LPS-induced suppression of macrophage cholesterol efflux is mediated by adipocyte enhancer-binding protein 1

Macrophages facilitate clearance of cholesterol from the body via reverse cholesterol transport (RCT). The first event in RCT is internalization of modified low density lipoprotein by macrophages, upon which PPARγ1 and LXRα signaling pathways are turned on, leading to the transactivation of a cascade of genes (e.g. ABCA1 and ABCG1), whose products promote macrophage cholesterol efflux. Down-regulation of macrophage cholesterol efflux mediators leads to an imbalance in cholesterol homeostasis, promoting foam cell formation. Lipopolysaccharide (LPS) has been shown to suppress PPARγ1 and its downstream target genes in macrophages, inducing foam cell formation; a key mechanism proposed to underlie bacterial infection-induced atherosclerosis. Herein, we show that adipocyte enhancer-binding protein 1 (AEBP1) is up-regulated during monocyte differentiation. Moreover, we provide experimental evidence suggesting that AEBP1 expression is induced by LPS, and that LPS-induced down-regulation of pivotal macrophage cholesterol efflux mediators, leading to foam cell formation, is largely mediated by AEBP1. Although AEBP1-independent pathways seem to contribute to these LPS effects, such pathways can only mediate lesser and delayed effects of LPS on macrophage cholesterol efflux and development of foam cells. We speculate that AEBP1 may serve as a potential therapeutic target for the prevention/treatment of bacterial infection-induced atherosclerosis.     

Wogonin promotes cholesterol efflux by increasing protein phosphatase 2B-dependent dephosphorylation at ATP-binding cassette transporter-A1 in macrophages

Wogonin, one component in Scutellaria baicalensis Georgi extracts, has several beneficial properties for cancers and inflammatory diseases. However, the efficacy of wogonin in cholesterol metabolism of macrophages remains unknown. In macrophages, cholesterol uptake is controlled by scavenger receptors (SR-A and CD36) and cholesterol efflux by SR-BI, ATP-binding cassette transporter-A1 (ABCA1) and ABCG1. In the present study, we investigated the effect and underlying molecular mechanism of wogonin on the formation of macrophage foam cells by murine J774.A1 macrophages. Wogonin attenuated oxidized low-density lipoprotein (oxLDL)-induced cholesterol accumulation in macrophages. The binding of oxLDL to macrophages and protein expression of SR-A and CD36 were not affected by wogonin. Wogonin enhanced cholesterol efflux and increased the protein level of ABCA1 without affecting the protein expression of SR-BI or ABCG1. Inhibition of ABCA1 by pharmacological inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt or neutralizing antibody abolished this suppressive effect of wogonin on lipid accumulation. Moreover, the up-regulation of ABCA1 protein by wogonin resulted from a decrease in degradation rate of ABCA1 protein, with no effect on ABCA1 mRNA expression. This reduction in ABCA1 degradation was due to increased protein phosphatase 2B (PP2B)-mediated ABCA1 dephosphorylation, as evidenced by increased interaction between ABCA1 and PP2B; pharmacological inhibition of PP2B would prevent wogonin-induced ABCA1 protein expression, dephosphorylation and attenuation of lipid accumulation. Collectively, wogonin increases the protein stability of ABCA1 via PP2B-mediated dephosphorylation, thus leading to reduced cholesterol accumulation in macrophage foam cells.     

OxLDL-mediated survival of macrophages does not require LDL internalization or signalling by major pattern recognition receptors

Macrophages play a key role in the pathogenesis of atherosclerosis, in part by destabilizing plaques. We and others have shown that low concentrations of oxidized LDL (oxLDL) inhibit macrophage apoptosis. As oxLDL is present in lesions, this may be a mechanism by which macrophage populations in the intima are expanded. We have previously shown that oxLDL activates prosurvival signalling pathways such as the phosphoinositide 3-kinase (PI3K) pathway in bone marrow derived macrophages (BMDMs). However, little is known about more upstream signalling events especially at the receptor level. The endocytic pattern recognition receptors (PRRs), scavenger receptor A (SR-A) and CD36, are the main receptors on macrophages for uptake of oxLDL and are therefore important in foam cell formation. The signalling PRRs such as toll-like receptor (TLR) 2 and 4 also bind some types of oxLDL. This study was done to determine if any of the known PRRs are required for the anti-apoptotic effects of oxLDL in BMDMs. To do this, we tested the effect of oxLDL on viability of BMDMs lacking both SR-A and CD36 or lacking TLR2, TLR4, CD14, FcγRIIb, or RAGE. Our results indicate that none of these receptors are essential for activating the oxLDL prosurvival pathway. Furthermore, we show that the anti-apoptotic effect is not dependent on the uptake of oxLDL.     

ER stress is associated with reduced ABCA-1 protein levels in macrophages treated with advanced glycated albumin - reversal by a chemical chaperone

ATP-binding cassette transporter A1 mediates the export of excess cholesterol from macrophages, contributing to the prevention of atherosclerosis. Advanced glycated albumin (AGE-alb) is prevalent in diabetes mellitus and is associated with the development of atherosclerosis. Independently of changes in ABCA-1 mRNA levels, AGE-alb induces oxidative stress and reduces ABCA-1 protein levels, which leads to macrophage lipid accumulation. These metabolic conditions are known to elicit endoplasmic reticulum (ER) stress. We sought to determine if AGE-alb induces ER stress and unfolded protein response (UPR) in macrophages and how disturbances to the ER could affect ABCA-1 content and cholesterol efflux in macrophages. AGE-alb induced a time-dependent increase in ER stress and UPR markers. ABCA-1 content and cellular cholesterol efflux were reduced by 33% and 47%, respectively, in macrophages treated with AGE-alb, and both were restored by treatment with 4-phenyl butyric acid (a chemical chaperone that alleviates ER stress), but not MG132 (a proteasome inhibitor). Tunicamycin, a classical ER stress inductor, also impaired ABCA-1 expression and cholesterol efflux (showing a decrease of 61% and 82%, respectively), confirming the deleterious effect of ER stress in macrophage cholesterol accumulation. Glycoxidation induces macrophage ER stress, which relates to the reduction in ABCA-1 and in reverse cholesterol transport, endorsing the adverse effect of macrophage ER stress in atherosclerosis. Thus, chemical chaperones that alleviate ER stress may represent a useful tool for the prevention and treatment of atherosclerosis in diabetes.     

4-hydroxynonenal contributes to macrophage foam cell formation through increased expression of class A scavenger receptor at the level of translation

4-Hydroxynonenal (HNE) is known to be atherogenic, but its mechanism of action in atherogenesis is not clear. Therefore, this study investigated the role of HNE in macrophage foam cell formation and the underlying mechanism involved in HNE-induced expression of scavenger receptors (SRs). In the aortic sinus of ApoE-deficient mice fed a high-fat diet, multiple plaque lesions were accompanied by increased accumulation of HNE adducts in the enhanced Mac-2 stained area. In an in vitro study, HNE exposure to J774A.1 macrophages led to increased expression of class A SR (SR-A) and CD36 at the protein level with a concomitant increase in endocytic uptake of oxLDL. In contrast to CD36 protein expression, which was associated with an increase in mRNA expression, the HNE-enhanced SR-A protein expression was neither accompanied by its mRNA expression nor affected by actinomycin D. HNE enhanced the incorporation rates of ³⁵S-Met/Cys into SR-A, and HNE-induced SR-A protein expression was effectively attenuated by translation inhibitors such as cycloheximide and rapamycin. Taken together, these data suggest that HNE contributes to macrophage foam cell formation through increased synthesis of SR-A at the level of mRNA translation, consequently leading to the progression of atherosclerosis.     

Vitamin D Deficiency Induces High Blood Pressure and Accelerates Atherosclerosis in Mice

Multiple epidemiological studies link vitamin D deficiency to increased cardiovascular disease (CVD), but causality and possible mechanisms underlying these associations are not established. To clarify the role of vitamin D-deficiency in CVD in vivo, we generated mouse models of diet-induced vitamin D deficiency in two backgrounds (LDL receptor- and ApoE-null mice) that resemble humans with diet-induced hypertension and atherosclerosis. Mice were fed vitamin D-deficient or -sufficient chow for 6 weeks and then switched to high fat (HF) vitamin D-deficient or –sufficient diet for 8–10 weeks. Mice with diet-induced vitamin D deficiency showed increased systolic and diastolic blood pressure, high plasma renin, and decreased urinary sodium excretion. Hypertension was reversed and renin was suppressed by returning chow-fed vitamin D-deficient mice to vitamin D-sufficient chow diet for 6 weeks. On a HF diet, vitamin D-deficient mice had ∼2-fold greater atherosclerosis in the aortic arch and ∼2–8-fold greater atherosclerosis in the thoracic and abdominal aorta compared to vitamin D-sufficient mice. In the aortic root, HF-fed vitamin D-deficient mice had increased macrophage infiltration with increased fat accumulation and endoplasmic reticulum (ER) stress activation, but a lower prevalence of the M1 macrophage phenotype within atherosclerotic plaques. Similarly, peritoneal macrophages from vitamin D-deficient mice displayed an M2-predominant phenotype with increased foam cell formation and ER stress. Treatment of vitamin D-deficient mice with the ER stress reliever PBA during HF feeding suppressed atherosclerosis, decreased peritoneal macrophage foam cell formation, and downregulated ER stress proteins without changing blood pressure. Thus, we suggest that vitamin D deficiency activates both the renin angiotensin system and macrophage ER stress to contribute to the development of hypertension and accelerated atherosclerosis, highlighting vitamin D replacement as a potential therapy to reduce blood pressure and atherosclerosis.     

Inhibition of the NLRP3 inflammasome attenuates foam cell formation of THP-1 macrophages by suppressing ox-LDL uptake and promoting cholesterol efflux

The NOD-like receptor family, pyrin domain–containing protein 3 (NLRP3) inflammasome plays an important role in the development of atherosclerosis. The activated NLRP3 inflammasome has been reported to promote macrophage foam cell formation, but not all studies have obtained the same result, and how NLRP3 inflammasome is involved in the formation of foam cells remains elusive. We used selective NLRP3 inflammasome inhibitors and NLRP3-deficient THP-1 cells to assess the effect of NLRP3 inflammasome inhibition on macrophage foam cell formation, oxidized low-density lipoprotein (ox-LDL) uptake, esterification, and cholesterol efflux, as well as the expression of associated proteins. Inhibition of the NLRP3 inflammasome attenuated foam cell formation, diminished ox-LDL uptake, and promoted cholesterol efflux from THP-1 macrophages. Moreover, it downregulated CD36, acyl coenzyme A: cholesterol acyltransferase-1 and neutral cholesterol ester hydrolase expression; upregulated ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI) expression; but had no effect on the expression of scavenger receptor class A and ATP-binding cassette transporter G1. Collectively, our findings show that inhibition of the NLRP3 inflammasome decreases foam cell formation of THP-1 macrophages via suppression of ox-LDL uptake and enhancement of cholesterol efflux, which may be due to downregulation of CD36 expression and upregulation of ABCA1 and SR-BI expression, respectively.     

Atherogenic lipids and lipoproteins trigger CD36-TLR2-dependent apoptosis in macrophages undergoing endoplasmic reticulum stress

Macrophage apoptosis in advanced atheromata, a key process in plaque necrosis, involves the combination of ER stress with other proapoptotic stimuli. We show here that oxidized phospholipids, oxidized LDL, saturated fatty acids (SFAs), and lipoprotein(a) trigger apoptosis in ER-stressed macrophages through a mechanism requiring both CD36 and Toll-like receptor 2 (TLR2). In vivo, macrophage apoptosis was induced in SFA-fed, ER-stressed wild-type but not Cd36?(/)? or Tlr2?(/)? mice. For atherosclerosis, we combined TLR2 deficiency with that of TLR4, which can also promote apoptosis in ER-stressed macrophages. Advanced lesions of fat-fed Ldlr?(/)? mice transplanted with Tlr4?(/)?Tlr2?(/)? bone marrow were markedly protected from macrophage apoptosis and plaque necrosis compared with WT →Ldlr?(/)? lesions. These findings provide insight into how atherogenic lipoproteins trigger macrophage apoptosis in the setting of ER stress and how TLR activation might promote macrophage apoptosis and plaque necrosis in advanced atherosclerosis.     

Regulation of cholesterol homeostasis in macrophages and consequences for atherosclerotic lesion development

Foam cell formation due to excessive accumulation of cholesterol by macrophages is a pathological hallmark of atherosclerosis. Macrophages cannot limit the uptake of cholesterol and therefore depend on cholesterol efflux pathways for preventing their transformation into foam cells. Several ABC-transporters, including ABCA1 and ABCG1, facilitate the efflux of cholesterol from macrophages. These transporters, however, also affect membrane lipid asymmetry which may have important implications for cellular endocytotic pathways. We propose that in addition to the generally accepted role of these ABC-transporters in the prevention of foam cell formation by induction of cholesterol efflux from macrophages, they also influence the macrophage endocytotic uptake.     

Oxidation as a crucial reaction for cholesterol to induce tissue degeneration: CD36 overexpression in human promonocytic cells treated with a biologically relevant oxysterol mixture

Oxidative stress, inflammation and altered cholesterol metabolism and levels are among the pathogenetic mechanisms of cognitive impairment that may accompany aging. Within the research area of hypercholesterolemia and age-related disease processes, the molecular mechanisms of cholesterol interaction with the inflammatory cells of the macrophage lineage are yet to be elucidated. We thus investigated the effect of both non-oxidized and oxidized cholesterol on monocytic cell differentiation and foam cell formation, as it occurs within vascular lesions during progression of atherosclerosis. In vitro experiments performed on human U937 promonocytic cells showed that a biologically representative mixture of oxysterols markedly stimulated CD36 expression and synthesis. In contrast, non-oxidized cholesterol did not exert any effect on CD36 mRNA and protein levels. Furthermore, the oxysterol-induced up-regulation of CD36 appeared to be based on the subsequent activation of protein kinase Cdelta (PKCdelta), extracellular signal-regulated kinase 1/2 (ERK1/2) and peroxisome proliferator-activated receptor gamma (PPARgamma). Cells overexpressing CD36 were indeed able to actively take up oxidized low-density lipoproteins, and become foam cells. The essential role of ERK pathway and CD36 receptor in oxysterol-induced foam cell formation was proved by the prevention of the latter event when monocytic cells were incubated in the presence of MEK1/2 selective inhibitor or anti-CD36 specific antibody. These experimental findings point to cholesterol oxidation as an essential reaction for this sterol to exert cellular stress and tissue damage in age-related diseases in which inflammation represents a main driving force.     

Macrophage‐mediated cholesterol handling in atherosclerosis

Formation of foam cells is a hallmark at the initial stages of atherosclerosis. Monocytes attracted by pro‐inflammatory stimuli attach to the inflamed vascular endothelium and penetrate to the arterial intima where they differentiate to macrophages. Intimal macrophages phagocytize oxidized low‐density lipoproteins (oxLDL). Several scavenger receptors (SR), including CD36, SR‐A1 and lectin‐like oxLDL receptor‐1 (LOX‐1), mediate oxLDL uptake. In late endosomes/lysosomes of macrophages, oxLDL are catabolysed. Lysosomal acid lipase (LAL) hydrolyses cholesterol esters that are enriched in LDL to free cholesterol and free fatty acids. In the endoplasmic reticulum (ER), acyl coenzyme A: cholesterol acyltransferase‐1 (ACAT1) in turn catalyses esterification of cholesterol to store cholesterol esters as lipid droplets in the ER of macrophages. Neutral cholesteryl ester hydrolases nCEH and NCEH1 are involved in a secondary hydrolysis of cholesterol esters to liberate free cholesterol that could be then out‐flowed from macrophages by cholesterol ATP‐binding cassette (ABC) transporters ABCA1 and ABCG1 and SR‐BI. In atherosclerosis, disruption of lipid homoeostasis in macrophages leads to cholesterol accumulation and formation of foam cells.     

ClC-2 inhibition prevents macrophage foam cell formation by suppressing Nlrp3 inflammasome activation

ABSTRACT

Macrophage foam cell formation and inflammation are a pathological hallmark of atherosclerosis. ClC-2 has been implicated in various pathological processes, including inflammation and lipid metabolic disorder. However, the functional role of ClC-2 in macrophage foam cell formation and inflammation is unclear. Here, we found that ClC-2 was dominantly expressed in macrophages of atherosclerotic plaque and increased in atherogenesis. Knockdown of ClC-2 inhibited ox-LDL -induced lipid uptake and deposition in macrophages. The increase in CD36 expression and the decrease in ABCA1 expression induced by ox-LDL were alleviated by ClC-2 downregulation. Further, ClC-2 lacking limited the ox-LDL-induced secretion of inflammatory cytokines and chemokine, and suppressed Nlrp3 inflammasome activation. Restoration of Nlrp3 expression reversed the effect of ClC-2 downregulation on macrophage lipid accumulation and inflammation. Collectively, our study demonstrates that ClC-2 knockdown ameliorates ox-LDL-induced macrophage foam cell formation and inflammation by inhibiting Nlrp3 inflammasome activation.     

Vitamin D Suppression of Endoplasmic Reticulum Stress Promotes an Antiatherogenic Monocyte/Macrophage Phenotype in Type 2 Diabetic Patients

Cardiovascular disease is the leading cause of morbidity/mortality in patients with type 2 diabetes mellitus (T2DM), but there is a lack of knowledge about the mechanism(s) of increased atherosclerosis in these patients. In patients with T2DM, the prevalence of 25-hydroxy vitamin D (25(OH)D) deficiency is almost twice that for nondiabetics and doubles the relative risk of developing cardiovascular disease compared with diabetic patients with normal 25(OH)D. We tested the hypothesis that monocytes from vitamin D-deficient subjects will have a proatherogenic phenotype compared with vitamin D-sufficient subjects in 43 patients with T2DM. Serum 25(OH)D level inversely correlated with monocyte adhesion to endothelial cells even after adjustment for demographic and comorbidity characteristics. Vitamin D-sufficient patients (≥30 ng/ml 25(OH)D) had lower monocyte endoplasmic reticulum (ER) stress, a predominance of M1 over M2 macrophage membrane receptors, and decreased mRNA expression of monocyte adhesion molecules PSGL-1, β₁-integrin, and β₂-integrin compared with patients with 25(OH)D levels of <30 ng/ml. In vitamin D-deficient macrophages, activation of ER stress increased adhesion and adhesion molecule expression and induced an M2-predominant phenotype. Moreover, adding 1,25(OH)₂D₃ to vitamin D-deficient macrophages shifted their phenotype toward an M1-predominant phenotype with suppressed adhesion. Conversely, deletion of the vitamin D receptor in macrophages from diabetic patients activated ER stress, accelerated adhesion, and increased adhesion molecule expression. The absence of ER stress protein CCAAT enhancer-binding protein homologous protein suppressed monocyte adhesion, adhesion molecule expression, and the M2-predominant phenotype induced by vitamin D deficiency. Thus, vitamin D is a natural ER stress reliever that induced an antiatherogenic monocyte/macrophage phenotype.     

巨噬细胞极化的研究进展

巨噬细胞是一群表型和功能均具有高度异质性的免疫细胞。巨噬细胞通过清除并修复受损的细胞和基质来维护组织完整性。巨噬细胞在不同的组织微环境、不同病理条件下,可极化成不同的表型即M1型巨噬细胞(经典活化的巨噬细胞)和M2型巨噬细胞(替代活化的巨噬细胞)。本文将对不同巨噬细胞亚群在抗细菌感染、抗寄生虫感染、哮喘、动脉粥样硬化和肿瘤产生中起到的的保护或致病作用,以及调控巨噬细胞极化的机制进行综述。掌握巨噬细胞极化在不同疾病中的作用以及调控巨噬细胞极化的具体机制,将为疾病的预防、诊断、治疗及药物研发提供新策略。