RESUMEN
The nuclear receptors Liver X receptors, LXRα and LXRß, regulate cholesterol and triglyceride metabolism. We and others have previously reported that synthetic LXR agonists reduced atherosclerosis in models of mouse with no detectable plasma cholesteryl ester transfer protein (CETP) activity, which plays an important role in reverse cholesterol transport. In the present study, we investigated the effect of LXR activation in rabbits to elucidate the influence of CETP activity. First, we cloned rabbit LXRs cDNA. The data indicated that rabbit LXRα was mostly highly expressed in the liver, whereas LXRß expression was ubiquitous. Next, we investigated the effect of LXR agonist on lipid levels. Treatment with LXR agonist T0901317 increased plasma CETP activity and consequently elevated LDL, but no change in HDL. High cholesterol (HC) diet-feeding, which is thought to provide oxysterols as the natural agonists, could also increase expression of CETP and other LXR target genes. Finally, we tested T0901317 in the atherosclerosis intervention study. Chronic administration of T0901317 significantly reduced atherosclerosis in HC diet-fed rabbits despite less favorable lipid profiles, i.e. increases of plasma triglycerides and no change of HDL. T0901317 induced ATP-binding cassette transporters ABCA1 and ABCG1 and suppressed inflammatory genes expression in the aorta, suggesting that direct actions of LXR agonist on vascular gene expression are likely to contribute to the antiatherogenic effect. The present work strongly supports the idea that LXR agonists could be beneficial as therapeutic agents for treatment of atherosclerosis.
Asunto(s)
Aterosclerosis/sangre , Proteínas de Transferencia de Ésteres de Colesterol/sangre , Expresión Génica , Hidrocarburos Fluorados/farmacología , Receptores Nucleares Huérfanos/agonistas , Transducción de Señal , Sulfonamidas/farmacología , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Animales , Anticolesterolemiantes/farmacología , Aorta/efectos de los fármacos , Aorta/metabolismo , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/etiología , Aterosclerosis/genética , Colesterol/sangre , Colesterol/farmacología , Proteínas de Transferencia de Ésteres de Colesterol/genética , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Expresión Génica/efectos de los fármacos , Lipoproteínas HDL/sangre , Lipoproteínas LDL/sangre , Hígado/efectos de los fármacos , Hígado/metabolismo , Receptores X del Hígado , Datos de Secuencia Molecular , Receptores Nucleares Huérfanos/metabolismo , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Triglicéridos/sangreRESUMEN
Liver X receptors (LXRs), LXRalpha and LXRbeta, are members of the nuclear receptor superfamily and regulate the expression of genes involved in the regulation of cholesterol and fatty acid metabolism. Human plasma, unlike mouse plasma, contains cholesteryl ester transfer protein (CETP), which plays an important role in reverse cholesterol transport (RCT). LXRs induce CETP transcription via a direct repeat 4 element in the CETP promoter. However, the specific roles of the individual LXR subtypes in CETP expression and their consequences on plasma lipoprotein metabolism are still unclear. Here we showed that synthetic LXR agonist enhanced plasma CETP activity and resulted in non-high density lipoprotein (non-HDL) increase and HDL decrease in cynomolgus monkeys and human CETP transgenic mice. To address the relative importance of the two LXR subtypes, we investigated the effect of the suppression of both LXR subtypes on CETP expression in HepG2 cells. CETP expression induced by the LXR agonist was significantly reduced by LXRalpha knock-down, but not by LXRbeta. Consistent with these data, CETP promoter activity was enhanced by LXRalpha activation, whereas LXRbeta activation had only a minor effect. Furthermore, we investigated the effect of genetic deficiency of both LXR subtypes in human CETP transgenic mice. LXRalpha deficiency abolished the augmentation of plasma CETP activity and hepatic CETP expression induced by the synthetic LXR agonist, consequently increasing HDL and decreasing non-HDL, whereas LXRbeta deficiency did not affect CETP activation. These findings indicate that LXRalpha has an essential role in the regulation of CETP expression and maintaining RCT.
Asunto(s)
Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Lípidos/sangre , Receptores Nucleares Huérfanos/metabolismo , Animales , Colesterol/sangre , Proteínas de Transferencia de Ésteres de Colesterol/genética , Relación Dosis-Respuesta a Droga , Ayuno/sangre , Regulación de la Expresión Génica , Células Hep G2 , Humanos , Hidrocarburos Fluorados/farmacología , Lipoproteínas HDL/sangre , Receptores X del Hígado , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores Nucleares Huérfanos/agonistas , Receptores Nucleares Huérfanos/deficiencia , Receptores Nucleares Huérfanos/genética , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Mensajero/metabolismo , Sulfonamidas/farmacología , TransfecciónRESUMEN
The objective of the present study was to determine whether a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, pactimibe sulfate (CS-505), could reduce atherosclerotic lesions beyond and independent of the reduction achieved by cholesterol lowering alone from two different types of lesions. (1) Early lesion model. Twelve-week-old apolipoprotein E (apoE)(-/-) mice were treated with 0.03 or 0.1% (w/w) CS-505, 0.1 or 0.3% avasimibe (CI-1011), or 3% cholestyramine for 12 weeks. Each treatment significantly reduced plasma cholesterol by a similar degree (43-48%). The antiatherosclerotic activity of 0.1% CS-505, however, was more efficacious than the effects of the other treatments (90% versus 40-50%). (2) Advanced lesion model. Twenty-four-week-old apoE(-/-) mice were treated with 0.03 or 0.1% CS-505 or 0.1% CI-1011 for 12 weeks. CS-505 at 0.1% revealed enhanced lesion reduction compared with 0.1% CI-1011 (77% versus 54%), whereas the plasma cholesterol-lowering effect of 0.1% CS-505 was almost the same as that of 0.1% CI-1011. Furthermore, immunohistochemical analysis demonstrated that CS-505 significantly reduced the number of macrophages and expression of matrix metalloproteinase (MMP)-2, MMP-9, and MMP-13. These data indicate that CS-505 can reduce and stabilize atherosclerotic lesions. This antiatherosclerotic activity is exerted via both cholesterol lowering and direct ACAT inhibition in plaque macrophages.
Asunto(s)
Anticolesterolemiantes/uso terapéutico , Apolipoproteínas E/deficiencia , Aterosclerosis/tratamiento farmacológico , Ácidos Indolacéticos/uso terapéutico , Esterol O-Aciltransferasa/antagonistas & inhibidores , Animales , Anticolesterolemiantes/farmacología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Línea Celular , Humanos , Ácidos Indolacéticos/farmacología , Lípidos/sangre , Ratones , Ratones Noqueados , Monocitos/efectos de los fármacosRESUMEN
Exposure of blood to tissue factor (TF) rapidly initiates the coagulation serine protease cascades. TF is expressed by macrophages and other types of cell within atherosclerotic lesions and plays an important role in thrombus formation after plaque rupture. Macrophage TF expression is induced by pro-inflammatory stimuli including lipopolysaccharide (LPS), interleukin-1beta and tumor necrosis factor-alpha. Here we demonstrate that activation of liver X receptors (LXRs) LXRalpha and LXRbeta suppresses TF expression. Treatment of mouse peritoneal macrophages with synthetic LXR agonist T0901317 or GW3965 reduced TF expression induced by pro-inflammatory stimuli. LXR agonists also suppressed TF expression and its activity in human monocytes. Human and mouse TF promoters contain binding sites for the transcription factors AP-1, NFkappaB, Egr-1 and Sp1, but no LXR-binding sites could be found. Cotransfection assays with LXR and TF promoter constructs in RAW 264.7 cells revealed that LXR agonists suppressed LPS-induced TF promoter activity. Analysis of TF promoter also showed that inhibition of TF promoter activity by LXR was at least in part through inhibition of the NFkappaB signaling pathway. In addition, in vivo, LXR agonists reduced TF expression within aortic lesions in an atherosclerosis mouse model as well as in kidney and lung in mice stimulated with LPS. These findings indicate that activation of LXR results in reduction of TF expression, which may influence atherothrombosis in patients with vascular disease.
Asunto(s)
Macrófagos Peritoneales/fisiología , Receptores Citoplasmáticos y Nucleares/metabolismo , Tromboplastina/genética , Animales , Células Cultivadas , Cartilla de ADN , Proteínas de Unión al ADN , Humanos , Receptores X del Hígado , Ratones , Ratones Endogámicos C57BL , Receptores Nucleares Huérfanos , Mapeo Restrictivo , Tromboplastina/antagonistas & inhibidores , TransfecciónRESUMEN
In a recent paper, we reported that pravastatin sodium (pravastatin), an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme. A reductase, decreases the concentrations of low density lipoprotein (LDL) cholesterol through an LDL receptor pathway in Japanese White (JW) rabbits, whereas this agent lowers high density lipoprotein (HDL) cholesterol in a manner correlated with a reduction of very low density lipoprotein (VLDL) cholesterol secretion from the liver. In the present study, we administered pravastatin to JW rabbits at 30 mg/kg for 14 days and examined further the mechanisms for the reduction of HDL cholesterol. A striking finding was that the 4-day administration of pravastatin at 30 mg/kg selectively decreased the concentration of HDL cholesterol. Since 4-day administration of pravastatin to JW rabbits did not change the concentrations of hepatic LDL receptor proteins, these receptors were not likely to be involved in the reduction of HDL cholesterol. Another important finding was that pravastatin suppressed VLDL cholesteryl ester (CE) secretion from the liver, but not that of other VLDL lipids and VLDL proteins, indicating that the CE-poor VLDL particles were secreted by the consecutive administration of pravastatin. There were, however, no differences in the levels of VLDL cholesterol between the control and pravastatin-treated groups over the experimental period of 14 days. These observations raised the possibility that the reduction of HDL cholesterol in the pravastatin-treated group was due to the transfer of CE molecules from HDL particles to these CE-poor VLDL particles. Molecular species analysis supported this notion that the VLDL-CE in the pravastatin-treated group was rich in cholesteryl linoleate, indicating that the CE in this group mainly originated from HDL, whereas the VLDL-CE in the control group was rich in cholesteryl oleate, indicating that the CE in this group originated from the liver. The present study suggests that pravastatin lowers HDL cholesterol by transferring CE from these lipoproteins to VLDL in JW rabbits.
Asunto(s)
Ésteres del Colesterol/metabolismo , HDL-Colesterol/metabolismo , VLDL-Colesterol/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Hígado/efectos de los fármacos , Pravastatina/farmacología , Animales , Hígado/metabolismo , Masculino , ConejosRESUMEN
Liver X receptors (LXR alpha and LXR beta) are nuclear receptors, which are important regulators of cholesterol and lipid metabolism. LXRs control genes involved in cholesterol efflux in macrophages, bile acid synthesis in liver and intestinal cholesterol absorption. LXRs also regulate genes participating in lipogenesis. To determine whether the activation of LXR promotes or inhibits development of atherosclerosis, T-0901317, a synthetic LXR ligand, was administered to low density lipoprotein receptor (LDLR)(-/-) mice. T-0901317 significantly reduced the atherosclerotic lesions in LDLR(-/-) mice without affecting plasma total cholesterol levels. This anti-atherogenic effect correlated with the plasma concentration of T-0901317, but not with high density lipoprotein cholesterol, which was increased by T-0901317. In addition, we observed that T-0901317 increased expression of ATP binding cassette A1 in the lesions in LDLR(-/-) mice as well as in mouse peritoneal macrophages. T-0901317 also significantly induced cholesterol efflux activity in peritoneal macrophages. These results suggest that LXR ligands may be useful therapeutic agents for the treatment of atherosclerosis.
Asunto(s)
Anticolesterolemiantes/uso terapéutico , Arteriosclerosis/prevención & control , Receptores Citoplasmáticos y Nucleares/agonistas , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/genética , Animales , Arteriosclerosis/metabolismo , Arteriosclerosis/patología , Transporte Biológico , Colesterol/metabolismo , Proteínas de Unión al ADN , Hidrocarburos Fluorados , Ligandos , Lípidos/sangre , Lipoproteínas/sangre , Receptores X del Hígado , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Receptores Nucleares Huérfanos , ARN Mensajero/biosíntesis , Receptores de LDL/genética , SulfonamidasRESUMEN
In experimental animals and humans, the concentration of serum mevalonate (MVA), a direct product of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is considered to reflect the activity of whole-body sterol synthesis. The relationship between the concentration of serum MVA and the activity of sterol synthesis in tissues, however, has not been fully clarified. In the present study, we examined MVA metabolism by using pravastatin, a liver-selective inhibitor of HMG-CoA reductase, and common marmosets, a good model animal for studying lipid metabolism. In the time course study, the maximal reduction in the concentration of serum MVA was observed 2 h after a single oral administration of 30 mg/kg pravastatin to common marmosets. We, therefore, examined the relationship between the concentrations of serum and hepatic MVA, and sterol synthesis in some tissues at this time point. Sterol synthesis was determined ex vivo in tissue slices by measuring the incorporation of [14C]acetate into digitonin-precipitable [14C]sterols. Pravastatin at 0.03-30 mg/kg reduced dose-dependently the activity of hepatic sterol synthesis, whereas no significant reduction of sterol synthesis was observed in other tissues such as intestine, kidney, testis and spleen, even with the highest dose (30 mg/kg). The liver-specific inhibition of sterol synthesis caused parallel reductions in the concentrations of both serum and liver MVA. In addition, there were good correlations between the concentration of either serum or hepatic MVA and the activity of hepatic sterol synthesis. These data indicate that the major origin of serum MVA is the liver, and that the concentration of serum MVA reflects the concentration of hepatic MVA and the activity of hepatic sterol synthesis 2 h after a single oral administration of pravastatin in common marmosets.