Lipid glyceryl ester homeostasis in macrophages and perturbation by environmental

描述（由申请人提供）：动脉粥样硬化导致心血管疾病，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受体拮抗剂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）在血管稳态中起重要作用，其扰动可能导致临床疾病。ECS由几种成分组成，包括CB受体、配体（2-花生四烯醇甘油，2AG; anandamide， AEA）、内源性大麻素生物合成酶、转运体以及降解2AG和AEA从而终止其作用的水解酶。除了这种复杂性之外，2AG还可以被环加氧酶氧化生成前列腺素甘油酯（PG-Gs），这可以引发促炎活性。虽然已知单酰基甘油脂肪酶（MAGL）和脂肪酸酰胺水解酶（FAAH）分别催化2AG和AEA的水解，但我们最近发现羧酸酯酶（CES） 1和2也能水解2AG和PG-Gs，但不能水解AEA。此外，在THP1巨噬细胞中，CES1分别占2AG和PG-G水解活性的40-50%和80-95%。丝氨酸水解酶活性谱还检测到一种功能未知的酶（Mr 31-32kDa），可能与剩余的甘油酯水解活性有关。农药，如有机磷（OPs），是环境有毒物质，可以通过电子烟解毒；然而，在试图催化转化的过程中，CES被OPs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的中性脂滴中释放游离胆固醇，在上一个资助周期中，我们发现该功能可以被CES1抑制剂阻断。此外，我们最近发现人类THP1巨噬细胞泡沫细胞合成并释放2AG和PG-Gs到培养基中，这些生物活性脂质的水平被OP农药的生物活性代谢物增加。因此，内源性大麻素可能在调节动脉粥样硬化发展的血管系统中具有生理作用。由于血管壁上的巨噬细胞表达CB1和CB2，并且当巨噬细胞受到外部信号刺激时，2AG的生物合成增强，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，血管壁上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浓度受到长期暴露于oxLDL和OP杀虫剂的生物活性代谢物的显著干扰，2AG及其COX衍生代谢物PGE2-G水平的升高调节了巨噬细胞的胆固醇代谢。我们将用以下3个目标来检验我们的假设：比较2AG（内源性大麻素）和PGE2-G（前列腺素E2甘油酯）在人血源性单核/巨噬细胞和THP-1细胞系中的代谢。鉴定推定的内源性大麻素水解酶（Mr 31-32 kDa），我们之前通过丝氨酸水解酶活性谱检测到。目的1的工作假设是，在原代和培养的人巨噬细胞中，脂质甘油酯的代谢将是相似的，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，我们假设其具有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的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，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。测定暴露于2AG和PGE2-G是否能减少野生型小鼠和CB1-和cb2 -缺失小鼠巨噬细胞的胆固醇外排。工作假设是CB1和CB2的激活对巨噬细胞的胆固醇酯转换和胆固醇外排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）共同失调巨噬细胞内源性大麻素系统的机制细节，从而促进泡沫细胞的发育。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。

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.