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Liver disease is the second most common cause of mortality in HIV-infected persons. Exactly how HIV infection per se affects liver disease progression is unknown. Here we have investigated mRNA expression of 49 nuclear hormone receptors (NRs) and 35 transcriptional coregulators in HepG2 cells upon stimulation with the HIV matrix protein p17. This viral protein regulated mRNA expression of some NRs among which LXRα and its transcriptional co-activator MED1 were highly induced at mRNA level. Dissection of p17 downstream intracellular pathway demonstrated that p17 mediated activation of Jak/STAT signaling is responsible for the promoter dependent activation of LXR. The treatment of both HepG2 as well as primary hepatocytes with HIV p17 results in the transcriptional activation of LXR target genes (SREBP1c and FAS) and lipid accumulation. These effects are lost in HepG2 cells pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide as well as in HepG2 cells pre-incubated with the natural LXR antagonist gymnestrogenin. These results suggest that HIV p17 affects NRs and their related signal transduction thus contributing to the progression of liver disease in HIV infected patients. Show less

Alpinetin is a natural flavonoid abundantly present in the ginger family. Here, we investigated the effect of alpinetin on cholesterol efflux and lipid accumulation in oxidized low-density lipoprotein (ox-LDL)-treated THP-1 macrophages and human peripheral blood monocyte-derived macrophages (HMDMs). After exposing THP-1 macrophages to alpinetin, cholesterol efflux was determined by liquid scintillator. The mRNA and protein levels of peroxisome proliferator-activated receptor gamma (PPAR-γ), liver X receptor alpha (LXR-α), ATP-binding cassette transporter A1 (ABCA1), and ABCG1 and scavenger receptor class B member 1 were determined by reverse-transcriptase PCR (RT-PCR) and Western blot analysis, respectively. Alpinetin promoted apolipoprotein A-I- and high-density-lipoprotein-mediated cholesterol efflux and elevated PPAR-γ and LXR-α mRNA and protein expression in a dose-dependent fashion in ox-LDL-treated THP-1 macrophages and HMDMs. Small interfering RNA-mediated silencing of PPAR-γ or LXR-α dose dependently reversed alpinetin-increased cholesterol efflux in THP-1 macrophages, indicating the involvement of PPAR-γ and LXR-α in alpinetin-promoted cholesterol efflux. Alpinetin inhibited ox-LDL-induced lipid accumulation and enhanced the expression of ABCA1 and ABCG1 mRNA and protein, which was reversed by specific knockdown of PPAR-γ or LXR-α. Taken together, our results reveal that alpinetin exhibits positive effects on cholesterol efflux and inhibits ox-LDL-induced lipid accumulation, which might be through PPAR-γ/LXR-α/ABCA1/ABCG1 pathway. Show less

The schweinfurthins have potent antiproliferative activity in multiple glioblastoma multiforme (GBM) cell lines; however, the mechanism by which growth is impeded is not fully understood. Previously, we demonstrated that the schweinfurthins reduce the level of key isoprenoid intermediates in the cholesterol biosynthetic pathway. Herein, we describe the effects of the schweinfurthins on cholesterol homeostasis. Intracellular cholesterol levels are greatly reduced in cells incubated with 3-deoxyschweinfurthin B (3dSB), an analog of the natural product schweinfurthin B. Decreased cholesterol levels are due to decreased cholesterol synthesis and increased cholesterol efflux; both of these cellular actions can be influenced by liver X-receptor (LXR) activation. The effects of 3dSB on ATP-binding cassette transporter 1 levels and other LXR targets are similar to that of 25-hydroxycholesterol, an LXR agonist. Unlike 25-hydroxycholesterol, 3dSB does not act as a direct agonist for LXR α or β. These data suggest that cholesterol homeostasis plays a significant role in the growth inhibitory activity of the schweinfurthins and may elucidate a mechanism that can be targeted in human cancers such as GBM. Show less

The expression of the epidermal growth factor receptor (EGFR) is highly regulated in normal cells, whereas some cancer cells have high constitutive levels. Understanding naturally-occurring ways of downregulating EGFR in cancer cells was investigated. Phosphatidic acid (PA) or Nuclear Receptors (NR) PPARα/RXRα/LXRα, enhance EGFR expression, mediated by the promoter region -856(A) to -226(T). Unexpectedly, the combination of NRs and PA caused repression. PA induces a conformational change in the nuclear receptor PPARα (increase of alpha-helices at the expense of decreasing beta-sheets), as evidenced by circular dichroism. This represses the naturally-enhancing capability of PPARα on EGFR transcription. PPARα-overexpressing cells in the presence of PA > 300 nM or the enzyme that produces it, phospholipase D (PLD), downregulate EGFR expression. The reasons are two-fold. First, PA displaces PPARα binding to the EGFR promoter at those concentrations. Second, NR heterodimer-dependent promoter activity is weakened in the presence of PA in vivo. Since other genes considered (β-catenin, cyclin D3, PLD2 and ACOX-1) are also downregulated with a PA + PPARα combination, the transrepression appears to be a global phenomenon. Lastly, the reported effect is greater in MCF-7 than in MDA-MB-231 breast cancer cells, which could provide a novel basis for regulating excessive expression of EGFR in luminal cancer cells. Show less

Adequate intake of dietary fibers has proven metabolic and cardiovascular benefits, molecular mechanisms remain still limited. This study was aimed to investigate the effects of cereal dietary fiber on obesity-related liver lipotoxicity in C57BL/6J mice fed a high-fat/cholesterol (HFC) diet and underlying mechanism. Forty-eight adult male C57BL/6J mice were randomly given a reference chow diet, or a high fat/cholesterol (HFC) diet supplemented with or without oat fiber or wheat bran fiber for 24 weeks. Our results showed mice fed oat or wheat bran fiber exhibited lower weight gain, lipid profiles and insulin resistance, compared with HFC diet. The two cereal dietary fibers potently decreased protein expressions of sterol regulatory element binding protein-1 and key factors involved in lipogenesis, including fatty acid synthase and acetyl-CoA carboxylase in target tissues. At molecular level, the two cereal dietary fibers augmented protein expressions of peroxisome proliferator-activated receptor alpha and gamma, liver X receptor alpha, and ATP-binding cassette transporter A1 in target tissues. Our findings indicated that cereal dietary fiber supplementation abrogated obesity-related liver lipotoxicity and dyslipidemia in C57BL/6J mice fed a HFC diet. In addition, the efficacy of oat fiber is greater than wheat bran fiber in normalizing these metabolic disorders and pathological profiles. Show less

Dickkopf1 (DKK1), a canonical Wnt/β-catenin pathway antagonist, is closely associated with cardiovascular disease and adipogenesis. We performed an in vitro study to determine whether oxidized low-density lipoprotein (ox-LDL) increased the expression of DKK1 in macrophages and whether β-catenin and liver X receptor α (LXRα) were involved in this regulation. Induction of DKK1 expression by ox-LDL decreased the level of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) via a Wnt/β-catenin pathway and increased ATP-binding cassette transporter A/G1 (ABCA/G1) levels via a signal transducer and activator of transcription 3 (STAT3) pathway. Lower LOX-1 and higher ABCA/G1 levels inhibited cholesterol loading in macrophages. In conclusion, ox-LDL may induce DKK1 expression in macrophages to inhibit the accumulation of lipids through a mechanism that involves downregulation of LOX-1-mediated lipid uptake and upregulation of ABCA/G1-dependent cholesterol efflux. Show less

It has been suggested that retinoic acid (RA) has a potential role in the prevention of atherosclerotic CVD. In the present study, we used J774A.1 cell lines and primary peritoneal macrophages to investigate the protective effects of RA on foam cell formation and atherogenesis in apoE-deficient (apoE- / -) mice. A total of twenty male apoE- / - mice (n 10 animals per group), aged 8 weeks, were fed on a high-fat diet (HFD) and treated with vehicle or 9-cis-RA for 8 weeks. The atherosclerotic plaque area in the aortic sinus of mice in the 9-cis-RA group was 40·7 % less than that of mice in the control group (P< 0·01). Mouse peritoneal macrophages from the 9-cis-RA group had higher protein expression levels of ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) than those from the control group. Serum total and LDL-cholesterol concentrations were lower in the 9-cis-RA group than in the control group (P< 0·05). In vitro studies showed that incubation of cholesterol-loaded J774A.1 macrophages with 9-cis-RA (0·1, 1 and 10 μmol/l) induced cholesterol efflux in a dose-dependent manner. The 9-cis-RA treatment markedly attenuated lipid accumulation in macrophages exposed to oxidised LDL. Moreover, treatment with 9-cis-RA significantly increased the protein expression levels of ABCA1 and ABCG1 in J774A.1 macrophages in a dose-dependent manner. Furthermore, 9-cis-RA dose-dependently enhanced the protein expression level of liver X receptor-α (LXRα), the upstream regulator of ABCA1 and ABCG1. Taken together, the present results show that 9-cis-RA suppresses foam cell formation and prevents HFD-induced atherogenesis via the LXRα-dependent up-regulation of ABCA1 and ABCG1. Show less

The liver X receptors (LXRs), LXRα and LXRβ, are transcription factors with well-established roles in the regulation of lipid metabolism and cholesterol homeostasis. In addition, LXRs influence innate and adaptive immunity, including responses to inflammatory stimuli, proliferation and differentiation, migration, apoptosis and survival. However, the majority of work describing the role of LXRs in immune cells has been carried out in mouse models, and there are a number of known species-specific differences concerning LXR function. Here we review what is known about the role of LXRs in human immune cells, demonstrating the importance of these receptors in the integration of lipid metabolism and immune function, but also highlighting the need for a better understanding of the species, isoform, and cell-type specific effects of LXR activation. Show less

A promising treatment for obesity involves the use of therapeutic agents that increase the level of the glucagon-like peptide (GLP-1) which reduces appetite and food intake. Native GLP-1 is rapidly metabolized by the dipeptidyl peptidase-4 (DPP-4) enzyme and, as such, GLP-1 mimetics or DPP-4 inhibitors represent promising treatment approaches. Interestingly, obese patient receiving such medications showed improved lipid profiles and cholesterol homeostasis, however the mechanism(s) involved are not known. Members of the ATP-binding cassette (ABC) transporters, including ABCA1 and ABCG1, play essential roles in reverse cholesterol transport and in high density lipoprotein (HDL) formation. These transporters are under the transcriptional regulation of liver X receptor alpha (LXR-α). We hypothesize that GLP-1 mimetics and/or DPP-4 inhibitors modulate ABCA1/ABCG1 expression in adipocytes through an LXR-α mediated process and thus affecting cholesterol homeostasis. 3T3-L1 adipocytes were treated with the DPP-4 inhibitor vildagliptin (2 nM) or the GLP-1 mimetic exendin-4 (5 nM). Gene and protein expression of ABCA1, ABCG1 and LXR-α were determined and correlated with cholesterol efflux. Expression levels of interleukin-6 (IL-6), leptin and the glucose transporter-4 (GLUT-4) were also determined. Treatment with both medications significantly increased the expression of ABCA1, ABCG1, LXR-α and GLUT-4, decreased IL-6 and leptin, and improved cholesterol efflux from adipocytes (P < 0.05). Our data suggest that GLP-1-based therapy modulate ABCA1/ABCG1 expression in adipocytes potentially through an LXR-α mediated process. Show less

Inflammation and lipids play significant roles in the progression of chronic kidney disease. This study was designed to investigate whether inflammation disrupts cellular cholesterol homeostasis and causes the lipid nephrotoxicity in vitro and in vivo, and explored its underlying mechanisms. Inflammatory stress was induced by cytokines (interleukin-1β (IL-1β); tumor necrosis factor α (TNF-α)) to human mesangial cells (HMCs) in vitro and by subcutaneous casein injection in C57BL/6J mice in vivo. The data showed that inflammatory stress exacerbated renal cholesterol ester accumulation in vitro and in vivo. Inflammation increased cellular cholesterol uptake and synthesis via upregulating the expression of low-density lipoprotein receptor (LDLr) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoA-R), while it decreased cholesterol efflux via downregulating the expression of liver X receptor alpha and ATP-binding cassette transporter A1. The increased lipid accumulation by inflammatory stress induced reactive oxygen species (ROS) and increased levels of endoplasmic reticulum (ER) stress markers (inositol-requiring protein 1 and activating transcription factor 6) in HMCs and kidneys of C57BL/6J mice. This study implied that inflammation promoted renal lipid accumulation and foam cell formation by disrupting cellular cholesterol homeostasis. Increased intracellular lipids under inflammatory stress caused oxidative stress and ER stress in vitro and in vivo which may contribute to renal injury and progression of chronic kidney disease. Show less

The accumulation of cholesterol in macrophages could induce the formation of foam cells and increase the risk of developing atherosclerosis. We wonder if quercetin, one of flavonoids with anti-inflammation functions in different cell types, could elevate the development of foam cells formation in atherosclerosis. We treated foam cells derived from oxLDL induced THP-1 cells with quercetin, and evaluated the foam cells formation, cholesterol content and apoptosis of the cells. We found that quercetin induced the expression of ABCA1 in differentiated THP-1 cells, and increased the cholesterol efflux from THP-1 cell derived foam cells. Eventually, cholesterol level and the formation of foam cell derived from THP-1 cells decreased after quercetin treatment. In addition, quercetin activated PPARγ-LXRα pathway to upregulate ABCA1 expression through increasing protein level of PPARγ and its transcriptional activity. Inhibition of PPARγ activity by siRNA knockdown or the addition of chemical inhibitor, GW9662, abolished quercetin induced ABCA1 expression and cholesterol efflux in THP-1 derived macrophages. Our data demonstrated that quercetin increased cholesterol efflux from macrophages through upregulating the expressions of PPARγ and ABCA1. Taken together, increasing uptake of quercetin or quercetin-rich foods would be an effective way to lower the risk of atherosclerosis. Show less

Niemann-Pick C1-like 1 (NPC1L1), ATP-binding cassette (ABC)G5, and ABCG8 mediate intestinal cholesterol absorption. It is unclear whether pravastatin (PR) or ezetimibe (EZ) affect expression of these transporters. We examined the effects of PR and EZ on NPC1L1, ABCG5, and ABCG8 expression in human hepatoma HepG2 cells and the murine small intestine. We also assessed expression of the transcription factors liver X receptor (LXR)a, LXRb and sterol regulatory element-binding protein. Transporter mRNA levels were determined in murine small intestines 6 and 24 h after oral PR and EZ administration by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). In PR- and EZ-treated HepG2 cells, transporter and transcription factor mRNA and protein levels were examined by RT-PCR and western blot, respectively. Significant decreases in NPC1L1, ABCG5, and ABCG8 mRNA expression were observed in the duodenum, but not jejunum and ileum, of mice 24 h after treatment with PR, but not EZ. In HepG2 cells, PR but not EZ treatment for 24 h also significantly decreased NPC1L1 protein and ABCG5, and ABCG8 mRNA expression, while increasing LXRa mRNA levels. PR but not EZ treatment reduced duodenal cholesterol transporter expression in mice. PR-induced increases in LXRa mRNA levels may be involved in attenuation of NPC1L1 expression, subsequently decreasing intestinal cholesterol absorption. Show less

Cholesterol is the template for steroid hormone biosynthesis. Cholesterol homeostasis is regulated by Cyt-P450 oxygenated cholesterols acting as ligands on LXR-α and LXR-β transcription factors that are now emerging as drug targets. Heterodimerization of LXRs with retinoic acid receptor is considered a prerequisite for target gene activation. Dietary plant oxysterol 28-homobrassinolide (28-HB) is a proven antihyperglycemic and a pro-steroidogenic agent in the rat. Whether 28-HB has a role in LXR gene expression was therefore investigated using oral gavage (15 days) of 28-HB (333 µg/kg b w) to normal and diabetic rat. PCR amplified LXR-α and β mRNA transcripts from treated rat liver and testis exhibited quantitative differences in their expression. Conformational differences in 28-HB docking to LXR-α and β binding domains were also noted through in silico studies, LXR-β adopting lesser specificity. We report that 28-HB transactivates LXR genes in the rat tissues. Show less

Liver X receptor (LXR) is an oxysterol-activated nuclear receptor involved in the control of major metabolic pathways for cholesterol homeostasis and lipogenesis. Although the role of LXR in hepatic steatosis is well known, its correlation with intrahepatic inflammation and fibrosis has not been thoroughly studied. We investigated the association between LXRα, hepatic inflammation, and fibrosis, as well as its correlation with other intrahepatic lipid transporters in patients with nonalcoholic fatty liver disease (NAFLD). We evaluated clinical characteristics including sex, age, body mass index, and laboratory findings from 40 NAFLD and 16 control patients. Immunohistochemical staining was carried out on liver biopsy samples from all patients. The positive rate of LXRα expression was 30 % in the control group, 50 % in the NAFLD group, and 97 % in NASH groups. LXRα expression was positively correlated with not only the amount of intrahepatic fat, but also with intrahepatic inflammation and hepatic fibrosis. LXRα expression showed positive correlation with intrahepatic expression of ABCG5/8, CD36, and SREBP-1c. The expression of ABCA1, ABCG5/8, SREBP-1c, and CD36 was higher in NAFLD than in controls and there was no further increase in the NASH group. NPC1L1 was abundant in human liver. Expression of NPC1L1 was negatively correlated with intrahepatic inflammation and LXRα intensity. LXR expression correlated with the degree of hepatic fat deposition, as well as with hepatic inflammation and fibrosis in NAFLD patients. Our research suggests that LXR is an attractive target for treatment and regulation of hepatic inflammation and fibrosis. Show less

We evaluated in vitro anti-diabetic activities of 497 native plants of Jeju Island (South Korea) by measuring the induction of adipocyte differentiation. Among the plants, Daphniphyllum macropodum fruit extract (DME) had the highest peroxisome proliferator-activated receptor γ (PPARγ) agonist activity and was therefore selected as a potential source of anti-diabetic agents. To elucidate the active components of DME, constituent compounds were purified and their effects on the adipocyte differentiation were studied. Using activity-guided fractionation, four compounds were isolated from DME and their adipogenic effects were evaluated. Among the compounds isolated, 5,7-dihydroxychromone potently induced the differentiation of mouse 3T3-L1 preadipocytes. DME and 5,7-dihydroxychromone increased PPARγ and liver X receptor α (LXRα) mRNA expression levels. To determine whether the adipogenic effects we observed might affect serum glucose levels, we undertook in vivo experiment using streptozotocin-/high-fat diet-induced type 2 diabetes mouse model. DME supplementation reduced serum glucose, total cholesterol, and triacylglycerol levels in diabetes mice. These results suggest that DME may be useful for the prevention and treatment of type 2 diabetes mellitus. Moreover, it was proposed that 5,7-dihydroxychromone isolated from DME is one of the active compounds that may contribute to regulate blood glucose levels. Show less

Chlorogenic acid (CGA) is one of the most abundant polyphenols in the human diet and is suggested to be a potential antiatherosclerotic agent due to its proposed hypolipidemic, anti-inflammatory and antioxidative properties. The aim of this study was to evaluate the effect of CGA on atherosclerosis development in ApoE(-/-) mice and its potential mechanism. ApoE(-/-) mice were fed a cholesterol-rich diet without (control) or with CGA (200 and 400 mg/kg) or atorvastatin (4 mg/kg) for 12 weeks. During the study plasma lipid and inflammatory parameters were determined. Treatment with CGA (400 mg/kg) reduced atherosclerotic lesion area and vascular dilatation in the aortic root, comparable to atorvastatin. CGA (400 mg/kg) also significantly decreased plasma levels of total cholesterol, triglycerides and low-density lipoprotein-cholesterol as well as inflammatory markers. Supplementation with CGA or CGA metabolites-containing serum suppressed oxidized low-density lipoprotein (oxLDL)-induced lipid accumulation and stimulated cholesterol efflux from RAW264.7 cells. CGA significantly increased the mRNA levels of PPARγ, LXRα, ABCA1 and ABCG1 as well as the transcriptional activity of PPARγ. Cholesterol efflux assay showed that three major metabolites, caffeic, ferulic and gallic acids, significantly stimulated cholesterol efflux from RAW264.7 cells. These results suggest that CGA potently reduces atherosclerosis development in ApoE(-/-) mice and promotes cholesterol efflux from RAW264.7 macrophages. Caffeic, ferulic and gallic acids may be the potential active compounds accounting for the in vivo effect of CGA. Show less

Adenosine triphosphate-binding cassette transporter A1 (ABCA1) and ABCG1 play crucial roles in reverse cholesterol transport, and have anti-atherosclerosis effects, and liver X receptor alpha (LXRα) can stimulate cholesterol efflux through these transporters. Angiotensin (Ang)-(1-7) can protect endothelial cells, inhibit smooth muscle cell growth, ameliorate inflammation and exert anti-atherosclerotic effects. In the present study, we attempted to clarify the effect of Ang-(1-7) on expression of ABCA1 and ABCG1, and explored the role of LXRα in the regulation of ABCA1 and ABCG1 in THP-1 macrophages that had been incubated with angiotensin-II (AngII). Ang-(1-7) increased ABCA1 and ABCG1 expression in a concentration-dependent manner at both the mRNA and protein levels, promoted cholesterol efflux, and decreased cholesterol content in THP-1 macrophages treated with AngII. Furthermore, Ang-(1-7) upregulated the expression of LXRα in a concentration-dependent manner in these cells. LXRα small interfering RNA, as well as the Mas receptor antagonist A-779, completely abolished these effects of Ang-(1-7). In summary, Ang-(1-7) upregulates ABCA1 and ABCG1 expression in THP-1 macrophages treated with AngII through the Mas receptor, via the LXRα pathway. This novel insight into the molecular mechanism underlying Ang-(1-7) and AngII interaction could prove useful for developing new strategies for treatment of cardiovascular diseases. Show less

This study was designed to investigate the direct effects of fatty acids (FAs) on the cell viability and the expression levels of genes involved in lipid metabolism in LO2 human liver cells. Palmitate (PA), oleate (OA) and docosahaexenoic acid (DHA) were used to represent saturated, mono-unsaturated and polyunsaturated FAs, respectively. At concentrations of ≤3.2 µg/ml, treatment with single FAs increased the viability of the LO2 cells. At FA concentrations of >3.2 µg/ml, cell viability following OA treatment was increased, but PA or DHA treatment at these concentrations reduced cell viability. Administration of mixtures of these FAs in three ratios (PA:OA:DHA = 1:2:1, 1:1:1 and 1:1:2, respectively) increased the cell viability compared with the control group. The intracellular triglyceride (TG) levels following all types of treatment were significantly increased and the accumulation of TGs was markedly increased with high doses of DHA. In addition, peroxisome proliferator-activated receptor-γ was significantly upregulated in all groups, with the exception of the 1:1:1 group at 3.2 µg/ml and the 1:1:2 group at 12.8 µg/ml. The expression levels of sterol regulatory-element binding protein‑1c, liver X receptor α and apolipoprotein C‑I were significantly reduced in all groups with the exception of the DHA‑treated group and the 1:2:1 groups at 3.2 and 12.8 µg/ml. In conclusion, these results indicate that the type, concentration and mixture ratios of FAs are all important in determining the cell viability and lipid metabolism-related gene expression in LO2 hepatocytes. Show less

Inflammation marks all stages of atherogenesis. DNA hypermethylation in the whole genome or specific genes is associated with inflammation and cardiovascular diseases. Therefore, we aimed to study whether inhibiting DNA methylation by DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) ameliorates atherosclerosis in low-density lipoprotein receptor knockout (Ldlr(-/-)) mice. Ldlr(-/-) mice were fed an atherogenic diet and adminisered saline or 5-aza-dC (0.25 mg/kg) for up to 30 weeks. 5-aza-dC treatment markedly decreased atherosclerosis development in Ldlr(-/-) mice without changes in body weight, plasma lipid profile, macrophage cholesterol levels and plaque lipid content. Instead, this effect was associated with decreased macrophage inflammation. Macrophages with 5-aza-dC treatment had downregulated expression of genes involved in inflammation (TNF-α, IL-6, IL-1β, and inducible nitric oxidase) and chemotaxis (CD62/L-selectin, chemokine [C-C motif] ligand 2/MCP-1 [CCL2/MCP-1], CCL5, CCL9, and CCL2 receptor CCR2). This resulted in attenuated macrophage migration and adhesion to endothelial cells and reduced macrophage infiltration into atherosclerotic plaques. 5-aza-dC also suppressed macrophage endoplasmic reticulum stress, a key upstream signal that activates macrophage inflammation and apoptotic pathways. Finally, 5-aza-dC demethylated liver X receptor α (LXRα) and peroxisome proliferator-activated receptor γ1 (PPARγ1) promoters, which are both enriched with CpG sites. This led to overexpression of LXRα and PPARγ, which may be responsible for 5-aza-dC's anti-inflammatory and atheroprotective effect. Our findings provide strong evidence that DNA methylation may play a significant role in cardiovascular diseases and serve as a therapeutic target for prevention and treatment of atherosclerosis. Show less

Stearoyl-CoA desaturase 1 (SCD1) is the rate limiting enzyme in unsaturated fatty acid biosynthesis. This enzyme has an important role in the regulation of hepatic lipogenesis and lipid oxidation, and alterations in these pathways may lead to several diseases. We examined, in HepG2 cell cultures, the mechanism of SCD1 regulation considering the involvement of two transcription factors: liver X receptor alpha (LXRα) and sterol regulatory element-binding protein-1 (SREBP-1), also investigating the effect of dietary polyunsaturated fatty acids (PUFAs) on this process. The analysis of SCD1 promoter allowed to identify a functional SREBP-1 binding site (SRE 1). LXRα activation increased SCD1 protein level through upregulation of SREBP-1 and its consequent binding to SRE 1 sequence. Polyunsaturated docosahexaenoic acid (DHA, C22:6), eicosapentaenoic acid (EPA, C20:5) and arachidonic acid (AA, C20:4) were able to reduce SREBP-1 binding to SCD1 promoter, while saturated stearic acid (SA, C18:0) did not give any effect. Surface plasmon resonance analysis showed a direct binding of DHA, EPA and AA to LXRα. These data indicate a direct inhibitory interaction of PUFAs with LXRα, a consequent reduction of SREBP-1 and of its binding to SCD1 promoter. This information provides a mechanism to explain the regulation of lipogenic pathways induced by PUFAs. Show less

Arsenic exposure has been linked to an increased incidence of atherosclerosis. Previously, we have shown in vitro and in vivo that arsenic inhibits transcriptional activation of the liver X receptors (LXRs), key regulators of lipid homeostasis. Therefore, we evaluated the role of LXRα in arsenic-induced atherosclerosis using the apoE(-/-) mouse model. Indeed, deletion of LXRα protected apoE(-/-) mice against the proatherogenic effects of arsenic. We have previously shown that arsenic changes the plaque composition in apoE(-/-) mice. Arsenic decreased collagen content in the apoE(-/-) model, and we have observed the same diminution in LXRα(-/-)apoE(-/-) mice. However, the collagen-producing smooth muscle cells (SMCs) were decreased in apoE(-/-), but increased in LXRα(-/-)apoE(-/-). Although transcriptional activation of collagen remained the same in SMC from both genotypes, arsenic-exposed LXRα(-/-)apoE(-/-) plaques had increased matrix metalloproteinase activity compared with both control LXRα(-/-)apoE(-/-) and apoE(-/-), which could be responsible for both the decrease in plaque collagen and the SMC invasion. In addition, arsenic increased plaque lipid accumulation in both genotypes. However, macrophages, the cells known to retain lipid within the plaque, were unchanged in arsenic-exposed apoE(-/-) mice, but decreased in LXRα(-/-)apoE(-/-). We confirmed in vitro that these cells retained more lipid following arsenic exposure and are more sensitive to apoptosis than apoE(-/-). Mice lacking LXRα are resistant to arsenic-enhanced atherosclerosis, but arsenic-exposed LXRα(-/-)apoE(-/-) mice still present a different plaque composition pattern than the arsenic-exposed apoE(-/-) mice. Show less

Liver-X-receptors, LXRα (NR1H3) and LXRβ (NR1H2), encode 2 different but highly homologous isoforms of transcription factors belonging to the nuclear receptor superfamily. Whether LXRα and LXRβ subtypes have discrete roles in the regulation of cardiac physiology/pathology is unknown. We determine the role of each LXR subtype in myocardial ischemia/reperfusion (MI/R) injury. Mice (wild type; those genetically depleted of LXRα, LXRβ, or both; and those overexpressing LXRα or LXRβ by in vivo intramyocardial adenoviral vector) were subjected to MI/R injury. Both LXRα and LXRβ were detected in wild-type mouse heart. LXRα, but not LXRβ, was significantly upregulated after MI/R. Dual activation of LXRα and LXRβ by natural and synthetic agonists reduced myocardial infarction and improved contractile function after MI/R. Mechanistically, LXR activation inhibited MI/R-induced oxidative stress and nitrative stress, attenuated endoplasmic reticulum stress and mitochondrial dysfunction, and reduced cardiomyocyte apoptosis in ischemic/reperfused myocardium. The aforementioned cardioprotective effects of LXR agonists were impaired in the setting of cardiac-specific gene silencing of LXRα, but not LXRβ subtype. Moreover, LXRα/β double-knockout and LXRα-knockout mice, but not LXRβ-knockout mice, increased MI/R injury, exacerbated MI/R-induced oxidative/nitrative stress, and aggravated endoplasmic reticulum stress and mitochondrial dysfunction. Furthermore, cardiac LXRα, not LXRβ, overexpression via adenoviral transfection suppressed MI/R injury. Our study provides the first direct evidence that the LXRα, but not LXRβ, subtype is a novel endogenous cardiac protective receptor against MI/R injury. Drug development strategies specifically targeting LXRα may be beneficial in treating ischemic heart disease. Show less

Recent studies have revealed critical roles that nuclear receptors like LXR-α (Liver X Receptor- alpha) plays as a class of post-transcriptional gene regulator in skin development and diseases. Keeping in view the fact that LXR-α plays crucial role in keratinocyte proliferation and differentiation, it becomes imperative to dissect the pathways and role of LXR-α genomics in the pathogenesis of psoriasis with ultimate aim to explore novel preventive/therapeutic strategies as treatment options. To explore the effects of agonists and activators of LXR-α on its own gene expression and the putative targets in psoriatic keratinocytes. Identification of promoter sequences for (vitamin D receptor) VDR and Catalase were done using in silico analysis followed by β-galactosidase (β-gal) reporter plasmid assay in keratinocytes from clinically heathy subjects. Determination of relative levels of LXR-α,VDR and catalase in control versus treated cells upon activation of LXR-α with Atorvastatin + 22R hydroxycholestrol and Ascorbic acid + 22R hydroxycholestrol was done by PCR and Cell Proliferation Assay. The cells transfected with the reporter plasmid element for VDR and catalase showed more than 5 and 4 fold increase respectively in the β-gal activity compared to the control. An increase of 55% in LXR-α gene expression at RNA level was observed in Atorvastatin + 22-R hydroxycholestrol compared to 24% in Ascorbic acid + 22-ROH cholesterol. The expression of the VDR and Catalase was significantly increased in both treated keratinocytes compared to its normal counterpart. Show less

We aimed to elucidate the effect of bilirubin on dyslipidemia and nephropathy in a diabetes mellitus (DM) type I animal model. Sprague-Dawley rats were separated into control, DM, and bilirubin-treated DM (Bil) groups. The Bil group was injected intraperitoneally with 60 mg/kg bilirubin 3 times per week and hepatoma cells were cultured with bilirubin at a concentration of 0.3 mg/dL. The Bil group showed lower serum creatinine levels 5 weeks after diabetes onset. Bilirubin treatment also decreased the amount of mesangial matrix, lowered the expression of renal collagen IV and transforming growth factor (TGF)-β1, and reduced the level of apoptosis in the kidney, compared to the DM group. These changes were accompanied by decreased tissue levels of hydrogen superoxide and NADPH oxidase subunit proteins. Bilirubin decreased serum total cholesterol, high-density lipoprotein cholesterol (HDL-C), free fatty acids, and triglycerides (TGs), as well as the TG content in the liver tissues. Bilirubin suppressed protein expression of LXRα, SREBP-1, SCD-1, and FAS, factors involved in TG synthesis that were elevated in the livers of DM rats and hepatoma cells under high-glucose conditions. In conclusion, bilirubin attenuates renal dysfunction and dyslipidemia in diabetes by suppressing LXRα and SREBP-1 expression and oxidative stress. Show less

Lipid deposition in artery walls is implied in the pathogenesis of atherosclerosis and imbalance between uptake and efflux of cholesterol favors the deposition. We investigated the effect of vitamin E with the same dose and duration on the different stages of atherosclerosis in Apolipoprotein E knockout (ApoE KO) mice and explored the potential mechanisms. The results showed that the ApoE KO mouse spontaneously develops atherosclerosis in an age-dependent manner from 14 to 46 weeks on the regular chow. Vitamin E (100 mg/kg) supplementation to ApoE KO mice at 6, 14, and 22 weeks for 8 weeks significantly reduced the atherosclerotic lesion area by 41, 29 and 19% respectively compared to the age-matched control mice; however had no significant effect on the lesion when given at 30 and 38 weeks. In addition, vitamin E supplemented at the ages from 6 to 30 weeks decreased the contents of serum oxLDL and TBARS without affecting the TC and TAG contents in serum and liver. Furthermore, vitamin E supplemented at 6, 14 and 22 weeks down-regulated vasculature mRNA expressions of scavenger receptor CD36 and up-regulated mRNA expressions of PPARγ, LXRα and ABCA1 which are involved in reverse cholesterol transportation; however had no significant effects on these genes when given at 30 and 38 weeks. In conclusion, vitamin E with same dose and duration inhibits the early but not advanced atherosclerotic lesion in ApoE KO mice by anti-oxidation and regulation of mRNA expression of genes involved in cholesterol uptake and efflux, which favors the improvement of atherosclerosis. Show less

Ezetimibe is a potent inhibitor of Niemann-Pick type C1-Like 1 and has been approved for the treatment of hypercholesterolemia. Our preliminary study showed that ezetimibe promotes cholesterol efflux from vascular smooth muscle cells (VSMCs). Our aim was to investigate the cellular mechanisms underlying the ezetimibe actions. Rat VSMCs were converted to foam cells by incubation with cholesterol:methyl-β-cyclodextrin. The intracellular free cholesterol, total cholesterol, and the ratio of cholesteryl ester to total cholesterol were decreased after the incubation of VSMCs with different concentrations of ezetimibe (3, 10, 30, and 30 μmol/l) or treated with 30 μmol/l of ezetimibe for different time periods (6, 12, 24, and 48 h). Our results also showed that the expression of caveolin-1, liver X receptor α, and ATP-binding cassette transporter ABCA1 was enhanced, but the expression of nSREBP-1c was decreased in a concentration- and time-dependent manner. RNA interference was used to determine the roles of caveolin-1 and SREBP-1 in the lipid-lowering effect of ezetimibe. The results showed that caveolin-1 was involved in the regulation of intracellular cholesterol content, and the expression of caveolin-1 was repressed by SREBP-1. The present study indicates that ezetimibe protects VSMCs from cholesterol accumulation by regulating the expression of lipid metabolism-related genes. Show less

Liver X receptor α (LXRα) plays an important role in the cholesterol metabolism process, and LXRα activation can reduce atherosclerosis. In the present study, using an LXRα-GAL4 luciferase reporter screening, we discovered IMB-170, a structural analog of quinazolinone, which showed potent LXRα agonistic activity. IMB-170 significantly activated LXRα, with an EC50 value of 0.27μM. Interestingly, IMB-170 not only increased the expression of ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1), which are related to the reverse cholesterol transport (RCT) process, but also influenced the expression levels of other genes involved in the cholesterol metabolism pathway in many cell lines. Moreover, IMB-170 significantly reduced cellular lipid accumulation and increased cholesterol efflux from RAW264.7 and THP-1 macrophages. Interestingly, compared with TO901317, IMB-170 only slightly increased protein expression levels of lipogenesis-related genes in HepG2 cells, indicating that IMB-170 may have a lower lipogenesis side effect in vivo. These results suggest that IMB-170 showed the selective agonistic activity for LXRα. Moreover, compared with full LXR-agonists, IMB-170 possesses a differential ability to recruit coregulators. This suggests that IMB-170 has distinct interactions with the active sites in the LXRα ligand-binding domain. In summary, IMB-170 is a novel partial LXRα agonist without the classical lipogenesis side effects, which could be used as a potential anti-atherosclerotic leading compound in the future. Show less

The objective of this study was to determine the effects of pentraxin3 (PTX3) on human oxidized low density lipoprotein (oxLDL) uptake and cholesterol efflux from human macrophage foam cells, which may play a critical role in atherogenesis. The effects of PTX3 on oxLDL uptake and cholesterol efflux were determined after transfection of human THP-1 macrophages with pSG5hPTX3 or PTX3siRNA plasmids. To evaluate the role of specific signaling pathways, human THP-1 cells were pre-treated with inhibitors of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), phosphatidylinositide 3-kinases (PI3-K), and p38 mitogen-activated protein kinase (MAPK) pathways (PD98059, LY294002, and SB203580, respectively), and then exposed to oxLDL for the uptake assay or oxLDL and [(3)H]-cholesterol and apolipoprotein A-I (apoA-I) for the cholesterol efflux assay. PTX3 overexpression not only promoted oxLDL uptake but also significantly reduced cholesterol efflux to apoA-I; it also significantly decreased the expression of peroxisome proliferator-activated receptor-γ (PPARγ), liver X receptor alpha (LXRα) and ATP-binding membrane cassette transporter A-1 (ABCA1), which was increased with PTX3 silencing. Furthermore, PTX3 significantly increased p-ERK1/2 levels in THP-1-derived foam cells, and inhibition of ERK1/2 by PD98059 significantly reduced the oxLDL uptake and promoted the cholesterol efflux induced by PTX3 overexpression. Here, we demonstrate that PTX3 affects lipid accumulation in human macrophages, increasing oxLDL uptake and inhibiting cholesterol efflux. That is the underlying possible mechanisms of PTX3 contribution to the progression of atherosclerosis. Show less

To examine the cholesterol efflux and the expressions of ATP-binding cassette transporter G1 (ABCG1) in macrophages of diabetic patients and the roles of liver-X receptor (LXR) in regulation of ABCG1 expressions. Blood was collected from patients with type 2 diabetes mellitus and healthy controls. The peripheral blood monocytes were differentiated into macrophages with macrophage colony stimulating factor (M-CSF). The cells were radio labeled with [(3)H] cholesterol and were performed with cholesterol efflux assays. Quantitative real-time PCR (qRT PCR) and Western blot were performed to measure the mRNA and protein expressions of ABCA1 and ABCG1. To test the effects of LXR on ABCG1 expressions, inhibition of LXRα and LXRβ by siRNA were performed. The DNA-protein complex of LXR and LXR element (LXRE) located in the promoter region of ABCG1 gene were detected with electrophery mobility supershift assay (EMSA). Macrophage ABCG1 expressions and high-density lipoprotein (HDL) induced cholesterol efflux were significantly reduced (19.0%±1.2% vs. 12.8%±3.6%, t=2.532, P=0.016) in the diabetic subjects whereas ABCA1 expressions and apolipoprotein A1 (ApoA1) induced cholesterol efflux were comparable (12.0%±1.2% vs. 10.2%±2.3%, t=1.771, P=0.109) between the diabetic patients and healthy subjects. The mRNA expressions of LXRα and LXRβ had no changes between the diabetes subjects and healthy controls (t=1.025, P=0.315; t=-0.531, P=0.600). The LXR-LXRE DNA-protein complex detected by EMSA were also similar between the diabetes subjects and healthy controls (t=1.483, P=0.164). Moreover, ABCG1 expressions were not altered by inhibition of LXRα/β siRNA (t=2.143, P=0.061). Our data indicated that expression of ABCG1 and HDL induced cholesterol efflux were reduced in type 2 diabetic patients. However, the LXR mRNA expression and binding complex of LXR and ABCG1 promoter were not changed. The impairment of cholesterol efflux and ABCG1 gene expressions might be regulated via an LXR-independent pathway. Show less

Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Although PPARγ is known to be a potent sterol sensor that play a fundamental role in cholesterol metabolism, the potential effects of PPARγ responsive miRNA still need to be revealed. In this study, we found that miR-613 is inversely correlated with LXRα and ABCA1 in PPARγ activated THP-1 cells. PPARγ negatively regulates the expression of miR-613 at transcriptional level, and miR-613 suppressed LXRα and ABCA1 by targeting the 3'-UTR of their mRNAs. Furthermore, downregulation of LXRα and ABCA1 by miR-613 inhibited cholesterol efflux from PPARγ activated THP-1 macrophages. These results revealed an alternative mechanism for PPARγ regulation and provided a potential target for the treatment of cholesterol metabolic diseases. Show less