Arctigenin promotes cholesterol efflux from THP-1 macrophages through PPAR-γ/LXR-α signaling pathway

Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Here, we sought to investigate the effects of arctigenin, a bioactive component of Arctium lappa, on the cholesterol efflux in oxidized low-density lipoprotein (oxLDL)-loaded THP-1 macrophages. Our data showed that arctigenin significantly accelerated apolipoprotein A-I- and high-density lipoprotein-induced cholesterol efflux in both dose- and time-dependent manners. Moreover, arctigenin treatment enhanced the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, and apoE, all of which are key molecules in the initial step of cholesterol efflux, at both mRNA and protein levels. Arctigenin also caused a concentration-dependent elevation in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α). The arctigenin-mediated induction of ABCA1, ABCG1, and apoE was abolished by specific inhibition of PPAR-γ or LXR-α using small interfering RNA technology. Our results collectively indicate that arctigenin promotes cholesterol efflux in oxLDL-loaded THP-1 macrophages through upregulation of ABCA1, ABCG1 and apoE, which is dependent on the enhanced expression of PPAR-γ and LXR-α.     

Induction of ABCA1 and ABCG1 expression by the liver X receptor modulator cineole in macrophages

We investigated the effect of cineole on the expression of genes related to reverse cholesterol transport and hepatic fatty acid metabolism. Cineole, a small aroma compound in teas and herbs, significantly stimulated the transactivation of liver X receptor modulator (LXR)-α and LXR-β. The mRNA and protein expression of LXRs and their target genes, including ABCA1 and ABCG1, was significantly increased in macrophages stimulated with cineole. This led to the subsequent removal of cholesterol from the cells. Interestingly, cineole showed tissue-selective LXR induction: hepatocytes stimulated with cineole showed significantly reduced expression of LXR-α and LXR-α-responsive genes, including FAS and SCD-1 (P <0.05). Accordingly, hepatocytes treated with cineole displayed reduced cellular lipid accumulation compared with control cells, as assessed by Oil Red O lipid staining and cholesterol quantification. These results suggest that cineole is a selective LXR modulator that regulates the expression of key genes in reverse cholesterol transport in macrophages without inducing lipogenesis in hepatocytes. This selective LXR modulator may have practical implications for the development of hypocholesterolemic or anti-atherosclerotic agents and also suggests.     

Resveratrol inhibits lipid accumulation in the intestine of atherosclerotic mice and macrophages

Disordered intestinal metabolism is highly correlated with atherosclerotic diseases. Resveratrol protects against atherosclerotic diseases. Accordingly, this study aims to discover novel intestinal proatherosclerotic metabolites and potential therapeutic targets related to the anti‐atherosclerotic effects of resveratrol. An untargeted metabolomics approach was employed to discover novel intestinal metabolic disturbances during atherosclerosis and resveratrol intervention. We found that multiple intestinal metabolic pathways were significantly disturbed during atherosclerosis and responsive to resveratrol intervention. Notably, resveratrol abolished intestinal fatty acid and monoglyceride accumulation in atherosclerotic mice. Meanwhile, oleate accumulation was one of the most prominent alterations in intestinal metabolism. Moreover, resveratrol attenuated oleate‐triggered accumulation of total cholesterol, esterified cholesterol and neutral lipids in mouse RAW 264.7 macrophages by activating ABC transporter A1/G1‐mediated cholesterol efflux through PPAR (peroxisome proliferator‐activated receptor) α/γ activation. Furthermore, we confirmed that PPARα and PPARγ activation by WY14643 and pioglitazone, respectively, alleviated oleate‐induced accumulation of total cholesterol, esterified cholesterol and neutral lipids by accelerating ABC transporter A1/G1‐mediated cholesterol efflux. This study provides the first evidence that resveratrol abolishes intestinal fatty acid and monoglyceride accumulation in atherosclerotic mice, and that resveratrol suppresses oleate‐induced accumulation of total cholesterol, esterified cholesterol and neutral lipids in macrophages by activating PPARα/γ signalling.     

Tryptase promotes human monocyte-derived macrophage foam cell formation by suppressing LXRα activation

Accumulated mast cells in atherosclerotic plaques secrete a high level of tryptase that may participate in the pathogenesis of atherosclerotic disease by diverse pathways. However, the role of tryptase in the lipid metabolism of macrophages remains to be defined. In the present study, we found that the addition of tryptase into THP-1-derived macrophages increased both intracellular lipid accumulation and total cholesterol level. Tryptase promoting foam cell formation was also observed by transmission electron microscope. These effects were resisted by APC366, a selective inhibitor of mast cell tryptase. Tryptase dramatically resisted 22RHC induced activation of LXRα protein expression, which can be reversed by SAM-11 (a PAR-2-specific neutralizing antibody) and reduced LXRα, ABCG1, ABCA1 and SREBP-1c mRNA levels and ABCG1 protein level, which were all blocked by APC366. PAR-2 agonist also redeemed 22RHC stimulation to activate LXRα, ABCG1 protein expression, and mRNA levels of LXRα and its target genes in both THP-1-derived macrophages and primary human monocyte-derived macrophages. In primary macrophages that were first transfected with PAR-2 siRNA and then treated with tryptase, both the ABCG1 protein level and mRNA levels of LXRα and ABCG1 were higher than those in the control siRNA-treated cells. Taken together, our data clarified the PAR-2 expression of human macrophages and suggested that tryptase might promote lipid accumulation in macrophages and foam cell formation by suppressing LXRα activation via PAR-2/LXRα/LXRα target genes signaling pathway. This investigation sheds a new light on the role of tryptase in foam cell formation and pathogenesis of atherosclerosis.     

Cloning of cDNAs with PDCD2C domain and their expressions during apoptosis of HEK293T cells

Modulation of the expression of genes involved in the control of cholesterol homeostasis by sterols in macrophages is crucial to foam cell formation. To characterize this regulation in THP-1 macrophages, we examined the effect of sterol loading and unloading on the expression of a number of genes that participate in lipoprotein uptake and cholesterol efflux. Sterol loading by exposure to acetylated LDL for 24 h resulted in an increase in free and esterified cholesterol of 1.4 and 1.8-fold, respectively. Under these conditions, the mRNA levels for SR-A were reduced a 59%, while those of CYP27 were increased by 4.6-fold. However, the expression of other genes involved in cholesterol efflux (ABCA1, ABCG1 and CLA-1) was not modified, despite a high intracellular cholesterol accumulation specially in the form of esterified cholesterol.On the other hand, HDL exposure reduced intracellular cholesterol content to 70%, and caused an increase in the expression of CD36 (78%), SR-A (51%) and CLA-1 (136%). Conversely, the expression of ABCA1, ABCG1 and CYP27 was decreased by 49, 67 and 57%, respectively. These findings indicate that in THP-1 macrophages, the expression of genes for receptors involved in lipoprotein binding and uptake tends to decrease upon cholesterol loading and to increase by cholesterol depletion, while the opposite pattern is found regarding the mRNA levels for proteins involved in cholesterol efflux.     

Polyphenol-rich black chokeberry (Aronia melanocarpa) extract regulates the expression of genes critical for intestinal cholesterol flux in Caco-2 cells

Black chokeberry (Aronia melanocarpa) is a rich source of polyphenols. The hypolipidemic effects of polyphenol-rich black chokeberry extract (CBE) have been reported, but underlying mechanisms have not been well characterized. We investigated the effect of CBE on the expression of genes involved in intestinal lipid metabolism. Caco-2 cells were incubated with 50 or 100 μg/ml of CBE for 24 h for quantitative realtime polymerase chain reaction analysis. Expression of genes for cholesterol synthesis (3-hydroxy-3-methylglutaryl coenzyme A reductase and sterol regulatory element binding protein 2), apical cholesterol uptake (Niemann-Pick C1 Like 1 and scavenger receptor class B Type 1) and basolateral cholesterol efflux [ATP-binding cassette transporter A1 (ABCA1)] was significantly decreased by CBE compared with control. Western blot analysis confirmed that CBE inhibited expression of these proteins. In contrast, CBE markedly induced mRNA and/or protein levels of ABCG5 and ABCG8 that mediate apical cholesterol efflux to the intestinal lumen. Furthermore, CBE significantly increased mRNA and protein levels of low-density lipoprotein (LDL) receptor, and cellular LDL uptake. Expression of genes involved in lipid metabolism and lipoprotein assembly, including sterol regulatory element-binding protein 1c, fatty acid synthase and acyl-CoA oxidase 1, was significantly decreased by CBE in a dose-dependent manner. Concomitantly, CBE significantly increased sirtuin 1, 3 and 5 mRNA levels, while it decreased SIRT-2. Our data suggest that hypolipidemic effects of CBE may be attributed, at least in part, to increased apical efflux of LDL-derived cholesterol and to decreased chylomicron formation in the intestine; and specific isoforms of SIRT may play an important role in this process.     

Adiponectin prevents atherosclerosis by increasing cholesterol efflux from macrophages

Plasma high density lipoprotein (HDL)-cholesterol levels are inversely correlated to the risk of atherosclerotic cardiovascular diseases. Reverse cholesterol transport (RCT) is one of the major protective systems against atherosclerosis, in which HDL particles play a crucial role to carry cholesterol derived from peripheral tissues to the liver. Recently, ATP-binding cassette transporters (ABCA1, ABCG1) and scavenger receptor (SR-BI) have been identified as important membrane receptors to generate HDL by removing cholesterol from foam cells. Adiponectin (APN) secreted from adipocytes is one of the important molecules to inhibit the development of atherosclerosis. Epidemiological studies have revealed a positive correlation between plasma HDL-cholesterol and APN concentrations in humans, although its mechanism has not been clarified. Therefore, in the present study, we investigated the role of APN on RCT, in particular, cellular cholesterol efflux from human monocyte-derived and APN-knockout (APN-KO) mice macrophages. APN up-regulated the expression of ABCA1 in human macrophages, respectively. ApoA-1-mediated cholesterol efflux from macrophages was also increased by APN treatment. Furthermore, the mRNA expression of LXRα and PPARγ was increased by APN. In APN-KO mice, the expression of ABCA1, LXRα, PPARγ, and apoA-I-mediated cholesterol efflux was decreased compared with wild-type mice. In summary, APN might protect against atherosclerosis by increasing apoA-I-mediated cholesterol efflux from macrophages through ABCA1-dependent pathway by the activation of LXRα and PPARγ.     

OxLDL up-regulates Niemann-Pick type C1 expression through ERK1/2/COX-2/PPARα-signaling pathway in macrophages

The Niemann-Pick type C1 (NPC1) is located mainly in the membranes of the late endosome/lysosome and controls the intracellular cholesterol trafficking from the late endosome/lysosome to the plasma membrane. It has been reported that oxidized low-density lipoprotein (oxLDL) can up-regulate NPC1 expression. However, the detailed mechanisms are not fully understood. In this study, we investigated the effect of oxLDL stimulation on NPC1 expression in THP-1 macrophages. Our results showed that oxLDL up-regulated NPC1 expression at both mRNA and protein levels in a dose-dependent and time-dependent manner. In addition, oxLDL also induced the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). Treatment with oxLDL significantly increased cyclooxygenase-2 (COX-2) mRNA and protein expression in the macrophages, and these increases were suppressed by the ERK1/2 inhibitor PD98059 or ERK1/2 small interfering RNA (siRNA) treatment. OxLDL up-regulated the expression of peroxisome proliferator-activated receptor α (PPARα) at the mRNA and protein levels, which could be abolished by COX-2 siRNA or COX-2 inhibitor NS398 treatment in these macrophages. OxLDL dramatically elevated cellular cholesterol efflux, which was abrogated by inhibiting ERK1/2 and/or COX-2. In addition, oxLDL-induced NPC1 expression and cellular cholesterol efflux were reversed by PPARα siRNA or GW6471, an antagonist of PPARα. Taken together, these results provide the evidence that oxLDL can up-regulate the expression of the NPC1 through ERK1/2/COX-2/PPARα-signaling pathway in macrophages.