Low-dose expression of a human apolipoprotein E transgene in macrophages restores cholesterol efflux capacity of apolipoprotein E-deficient mouse plasma

Apolipoprotein E- (apoE) deficient (E−/−) mice develop severe hyperlipidemia and diffuse atherosclerosis. Low-dose expression of a human apoE3 transgene in macrophages of apoE-deficient mice (E−/−hTgE+/0), which results in about 5% of wild-type apoE plasma levels, did not correct hyperlipidemia but significantly reduced the extent of atherosclerotic lesions. To investigate the contribution of apoE to reverse cholesterol transport, we compared plasmas of wild-type (E+/+), E−/−, and E−/−hTgE+/0 mice for the appearance of apoE-containing lipoproteins by electrophoresis and their capacity to take up and esterify 3H-labeled cholesterol from radiolabeled fibroblasts or J774 macrophages. Wild-type plasma displayed lipoproteins containing apoE that were the size of high density lipoprotein and that had either electrophoretic α or γ mobilities. Similar particles were also present in E−/−hTgE+/0 plasma. Depending on incubation time, E−/− plasma released 48–74% less 3H-labeled cholesterol from fibroblasts than E+/+ plasma, whereas cholesterol efflux into E−/−hTgE+/0 plasma was only 11–25% lower than into E+/+ plasma. E−/−hTgE+/0 plasma also released 10% more 3H-labeled cholesterol from radiolabeled J774 macrophages than E−/− plasma. E+/+ and E−/−hTgE+/0 plasma each esterified significantly more cell-derived 3H-labeled cholesterol than E−/− plasma. Moreover, E−/− plasma accumulated much smaller proportions of fibroblast-derived 3H-labeled cholesterol in fractions with electrophoretic γ and α mobility than E+/+ and E−/−hTgE+/0 plasma. Thus, low-dose expression of apoE in macrophages nearly restored the cholesterol efflux capacity of apoE-deficient plasma through the formation of apoE-containing particles, which efficiently take up cell-derived cholesterol, and through the increase of cholesterol esterification activity. Thus, macrophage-derived apoE may protect against atherosclerosis by increasing cholesterol efflux from arterial wall cells.

Cholesterol acceptor capacity is preserved by different mechanisms in preterm and term fetuses.

Fetal serum cholesterol and lipoprotein concentrations differ between preterm and term born neonates. An imbalance of the flow of cholesterol from the sites of synthesis or efflux from cells of peripheral organs to the liver, the reverse cholesterol transport (RCT), is linked to atherosclerosis and cardiovascular disease (CVD). Preterm delivery is a risk factor for the development of CVD. Thus, we hypothesized that RCT is affected by a diminished cholesterol acceptor capacity in preterm as compared to term fetuses. Cholesterol efflux assays were performed in RAW264.7, HepG2, and HUVEC cell lines. In the presence and absence of ABC transporter overexpression by TO-901317, umbilical cord sera of preterm and term born neonates (n = 28 in both groups) were added. Lipid components including high density lipoprotein (HDL), low density lipoprotein (LDL), apolipoprotein A1, and apolipoprotein E were measured and related to fractional cholesterol efflux values. We found overall, fractional cholesterol efflux to remain constant between the study groups, and over gestational ages at delivery, respectively. However, correlation analysis revealed cholesterol efflux values to be predominantly related to HDL concentration at term, while in preterm neonates, cholesterol efflux was mainly associated with LDL In conclusion cholesterol acceptor capacity during fetal development is kept in a steady state with different mechanisms and lipid fractions involved at distinct stages during the second half of fetal development. However, RCT mechanisms in preterm neonates seem not to be involved in the development of CVD later in life suggesting rather changes in the lipoprotein pattern causative.

Cellular cholesterol efflux mediated by HDL isolated from subjects with low HDL levels and coronary artery disease

OBJECTIVE: The aim of this study was to verify whether HDL particles isolated from patients with coronary artery disease (CAD) and low HDL-C had diminished ability to promote cholesterol efflux from cultured cells compared with HDL isolated from subjects without CAD and with normal HDL-C. METHODS: Smooth muscle cells isolated from human aortas cultured and radiolabeled with ³H-cholesterol were loaded with cholesterol and incubated with increasing concentrations of HDL isolated from 13 CAD patients with low HDL-C (CAD group) or from 5 controls without CAD (C group). Efflux of cellular cholesterol was measured by cellular depletion of radiolabeled cholesterol and by the appearance of ³H-cholesterol into experimental medium expressed as a percentage of total labeled cholesterol. RESULTS: Cholesterol efflux increased with the amount of HDL present in the medium, and no difference was found between groups at various HDL protein concentrations: efflux was 28 ± 6.3% (C) and 25.5 ± 8.9% (CAD) with 25 mg/mL; 34 ± 4.3% (C) and 31.9 ± 6.6% (CD) with 50 mg/mL and 39.5 ± 3.5% (C) and 37.1 ± 4.4% (CAD) with 100 mg/mL, HDL. CONCLUSION: Because the HDL fraction of CAD patients with low HDL-C have normal ability to extract cholesterol from cells of the vessel wall, it is suggested that low HDL-C atherogenicity should be ascribed to diminished concentrations of HDL particles rather than to the qualitative properties of the HDL fraction.

A growth hormone-releasing peptide that binds scavenger receptor CD36 and ghrelin receptor up-regulates sterol transporters and cholesterol efflux in macrophages through a peroxisome proliferator-activated receptor gamma-dependent pathway.

Macrophages play a central role in the pathogenesis of atherosclerosis by accumulating cholesterol through increased uptake of oxidized low-density lipoproteins by scavenger receptor CD36, leading to foam cell formation. Here we demonstrate the ability of hexarelin, a GH-releasing peptide, to enhance the expression of ATP-binding cassette A1 and G1 transporters and cholesterol efflux in macrophages. These effects were associated with a transcriptional activation of nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma in response to binding of hexarelin to CD36 and GH secretagogue-receptor 1a, the receptor for ghrelin. The hormone binding domain was not required to mediate PPARgamma activation by hexarelin, and phosphorylation of PPARgamma was increased in THP-1 macrophages treated with hexarelin, suggesting that the response to hexarelin may involve PPARgamma activation function-1 activity. However, the activation of PPARgamma by hexarelin did not lead to an increase in CD36 expression, as opposed to liver X receptor (LXR)alpha, suggesting a differential regulation of PPARgamma-targeted genes in response to hexarelin. Chromatin immunoprecipitation assays showed that, in contrast to a PPARgamma agonist, the occupancy of the CD36 promoter by PPARgamma was not increased in THP-1 macrophages treated with hexarelin, whereas the LXRalpha promoter was strongly occupied by PPARgamma in the same conditions. Treatment of apolipoprotein E-null mice maintained on a lipid-rich diet with hexarelin resulted in a significant reduction in atherosclerotic lesions, concomitant with an enhanced expression of PPARgamma and LXRalpha target genes in peritoneal macrophages. The response was strongly impaired in PPARgamma(+/-) macrophages, indicating that PPARgamma was required to mediate the effect of hexarelin. These findings provide a novel mechanism by which the beneficial regulation of PPARgamma and cholesterol metabolism in macrophages could be regulated by CD36 and ghrelin receptor downstream effects.

Triglyceride-rich lipoproteins inhibit cholesterol efflux to apolipoprotein (apo) A1 from human macrophage foam cells

High circulating levels of triglyceride-rich lipoproteins (TGRL) represent an independent risk factor for coronary artery disease. Here, we show that TGRL inhibit the efflux of cholesterol from 'foam cell' macrophages to lipid-poor apolipoprotein (apo) A1, and may thereby inhibit arterial reverse cholesterol transport and promote the formation of atherosclerotic lesions. Human (THP-1) monocyte-derived macrophages were pre-incubated (48h) with acetylated low-density lipoprotein (AcLDL) to provide a foam cell model of cholesterol efflux to apoA1. Pre-incubation of macrophage 'foam cells' with TGRL (0-200 mug/ml, 0-24 h) inhibited the efflux of exogenously radiolabelled ([H-3]), endogenously synthesised ([C-14]) and cellular cholesterol mass to lipid-poor apoA1, but not control medium, during a (subsequent) efflux period. This inhibition is dependent upon the length of prior exposure to, and concentration of, TGRL employed, but is independent of changes in intracellular triglyceride accumulation or turnover of the cholesteryl ester pool. Despite the negative impact of TGRL on cholesterol efflux, major proteins involved in this process-namely apoE, ABCA1, SR-B1 and caveolin-1-were unaffected by TGRL pre-incubation, suggesting that exposure to these lipoproteins inhibits an alternate, and possibly novel, anti-atherogenic pathway. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

Expression of caveolin-1 enhances cholesterol efflux in hepatic cells

HepG2 cells were stably transfected with human caveolin-1 (HepG2/cav cells). Transfection resulted in expression of caveolin-1 mRNA, a high abundance of caveolin-1 protein, and the formation of caveolae on the plasma membrane. Cholesterol efflux from HepG2/cav cells was 280 and 45% higher than that from parent HepG2 cells when human plasma and human apoA-I, respectively, were used as acceptors. The difference in efflux was eliminated by treatment of cells with progesterone. There was no difference in cholesterol efflux to cyclodextrin. Cholesterol efflux from plasma membrane vesicles was similar for the two cell types. Transfection led to a 40% increase in the amount of plasma membrane cholesterol in cholesterol-rich domains ( caveolae and/or rafts) and a 67% increase in the rate of cholesterol trafficking from intracellular compartments to these domains. Cholesterol biosynthesis in HepG2/cav cells was increased by 2-fold, and cholesterol esterification was reduced by 50% compared with parent HepG2 cells. The proliferation rate of transfected cells was significantly lower than that of non-transfected cells. Transfection did not affect expression of ABCA1 or the abundance of ABCA1 protein, but decreased secretion of apoA-I. We conclude that overexpression of caveolin-1 in hepatic cells stimulates cholesterol efflux by enhancing transfer of cholesterol to cholesterol-rich domains in the plasma membrane.

Les effets du dalcetrapib, un inhibiteur de la protéine de transfert des esters de cholestérol (CETP), sur la structure et la fonction des lipoprotéines de haute densité (HDL) dans l’étude dal-PLAQUE2

Introduction : Le dalcetrapib, inhibiteur de la glycoprotéine hydrophobe de transfert des esters de cholestérol (CETP), a été étudié dans le cadre de l’essai clinique de phase II dal-PLAQUE2 (DP2). L’objectif principal est d’étudier l’effet du dalcetrapib après 1 an de traitement sur la structure et la fonction des HDL dans une sous-population de la cohorte DP2. Méthode : Les sujets de la cohorte DP2 ayant une série de mesures de cIMT et des échantillons de plasma et sérum au baseline et à 1 an de traitement furent sélectionnés (379 sujets: 193 du groupe placebo (PCB) et 186 du groupe dalcetrapib (DAL)). Des données biochimiques prédéterminées, le profil des concentrations et tailles des sous-classes de HDL et LDL en résonance magnétique nucléaire (RMN) et 2 mesures de capacité d’efflux de cholestérol (CEC) du sérum ont été explorées. Les données statistiques furent obtenues en comparant les changements à un an à partir du « baseline » avec un ANOVA ou ANCOVA. La procédure normalisée de fonctionnement d’essai d’efflux de cholestérol permet de calculer l’efflux fractionnel (en %) de 3H-cholestérol des lignées cellulaires BHK-ABCA1 (fibroblastes) et J774 (macrophages, voie ABCA1) et HepG2 (hépatocytes, voie SR-BI), vers les échantillons sériques de la cohorte DP2. Résultats : Pour la biochimie plasmatique, un effet combiné des changements d’activité de CETP dans les 2 groupes a causé une réduction de 30% dans le groupe DAL. Après 1 an de traitement dans le groupe DAL, la valeur de HDL-C a augmenté de 35,5% (p < 0,001) et l’apoA-I a augmenté de 14,0% (p < 0,001). Au profil RMN, dans le groupe DAL après 1 an de traitement, il y a augmentation de la taille des HDL-P (5,2%; p < 0,001), des grosses particules HDL (68,7%; p < 0,001) et des grosses particules LDL (37,5%; p < 0,01). Les petites particules HDL sont diminuées (-9,1%; p < 0,001). Il n’y a aucune différence significative de mesure de cIMT entre les deux groupes après 1 an de traitement. Pour la CEC, il y a augmentation significative par la voie du SR-BI et une augmentation via la voie ABCA1 dans le groupe DAL après 1 an de traitement. Conclusion : Après un an de traitement au dalcetrapib, on note une hausse de HDL-C, des résultats plutôt neutres au niveau du profil lipidique par RMN et une CEC augmentée mais trop faible pour affecter la valeur de cIMT chez les échantillons testés.

Avaliação da aterosclerose subclínica em portadores de HDL-colesterol marcadamente elevado

O HDL-c é um fator de risco cardiovascular negativo e sua concentração plasmática apresenta relação inversa com a incidência de eventos cardiovasculares. Entretanto, as evidências relativas ao grupo de indivíduos com níveis de HDL-c acima do percentil 95 da população geral ainda são escassas e o impacto da hiperalfalipoproteinemia (HALP) sobre o risco cardiovascular continua representando motivo de controvérsia na literatura médica. Alguns estudos em populações específicas associam a HALP a aumento do risco cardiovascular. Ao mesmo tempo, outros estudos identificaram populações de indivíduos hipoalfalipoproteinêmicos com marcada longevidade. Assim, demonstrou-se aparente dissociação entre níveis de HDL-c e risco cardiovascular em determinadas populações, reconduzível a aspectos disfuncionais da HDL. O objetivo do presente estudo foi verificar o papel da HALP na determinação do risco cardiovascular; comparar a prevalência de doença cardiovascular subclínica, avaliada por meio da quantificação ultrassonográfica da Espessura Íntimo-Medial Carotídea (EIMC), entre portadores de HDL-c >= 90mg/dL (grupo HALP) e portadores de concentrações de HDL-c atualmente consideradas normais (entre 40 e 50mg/dL para os homens e entre 50 e 60mg/dL para as mulheres); e avaliar características e função da HDL em portadores de HALP por meio do estudo de sua composição, de sua capacidade de efluxo de colesterol, e de sua atividade anti-inflamatória e antioxidante, correlacionando estas características com a presença de doença cardiovascular subclínica avaliada por meio da determinação da EIMC, da Velocidade de Onda de Pulso (VOP) e da presença de Calcificação Arterial Coronariana (CAC) avaliada pela TCMD. Para responder estas perguntas, o presente estudo foi articulado em dois braços: Braço 1: Análise da coorte do estudo ELSA com o objetivo de determinar a prevalência de HALP em uma população geral; definir o perfil demográfico, antropométrico e metabólico dos portadores de HALP; e comparar a prevalência de doença vascular subclínica deste grupo com controles da mesma coorte com níveis normais de HDL-colesterol. Braço 2: Recrutamento de 80 voluntários hígidos e portadores de HALP para avaliação da correlação entre presença de doença vascular subclínica, e aspectos estruturais e funcionais da HDL. Em seus dois braços, o estudo levou a quatro conclusões principais: 1) Níveis marcadamente elevados de HDL-c estão associados a menor espessura íntimo-medial carotídea quando comparados a níveis de HDL-c considerados normais pelas diretrizes vigentes. Embora portadores do fenótipo HALP apresentem, como grupo, um perfil metabólico mais favorável que o encontrado em indivíduos com HDL-c normal, a associação entre EIMC e HALP foi independente dos fatores de risco tradicionais, indicando que a menor prevalência destes últimos em portadores de HDL-c marcadamente elevado justifica apenas parcialmente a menor prevalência de doença vascular subclínica neste grupo; 2) Embora a HALP se apresente como um fenótipo ateroprotetor, há indivíduos com níveis marcadamente elevados de HDL-c que evoluem com doença cardiovascular, clínica ou subclínica. Neste contexto, nossos resultados indicam correlação entre os três métodos avaliados para estudar doença vascular subclínica em portadores de HALP: EIMC, VOP e CAC; 3) Os fatores de risco tradicionais continuam exercendo seu peso na determinação do risco cardiovascular em portadores de HALP. Idade, tabagismo, hipertensão arterial, hipertrigliceridemia e altos níveis de LDL-c apresentaram associação estatisticamente significativa com a presença de doença vascular subclínica no grupo estudado; 4) A avaliação da composição e da função da HDL em portadores de HALP pode permitir identificar indivíduos especificamente mais suscetíveis à aterosclerose. Nossos resultados indicam que, em particular, a atividade anti-inflamatória da HDL, avaliada pela capacidade de inibição da produção de IL-6; o efluxo de colesterol e a capacidade de transferência de triglicérides apresentaram associação independente com menor espessura íntimo-medial carotídea em portadores de HALP, enquanto níveis mais altos de Apo A-IV se associaram a maior grau de doença cardiovascular subclínica

Les effets du dalcetrapib, un inhibiteur de la protéine de transfert des esters de cholestérol (CETP), sur la structure et la fonction des lipoprotéines de haute densité (HDL) dans l’étude dal-PLAQUE2

Introduction : Le dalcetrapib, inhibiteur de la glycoprotéine hydrophobe de transfert des esters de cholestérol (CETP), a été étudié dans le cadre de l’essai clinique de phase II dal-PLAQUE2 (DP2). L’objectif principal est d’étudier l’effet du dalcetrapib après 1 an de traitement sur la structure et la fonction des HDL dans une sous-population de la cohorte DP2. Méthode : Les sujets de la cohorte DP2 ayant une série de mesures de cIMT et des échantillons de plasma et sérum au baseline et à 1 an de traitement furent sélectionnés (379 sujets: 193 du groupe placebo (PCB) et 186 du groupe dalcetrapib (DAL)). Des données biochimiques prédéterminées, le profil des concentrations et tailles des sous-classes de HDL et LDL en résonance magnétique nucléaire (RMN) et 2 mesures de capacité d’efflux de cholestérol (CEC) du sérum ont été explorées. Les données statistiques furent obtenues en comparant les changements à un an à partir du « baseline » avec un ANOVA ou ANCOVA. La procédure normalisée de fonctionnement d’essai d’efflux de cholestérol permet de calculer l’efflux fractionnel (en %) de 3H-cholestérol des lignées cellulaires BHK-ABCA1 (fibroblastes) et J774 (macrophages, voie ABCA1) et HepG2 (hépatocytes, voie SR-BI), vers les échantillons sériques de la cohorte DP2. Résultats : Pour la biochimie plasmatique, un effet combiné des changements d’activité de CETP dans les 2 groupes a causé une réduction de 30% dans le groupe DAL. Après 1 an de traitement dans le groupe DAL, la valeur de HDL-C a augmenté de 35,5% (p < 0,001) et l’apoA-I a augmenté de 14,0% (p < 0,001). Au profil RMN, dans le groupe DAL après 1 an de traitement, il y a augmentation de la taille des HDL-P (5,2%; p < 0,001), des grosses particules HDL (68,7%; p < 0,001) et des grosses particules LDL (37,5%; p < 0,01). Les petites particules HDL sont diminuées (-9,1%; p < 0,001). Il n’y a aucune différence significative de mesure de cIMT entre les deux groupes après 1 an de traitement. Pour la CEC, il y a augmentation significative par la voie du SR-BI et une augmentation via la voie ABCA1 dans le groupe DAL après 1 an de traitement. Conclusion : Après un an de traitement au dalcetrapib, on note une hausse de HDL-C, des résultats plutôt neutres au niveau du profil lipidique par RMN et une CEC augmentée mais trop faible pour affecter la valeur de cIMT chez les échantillons testés.

Characteristics and functional relevance of apolipoprotein-A1 and cholesterol binding in mammary gland tissues and epithelial cells

Cholesterol in milk is derived from the circulating blood through a complex transport process involving the mammary alveolar epithelium. Details of the mechanisms involved in this transfer are unclear. Apolipoprotein-AI (apoA-I) is an acceptor of cellular cholesterol effluxed by the ATP-binding cassette (ABC) transporter A1 (ABCA1). We aimed to 1) determine the binding characteristics of (125)I-apoA-I and (3)H-cholesterol to enriched plasma membrane vesicles (EPM) isolated from lactating and non-lactating bovine mammary glands (MG), 2) optimize the components of an in vitro model describing cellular (3)H-cholesterol efflux in primary bovine mammary epithelial cells (MeBo), and 3) assess the vectorial cholesterol transport in MeBo using Transwell(®) plates. The amounts of isolated EPM and the maximal binding capacity of (125)I-apoA-I to EPM differed depending on the MG's physiological state, while the kinetics of (3)H-cholesterol and (125)I-apoA-I binding were similar. (3)H-cholesterol incorporated maximally to EPM after 25±9 min. The time to achieve the half-maximum binding of (125)I-apoA-I at equilibrium was 3.3±0.6 min. The dissociation constant (KD) of (125)I-apoA-I ranged between 40-74 nmol/L. Cholesterol loading to EPM increased both cholesterol content and (125)I-apoA-I binding. The ABCA1 inhibitor Probucol displaced (125)I-apoA-I binding to EPM and reduced (3)H-cholesterol efflux in MeBo. Time-dependent (3)H-cholesterol uptake and efflux showed inverse patterns. The defined binding characteristics of cholesterol and apoA-I served to establish an efficient and significantly shorter cholesterol efflux protocol that had been used in MeBo. The application of this protocol in Transwell(®) plates with the upper chamber mimicking the apical (milk-facing) and the bottom chamber corresponding to the basolateral (blood-facing) side of cells showed that the degree of (3)H-cholesterol efflux in MeBo differed significantly between the apical and basolateral aspects. Our findings support the importance of the apoA-I/ABCA1 pathway in MG cholesterol transport and suggest its role in influencing milk composition and directing cholesterol back into the bloodstream.

Metabolic syndrome features small, apolipoprotein A-I-poor, triglyceride-rich HDL3 particles with defective anti-apoptotic activity

The metabolic syndrome (MetS) phenotype is typically characterized by visceral obesity, insulin resistance, atherogenic dyslipidemia involving hypertriglyceridemia and subnormal levels of high density lipoprotein-cholesterol (HDL-C), oxidative stress and elevated cardiovascular risk. The potent antioxidative activity of small HDL3 is defective in MetS [Hansel B, et al. J Clin Endocrinol Metab 2004;89:4963-71]. We evaluated the functional capacity of small HDL3 particles from MetS subjects to protect endothelial cells from apoptosis induced by mildly oxidized low-density lipoprotein (oxLDL). MetS subjects presented an insulin-resistant obese phenotype, with hypertriglyceridemia, elevated apolipoprotein B and insulin levels, but subnormal HDL-C concentrations and chronic low grade inflammation (threefold elevation of C-reactive protein). When human microvascular endothelial cells (HMEC-1) were incubated with oxLDL (200 jig apolipoprotein B/ml) in the presence or absence of control HDL subfiractions (25 mu g protein/ml), small, dense HDL3b and 3c significantly inhibited cellular annexin V binding and intracellular generation of reactive oxygen species. The potent anti-apoptotic activity of small HDL3c particles was reduced (-35%; p < 0.05) in MetS subjects (n = 16) relative to normolipidemic controls (n = 7). The attenuated anti-apoptotic activity of HDL3c correlated with abdominal obesity, atherogenic dyslipidemia and systemic oxidative stress (p < 0.05), and was intimately associated with altered physicochemical properties of apolipoprotein A-I (apoA-I-poor HDL3c, involving core cholesteryl ester depletion and triglyceride enrichment. We conclude that in MetS, apoA-I-poor, small, dense HDL3c exert defective protection of endothelial cells from oxLDL-induced apoptosis, potentially reflecting functional anomalies intimately associated with abnormal neutral lipid core content. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

A caveolin dominant negative mutant associates with lipid bodies and induces intracellular cholesterol imbalance

Recent studies have indicated a role for caveolin in regulating cholesterol-dependent signaling events. In the present study we have analyzed the role of caveolins in intracellular cholesterol cycling using a dominant negative caveolin mutant. The mutant caveolin protein, cav-3(DGV) specifically associates with the membrane surrounding large lipid droplets. These structures contain neutral lipids, and are accessed by caveolin 1-3 upon overexpression. Fluorescence, electron, and video microscopy observations are consistent with formation of the membrane-enclosed lipid rich structures by maturation of subdomains of the ER. The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis. The amphiphile U18666A acts synergistically with cav(DGV) to increase intracellular accumulation of FC. Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets. We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts.

Les transporteurs sélectifs de cholestérol SR-BI, SR-BII, CD36 et ABCA1 contribuent au maintien de l’homéostasie du cholestérol intratesticulaire

Le testicule assure la production des spermatozoïdes et la sécrétion de la testostérone. Chaque fonction est assumée par un compartiment cellulaire distinct: l’épithélium séminifère et le tissu interstitiel. Le cholestérol, présent dans les deux compartiments, est un composé indispensable aux membranes cellulaires et un précurseur essentiel de la testostérone. Dans le compartiment interstitiel, environ 40 % du cholestérol utilisé pour la production hormonale est importé du sang à partir des lipoprotéines HDL et/ou LDL. Dans l’épithélium séminifère, la cellule de Sertoli assure le contrôle et le maintien de la spermatogenèse. Elle a la capacité de synthétiser du cholestérol à partir de l’acétate in vitro, néanmoins, il n’y a pas d’évidence qu’elle le fait in vivo. De plus il existe, au niveau des tubules séminifères, une barrière hémato-testiculaire qui empêche le libre passage de plusieurs composés sanguins, y compris le cholestérol. Nous avons testé l’hypothèse qu’il existe des moyens d’importation du cholestérol sanguin, mais aussi l’exportation du cholestérol intra-tissulaire, qui contourneraient cette barrière et qui contribueraient au maintien du taux intratubulaire du cholestérol compatible avec le bon déroulement de la spermatogenèse. Nous avons comparé les taux de variation de l’expression de l’ARNm et de la protéine des transporteurs sélectifs de cholestérol SR-BI, SR-BII, CD36 et ABCA1 aux taux de variation du cholestérol libre et estérifié au cours de la spermatogenèse chez les souris normales durant le développement postnatal. Afin de mieux apprécier le niveau d’implication de chacun de ces récepteurs, nous avons examiné comment la suppression du gène d’une enzyme comme la lypase hormono-sensible (HSL) ou de celui d’un transporteur de cholestérol comme SR-BI, CD36 ou NPC1 était compensée et comment cette suppression affectait le taux de cholestérol libre et estérifié dans chacun des deux compartiments cellulaires du testicule. Nous avons dans un premier temps mis au point une nouvelle technique d’isolation des testicules en fraction enrichie en tissu interstitiel (ITf) et en tubules séminifères (STf) qui a l’avantage de mieux préserver l’intégrité des formes phosphorylées et glycosylées des protéines comparée aux techniques préexistantes. Les résultats de nos analyses ont montré que l’expression de SR-BI et CD36 étaient maximales dans les ITf au moment où les souris ont complété leur maturité sexuelle et où le niveau de synthèse de la testostérone était maximal. Dans les tubules séminifères, l’expression maximale de SR-BI et le taux le plus élevé de cholestérol estérifié étaient mesurés de façon concomitante à 35 jours après la naissance, au moment où la première vague de l’activité spermatogénétique était complétée. L’expression de l’ABCA1 était maximale au moment où le taux de cholestérol était élevé et minimale au moment où le taux de cholestérol était le plus bas, alors que le niveau d’expression de CD36 était maximal chez l’adulte au moment où le taux de spermiation était le plus élevé. L’expression de SR-BII variait peu dans les deux compartiments cellulaires durant le développement. La suppression génétique de la HSL et de NPC1, qui cause une infertilité chez les souris mâles, était accompagnée d’une accumulation de cholestérol libre et estérifié dans les tubules séminifères. Par contre, la suppression génétique de SR-BI et CD36, qui ne causent pas d’infertilité chez les souris mâles était sans impact significatif sur le taux de cholestérol intratubulaire. Nous avons montré que l’invalidation génétique d’un transporteur sélectif ou d’une enzyme du métabolisme du cholestérol était accompagnée d’un ensemble de mécanismes de compensation visant à maintenir le taux de cholestérol libre aux niveaux semblables à ceux mesurés dans les fractions tissulaires de souris normales. Ensemble, nos résultats ont montré que l’expression des transporteurs sélectifs de cholestérol SR-BI, SR-BII, CD36 et ABCA1 variait en fonction de la spermatogenèse et du taux intratesticulaire du cholestérol suggérant leur contribution au maintien de l’homéostasie du cholestérol intratesticulaire.

Advanced glycated albumin impairs HDL anti-inflammatory activity and primes macrophages for inflammatory response that reduces reverse cholesterol transport

Objective: We investigated the effect of advanced glycated albumin (AGE-albumin) on macrophage sensitivity to inflammation elicited by S100B calgranulin and lipopolysaccharide (LPS) and the mechanism by which HDL modulates this response. We also measured the influence of the culture medium, isolated from macrophages treated with AGE-albumin, on reverse cholesterol transport (RCT). Methods and results: Macrophages were incubated with control (C) or AGE-albumin in the presence or absence of HDL, followed by incubations with S100B or LPS. Also, culture medium obtained from cells treated with C- or AGE-albumin, following S100B or LPS stimulation was utilized to treat naive macrophages in order to evaluate cholesterol efflux and the expression of HDL receptors. In comparison with C-albumin, AGE-albumin, promoted a greater secretion of cytokines after stimulation with S100B or LPS. A greater amount of cytokines was also produced by macrophages treated with AGE-albumin even in the presence of HDL Cytokine-enriched medium, drawn from incubations with AGE-albumin and S100B or LPS impaired the cholesterol efflux mediated by apoA-I (23% and 37%, respectively), HDL2 (43% and 47%, respectively) and HDL3 (20% and 8.5%, respectively) and reduced ABCA-1 protein level (16% and 26%, respectively). Conclusions: AGE-albumin primes macrophages for an inflammatory response impairing the RCT. Moreover, AGE-albumin abrogates the anti-inflammatory role of HDL, which may aggravate the development of atherosclerosis in DM. (C) 2012 Elsevier BM. All rights reserved.