RESUMEN
INTRODUCTION: A low-sodium diet (LSD) was shown to increase both angiotensin II (AngII) and aldosterone levels, and to accelerate atherosclerosis in apolipoprotein E-deficient (E0) mice. The aim of the present study was to examine whether accelerated atherosclerosis in E0 mice fed a LSD is mediated by aldosterone, using the mineralocorticoid receptor blocker, eplerenone (Epl). METHODS AND RESULTS: Mice were divided into three groups: normal diet (ND), LSD and LSD treated with Epl at 100 mg/kg per day (LSD+Epl) for 10 weeks. LSD significantly enhanced plasma renin and aldosterone levels, which were further increased in mice fed LSD+Epl. The aortic lesion area increased three-fold with LSD, while LSD+Epl significantly reduced the lesion area to values similar to ND. Serum and peritoneal macrophages obtained from LSD-fed mice exhibited pro-atherogenic properties including increased inflammation, oxidation and cholesterol accumulation, which were inhibited in mice fed LSD+Epl. In a J774A.1 macrophage-like cell line stimulated with lipopolysaccharide, Epl was shown to have a direct anti-inflammatory effect. CONCLUSION: In E0 mice, Epl inhibited LSD-accelerated atherosclerosis, despite the elevation of renin and aldosterone levels. It is therefore suggested that the atherogenic action of LSD could be mediated, at least in part, by activation of the mineralocorticoid receptor. In addition, eplerenone may have direct anti-inflammatory actions.
Asunto(s)
Apolipoproteínas E/deficiencia , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/patología , Dieta Hiposódica , Antagonistas de Receptores de Mineralocorticoides/uso terapéutico , Receptores de Mineralocorticoides/metabolismo , Aldosterona/metabolismo , Animales , Aorta/efectos de los fármacos , Aorta/patología , Aterosclerosis/sangre , Biomarcadores/sangre , Eplerenona , Humanos , Inflamación/patología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/patología , Ratones , Antagonistas de Receptores de Mineralocorticoides/farmacología , Renina/metabolismo , Sistema Renina-Angiotensina/efectos de los fármacos , Espironolactona/análogos & derivados , Espironolactona/farmacología , Espironolactona/uso terapéuticoRESUMEN
The glycosylation of recombinant ß-glucocerebrosidase, and in particular the exposure of mannose residues, has been shown to be a key factor in the success of ERT (enzyme replacement therapy) for the treatment of GD (Gaucher disease). Macrophages, the target cells in GD, internalize ß-glucocerebrosidase through MRs (mannose receptors). Three enzymes are commercially available for the treatment of GD by ERT. Taliglucerase alfa, imiglucerase and velaglucerase alfa are each produced in different cell systems and undergo various post-translational or post-production glycosylation modifications to expose their mannose residues. This is the first study in which the glycosylation profiles of the three enzymes are compared, using the same methodology and the effect on functionality and cellular uptake is evaluated. While the major differences in glycosylation profiles reside in the variation of terminal residues and mannose chain length, the enzymatic activity and stability are not affected by these differences. Furthermore, the cellular uptake and in-cell stability in rat and human macrophages are similar. Finally, in vivo studies to evaluate the uptake into target organs also show similar results for all three enzymes. These results indicate that the variations of glycosylation between the three regulatory-approved ß-glucocerebrosidase enzymes have no effect on their function or distribution.
Asunto(s)
Glucosilceramidasa/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Transporte Biológico , Conformación de Carbohidratos , Secuencia de Carbohidratos , Línea Celular , Estabilidad de Enzimas , Glucosilceramidasa/química , Glucosilceramidasa/farmacocinética , Glicosilación , Humanos , Cinética , Macrófagos Alveolares/enzimología , Masculino , Ratones , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Ratas , Proteínas Recombinantes/metabolismo , Distribución TisularRESUMEN
INTRODUCTION: Aldosterone is known to be involved in atherosclerosis and cardiovascular disease and blockade of its receptor was shown to improve cardiovascular function. It was, therefore, hypothesized that inhibition of aldosterone synthesis would also reduce atherosclerosis development. METHOD: To test this hypothesis, we examined the effect of FAD286 (FAD), an aldosterone synthase inhibitor, on the development of atherosclerosis in spontaneous atherosclerotic apolipoprotein E-deficient mice. Mice were divided into three treatment groups: normal diet, low-salt diet (LSD) and LSD treated with FAD at 30 mg/kg per day (LSD + FAD) for 10 weeks. RESULTS AND CONCLUSION: Histomorphometry of the aortas obtained from these mice showed that atherosclerotic lesion area increased by three-fold under LSD compared with normal diet and FAD significantly reduced lesion area to values similar to normal diet. Changes in atherosclerosis were paralleled by changes in the expression of the inflammation markers (C-reactive protein, monocyte chemotactic protein-1, interleukin-6, nuclear factor kappa B and intercellular adhesion molecule-1) in peritoneal macrophages obtained from these mice. Surprisingly, whereas LSD increased serum or urine aldosterone levels, FAD did not alter these levels when evaluated at the end of the study. In J774A.1 macrophage-like cell line stimulated with lipopolysaccharide, FAD was shown to have a direct dose-dependent anti-inflammatory effect. In apolipoprotein E-deficient mice, FAD reduces atherosclerosis and inflammation. However, these actions appeared to be dissociated from its effect on inhibition of aldosterone synthesis.
Asunto(s)
Apolipoproteínas E/deficiencia , Aterosclerosis/prevención & control , Citocromo P-450 CYP11B2/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Inflamación/prevención & control , Piridinas/farmacología , Aldosterona/sangre , Animales , Apolipoproteínas E/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Línea Celular , Dieta Hiposódica , Fadrozol , Inflamación/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones NoqueadosRESUMEN
OBJECTIVE: Selective uptake of high density lipoprotein (HDL) cholesteryl ester (CE) is considered as the major source of cholesterol for production of steroids in the adrenal gland in rodents. As paraoxonase 1 (PON1) is an HDL-associated lipo-lactonase that has been shown to increase binding of HDL to macrophages, we used PON1 knock-out (PON1KO) mice to test the possible role of PON1 in corticosterone (CS) biosynthesis. METHODS AND RESULTS: PON1 deficiency was associated with reduced serum CS concentration. Adrenal glands obtained from PON1KO mice had significantly lower CE content compared to adrenals from C57Bl6 control mice. Binding of HDL obtained from PON1KO mice to human adrenocortical carcinoma cell line was found to be significantly lower than that of control HDL, and was associated with decreased CS biosynthesis. Addition of purified PON1 to HDL from PON1KO mice increased HDL binding and CS synthesis. Furthermore, the expression of the HDL receptor, SR-BI, protein and mRNA, was reduced in adrenals from PON1KO mice compared to control mice. When challenged with low salt diet, PON1KO mice demonstrated an increase in adrenal SR-BI gene expression and in serum corticosterone which reached levels similar to those obtained in control mice. CONCLUSION: PON1 regulates adrenal CS biosynthesis at two levels: (a) via an accessory role in HDL binding properties, and (b) a supportive role in SR-BI expression and CE supply to the cells.
Asunto(s)
Arildialquilfosfatasa/deficiencia , Ésteres del Colesterol/metabolismo , Corticosterona/sangre , Lipoproteínas HDL/metabolismo , Receptores Depuradores de Clase B/biosíntesis , Glándulas Suprarrenales/metabolismo , Animales , Corticosterona/biosíntesis , Dieta Hiposódica , Humanos , Ratones , Ratones Noqueados , Células Tumorales CultivadasRESUMEN
BACKGROUND: Paraoxonase 1 (PON1) was shown to stimulate HDL binding and HDL-mediated cholesterol efflux from macrophages. This study examined the role of PON1 in the expression of proteins that enhance macrophage HDL binding, i.e. ABCA1 and SR-BI. METHODS AND RESULTS: ABCA1 expression was similar, whereas SR-BI expression (mRNA and protein determined by FACS, Western blot, or immunocytochemistry) was significantly decreased in peritoneal macrophages from PON1 deficient (MPM-PON1(0)) in comparison to C57Bl/6 (MPM-Control) mice. PON1 deficiency correction with HDL-control, recombinant PON1 (rePON1), or by transfection with a plasmid containing the rePON1 gene, increased SR-BI expression in MPM-PON1(0), whereas rePON1/H115Gln mutant, or the H115Q/H134Q double mutant, which lack catalytic activity, did not stimulate SR-BI expression. Lysophosphatidyl choline (LPC) resulting from PON1 action on macrophage PC, upregulated SR-BI expression in MPM-PON1(0) via activation of ERK1/2 and PI3K. Functionally, HDL bound to MPM-PON1(0) significantly less than to MPM-Control, and failed to inhibit tunicamycin-induced apoptosis, but had no significant effect on HDL-mediated cholesterol efflux from macrophages. CONCLUSIONS: PON1 deficiency in mice is associated with decreased macrophage SR-BI expression, decreased cellular HDL binding, and consequently the loss of HDL-mediated cytoprotection against apoptosis, which may contribute to the accelerated atherosclerosis observed in PON1(0) mice. These findings add new insights into the function of SR-BI in macrophages, and define the potential role of PON1 in regulating SR-BI-mediated HDL protection against macrophages apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Arildialquilfosfatasa/deficiencia , Receptores Depuradores de Clase B/biosíntesis , Animales , Arildialquilfosfatasa/genética , Línea Celular , Humanos , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/farmacología , Lisofosfatidilcolinas/farmacología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Regulación hacia ArribaRESUMEN
OBJECTIVE: Serum paraoxonase-1 (PON1) expression is regulated by polyphenols, shown to activate the peroxisome proliferator-activated receptor gamma (PPARgamma). Pomegranate juice (PJ) is a polyphenol-rich fruit. Because promoter sequence of PON1 gene indicates that it could be regulated by nuclear receptors, we investigated the effect of PJ polyphenols on PON1 gene expression in HuH7 hepatocytes. METHODS AND RESULTS: PON1 protein or mRNA expression, determined by immunocytochemistry, or quantitative PCR, respectively, as well as PON1 gene promoter activation, was significantly increased in hepatocytes incubated with PJ or with its major polyphenols punicalagin, or gallic acid (GA). Ellagic acid (EA) elicited only modest stimulatory effect. Accordingly, PJ, punicalagin, GA, and less so EA, dose-dependently increased cell-associated and hepatocyte-secreted PON1 arylesterase activity. Functionally, the secreted PON1 exhibited biological activity by protecting LDL and HDL from oxidation. Finally, PJ polyphenols upregulated the hepatocyte PON1 expression, at least in part, via the intracellular signaling cascade PPARgamma-PKA-cAMP. CONCLUSIONS: This study shows for the first time that PJ polyphenols have a specific transcriptional role in hepatocyte PON1 expression upregulation, and its secretion to the medium. We conclude that the anti-atherogenic characteristics of PJ polyphenols are modulated, at least in part, via hepatocyte PON1 upregulation and its subsequent release to the medium.
Asunto(s)
Arildialquilfosfatasa/biosíntesis , Ácido Elágico/farmacología , Ácido Gálico/farmacología , Hepatocitos/enzimología , Taninos Hidrolizables/farmacología , Lythraceae , PPAR gamma/fisiología , Regulación hacia Arriba , Células Cultivadas , HumanosRESUMEN
AIMS: Diabetes is associated with atherogenesis and macrophage-foam cell formation, due in part to a decrease in HDL-mediated cholesterol efflux from macrophages. This study examined the expression of proteins involved in cholesterol transport, i.e. ABCA1 and SR-BI, under diabetic conditions. METHODS AND RESULTS: ABCA1 expression was similar, whereas SR-BI expression (mRNA and protein) was significantly increased in mouse peritoneal macrophages (MPM) harvested from C57Bl/6 diabetic mice, compared to MPM from control non-diabetic mice. Similar results were obtained in vitro in J-774A.1 macrophage-like cell line incubated with high (30 mM) vs. low (5mM) glucose concentrations. Accordingly, association and internalization of HDL to MPM from diabetic mice, or to J-774A.1 macrophages grown under diabetic conditions was significantly higher compared to control cells. Unexpectedly, however, increased macrophage SR-BI expression was associated with a substantial reduction in HDL-mediated cholesterol efflux from the macrophages. Moreover, total cellular cholesterol content was increased by 28% in macrophages incubated with HDL under high glucose concentrations, compared to low glucose concentrations. This effect was abolished by a rabbit polyclonal anti-SR-BI, which blocks binding to the receptor, or alternatively by using BLT1, a specific inhibitor of lipid transport via the SR-BI. CONCLUSIONS: Diabetes stimulates the expression of SR-BI in macrophages and leads to a shift in its activity from HDL-mediated cholesterol efflux to HDL-mediated cholesterol influx. These effects may lead to increased foam cell formation and atherosclerosis development.
Asunto(s)
HDL-Colesterol/metabolismo , Diabetes Mellitus Experimental/metabolismo , Glucosa/metabolismo , Hiperglucemia/metabolismo , Macrófagos Peritoneales/metabolismo , Receptores Depuradores de Clase B/biosíntesis , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/biosíntesis , Animales , Aterosclerosis/etiología , Ciclopentanos/farmacología , Diabetes Mellitus Experimental/complicaciones , Glucosa/farmacología , Hiperglucemia/complicaciones , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Receptores Depuradores de Clase B/antagonistas & inhibidores , Tiosemicarbazonas/farmacologíaRESUMEN
AIMS: We have recently shown that urokinase plasminogen activator (uPA) increases oxidative stress (OS), cholesterol biosynthesis, and paraoxonase 2 (PON2) expression in macrophages via binding to its receptor, the uPAR. Since PON2 is regulated by both OS and cholesterol content, we hypothesized that uPA elicits a cascade of signal transduction events shared by NADPH oxidase and cholesterol biosynthesis that culminates in PON2 gene expression. Here, we investigated the signalling pathway that leads to the expression of PON2 in macrophages in response to uPA. METHODS AND RESULTS: The increase in macrophage PON2 mRNA levels in response to uPA was shown to depend on PON2 gene promoter activation and mRNA transcription. LDL abolished these effects, suggesting a possible role for a transcription factor involved in cellular cholesterogenesis. Indeed, uPA upregulated PON2 expression in a sterol regulatory binding protein-2 (SREBP-2)-dependent manner, since blocking SREBP-2 maturation by 4-(2-aminoethyl)-benzenesulfonyl fluoride abolished uPA-stimulation of PON2, whereas inhibition of SREBP-2 catabolism by N-acetyl-leucyl-norleucinal had an opposite effect. The upstream signalling mechanisms include uPA activation of extracellular signal-regulated kinases (ERK1/2), which was dependent on NADPH oxidase and phosphatidylinositol 3-kinase activation, and these latter effects were mediated by the tyrosine kinase activity of the platelet-derived growth factor receptor-beta. CONCLUSION: These findings provide a framework linking interactions among cellular signalling pathways associated with reactive oxygen species production, macrophage cholesterol biosynthesis, and cellular PON2 expression in vascular pathophysiology.
Asunto(s)
Arildialquilfosfatasa/genética , Macrófagos/enzimología , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Especies Reactivas de Oxígeno/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/fisiología , Transducción de Señal/fisiología , Proteína 2 de Unión a Elementos Reguladores de Esteroles/fisiología , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Regulación de la Expresión Génica , Humanos , NADPH Oxidasas/fisiología , Activador de Tejido Plasminógeno/farmacología , Transcripción GenéticaRESUMEN
Pak (p21-activated kinase) serine/threonine kinases have been shown to mediate directional sensing of chemokine gradients. We hypothesized that Pak may also mediate chemokine-induced shape changes, to facilitate leucocyte chemotaxis through restrictive barriers, such as the extracellular matrix. A potent inhibitor, Pak(i), was characterized and used to probe the role of Pak-family kinases in SDF-1alpha (stromal-cell derived factor-1alpha/CXCL12)-induced chemotaxis in a T cell model. Pak(i) potently inhibited SDF-1alpha-induced Pak activation by a bivalent mechanism, as indicated by its complete inactivation upon point mutation of two binding sites, but partial inactivation upon mutation of either site alone. Importantly, Pak(i) was not toxic to cells over the time frame of our experiments, since it did not substantially affect cell surface expression of CXCR4 (CXC chemokine receptor 4) or integrins, cell cycle progression, or a number of ligand-induced responses. Pak(i) produced dose-dependent inhibition of SDF-1alpha-induced migration through rigid filters bearing small pores; but unexpectedly, did not substantially affect the magnitude or kinetics of chemotaxis through filters bearing larger pores. SDF-1alpha-induced Pak activation was partly dependent on PIX (Pak-interactive exchange factor); correspondingly, an allele of beta-PIX that cannot bind Pak inhibited SDF-1alpha-induced chemotaxis through small, but not large pores. By contrast, other key players in chemotaxis: G(i), PI3K (phosphoinositide 3-kinase), and the Rho-family G-proteins, Rac and Cdc42 (cell division cycle 42), were required for SDF-1alpha-induced migration regardless of the barrier pore-size. These studies have revealed a distinct branch of the SDF-1alpha signalling pathway, in which the Rac/Cdc42 effector, Pak, and its partner, PIX, specifically regulate the cellular events required for chemokine-induced migration through restrictive barriers.