Cholesterol Influx Research Articles

Novel Mechanism of Cholesterol Transport by ABCA5 in Macrophages and Its Role in Dyslipidemia

Cholesterol homeostasis results from a delicate interplay between influx and efflux of free cholesterol primarily mediated by ABCA1. Here we report downregulation of ABCA1 in hyper-cholesterol conditions in macrophages, which might be responsible for compromised reverse cholesterol transport and hyperlipidemia. Surprisingly, this is countered by the upregulation of a lesser known family member ABCA5 to maintain cholesterol efflux. The relative contribution of ABCA1 and ABCA5 toward cholesterol efflux was evaluated and revealed ABCA5 as the primary efflux mediator under high cholesterol load. These observations were correlated to cholesterol load in circulation in vivo, and we observed an inverse expression profile in mice models of atherosclerosis (ApoE−/−) and hyperlipidemia (PPARα−/−) in response to high cholesterol diet. Observations were further validated in human plasma samples. Simulation studies revealed a unique conformation of ABCA5 proposing a favored route for cholesterol loading onto high-density lipoproteins for reverse cholesterol transport. Thus, our study implicates a functional complementation between these two transporters, formulating an efficient strategy to maintain efflux in cholesterol excess conditions in macrophages.

ALK7 Acts as a Positive Regulator of Macrophage Activation through Down-Regulation of PPARγ Expression.

Aim: Activin receptor-like kinase 7 (ALK7) acts as a key receptor for TGF-β family members, which play important roles in regulating cardiovascular activity. However, ALK7's potential role, and underlying mechanism, in the macrophage activation involved in atherogenesis remain unexplored.Methods: ALK7 expression in macrophages was tested by RT-PCR, western blot, and immunofluorescence co-staining. The loss-of-function strategy using AdshALK7 was performed for functional study. Oil Red O staining was used to observe the foam cell formation, while inflammatory mediators and genes related to cholesterol efflux and influx were determined by RT-PCR and western blot. A PPARγ inhibitor (G3335) was used to reveal whether PPARγ was required for ALK7 to affect macrophage activation.Results: The results exhibited upregulated ALK7 expression in oxidized low-density lipoprotein (Ox-LDL) induced bone marrow derived macrophages (BMDMs) and mouse peritoneal macrophages (MPMs), isolated from ApoE-deficient mice, while ALK7's strong immunoreactivity in BMDMs was observed. ALK7 knockdown significantly attenuated pro-inflammatory, but promoted anti-inflammatory, macrophage markers expression. Additionally, ALK7 silencing decreased foam cell formation, accompanied by the up-regulation of ABCA1 and ABCG1 involved in cholesterol efflux but the down-regulation of CD36 and SR-A implicated in cholesterol influx. Mechanistically, ALK7 knockdown upregulated PPARγ expression, which was required for the ameliorated effect of ALK7 silencing macrophage activation.Conclusions: Our study demonstrated that ALK7 was a positive regulator for macrophage activation, partially through down-regulation of PPARγ expression, which suggested that neutralizing ALK7 might be promising therapeutic strategy for treating atherosclerosis.

Loss of core fucosylation reduces low-density lipoprotein receptor expression in hepatocytes by inducing PCSK9 production

Fucosylation is a type of glycosylation, a form of post-transcriptional regulation of proteins, involved in cancer and inflammation. It involves the attachment of a fucose residue to N-glycans, O-glycans, and glycolipids, which is catalyzed by a family of enzymes called fucosyltransferases (Futs). Among the many Futs, α-1,6-fucosyltransferase (Fut8) is the only enzyme that produces α-1,6-fucosylated oligosaccharides (core fucose). In the human liver, the expression and activity of Fut8 are frequently elevated during progression of chronic liver diseases. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a well-known negative regulator of the low-density lipoprotein receptor (LDLR). Here, we found that loss of core fucose in immortalized hepatocytes led to LDLR downregulation through a dramatic induction of PCSK9. We used the immortalized hepatocytes derived from Fut8 knockout mice or a Fut8 knockdown AML12 hepatocyte cell line. Using these cells, we investigated the effects of Fut8 on hepatocyte cholesterol influx. Both cell lines had reduced LDLR protein levels, resulting from marked increases in PCSK9 expression. Intracellular cholesterol levels were significantly lower and LDL cholesterol uptake was suppressed in Fut8-KO cells. Hepatocyte nuclear factor 1α accumulated in nuclei of Fut8-KO hepatocytes, which mediated increases in PCSK9 mRNA expression. Our findings demonstrated that loss of core fucosylation promoted degradation of LDLR and impaired cholesterol uptake, which is a novel mechanism that regulates cholesterol influx, suggesting that Fut8 might be a novel causative gene for familial hypercholesterolemia.

4β-Hydroxycholesterol Signals From the Liver to Regulate Peripheral Cholesterol Transporters.

Activation of pregnane X receptor (PXR) elevates circulating 4β-hydroxycholesterol (4βHC), an agonist of liver X receptor (LXR). PXR may also regulate 25-hydroxycholesterol and 27-hydroxycholesterol. Our aim was to elucidate the roles of PXR and oxysterols in the regulation of cholesterol transporters. We measured oxysterols in serum of volunteers dosed with PXR agonist rifampicin 600 mg/day versus placebo for a week and analyzed the expression of cholesterol transporters in mononuclear cells. The effect of 4βHC on the transport of cholesterol and the expression of cholesterol transporters was studied in human primary monocyte-derived macrophages and foam cells in vitro. The expression of cholesterol transporters was measured also in rat tissues after dosing with a PXR agonist. The levels of 4βHC were elevated, while 25-hydroxycholesterol and 27-hydroxycholesterol remained unchanged in volunteers dosed with rifampicin. The expression of ATP binding cassette transporter A1 (ABCA1) was induced in human mononuclear cells in vivo. The influx of cholesterol was repressed by 4βHC, as was the expression of influx transporter lectin-like oxidized LDL receptor-1 in vitro. The cholesterol efflux and the expression of efflux transporters ABCA1 and ABCG1 were induced. The expression of inducible degrader of the LDL receptor was induced. In rats, PXR agonist increased circulating 4βHC and expression of LXR targets in peripheral tissues, especially ABCA1 and ABCG1 in heart. In conclusion, PXR activation-elevated 4βHC is a signaling molecule that represses cholesterol influx and induces efflux. The PXR-4βHC-LXR pathway could link the hepatic xenobiotic exposure and the regulation of cholesterol transport in peripheral tissues.

Cholesterol, Oxysterols and LXRs in Breast Cancer Pathophysiology.

Breast cancer is the most frequent cancer among women. In 2018, it is estimated that 627,000 women died from breast cancer. This is approximately 15% of all cancer deaths among women (WHO 2018). Breast cancer is a multifactorial chronic disease. While important progress has been made to treat patients, many questions regarding aspects of this disease relating to carcinogenesis are still open. During carcinogenesis, cells exhibit cholesterol homeostasis deregulation. This results in an accumulation of intracellular cholesterol, which is required to sustain their high growth rate. Cholesterol efflux and influx are two metabolic pathways that are necessary to prevent cholesterol accumulation in the cells. Liver X receptors (LXRs) are nuclear receptors that, upon activation, induce the expression of ABC transporters, responsible for promoting cholesterol efflux, and the expression of IDOL (inducible degrader of low-density lipoprotein receptor), in charge of reducing cholesterol influx. Oxysterols, oxygenated derivatives of cholesterol formed through different pathways, have been discovered as LXR-specific ligands. Some oxysterols are involved in tumor formation while others are considered anti-tumor agents. In the present review, we discuss the involvement of cholesterol, oxysterols and LXRs in breast cancer pathophysiology, with an emphasis on the biological effects of LXR ligands.

HDL cholesterol is associated with PBMC expression of genes involved in HDL metabolism and atherogenesis.

To reveal the association of plasma level of high density lipoprotein cholesterol (HDL-C) level with the transcript level of annotated genes in peripheral blood mononuclear cells (PBMC) and involved in HDL metabolism and atherogenesis at the absence of morphologically evident coronary stenosis. Transcript levels of 63 genes in PBMC from 38 male patients 40-60 years without coronary atherosclerosis with widely varied HDL-C level were measured. The protein interactions were analyzed with STRING database. Among 22 HDL-related genes, the transcript levels for 10 genes (ABCA1, BMP1, CUBN, HDLBP, LCAT, LDLR, PRKACB, PRKACG, SCARB1 and ZDHHC8) negatively correlated with HDL-C, while positively for APOA1 gene. Among 41 atherosclerosis-prone genes, the transcript levels for 11 genes (CSF1R, CSF2RB, IL18R1, ITGAM, ITGB3, PRKCQ, SREBF1, TLR5, TLR8, TNFRSF1A and TNFRSF1B) negatively correlated with HDL-C only, not with LDL-C and plasma TG. The protein products efficiently interacted within each cluster while only two intersection nodes existed between clusters. Coordinate regulation of cholesterol influx and efflux in PBMC in atherosclerosis-free subjects with widely varied HDL-C level is suggested. The decreased synthesis and transport of cholesteryl ester to the liver may contribute to hyperalphalipoproteinemia. HDL-C increase is associated with the decrease of expression of innate immunity and inflammation genes. Visualization of 22 responder genes is suggested to be useful in the validation of HDL functionality and atherogenesis even at the absence of morphologically evident coronary stenosis.

Maternal betaine suppresses adrenal expression of cholesterol trafficking genes and decreases plasma corticosterone concentration in offspring pullets

BackgroundLaying hens supplemented with betaine demonstrate activated adrenal steroidogenesis and deposit higher corticosterone (CORT) in the egg yolk. Here we further investigate the effect of maternal betaine on the plasma CORT concentration and adrenal expression of steroidogenic genes in offspring pullets.ResultsMaternal betaine significantly reduced (P < 0.05) plasma CORT concentration and the adrenal expression of vimentin that is involved in trafficking cholesterol to the mitochondria for utilization in offspring pullets. Concurrently, voltage-dependent anion channel 1 and steroidogenic acute regulatory protein, the two mitochondrial proteins involved in cholesterol influx, were both down-regulated at mRNA and protein levels. However, enzymes responsible for steroid syntheses, such as cytochrome P450 family 11 subfamily A member 1 and cytochrome P450 family 21 subfamily A member 2, were significantly (P < 0.05) up-regulated at mRNA or protein levels in the adrenal gland of pullets derived from betaine-supplemented hens. Furthermore, expression of transcription factors, such as steroidogenic factor-1, sterol regulatory element-binding protein 1 and cAMP response element-binding protein, was significantly (P < 0.05) enhanced, together with their downstream target genes, such as 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, LDL receptor and sterol regulatory element-binding protein cleavage-activating protein. The promoter regions of most steroidogenic genes were significantly (P < 0.05) hypomethylated, although methyl transfer enzymes, such as AHCYL, GNMT1 and BHMT were up-regulated.ConclusionsThese results indicate that the reduced plasma CORT in betaine-supplemented offspring pullets is linked to suppressed cholesterol trafficking into the mitochondria, despite the activation of cholesterol and corticosteroid synthetic genes associated with promoter hypomethylation.

Polychlorinated Biphenyl Quinone Promotes Macrophage-Derived Foam Cell Formation.

Polychlorinated biphenyls (PCBs) are organic environmental pollutants that are accused of various toxic effects. PCB exposure is widely believed to be associated with atherosclerosis, but the underlying mechanisms are unclear. Although PCBs are easily metabolized, there is rarely information on the effects of their metabolites on atherosclerosis. Currently, we evaluate the effect of 2,3,5-trichloro-6-phenyl-[1,4]-benzoquinone (PCB29-pQ) on the critical phase of atherosclerosis development, that is, the formation of macrophage-derived foam cells. We exposed Ox-LDL-induced RAW264.7 cells to 2.5 μM and 5 μM PCB29-pQ. Varieties of evidence have demonstrated that PCB29-pQ promotes foam cell formation and develops proinflammatory cascade and cell necroptosis. In detail, we observed that PCB29-pQ increased levels of total cholesterol (TC), free cholesterol (FC), triglyceride (TG), and cholesteryl ester (CE) by increasing the cholesterol influx and reducing the cholesterol efflux. Moreover, we found that PCB29-pQ induced inflammatory cytokines, such as tumor necrosis factor (TNF-α), interleukin 6 (IL-6), and IL-1β, released by activating the mitogen-activated protein kinase (MAPK)-nuclear factor kappa B (NF-κB) inflammatory pathway. In addition, we demonstrated that PCB29-pQ induced cell necroptosis via receptor interacting protein kinases 1 and 3 (RIPK1/3) and a mixed-lineage kinase domain-like (MLKL) pathway. Finally, the overproduction of reactive oxygen species (ROS) by PCB29-pQ played significant roles in these processes, which could be reversed with an antioxidant. Overall, our results indicated that PCB29-pQ promoted the macrophage formation of foam cells, inflammation, and cell necroptosis.

The Impact of Mevastatin on HCV Replication and Autophagy of Non-Transformed HCV Replicon Hepatocytes Is Influenced by the Extracellular Lipid Uptake.

Statins efficiently inhibit cholesterol synthesis by blocking 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase in the mevalonate pathway. However, the effect of statins on intracellular cholesterol is partially counterbalanced by a consequent increased uptake of extracellular lipid sources. Hepatitis C virus (HCV) infection induces intracellular accumulation of cholesterol by promoting both new synthesis and uptake of circulating lipoproteins, which is required for HCV replication and release. Hepatocytes respond to the increase in intracellular cholesterol levels by inducing lipophagy, a selective type of autophagy mediating the degradation of lipid deposits within lysosomes. In a cellular system of HCV replication based on HuH7 hepatoma cells, statin treatment was shown to be sufficient to decrease intracellular cholesterol, which is accompanied by reduced HCV replication and decreased lipophagy, and has no apparent impact on endocytosis-mediated cholesterol uptake. To understand whether these results were influenced by an altered response of cholesterol influx in hepatoma cells, we analyzed the effect of statins in non-transformed murine hepatocytes (MMHD3) harboring subgenomic HCV replicons. Notably, we found that total amount of cholesterol is increased in MMHD3 cells upon mevastatin treatment, which is associated with increased HCV replication and lipophagy. Conversely, mevastatin is able to reduce cholesterol amounts only when cells are grown in the presence of delipidated serum to prevent extracellular lipid uptake. Under this condition, HCV replication is reduced and autophagy flux is severely impaired. Altogether, these results indicate that both de novo synthesis and extracellular uptake have to be targeted in non-transformed hepatocytes in order to decrease intracellular cholesterol levels and consequently limit HCV replication.

Targeting the transcription factor receptor LXR to treat clear cell renal cell carcinoma: agonist or inverse agonist?

Growing evidence indicates that clear cell renal cell carcinoma (ccRCC) is a metabolism-related disease. Changes in fatty acid (FA) and cholesterol metabolism play important roles in ccRCC development. As a nuclear transcription factor receptor, Liver X receptor (LXR) regulates a variety of key molecules associated with FA synthesis and cholesterol transport. Therefore, targeting LXR may provide new therapeutic targets for ccRCC. However, the potential regulatory effect and molecular mechanisms of LXR in ccRCC remain unknown. In the present study, we found that both an LXR agonist and an XLR inverse agonist could inhibit proliferation and colony formation and induce apoptosis in ccRCC cells. We observed that the LXR agonist LXR623 downregulated the expression of the low-density lipoprotein receptor (LDLR) and upregulated the expression of ABCA1, which resulted in reduced intracellular cholesterol and apoptosis. The LXR inverse agonist SR9243 downregulated the FA synthesis proteins sterol regulatory element-binding protein 1c (SREBP-1c), fatty acid synthase (FASN) and stearoyl-coA desaturase 1 (SCD1), causing a decrease in intracellular FA content and inducing apoptosis in ccRCC cells. SR9243 and LXR623 induced apoptosis in ccRCC cells but had no killing effect on normal renal tubular epithelial HK2 cells. We also found that SRB1-mediated high-density lipoprotein (HDL) in cholesterol influx is the cause of high cholesterol in ccRCC cells. In conclusion, our data suggest that an LXR inverse agonist and LXR agonist decrease the intracellular FA and cholesterol contents in ccRCC to inhibit tumour cells but do not have cytotoxic effects on non-malignant cells. Thus, LXR may be a safe therapeutic target for treating ccRCC patients.

Postprandial changes in gene expression of cholesterol influx and efflux mediators after intake of SFA compared with n-6 PUFA in subjects with and without familial hypercholesterolaemia: secondary outcomes of a randomised controlled trial.

Abstract

The TICE Pathway: Mechanisms and Lipid-Lowering Therapies.

Besides the well-known hepatobiliary pathway of cholesterol excretion into the feces, transintestinal cholesterol excretion (TICE) is a second major pathway through which cholesterol is disposed from the body. In the process of TICE, cholesterol is taken up from lipoprotein particles at the basolateral side of the enterocyte and translocates towards the apical side of the enterocyte. At the apical side, the ATP-binding cassette transporters G5 and G8 form a heterodimer that transports cholesterol into the intestinal lumen. A substantial amount of the secreted cholesterol is likely reabsorbed by the cholesterol influx transporter Niemann-Pick C1-Like 1 (NPC1L1) since recent data indicate that inhibition of NPC1L1 increases the efficacy of TICE for disposal of cholesterol via the feces. The pathways and proteins involved in intracellular cholesterol trafficking in the enterocyte have not yet been identified. Therefore, in addition to discussing known mediators of TICE, this review will also examine potential candidates involved in cholesterol translocation in the enterocyte. Both the cholesterol reuptake and efflux pathways can be influenced by pharmaceutical means; thus, the TICE pathway is a very attractive target to increase cholesterol excretion from the body and prevent or mitigate atherosclerotic cardiovascular disease.

HDL-Mediated Lipid Influx to Endothelial Cells Contributes to Regulating Intercellular Adhesion Molecule (ICAM)-1 Expression and eNOS Phosphorylation

Reverse cholesterol transport (RCT) is considered as the most important antiatherogenic role of high-density lipoproteins (HDL), but interventions based on RCT have failed to reduce the risk of coronary heart disease. In contrast to RCT, important evidence suggests that HDL deliver lipids to peripheral cells. Therefore, in this paper, we investigated whether HDL could improve endothelial function by delivering lipids to the cells. Internalization kinetics using cholesterol and apolipoprotein (apo) AI fluorescent double-labeled reconstituted HDL (rHDL), and human dermal microvascular endothelial cells-1 (HMEC-1) showed a fast cholesterol influx (10 min) and a slower HDL protein internalization as determined by confocal microscopy and flow cytometry. Sphingomyelin kinetics overlapped that of apo AI, indicating that only cholesterol became dissociated from rHDL during internalization. rHDL apo AI internalization was scavenger receptor class B type I (SR-BI)-dependent, whereas HDL cholesterol influx was independent of SR-BI and was not completely inhibited by the presence of low-density lipoproteins (LDL). HDL sphingomyelin was fundamental for intercellular adhesion molecule-1 (ICAM-1) downregulation in HMEC-1. However, vascular cell adhesion protein-1 (VCAM-1) was not inhibited by rHDL, suggesting that components such as apolipoproteins other than apo AI participate in HDL’s regulation of this adhesion molecule. rHDL also induced endothelial nitric oxide synthase eNOS S1177 phosphorylation in HMEC-1 but only when the particle contained sphingomyelin. In conclusion, the internalization of HDL implies the dissociation of lipoprotein components and a SR-BI-independent fast delivery of cholesterol to endothelial cells. HDL internalization had functional implications that were mainly dependent on sphingomyelin. These results suggest a new role of HDL as lipid vectors to the cells, which could be congruent with the antiatherogenic properties of these lipoproteins.

Single Cell Titration-Type Assay for Plasma Membrane Cholesterol Chemical Potential.

In this paper, a titration-type assay is described that determines the minimum concentration of cholesterol in solution that is required to drive net influx of cholesterol to the plasma membrane and thus increase the cholesterol concentration. The increase in cholesterol in the plasma membrane is detected by cholesterol diffusion at the site of contact by a cholesterol oxidase-modified microelectrode. In the presented thermodynamic model, the minimum solution phase cholesterol concentration that drives influx to the plasma membrane is a close approximation of the true solution-phase equilibrium concentration of cholesterol produced from cellular cholesterol efflux and as such it is a quantitative measure of the chemical potential of cholesterol in the cellular plasma membrane. This assay provides a measure of cholesterol chemical potential in the living cellular plasma membrane through reference to a solution concentration which avoids invoking classic kinetic theory to relate a rate to a specific thermodynamic activity and which avoids uncertainty associated with mass transfer phenomena.

Fish Oil Supplementation Alleviates the Altered Lipid Homeostasis in Blood, Liver, and Adipose Tissues in High-Fat Diet-Fed Rats.

This study investigated the effects of dietary supplementation of fish oil on the signals of lipid metabolism involved in hepatic cholesterol and triglyceride influx and excretion in high-fat diet (HFD)-fed rats. Fish oil (FO) repressed body (HFD, 533 ± 18.2 g; HFD+FO, 488 ± 28.0 g, p < 0.05) and liver weights (HFD, 5.7 ± 0.6 g/100 g of body weight; HFD+FO, 4.8 ± 0.4 g/100 g of body weight, p < 0.05) in HFD-fed rats. Fish oil could also improve HFD-induced imbalance of lipid metabolism in blood, liver, and adipose tissues including the significant decreases in plasma and liver total cholesterol (TC) (plasma-HFD, 113 ± 33.6 mg/dL; HFD+FO, 50.0 ± 5.95 mg/dL, p < 0.05; liver-HFD, 102 ± 13.0 mg/g liver; [corrected] HFD+FO, 86.6 ± 7.81 mg/g liver, [corrected] p < 0.05), blood, liver, and adipose triglyceride (TG) (blood-HFD, 52.5 ± 20.4 mg/dL; HFD+FO, 29.8 ± 4.30 mg/dL, p < 0.05; liver-HFD, 56.2 ± 10.0 mg/g liver; [corrected] HFD+FO, 30.3 ± 5.28 mg/g liver, [corrected] p < 0.05; adipose-HFD, 614 ± 73.2 mg/g liver, [corrected] HFD+FO, 409 ± 334 mg/g of adipose tissue, [corrected] p < 0.05), and low density (HFD, 79.8 ± 40.9 mg/dL; HFD+FO, 16.6 ± 5.47 mg/dL, p < 0.05) and very-low-density (HFD, 49.7 ± 33.3 mg/dL; HFD+FO, 10.4 ± 3.45 mg/dL, p < 0.05) lipoprotein and the significant increases in fecal TC (HFD, 12.2 ± 0.67 mg/g feces; [corrected] HFD+FO, 16.3 ± 2.04 mg/g feces, [corrected] < 0.05) and TG (HFD, 2.09 ± 0.10 mg/g feces; [corrected] HFD+FO, 2.38 ± 0.22 mg/g feces, [corrected] p < 0.05) and lipoprotein lipase activity of adipose tissues (HFD, 16.6 ± 3.64 μM p-nitrophenol; HFD+FO, 24.5 ± 4.19 μM p-nitrophenol, p < 0.05). Moreover, fish oil significantly activated the protein expressions of hepatic lipid metabolism regulators (AMPKα and PPARα) and significantly regulated the lipid-transport-related signaling molecules (ApoE, MTTP, ApoB, Angptl4, ApoCIII, ACOX1, and SREBPF1) in blood or liver of HFD-fed rats. These results suggest that fish oil supplementation improves HFD-induced imbalance of lipid homeostasis in blood, liver, and adipose tissues in rats.

Osteopontin deficiency ameliorates Alport pathology by preventing tubular metabolic deficits.

Alport syndrome is a rare hereditary renal disorder with no etiologic therapy. We found that osteopontin (OPN) is highly expressed in the renal tubules of the Alport mouse and plays a causative pathological role. OPN genetic deletion ameliorated albuminuria, hypertension, tubulointerstitial proliferation, renal apoptosis, and hearing and visual deficits in the Alport mouse. In Alport renal tubules we found extensive cholesterol accumulation and increased protein expression of dynamin-3 (DNM3) and LDL receptor (LDLR) in addition to dysmorphic mitochondria with defective bioenergetics. Increased pathological cholesterol influx was confirmed by a remarkably increased uptake of injected DiI-LDL cholesterol by Alport renal tubules, and by the improved lifespan of the Alport mice when crossed with the Ldlr-/- mice with defective cholesterol influx. Moreover, OPN-deficient Alport mice demonstrated significant reduction of DNM3 and LDLR expression. In human renal epithelial cells, overexpressing DNM3 resulted in elevated LDLR protein expression and defective mitochondrial respiration. Our results suggest a potentially new pathway in Alport pathology where tubular OPN causes DNM3- and LDLR-mediated enhanced cholesterol influx and impaired mitochondrial respiration.

Silica nanoparticles promote oxLDL-induced macrophage lipid accumulation and apoptosis via endoplasmic reticulum stress signaling

Oxidized low-density lipoprotein (oxLDL), a marker of hyperlipidemia, plays a pivotal role in the development of atherosclerosis through the induction of macrophage-derived foam cell formation and thereafter apoptosis. Previous studies have indicated that silica nanoparticle (SiNPs) may exert a proatherogenic role, which could induce endothelial dysfunction, and monocytes infiltration. However, little is known about SiNPs' effects on macrophage-derived foam cell formation and apoptosis in the pathogenesis of atherosclerosis. In this study, we investigated the effects of SiNPs and oxLDL coexposure on macrophage-derived lipid metabolism, foam cell and apoptosis by using Raw264.7 cells. As a result, SiNPs enhanced cytotoxicity, apoptosis, and lipid accumulation upon oxLDL stimulation. Furthermore, quantitative determination of the expression levels of genes involved in cholesterol influx or efflux showed significantly up-regulated expressions of CD36 and SRA, whereas down-regulated expressions of ATP-binding cassette A1 (ABCA1), ABCG1, and SRB1 in oxLDL-treated macrophages, especially upon the co-exposure with SiNPs. It indicated that SiNPs promoted lipid accumulation in macrophage cells through not only facilitating cholesterol influx but also inhibiting cholesterol efflux. Endoplasmic reticulum (ER) is specialized for the production, modification, even trafficking of lipids. Interestingly, ER response was triggered upon oxLDL treatment, while SiNPs coexposure augmented the ER stress. Taken together, our results revealed that SiNPs promoted oxLDL-induced macrophage foam cell formation and apoptosis, which may be mediated by ER stress signaling. Thus we propose future researches needed for a better understanding of NPs' toxicity and their interactions with various pathophysiological conditions.

Silencing carboxylesterase 1 in human THP-1 macrophages perturbs genes regulated by PPARγ/RXR and RAR/RXR: down-regulation of CYP27A1-LXRα signaling.

Macrophage foam cells store excess cholesterol as cholesteryl esters, which need to be hydrolyzed for cholesterol efflux. We recently reported that silencing expression of carboxylesterase 1 (CES1) in human THP-1 macrophages [CES1KD (THP-1 cells with CES1 expression knocked down) macrophages] reduced cholesterol uptake and decreased expression of CD36 and scavenger receptor-A in cells loaded with acetylated low-density lipoprotein (acLDL). Here, we report that CES1KD macrophages exhibit reduced transcription of cytochrome P45027A1 (CYP27A1) in nonloaded and acLDL-loaded cells. Moreover, levels of CYP27A1 protein and its enzymatic product, 27-hydroxycholesterol, were markedly reduced in CES1KD macrophages. Transcription of LXRα (liver X receptor α) and ABCA1 (ATP-binding cassette transporter A1) was also decreased in acLDL-loaded CES1KD macrophages, suggesting reduced signaling through PPARγ-CYP27A1-LXRα. Consistent with this, treatment of CES1KD macrophages with agonists for PPARγ, RAR, and/or RAR/RXR partially restored transcription of CYP27A1 and LXRα, and repaired cholesterol influx. Conversely, treatment of control macrophages with antagonists for PPARγ and/or RXR decreased transcription of CYP27A1 and LXRα Pharmacologic inhibition of CES1 in both wild-type THP-1 cells and primary human macrophages also decreased CYP27A1 transcription. CES1 silencing did not affect transcript levels of PPARγ and RXR in acLDL-loaded macrophages, whereas it did reduce the catabolism of the endocannabinoid 2-arachidonoylglycerol. Finally, the gene expression profile of CES1KD macrophages was similar to that of PPARγ knockdown cells following acLDL exposures, further suggesting a mechanistic link between CES1 and PPARγ. These results are consistent with a model in which abrogation of CES1 function attenuates the CYP27A1-LXRα-ABCA1 signaling axis by depleting endogenous ligands for the nuclear receptors PPARγ, RAR, and/or RXR that regulate cholesterol homeostasis.

Phenylpropanoid glucosides from Tadehagi triquetrum inhibit oxLDL-evoked foam cell formation through modulating cholesterol homeostasis in RAW264.7 macrophages

The phenylpropanoid glucosides from Tadehagi triquetrum were found to be beneficial to glucose and lipid metabolism in vitro. Herein, we investigated the effects of these compounds on oxidised low-density lipoprotein (oxLDL)-induced foam cell formation in RAW264.7 macrophages, aiming to evaluate their potential utility in prevention of atherosclerosis. Our results showed that three out of seven phenylpropanoid glucosides significantly inhibited oxLDL-evoked foam cell formation. These three compounds remarkably inhibited cholesterol influx and enhanced cholesterol efflux. Treatment with compounds 3, 4 and 7 significantly down-regulated the expression of scavenge receptors 1 (SR-1) and cluster of differentiation 36 (CD36) and increased the expression ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1). Analyses of structure–activity relationships revealed that cinnamyl group was the most pivotal group for their activities. This work provided phenomenon that these phenylpropanoid glucosides are effective regulator of cholesterol influx/efflux and may be useful in leading for development of anti-atherosclerotic agents.

Chronic administration of mitochondrion-targeted peptide SS-31 prevents atherosclerotic development in ApoE knockout mice fed Western diet.

BackgroundOxidative stress and inflammatory factors are deeply involved in progression of atherosclerosis. Mitochondrion-targeted peptide SS-31, selectively targeting to mitochondrial inner membrane reacting with cardiolipin, has been reported to inhibit ROS generation and mitigate inflammation. The present study was designed to investigate whether SS-31 could suppress the development of atherosclerosis in vivo.MethodsMale ApoE-/- mice (8 weeks old) fed with Western diet were treated with normal saline or SS-31 (1 mg/kg/d or 3 mg/kg/d) through subcutaneous injection for 12 weeks. Oil Red O staining was performed to evaluate area and sizes of the plaques. DHE staining and immunohistochemical staining of 8-OHDG was performed to assess the oxidative stress. The aorta ATP contents were assessed by the ATP bioluminescence assay kit. Immunohistochemical staining of CD68 and α-SMA and Masson’s trichrome staining were performed to evaluate the composition of atherosclerotic plaque. Biochemical assays were performed to determine the protein level and activity of superoxide dismutase (SOD). The levels of CD36, LOX-1 and ABCA1 were immunohistochemically and biochemically determined to evaluate the cholesterol transport in aorta and peritoneal macrophages. Inflammatory factors, including ICAM-1, MCP-1, IL-6 and CRP in serum, were detected through ELISA.ResultsSS-31 administration reduced the area and sizes of western diet-induced atherosclerotic plaques and changed the composition of the plaques in ApoE-/- mice. Oxidative stress was suppressed, as evidenced by the reduced DHE stain, down-regulated 8-OHDG expression, and increased SOD activity after chronic SS-31 administration. Moreover, systemic inflammation was ameliorated as seen by decreasing serum ICAM-1, MCP-1, and IL-6 levels. Most importantly, SS-31 administration inhibited cholesterol influx by down-regulating expression of CD36 and LOX-1 to prevent lipid accumulation to further suppress the foam cell formation and atherosclerotic progression.ConclusionAdministration of SS-31 prevents against atherosclerotic formation in ApoE-/- mice suggesting that SS-31 might be considered to be a potential drug to prevent atherosclerotic progression.