2AG (endocannabinoid) and PGE2-G (prostaglandin E2 glyceryl ester) in human blood-derived monocytes/macrophages and the THP-1 cell line. Identify the putative endocannabinoid hydrolytic enzyme (Mr 31-32 kDa) that we previously detected by serine hydrolase activity profiling. The working hypothesis for aim 1 is that the metabolism of lipid glyceryl esters will be similar in primary and cultured human macrophages because both cell types will likely have the same complement of endocannabinoid metabolizing enzymes, including the 31-32 kDa enzyme, which we postulate has 2AG hydrolytic activity. SA 2. Determine the mechanism by which pro-atherogenic factors activate the biosynthesis and catabolism of 2AG and PGE2-G in macrophages. The working hypothesis is that factors that contribute to atherosclerosis, such as oxidized LDL and toxicants, can modulate the metabolism of lipid glyceryl esters in macrophages. SA 3. Determine if exposure to 2AG and PGE2-G reduces cholesterol efflux from macrophages obtained from wild-type mice and CB1- and CB2-null mice. The working hypothesis is that activation of CB1 and CB2 have opposing functional effects on cholesteryl ester turnover and cholesterol efflux from macrophages. The research proposed in this application will make a major impact because it will elucidate the mechanistic details by which endogenous toxins (oxLDL) and exogenous toxicants (pesticides) can together dysregulate the endocannabinoid system in macrophages, thus enhancing foam cell development. Accordingly, combination therapies that utilize both CB1 receptor antagonists and CB2 receptor agonists could be developed in order to restore homeostasis within the vessel wall, thereby promoting human health. The studies outlined here will help to determine the feasibility of this approach. PUBLIC HEALTH RELEVANCE: The research proposed in this application will make a major impact because it will elucidate the mechanistic details by which endogenous toxins (oxLDL) and exogenous toxicants (pesticides) can together dysregulate the endocannabinoid system in macrophages, thus enhancing foam cell development. Accordingly, combination therapies that utilize both cannabinoid (CB)1 receptor antagonists and CB2 receptor agonists could be developed in order to restore homeostasis within the vessel wall, thereby promoting human health. The studies outlined here will help to determine the feasibility of this approach.

描述（由申请人提供）：动脉粥样硬化会导致心血管疾病，从而导致大量的发病率和死亡率。在动物模型中，大麻素 (CB)1 受体拮抗作用可以减少动脉粥样硬化，而激动剂激活 CB2 受体则具有动脉粥样硬化保护作用。这些发现表明内源性大麻素系统（ECS）在血管稳态中发挥重要作用，其扰动可能导致临床疾病。 ECS 由多种成分组成，包括 CB 受体、配体（2-花生四烯酰甘油，2AG；花生四烯酸乙醇胺，AEA）、内源性大麻素生物合成酶、转运蛋白以及降解 2AG 和 AEA 从而终止其作用的水解酶。除了这种复杂性之外，2AG 还可以被环氧合酶氧化产生前列腺素甘油酯 (PG-G)，从而引发促炎活性。尽管已知单酰基甘油脂肪酶 (MAGL) 和脂肪酸酰胺水解酶 (FAAH) 分别可催化 2AG 和 AEA 的水解，但我们最近表明，羧酸酯酶 (CES) 1 和 2 也能有效水解 2AG 和 PG-G，但不是 AEA。此外，CES1分别占THP1巨噬细胞中2AG和PG-G水解活性的40-50%和80-95%。通过丝氨酸水解酶活性分析还检测到一种功能未知的酶（Mr 31-32kDa），该酶可能与剩余的甘油酯水解活性有关。农药，如有机磷酸酯 (OP)，是环境毒物，可通过 CES 解毒；然而，在尝试催化转换期间，CES 会被 OP 的生物活性代谢物不可逆地抑制。 CES1 已被证明可以从巨噬细胞中的中性脂滴中释放游离胆固醇，并且在上一个资助周期中，我们表明该功能可以被 CES1 抑制剂阻断。此外，我们最近表明，人 THP1 巨噬细胞泡沫细胞合成并将 2AG 和 PG-G 释放到培养基中，并且这些生物活性脂质的水平因 OP 农药的生物活性代谢物而增加。因此，内源性大麻素可能在调节动脉粥样硬化发展的脉管系统中具有生理作用。由于血管壁中的巨噬细胞表达 CB1 和 CB2，并且当巨噬细胞受到外部信号刺激时 2AG 生物合成会增强，因此尚不清楚这些相反的信号通路如何整合以影响动脉粥样硬化的发展。因此，血管壁中2AG的局部浓度可能是泡沫细胞形成和动脉粥样硬化的重要决定因素。我们假设血管壁巨噬细胞的内源性大麻素基调受到长期暴露于 oxLDL 和 OP 杀虫剂生物活性代谢物的显着干扰，并且 2AG 及其 COX 衍生代谢物 PGE2-G 水平升高可调节巨噬细胞中的胆固醇代谢。我们将通过以下 3 个目标来检验我们的假设： SA 1. 比较人血单核细胞/巨噬细胞和 THP-1 细胞系中 2AG（内源性大麻素）和 PGE2-G（前列腺素 E2 甘油酯）的代谢。鉴定我们之前通过丝氨酸水解酶活性分析检测到的假定内源性大麻素水解酶 (Mr 31-32 kDa)。目标 1 的工作假设是，脂质甘油酯的代谢在原代和培养的人类巨噬细胞中相似，因为两种细胞类型可能具有相同的内源性大麻素代谢酶，包括 31-32 kDa 酶，我们假设该酶具有2AG水解活性。 SA 2. 确定促动脉粥样硬化因子激活巨噬细胞中 2AG 和 PGE2-G 生物合成和分解代谢的机制。目前的假设是，导致动脉粥样硬化的因素，例如氧化的低密度脂蛋白和有毒物质，可以调节巨噬细胞中脂质甘油酯的代谢。 SA 3. 确定暴露于 2AG 和 PGE2-G 是否会减少从野生型小鼠以及 CB1 和 CB2 缺失小鼠获得的巨噬细胞的胆固醇流出。工作假设是，CB1 和 CB2 的激活对胆固醇酯周转和巨噬细胞胆固醇流出具有相反的功能作用。本申请中提出的研究将产生重大影响，因为它将阐明内源性毒素（oxLDL）和外源性毒物（农药）共同失调巨噬细胞中的内源性大麻素系统，从而促进泡沫细胞发育的机制细节。因此，可以开发利用CB1受体拮抗剂和CB2受体激动剂的联合疗法，以恢复血管壁内的稳态，从而促进人类健康。这里概述的研究将有助于确定这种方法的可行性。公共健康相关性：本申请中提出的研究将产生重大影响，因为它将阐明内源性毒素 (oxLDL) 和外源性毒物（农药）共同失调巨噬细胞中的内源性大麻素系统，从而促进泡沫细胞发育的机制细节。因此，可以开发利用大麻素（CB）1受体拮抗剂和CB2受体激动剂的联合疗法，以恢复血管壁内的稳态，从而促进人类健康。这里概述的研究将有助于确定这种方法的可行性。