RESUMEN
Human biobanks are recognised as vital components of translational research infrastructure. With the growth in personalised and precision medicine, and the associated expansion of biomarkers and novel therapeutics under development, it is critical that researchers can access a strong collection of patient biospecimens, annotated with clinical data. Biobanks globally are undertaking transformation of their operating models in response to changing research needs; transition from a 'classic' model representing a largely retrospective collection of pre-defined specimens to a more targeted, prospective collection model, although there remains a research need for both models to co-exist. Here we introduce the Health Science Alliance (HSA) Biobank, established in 2012 as a classic biobank, now transitioning to a hybrid operational model. Some of the past and current challenges encountered are discussed including clinical annotation, specimen utilisation and biobank sustainability, along with the measures the HSA Biobank is taking to address these challenges. We describe new directions being explored, going beyond traditional specimen collection into areas involving bioimages, microbiota and live cell culture. The HSA Biobank is working in collaboration with clinicians, pathologists and researchers, piloting a sustainable, robust platform with the potential to integrate future needs.
RESUMEN
RATIONALE: High-density lipoprotein (HDL) is a heterogeneous population of particles. Differences in the capacities of HDL subfractions to remove cellular cholesterol may explain variable correlations between HDL-cholesterol and cardiovascular risk and inform future targets for HDL-related therapies. The ATP binding cassette transporter A1 (ABCA1) facilitates cholesterol efflux to lipid-free apolipoprotein A-I, but the majority of apolipoprotein A-I in the circulation is transported in a lipidated state and ABCA1-dependent efflux to individual HDL subfractions has not been systematically studied. OBJECTIVE: Our aims were to determine which HDL particle subfractions are most efficient in mediating cellular cholesterol efflux from foam cell macrophages and to identify the cellular cholesterol transporters involved in this process. METHODS AND RESULTS: We used reconstituted HDL particles of defined size and composition, isolated subfractions of human plasma HDL, cell lines stably expressing ABCA1 or ABCG1, and both mouse and human macrophages in which ABCA1 or ABCG1 expression was deleted. We show that ABCA1 is the major mediator of macrophage cholesterol efflux to HDL, demonstrating most marked efficiency with small, dense HDL subfractions (HDL3b and HDL3c). ABCG1 has a lesser role in cholesterol efflux and a negligible role in efflux to HDL3b and HDL3c subfractions. CONCLUSIONS: Small, dense HDL subfractions are the most efficient mediators of cholesterol efflux, and ABCA1 mediates cholesterol efflux to small dense HDL and to lipid-free apolipoprotein A-I. HDL-directed therapies should target increasing the concentrations or the cholesterol efflux capacity of small, dense HDL species in vivo.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/fisiología , HDL-Colesterol/metabolismo , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Macrófagos/metabolismo , Transportador 1 de Casete de Unión a ATP/antagonistas & inhibidores , Transportador 1 de Casete de Unión a ATP/deficiencia , Transportador 1 de Casete de Unión a ATP/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/deficiencia , Transportadoras de Casetes de Unión a ATP/fisiología , Animales , Apolipoproteína A-I/metabolismo , Transporte Biológico , Células CHO , Línea Celular , Cricetinae , Cricetulus , Células Espumosas/metabolismo , Silenciador del Gen , Humanos , Lipoproteínas/deficiencia , Lipoproteínas/fisiología , Lipoproteínas HDL2/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de la Partícula , Proteínas Recombinantes de Fusión/metabolismo , Enfermedad de Tangier/enzimología , Enfermedad de Tangier/genéticaRESUMEN
Vitamin E membrane transport has been shown to involve the cholesterol transporters SR-BI, ABCA1 and NPC1L1. Our aim was to investigate the possible participation of another cholesterol transporter in cellular vitamin E efflux: ABCG1. In Abcgl-deficient mice, vitamin E concentration was reduced in plasma lipoproteins whereas most tissues displayed a higher vitamin E content compared to wild-type mice. α- and γ-tocopherol efflux was increased in CHO cells overexpressing human ABCG1 compared to control cells. Conversely, α- and γ- tocopherol efflux was decreased in ABCG1-knockdown human cells (Hep3B hepatocytes and THP-1 macro- phages). Interestingly, α- and γ-tocopherol significantly downregulated ABCG1 and ABCA1 expression levels in Hep3B and THP-1, an effect confirmed in vivo in rats given vitamin E for 5 days. This was likely due to reduced LXR activation by oxysterols, as Hep3B cells and rat liver treated with vitamin E displayed a significantly reduced content in oxysterols compared to their respective controls. Overall, the present study reveals for the first time that ABCG1 is involved in cellular vitamin E efflux.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Lipoproteínas/metabolismo , Vitamina E/metabolismo , Transportador 1 de Casete de Unión a ATP/genética , Transportador 1 de Casete de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/deficiencia , Transportadoras de Casetes de Unión a ATP/genética , Animales , Transporte Biológico , Células CHO , Cromanos/metabolismo , Cricetinae , Cricetulus , Regulación hacia Abajo , Humanos , Lipoproteínas/deficiencia , Hígado/metabolismo , Receptores X del Hígado , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Especificidad de Órganos , Receptores Nucleares Huérfanos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , TransfecciónRESUMEN
Cholesterol-loaded foam cell macrophages are prominent in atherosclerotic lesions and play complex roles in both inflammatory signaling and lipid metabolism, which are underpinned by large scale reprogramming of gene expression. We performed a microarray study of primary human macrophages that showed that transcription of the sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) gene is up-regulated after cholesterol loading. SMPDL3A protein expression in and secretion from primary macrophages are stimulated by cholesterol loading, liver X receptor ligands, and cyclic AMP, and N-glycosylated SMPDL3A protein is detectable in circulating blood. We demonstrate for the first time that SMPDL3A is a functional phosphodiesterase with an acidic pH optimum. We provide evidence that SMPDL3A is not an acid sphingomyelinase but unexpectedly is active against nucleotide diphosphate and triphosphate substrates at acidic and neutral pH. SMPDL3A is a major source of nucleotide phosphodiesterase activity secreted by liver X receptor-stimulated human macrophages. Extracellular nucleotides such as ATP may activate pro-inflammatory responses in immune cells. Increased expression and secretion of SMPDL3A by cholesterol-loaded macrophage foam cells in lesions may decrease local concentrations of pro-inflammatory nucleotides and potentially represent a novel anti-inflammatory axis linking lipid metabolism with purinergic signaling in atherosclerosis.
Asunto(s)
Colesterol/metabolismo , Macrófagos/metabolismo , Nucleótidos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Animales , Western Blotting , Células CHO , Línea Celular Tumoral , Células Cultivadas , Colesterol/farmacología , Cricetinae , Cricetulus , AMP Cíclico/farmacología , Células Espumosas/efectos de los fármacos , Células Espumosas/metabolismo , Aparato de Golgi/metabolismo , Células HEK293 , Humanos , Receptores X del Hígado , Macrófagos/efectos de los fármacos , Microscopía Confocal , Nucleótidos/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores Nucleares Huérfanos/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esfingomielina Fosfodiesterasa/sangre , Esfingomielina Fosfodiesterasa/genética , Transcriptoma/efectos de los fármacos , Transcriptoma/genéticaRESUMEN
Type 2 diabetes and dyslipidemia are risk factors for cardiovascular disease. However, mechanisms by which hypertriglyceridemia influences atherogenesis remain unclear. We examined effects of dyslipidemic diabetic serum on macrophage lipid accumulation as a model of foam cell formation. Normal human macrophages were cultured in media supplemented with 10% serum from non-diabetic normolipidemic or non-diabetic hypercholesterolemic adults versus adults with Type 2 diabetes; diabetes and hypertriglyceridemia; or diabetes and hypercholesterolemia. Exposure to diabetic sera resulted in increased macrophage fatty acids (2-3 fold higher, both saturated and unsaturated). Macrophage expression of CD36, scavenger receptor A (SR-A) and stearoyl-CoA desaturase (SCD) was increased, most prominently in macrophages exposed to hypertriglyceridemic diabetic serum (twofold increase in CD36 and fourfold increase in SCD, p < 0.05). In these conditions, RNA inhibition of CD36 reduced macrophage free cholesterol (163.9 ± 10.5 vs. 221.9 ± 26.2 mmol free cholesterol/g protein, p = 0.04). RNA inhibition of SCD decreased macrophage fatty acid content, increased ABCA1 level and enhanced cholesterol efflux (18.0 ± 3.9 vs. 8.0 ± 0.8% at 48 h, p = 0.03). Diabetic dyslipidemia may contribute to accelerated atherosclerosis via alterations in macrophage lipid metabolism favoring foam cell formation. Increased expression of CD36 and SR-A would facilitate macrophage lipid uptake, while increased expression of SCD could block compensatory upregulation of ABCA1 and cholesterol efflux. Further studies are needed to clarify whether modulation of macrophage lipid metabolism might reduce progression of diabetic atherosclerosis.
Asunto(s)
Diabetes Mellitus Tipo 2/sangre , Dislipidemias/sangre , Metabolismo de los Lípidos , Macrófagos/enzimología , Estearoil-CoA Desaturasa/metabolismo , Adulto , Antígenos CD36/genética , Antígenos CD36/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 2/complicaciones , Dislipidemias/complicaciones , Ácidos Grasos/metabolismo , Células Espumosas/metabolismo , Humanos , ARN Interferente Pequeño/genética , Receptores Depuradores de Clase A/metabolismo , Estearoil-CoA Desaturasa/genéticaRESUMEN
Macrophages in arterial walls accumulate lipids leading to the development of atherosclerotic plaques. However, mechanisms underlying macrophage lipid accumulation and foam cell formation are often studied without accounting for risk factors such as dyslipidemia. We investigated the effect of varying concentrations of triglyceride (TG) within physiological range on macrophage fatty acid (FA) accumulation and expression of cholesterol efflux proteins. Human monocytes were cultured in media supplemented with 10% sera containing low (0.7 mmol/L) to high (1.4 mmol/L) TG. The resulting macrophages were harvested after 10 days for analysis of FA content and composition and expression of genes involved in lipid metabolism. Exposure to higher TG and lower HDL concentrations in media increased macrophage lipid content. Macrophages exposed to higher TG had increased total FA content compared with controls (876 µg/mg protein vs. 652 µg/mg protein) and greater proportions of C16:0, C18:1 and C18:2. Macrophage expression of both ABCA1 and ABCG1 cholesterol efflux proteins were reduced when higher TG concentrations were present in the media. Expression of scavenger receptor CD36, involved in lipoprotein uptake, was also downregulated in macrophages exposed to higher TG. Culturing macrophages in conditions of higher versus lower TG influenced macrophage FA content and composition, and levels of regulatory proteins. Replicating in vitro levels of dyslipidemia encountered in vivo may provide an informative model for investigation of atherogenesis.
Asunto(s)
Dislipidemias/sangre , Ácidos Grasos/metabolismo , Macrófagos/química , Macrófagos/metabolismo , Suero/química , Transportador 1 de Casete de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Antígenos CD36/genética , Antígenos CD36/metabolismo , Células Cultivadas , Medios de Cultivo/química , Ácidos Grasos/química , Humanos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Estearoil-CoA Desaturasa/genética , Estearoil-CoA Desaturasa/metabolismo , Triglicéridos/farmacologíaRESUMEN
OBJECTIVE: Maintenance of cholesterol homeostasis in human macrophages is essential to prevent foam cell formation. We evaluated the relative contribution of the ABCA1 and ABCG1 transporters to cholesterol efflux from human macrophages, and of the capacity of LXR agonists to reduce foam cell formation by stimulating export of cellular cholesterol. METHODS AND RESULTS: ABCG1 mRNA levels were strongly increased in acLDL-loaded THP-1 macrophages and in HMDM on stimulation with LXR agonists. However, silencing of ABCG1 expression using ABCG1-specific siRNA indicated that ABCG1 was not essential for cholesterol efflux to HDL in cholesterol-loaded human macrophages stimulated with LXR agonists. Indeed, ABCA1 was solely responsible for the stimulation of cholesterol efflux to HDL on LXR activation, as this effect was abolished in HMDM from Tangier patients. Furthermore, depletion of cellular ATP indicated that the LXR-induced export of cholesterol was an ATP-dependent transport mechanism in human macrophages. Finally, use of an anti-Cla-1 blocking antibody identified the Cla-1 receptor as a key component in cholesterol efflux to HDL from cholesterol-loaded human macrophages. CONCLUSIONS: Our data indicate that stimulation of cholesterol efflux to HDL by LXR agonists in human foam cells involves an ATP-dependent transport mechanism mediated by ABCA1 that it appears to be independent of ABCG1 expression.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Aterosclerosis/metabolismo , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Macrófagos/metabolismo , Receptores Nucleares Huérfanos/antagonistas & inhibidores , Transportador 1 de Casete de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Células Espumosas/citología , Células Espumosas/metabolismo , Homeostasis/fisiología , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/fisiología , Lipoproteínas HDL/farmacología , Receptores X del Hígado , Macrófagos/citología , Macrófagos/efectos de los fármacos , ProbabilidadRESUMEN
OBJECTIVE: ATP-binding cassette transporter G1 (Abcg1) and apolipoprotein E (Apoe) play a role in macrophage cholesterol efflux and consequently the development of atherosclerosis. A possible interaction between Abcg1 and Apoe in cholesterol efflux was postulated, but the potential combined action of these proteins on atherosclerotic lesion formation is unclear. METHODS: LDL receptor knockout (KO) mice were transplanted with bone marrow from Abcg1/Apoe double KO (dKO) mice, their respective single knockouts, and wild-type (WT) controls and challenged with a high-fat/high-cholesterol diet for 6 weeks to induce atherosclerosis. RESULTS: No differences were found in serum lipid levels. The mean atherosclerotic lesion area in dKO transplanted animals (187+/-18x10(3)microm(2)) was 1.4-fold (p<0.01) increased compared to single knockouts (Abcg1 KO: 138+/-5x10(3)microm(2); Apoe KO: 131+/-7x10(3)microm(2)) and 1.9-fold (p<0.001) as compared to WT controls (97+/-15x10(3)microm(2)). In vitro cholesterol efflux experiments established that combined deletion of Abcg1 and Apoe leads to a larger attenuation of macrophage cholesterol efflux to HDL as compared to single knockouts. CONCLUSIONS: Single deletion of macrophage Abcg1 or Apoe does lead to a moderate non-significant increase in atherosclerotic lesion development as tested by ANOVA, while combined deletion of Abcg1 and Apoe induces a more dramatic and significant increase in atherosclerosis. Our results indicate an additive, independent effect for both macrophage Abcg1 and Apoe in the prevention of atherosclerosis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Apolipoproteínas E/metabolismo , Aterosclerosis/patología , Lipoproteínas/metabolismo , Macrófagos/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/genética , Animales , Aorta/patología , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/metabolismo , Trasplante de Médula Ósea , Colesterol/metabolismo , Eliminación de Gen , Genotipo , Lipoproteínas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos BiológicosRESUMEN
The T lymphocyte plasma membrane condenses at the site of activation but the functional significance of this receptor-mediated membrane reorganization is not yet known. Here we demonstrate that membrane condensation at the T cell activation sites can be inhibited by incorporation of the oxysterol 7-ketocholesterol (7KC), which is known to prevent the formation of raft-like liquid-ordered domains in model membranes. We enriched T cells with 7KC, or cholesterol as control, to assess the importance of membrane condensation for T cell activation. Upon 7KC treatment, T cell antigen receptor (TCR) triggered calcium fluxes and early tyrosine phosphorylation events appear unaltered. However, signaling complexes form less efficiently on the cell surface, fewer phosphorylated signaling proteins are retained in the plasma membrane and actin restructuring at activation sites is impaired in 7KC-enriched cells resulting in compromised downstream activation responses. Our data emphasizes lipids as an important medium for the organization at T cell activation sites and strongly indicates that membrane condensation is an important element of the T cell activation process.
Asunto(s)
Activación de Linfocitos , Linfocitos T/inmunología , Membrana Celular/inmunología , Humanos , Células Jurkat , Cetocolesteroles/administración & dosificaciónRESUMEN
Certain oxysterols, when added to cultured cells, are potent regulators of cholesterol homeostasis, decreasing cholesterol synthesis and uptake and increasing cholesterol efflux. However, very little is known about whether or not endogenous oxysterol(s) plays a significant role in cholesterol homeostasis. 24(S),25-Epoxycholesterol (24,25EC) is unique among oxysterols in that it is produced in a shunt of the mevalonate pathway which also produces cholesterol. We investigated the role of endogenously produced 24,25EC using a novel strategy of overexpressing the enzyme 2,3-oxidosqualene cyclase in Chinese hamster ovary cells to selectively inhibit the synthesis of this oxysterol. First, loss of 24,25EC decreased expression of the LXR target gene, ABCA1, substantiating its role as an endogenous ligand for LXR. Second, loss of 24,25EC increased acute cholesterol synthesis, which was rationalized by a concomitant increase in HMG-CoA reductase gene expression at the level of SREBP-2 processing. Therefore, in the absence of 24,25EC, fine-tuning of the acute regulation of cholesterol homeostasis is lost, supporting the hypothesis that 24,25EC functions to protect the cell against the accumulation of newly synthesized cholesterol.
Asunto(s)
Colesterol/análogos & derivados , Colesterol/metabolismo , Hidroximetilglutaril-CoA Reductasas/metabolismo , Transferasas Intramoleculares/genética , Transferasas Intramoleculares/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Células CHO , Línea Celular , Colesterol/fisiología , Cricetinae , Cricetulus , Genes myc , Homeostasis , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
The serine palmitoyl transferase inhibitor myriocin potently suppresses the development of atherosclerosis in apolipoprotein E (apoE) gene knockout (apoE(-/-)) mice fed a high-fat diet. This is associated with reduced plasma sphingomyelin (SM) and glycosphingolipid levels. Furthermore, oral administration of myriocin decreases plasma cholesterol and triglyceride (TG) levels. Here, we aimed to determine whether myriocin could inhibit the progression (or stimulate the regression) of established atherosclerotic lesions and to examine potential changes in hepatic and plasma lipid concentrations. Adult apoE(-/-) mice were fed a high-fat diet for 30 days, and lesion formation was histologically confirmed. Replicate groups of mice were then transferred to either regular chow or chow containing myriocin (0.3 mg/kg/day) and maintained for a further 60 days. Myriocin significantly inhibited the progression of established atherosclerosis when combined lesion areas (aortic sinus, arch, and celiac branch point) were measured. Although the inhibition of lesion progression was observed mainly in the distal regions of the aorta, regression of lesion size was not detected. The inhibition of lesion progression was associated with reductions in hepatic and plasma SM, cholesterol, and TG levels and increased hepatic and plasma apoA-I levels, indicating that the modulation of pathways associated with several classes of atherogenic lipids may be involved.
Asunto(s)
Apolipoproteínas E/genética , Aterosclerosis/tratamiento farmacológico , Ácidos Grasos Monoinsaturados/administración & dosificación , Administración Oral , Animales , Anticolesterolemiantes/administración & dosificación , Anticolesterolemiantes/uso terapéutico , Apolipoproteínas E/deficiencia , Aterosclerosis/sangre , Aterosclerosis/patología , Progresión de la Enfermedad , Ácidos Grasos Monoinsaturados/uso terapéutico , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones NoqueadosRESUMEN
The ATP-binding cassette transporters, ABCA1 and ABCG1, are LXR-target genes that participate in the removal of cholesterol from lipid-laden macrophages, a crucial anti-atherogenic mechanism. Statins are currently the most efficacious therapy for the treatment of hypercholesterolemia and cardiovascular disease. We and others have shown that statins decrease ABCA1 and ABCG1 expression as well as cholesterol efflux from human macrophages. However, other studies have reported that statins produce no change, or even a modest increase in these variables. In an attempt to reconcile these conflicting reports, we investigated how the effect of statins on transcription of ABCA1 and ABCG1 is modulated by cellular cholesterol status and the extent of macrophage differentiation. We showed that supplementing human macrophages with cholesterol reversed the statin-mediated down-regulation of ABC transporter expression whereas depletion of cellular cholesterol tended to accentuate the statin effect. Down-regulation of ABC transporter expression was more pronounced with increased macrophage differentiation status and already evident at statin concentrations equivalent to those present in plasma. Addition of LXR agonists, which are currently on trial as anti-atherogenic agents, reversed the effects on ABC transporter expression while PPAR alpha and PPAR gamma agonists did not. The significance of these results in light of current and future combination therapies is discussed.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/efectos de los fármacos , Diferenciación Celular/fisiología , Colesterol/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Macrófagos/efectos de los fármacos , Transportador 1 de Casete de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/metabolismo , Benzoatos/farmacología , Bencilaminas/farmacología , Células Cultivadas , Proteínas de Unión al ADN/agonistas , Proteínas de Unión al ADN/efectos de los fármacos , Regulación hacia Abajo , Perfilación de la Expresión Génica , Humanos , Hidrocarburos Fluorados/farmacocinética , Receptores X del Hígado , Macrófagos/metabolismo , Receptores Nucleares Huérfanos , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Sulfonamidas/farmacocinéticaRESUMEN
Cholesterol is an essential component of the CNS and its metabolism in the brain has been implicated in various neurodegenerative diseases. The oxysterol produced from cholesterol, 24(S)-hydroxycholesterol, is known to be an important regulator of brain cholesterol homeostasis. In this study, we focussed on another oxysterol, 24(S),25-epoxycholesterol (24,25EC), which has not been studied before in a neurological context. 24,25EC is unique in that it is synthesized in a shunt in the mevalonate pathway, parallel to cholesterol and utilizing the same enzymes. Considering that all the cholesterol present in the brain is derived from de novo synthesis, we investigated whether or not primary human neurons and astrocytes can produce 24,25EC. We found that astrocytes produced more 24,25EC than neurons under basal conditions, but both cell types had the capacity to synthesize this oxysterol when the enzyme 2,3-oxidosqualene cyclase was partially inhibited. Furthermore, both added 24,25EC and stimulated cellular production of 24,25EC (by partial inhibition of 2,3-oxidosqualene cyclase) modulated expression of key cholesterol-homeostatic genes regulated by the liver X receptor and the sterol regulatory element-binding protein-2. Moreover, we found that 24,25EC synthesized in astrocytes can be taken up by neurons and exert downstream effects on gene regulation. In summary, we have identified 24,25EC as a novel neurosterol which plays a likely role in brain cholesterol homeostasis.
Asunto(s)
Astrocitos/metabolismo , Colesterol/análogos & derivados , Colesterol/metabolismo , Regulación de la Expresión Génica/fisiología , Apolipoproteína A-I/metabolismo , Astrocitos/química , Astrocitos/efectos de los fármacos , Encéfalo/citología , Células Cultivadas , Colesterol/farmacología , Medios de Cultivo Condicionados/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Neuronas/metabolismo , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismoRESUMEN
AIM: The effects of 24(S),25-epoxycholesterol (24,25EC) on aspects of cholesterol homeostasis is well-documented. When added to cells, 24,25EC decreases cholesterol synthesis and up-regulates cholesterol efflux genes, including ABCA1. Synthesis of 24,25EC occurs in a shunt of the mevalonate pathway which also produces cholesterol. Therefore, 24,25EC synthesis should be subject to the same negative feedback regulation as cholesterol synthesis. To date, no role has been ascribed to 24,25EC in light of the fact that increased accumulation of cholesterol should decrease formation of this oxysterol through feedback inhibition. This leads to the intriguing paradox: why inhibit production of an apparently important regulator of cholesterol homeostasis when it is needed most? METHODS: We used a combination of pharmacological and genetic approaches in Chinese Hamster Ovary cell-lines to investigate this paradox. Endogenous synthesis of 24,25EC was manipulated using partial inhibition of the enzyme, Oxidosqualene Cyclase. Changes in cholesterol and 24,25EC synthesis were determined using metabolic labelling with [1-14C]-acetate, thin-layer chromatography and phosphorimaging. Transcriptional effects mediated via SREBP and LXR were analysed by luciferase reporter assays. RESULTS: We showed that cholesterol addition to cells lead to a rapid and preferential inhibition of 24,25EC synthesis. Addition of 24,25EC resulted in parallel inhibition of 24,25EC and cholesterol synthesis. Furthermore, we used a variety of approaches to examine the relationship between cholesterol and 24,25EC synthesis, including cell-lines with different rates of cholesterol synthesis, varying cholesterol synthetic rates by pre-treatment with a statin, or lipoprotein cholesterol loading of macrophages. In all cases, we showed that 24,25EC synthesis faithfully tracked cholesterol synthesis. Moreover, changes in 24,25EC synthesis exerted downstream effects, reducing SREBP transcriptional activity whilst increasing ABCA1 and LXR transcriptional activity. CONCLUSION: Our results show that 24,25EC synthesis parallels cholesterol synthesis, consistent with this oxysterol functioning as a safety valve to protect against the accumulation of newly-synthesised cholesterol (as opposed to exogenously-derived cholesterol). Considering that 24,25EC is capable of being produced in all cholesterogenic cells, we propose that production of 24,25EC may represent a ubiquitous defence mechanism.
Asunto(s)
Colesterol/análogos & derivados , Colesterol/biosíntesis , Animales , Células CHO , Colesterol/farmacología , Cricetinae , Cricetulus , Proteínas de Unión al ADN/genética , Receptores X del Hígado , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Receptores Nucleares Huérfanos , Receptores Citoplasmáticos y Nucleares/genética , Proteínas de Unión a los Elementos Reguladores de Esteroles/genética , Transcripción Genética/genéticaRESUMEN
Glycosphingolipids (GSL) have been implicated as potential atherogenic lipids. Inhibition of hepatic serine palmitoyl transferase (SPT) reduces plasma sphingomyelin (SM) levels in the absence of changes in cholesterol or triglyceride (TG) concentration and this leads to a reduction of atherosclerosis in apolipoprotein-E gene knockout (apoE(-/-)) mice. The possibility that the reduced atherosclerosis resulting from SPT inhibition is associated with decreases in plasma GSL concentration has not been examined and was the primary aim of this investigation. We show that intraperitoneal delivery of the SPT inhibitor myriocin for 9 weeks inhibits atherosclerosis in apoE(-/-) mice fed a high fat diet. Lesion inhibition was most pronounced at the aortic arch and distal sites of the thoracic and abdominal aorta. There was also a trend towards a reduction in lesion area at the aortic root. Myriocin treatment resulted in significant reductions in both plasma SM and GSL concentration of 42% and 25%, as assessed by enzymatic and HPLC methods, respectively. Moreover, SM and GSL concentrations were significantly correlated, indicating that SPT inhibition suppresses the synthesis of both these sphingolipids concomitantly. The inhibition of atherosclerosis induced by myriocin was not associated with changes in plasma cholesterol or TG concentrations or lipoprotein profiles as determined by FPLC. These data indicate that therapeutic reduction of plasma SM and/or GSL concentrations may offer a novel treatment for atherosclerosis.
Asunto(s)
Aterosclerosis/prevención & control , Ácidos Grasos Monoinsaturados/uso terapéutico , Glicoesfingolípidos/sangre , Plasma/efectos de los fármacos , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/fisiología , Aterosclerosis/sangre , Ácidos Grasos Monoinsaturados/farmacología , Masculino , Ratones , Ratones Noqueados , Plasma/química , Serina C-Palmitoiltransferasa/fisiologíaRESUMEN
Cholesterol accumulation and removal are regulated by two different transcription factors. SREBP-2 (sterol-regulatory-element-binding protein-2) is best known to up-regulate genes involved in cholesterol biosynthesis and uptake, whereas LXR (liver X receptor) is best known for up-regulating cholesterol efflux genes. An important cholesterol efflux gene that is regulated by LXR is the ATP-binding cassette transporter, ABCA1 (ATP-binding cassette transporter-A1). We have previously shown that statin treatment down-regulated ABCA1 expression in human macrophages, probably by inhibiting synthesis of the LXR ligand 24(S),25-epoxycholesterol. However, it was subsequently reported that ABCA1 expression is down-regulated by SREBP-2 through binding of SREBP-2 to an E-box element in ABCA1's proximal promoter. As statin treatment induces SREBP-2 activation, this may provide an alternative explanation for the statin-mediated down-regulation of ABCA1. In the present study, we employed a set of CHO (Chinese-hamster ovary) mutant cell lines to investigate the role of SREBP-2 in the regulation of ABCA1. We observed increased ABCA1 mRNA levels in SREBP-2-overexpressing cells and decreased levels in cells lacking a functional SREBP-2 pathway, which were restored when the SREBP-2 pathway was reinstated. Moreover, ABCA1 gene expression was positively associated with synthesis of 24(S),25-epoxycholesterol in these cell lines. In studies using a human ABCA1 promoter reporter assay, mutation of the E-box motif had a similar response as the wild-type construct to either statin treatment or addition of 24(S),25-epoxycholesterol. By contrast, these responses were completely ablated when the DR4 element to which LXR binds was mutated. These results support the idea that 24(S),25-epoxycholesterol and statin treatment influence ABCA1 transcription via supply of an LXR ligand and not through an SREBP-2/E-box-related mechanism. In addition, our results indicate a critical role of SREBP-2 as a positive regulator of ABCA1 gene expression by enabling the generation of oxysterol ligands for LXR.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Colesterol/metabolismo , Proteínas de Unión al ADN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Transcripción Genética , Transportador 1 de Casete de Unión a ATP , Animales , Línea Celular , Colesterol/análogos & derivados , Colesterol/biosíntesis , Cricetinae , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/fisiología , Humanos , Ligandos , Receptores X del Hígado , Receptores Nucleares Huérfanos , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Esteroides/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Regulación hacia ArribaRESUMEN
Cellular glycosphingolipid (GSL) storage is known to promote cholesterol accumulation. Although physical interactions between GSLs and cholesterol are thought to cause intracellular cholesterol "trapping," it is not known whether cholesterol homeostatic mechanisms are also impaired under these conditions. ApoA-I-mediated cholesterol efflux via ABCA1 (ATP-binding cassette transporter A1) is a key regulator of cellular cholesterol balance. Here, we show that apoA-I-mediated cholesterol efflux was inhibited (by up to 53% over 8 h) when fibroblasts were treated with lactosylceramide or the glucocerebrosidase inhibitor conduritol B epoxide. Furthermore, apoA-I-mediated cholesterol efflux from fibroblasts derived from patients with genetic GSL storage diseases (Fabry disease, Sandhoff disease, and GM1 gangliosidosis) was impaired compared with control cells. Conversely, apoA-I-mediated cholesterol efflux from fibroblasts and cholesterol-loaded macrophage foam cells was dose-dependently stimulated (by up to 6-fold over 8 h) by the GSL synthesis inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). Unexpectedly, a structurally unrelated GSL synthesis inhibitor, N-butyldeoxynojirimycin, was unable to stimulate apoA-I-mediated cholesterol efflux despite achieving similar GSL depletion. PDMP was found to up-regulate ABCA1 mRNA and protein expression, thereby identifying a contributing mechanism for the observed acceleration of cholesterol efflux to apoA-I. This study reveals a novel defect in cellular cholesterol homeostasis induced by GSL storage and identifies PDMP as a new agent for enhancing cholesterol efflux via the ABCA1/apoA-I pathway.
Asunto(s)
1-Desoxinojirimicina/análogos & derivados , Transportadoras de Casetes de Unión a ATP/química , Apolipoproteína A-I/química , Colesterol/metabolismo , Glicoesfingolípidos/metabolismo , 1-Desoxinojirimicina/farmacología , Transportador 1 de Casete de Unión a ATP , Antígenos CD/metabolismo , Biotinilación , Western Blotting , Línea Celular , Membrana Celular/metabolismo , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Fibroblastos/metabolismo , Humanos , Lactosilceramidos/metabolismo , Lipoproteínas LDL/química , Monocitos/metabolismo , Morfolinas/química , Morfolinas/farmacología , Fosfolípidos/química , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Regulación hacia ArribaRESUMEN
CYP27A1 (sterol 27-hydroxylase) catalyses an important sterol elimination pathway in the human macrophage, and consequently may protect against atherosclerosis. We studied the expression and regulation of CYP27A1 in a human macrophage-like cell-line, THP-1, and primary HMDMs (human monocyte-derived macrophages). In both macrophage cell types, we found that CYP27A1 expression is independent of cellular cholesterol levels and of LXR (liver X receptor)-dependent control of transcription. However, the RXR (retinoid X receptor) ligand, 9-cis-retinoic acid, upregulates CYP27A1 expression. Of the RXR heterodimeric partners tested, PPAR (peroxisome-proliferator-activated receptor) gamma ligands significantly increased CYP27A1 mRNA levels. Its reversal by a PPARgamma antagonist demonstrated the specificity of this effect. Interestingly, HMDMs express markedly higher levels of CYP27A1 than THP-1 macrophages, and this difference was reflected in both protein levels and enzyme activities between the two cell types. In conclusion, stimulation of CYP27A1 by PPARgamma may represent a key previously unrecognized mechanism by which PPARgamma protects against atherosclerosis.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Macrófagos/enzimología , PPAR gamma/metabolismo , Receptores X Retinoide/metabolismo , Esteroide Hidroxilasas/metabolismo , Línea Celular Tumoral , Células Cultivadas , Colestanotriol 26-Monooxigenasa , Colesterol/metabolismo , Humanos , Ligandos , PPAR gamma/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores X Retinoide/química , Esteroide Hidroxilasas/genéticaRESUMEN
OBJECTIVE: Cholesterol efflux from macrophages in the artery wall, a key cardioprotective mechanism, is largely coordinated by the nuclear oxysterol-activated liver X receptor, LXRalpha. We investigated the effect of statins on LXR target gene expression and cholesterol efflux from human macrophages. METHODS AND RESULTS: In human macrophages (THP-1 cell line and primary cells), the archetypal statin, compactin, greatly reduced mRNA levels of 2 LXR target genes, ABCA1 and ABCG1 mRNA, as well as decreased cholesterol efflux. Commonly prescribed statins also downregulated LXR target gene expression in THP-1 cells. We provide several lines of evidence indicating that statins decrease expression of LXR target genes by inhibiting the synthesis of an oxysterol ligand for LXR, 24(S),25-epoxycholesterol. When THP-1 cells were cholesterol-loaded via incubation with acetylated low-density lipoprotein, synthesis of 24(S),25-epoxycholesterol was greatly reduced and the downregulatory effect of compactin on ABCA1 mRNA levels and cholesterol efflux was lost. CONCLUSIONS: Our results suggest that statins may downregulate cholesterol efflux from nonloaded human macrophages by inhibiting synthesis of an oxysterol ligand for LXR. Further work is needed to determine how relevant our observations are to arterial foam cells in vivo.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Colesterol/análogos & derivados , Colesterol/biosíntesis , Colesterol/metabolismo , Proteínas de Unión al ADN/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/metabolismo , Lovastatina/análogos & derivados , Macrófagos/química , Macrófagos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Transportador 1 de Casete de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Diferenciación Celular/fisiología , Línea Celular , Colesterol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Ligandos , Receptores X del Hígado , Lovastatina/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Monocitos/fisiología , Receptores Nucleares Huérfanos , Acetato de Tetradecanoilforbol/inmunologíaRESUMEN
Apolipoprotein A-I (apoA-I)-mediated cholesterol efflux involves the binding of apoA-I to the plasma membrane via its C terminus and requires cellular ATP-binding cassette transporter (ABCA1) activity. ApoA-I also stimulates secretion of apolipoprotein E (apoE) from macrophage foam cells, although the mechanism of this process is not understood. In this study, we demonstrate that apoA-I stimulates secretion of apoE independently of both ABCA1-mediated cholesterol efflux and of lipid binding by its C terminus. Pulse-chase experiments using (35)S-labeled cellular apoE demonstrate that macrophage apoE exists in both relatively mobile (E(m)) and stable (E(s)) pools, that apoA-I diverts apoE from degradation to secretion, and that only a small proportion of apoA-I-mobilized apoE is derived from the cell surface. The structural requirements for induction of apoE secretion and cholesterol efflux are clearly dissociated, as C-terminal deletions in recombinant apoA-I reduce cholesterol efflux but increase apoE secretion, and deletion of central helices 5 and 6 decreases apoE secretion without perturbing cholesterol efflux. Moreover, a range of 11- and 22-mer alpha-helical peptides representing amphipathic alpha-helical segments of apoA-I stimulate apoE secretion whereas only the C-terminal alpha-helix (domains 220-241) stimulates cholesterol efflux. Other alpha-helix-containing apolipoproteins (apoA-II, apoA-IV, apoE2, apoE3, apoE4) also stimulate apoE secretion, implying a positive feedback autocrine loop for apoE secretion, although apoE4 is less effective. Finally, apoA-I stimulates apoE secretion normally from macrophages of two unrelated subjects with genetically confirmed Tangier Disease (mutations C733R and c.5220-5222delTCT; and mutations A1046D and c.4629-4630insA), despite severely inhibited cholesterol efflux. We conclude that apoA-I stimulates secretion of apoE independently of cholesterol efflux, and that this represents a novel, ABCA-1-independent, positive feedback pathway for stimulation of potentially anti-atherogenic apoE secretion by alpha-helix-containing molecules including apoA-I and apoE.