RESUMO
It was documented that pomegranate has anti-inflammatory effects. In this study, we investigated a direct effect of pomegranate juice (PJ) and its polyphenols on macrophage inflammatory phenotype. In vitro, PJ and its major polyphenols dose-dependently attenuated macrophage response to M1 proinflammatory activation in J774.A1 macrophage-like cell line. This was evidenced by a significant decrease in TNFα and IL-6 secretion in response to stimulation by IFNγ and Lipopolysaccharide. In addition, PJ and punicalagin dose-dependently promoted the macrophages toward a M2 anti-inflammatory phenotype, as determined by a significant increase in the spontaneous secretion of IL-10. In mice, supplementation with dietary PJ substantially inhibited the M2 to M1 macrophage phenotypic shift associated with age, toward a favorable anti-inflammatory M2 phenotype. This effect was also reflected in the mice atherosclerotic plaques, as evaluated by the distinct expression of arginase isoforms. PJ consumption inhibited the increment of arginase II (Arg II, M1) mRNA expression during aging, and maintained the levels of Arg I (M2) expression similar to those in young mice aorta. This study demonstrates, for the first time, that pomegranate polyphenols directly suppress macrophage inflammatory responses and promote M1 to M2 switch in macrophage phenotype. Furthermore, this study indicates that PJ consumption may inhibit the progressive proinflammatory state in the aorta along atherosclerosis development with aging, due to a switch in macrophage phenotype from proinflammatory M1 to anti-inflammatory M2.
Assuntos
Envelhecimento/imunologia , Anti-Inflamatórios não Esteroides/farmacologia , Bebidas , Lythraceae/química , Macrófagos/efeitos dos fármacos , Placa Aterosclerótica/prevenção & controle , Envelhecimento/genética , Envelhecimento/patologia , Animais , Aorta/efeitos dos fármacos , Aorta/imunologia , Aorta/patologia , Arginase/antagonistas & inibidores , Arginase/genética , Arginase/imunologia , Linhagem Celular , Regulação da Expressão Gênica , Interferon gama/antagonistas & inibidores , Interferon gama/farmacologia , Interleucina-10/agonistas , Interleucina-10/genética , Interleucina-10/imunologia , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/imunologia , Masculino , Camundongos , Estresse Oxidativo , Placa Aterosclerótica/genética , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/patologia , Transdução de SinaisRESUMO
OBJECTIVE: The liver is the central organ of fatty acid and triglyceride metabolism. Oxidation and synthesis of fatty acids and triglycerides is under the control of peroxisome-proliferator-activated receptors (PPAR) α. Impairment of these receptors' function contributes to the accumulation of triglycerides in the liver resulting in non-alcoholic fatty liver disease. Urokinase-type plasminogen activator (uPA) was shown to regulate gene expression in the liver involving PPARγ transcriptional activity. In this study we questioned whether uPA modulates triglyceride metabolism in the liver, and investigated the mechanisms involved in the observed processes. METHODS AND RESULTS: Huh7 hepatoma cells were incubated with increasing concentrations of uPA for 24 h uPA dose-dependently increased the cellular triglyceride mass, and this effect resulted from increased de novo triglyceride synthesis mediated by the enzyme diglyceride acyltransferase 2 (DGAT2). Also, the amount of free fatty acids was highly up regulated by uPA through activation of the transcription factor SREBP-1. Chemical activation of PPARα further increased uPA-stimulated triglyceride synthesis, whereas inhibition of p38, an upstream activator of PPARα, completely abolished the stimulatory effect of uPA on both triglyceride synthesis and DGAT2 upregulation. The effect of uPA on triglyceride synthesis in Huh7 cells was mediated via binding to its receptor, the uPAR. In vivo studies in uPAR(-/-) mice demonstrated that no lipid droplets were observed in their livers compared to C57BL/6 mice and the triglyceride levels were significantly lower. CONCLUSION: This study presents a new biological function of the uPA/uPAR system in the metabolism of triglycerides and might present a new target for an early therapeutic intervention for NAFLD.
Assuntos
Carcinoma Hepatocelular/metabolismo , Diacilglicerol O-Aciltransferase/metabolismo , Neoplasias Hepáticas/metabolismo , Triglicerídeos/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ácidos Graxos/química , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Lipídeos/química , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Regulação para CimaRESUMO
OBJECTIVES: The aim of the present study was to investigate the effect of urokinase-type plasminogen activator (uPA) on the expression of the scavenger receptor class B type I (SR-BI) in hepatocytes, and its impact on the removal of HDL-cholesteryl ester (CE) in the liver. METHODS AND RESULTS: Huh7 hepatoma cell lines were incubated with increasing concentrations of uPA. uPA dose-dependently decreased SR-BI protein expression, as determined by flow cytometry (FACS) and by Western blot assays, and down-regulated SR-BI gene expression. Functionally, uPA decreased both the cellular binding of HDL to Huh7 hepatocytes, and the selective uptake of CE from HDL, as determined by several methods including BODIPY staining, cellular cholesterol determination and chasing radio-labeled CE transfer from HDL to the cells. These results were further confirmed using primary rat hepatocytes. The effect of uPA on hepatic SR-BI expression was mediated via binding to the uPA receptor (uPAR). In vivo, SR-BI protein and gene expressions were found to be increased in hepatocytes derived from the uPAR-KO mice compared to C57Bl/6 mice, and in parallel HDL-cholesterol levels in plasma derived from uPAR-KO mice were decreased. Moreover, deficiency of uPAR significantly accelerated the plasma decay of injected HDL-[(3)H]CE. CONCLUSIONS: The results of this study suggest that uPA decreases the removal of HDL-CE in the liver via suppression of the hepatic SR-BI expression. Impaired reverse cholesterol transport (RCT) may result in atherogenic dysfunctional HDL metabolism and may contribute to atherosclerosis development.
Assuntos
Ésteres do Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Receptores Depuradores Classe B/biossíntese , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Animais , Linhagem Celular Tumoral , Colesterol/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Ratos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
The pathogenesis of atherosclerosis involves an imbalanced lipid metabolism and a deregulated immune response culminating in chronic inflammation of the arterial wall. Recent studies show that endogenous ligands, such as modified plasma lipoproteins, can trigger pattern recognition receptors (PRR) of innate immunity for cellular and humoral reactions. The underlying molecular pathways remain less explored. In this study, we investigated the mechanisms of inflammatory effects of oxidized low-density lipoproteins (oxLDL) on human primary coronary artery smooth muscle cells (VSMC). We show that already low concentration of oxLDL initiated atherogenic signals triggering VSMC transition to proinflammatory phenotype. oxLDL impaired the expression of contractile proteins and myocardin in VSMC and initiated changes in cell functional responses, including expression of proinflammatory molecules. The effects of oxLDL were abolished by downregulation of the multifunctional urokinase receptor (uPAR). In response to oxLDL uPAR associated with CD36 and TLR4, the two main PRR for both pathogen and endogenous ligands. We demonstrate that uPAR association with CD36 and TLR4 mediated oxLDL-induced and NF-κB-dependent G-CSF and GM-CSF expression and changes in VSMC contractile proteins. uPAR-mediated release of G-CSF and GM-CSF by VSMC affected macrophage behavior and production of MCP-1. We provide evidence for functional relevance of our in vitro findings to in vivo human atherosclerotic tissues. Our data imply uPAR as a part of a PRR cluster interfering structurally and functionally with CD36 and TLR4 and responding to endogenous atherogenic ligands. They further point to specific function of each component of this cluster in mediating the ultimate signaling pattern.
Assuntos
Aterosclerose/metabolismo , Antígenos CD36/metabolismo , Lipoproteínas LDL/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Receptor 4 Toll-Like/metabolismo , Aterosclerose/genética , Aterosclerose/patologia , Antígenos CD36/genética , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiotaxia , Proteínas Contráteis/genética , Proteínas Contráteis/metabolismo , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Fator Estimulador de Colônias de Granulócitos/biossíntese , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Cultura Primária de Células , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptor 4 Toll-Like/genética , Transativadores/genética , Transativadores/metabolismoRESUMO
OBJECTIVE: Monocyte-to-macrophage differentiation and macrophage death play a pivotal role in atherogenesis. uPA and its receptor uPAR are expressed in atherosclerotic lesion macrophages and contribute to atherosclerosis progression. In the present study we investigated the effect and mechanisms of action of uPA on monocyte-to-macrophage differentiation and on macrophage apoptotic death. METHODS AND RESULTS: The number of mouse peritoneal macrophages (MPM) harvested from uPAR-deficient (uPAR(-/-)) mice was significantly lower by 30% in comparison to control C57BL/6 mice. In vitro, uPA intensified PMA-induced THP-1 monocyte differentiation, as determined by increased expression of the macrophage marker CD36. This effect was mediated via G1 arrest, downregulation of G2/S phase and inhibition of PMA-induced cell death. uPA attenuated MonoMac6 (MM6) macrophage-like cell line apoptosis induced by oxidized LDL (Ox-LDL) and by thapsigargin (inhibitor of sarco-endoplasmic reticulum Ca(2+)-ATPase), but not by staurosporine (protein kinase inhibitor), suggesting that uPA antiapoptotic activity is Ca(2+)-independent, but involves a kinase activation. The antiapoptotic activity of uPA was dependent on the presence of uPAR, and it involved ERK1/2 activation-dependent downregulation of the proapoptotic protein Bim in macrophages stimulated with Ox-LDL. CONCLUSIONS: The present study demonstrates, for the first time, that uPA stimulates the differentiation of monocytes into macrophages and attenuates Ox-LDL-induced macrophage apoptotic death via ERK1/2 activation-dependent Bim downregulation. These processes may result in prolonged macrophage survival in the lesion, increased lesion cellularity, and eventually necrosis, which accelerates lesion development.
Assuntos
Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Macrófagos/patologia , Monócitos/efeitos dos fármacos , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Animais , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/biossíntese , Humanos , Lipoproteínas LDL/antagonistas & inibidores , Macrófagos Peritoneais/fisiologia , Masculino , Camundongos , Camundongos Knockout , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
OBJECTIVES: Paraoxonase 1 (PON1) was suggested to play an anti-inflammatory role. In the present study we questioned whether PON1 has a direct impact on macrophage inflammatory responses, and the possible functional implications of such effects. METHODS AND RESULTS: Ex-vivo studies were performed with bone marrow-derived macrophages (BMDM) harvested from C57BL/6 and human-PON1 transgenic (PON1-Tg) mice, and for the in vitro studies the J774.A1 macrophage-like cell line was used. Pro-inflammatory (M1) activation was induced by LPS and INFγ. The spontaneous and M1-induced TNFα and IL-6 secretion were significantly reduced in BMDM derived from PON1-Tg vs. C57BL/6 mice. In vitro, PON1 dose-dependently attenuated both the spontaneous and M1-induced TNFα and IL-6 secretion, and contributed to the anti-inflammatory activity of HDL. Functionally, PON1 attenuated M1-induced production of reactive oxygen species (ROS), phagocytosis, and necrotic macrophage death. PON1 anti-inflammatory activity was mediated, at least in part, via binding to SR-BI, but was independent of the enzyme catalytic activity or of cholesterol efflux stimulation, and did not involve binding to ABCA1. CONCLUSIONS: The present study demonstrates, for the first time, that PON1 directly suppresses macrophage pro-inflammatory responses. These findings suggest that PON1 decreases sustained pro-inflammatory reactions, which subsequently can attenuate plaque progression.
Assuntos
Arildialquilfosfatase/metabolismo , Inflamação/prevenção & controle , Ativação de Macrófagos , Macrófagos/enzimologia , Animais , Arildialquilfosfatase/genética , Linhagem Celular , Humanos , Inflamação/enzimologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Interferon gama/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Necrose , Estresse Oxidativo , Fagocitose , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Receptores Depuradores Classe B/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Serum paraoxonase-1 (PON1) is a member of the paraoxonases family (PON1, PON2, and PON3). PON1 is synthesized and secreted by the liver, and in circulation it is associated with HDL. PON1 has antioxidative properties, which are associated with the enzyme's capability to decrease oxidative stress in atherosclerotic lesions and to attenuate atherosclerosis development. Epidemiological evidence demonstrates that low PON1 activity is associated with increased risk of cardiovascular events and cardiovascular disease and is an independent risk factor for coronary artery disease. Therefore, pharmacological modulation of PON1 activity or PON1 gene expression could constitute a useful approach for preventing atherosclerosis. A primary determinant of serum PON1 levels is the availability of the enzyme for release by the liver, the principal site of PON1 production. Together with the enzyme secretion rate, enzymatic turnover, and protein stability, the level of PON1 gene expression is a major determinant of PON1 status. This paper summarizes recent progress in understanding the regulation of PON1 expression in hepatocytes.
RESUMO
OBJECTIVE: The atherosclerotic lesion is characterized by lipid peroxide accumulation. Paraoxonase 1 (PON1) reduces atherosclerotic lesion oxidative stress, whereas urokinase-type plasminogen activator (uPA) increases oxidative stress in atherosclerotic lesions and contributes to the progression and complications of atherosclerosis. We hypothesized that uPA may promote oxidative stress in the arterial wall via modulation of PON1 activity. Because the liver is the main site for PON1 production, in the present study, we tested whether uPA influences PON1 expression in hepatocytes. METHODS AND RESULTS: HuH7 hepatocytes were incubated in culture with increasing concentrations of uPA. uPA decreased PON1 gene expression and activity in a dose-dependent manner and accordingly suppressed PON1 secretion from hepatocytes. This effect required uPA/uPA receptor interaction. uPA downregulated PON1 gene expression via inactivation of peroxisome proliferator-activated receptor-γ (PPARγ) activity, and this effect was dependent on uPA-mediated mitogen-activated protein kinase kinase activation. Mechanistic studies showed that uPA enhanced mitogen-activated protein kinase kinase-PPARγ interaction, resulting in PPARγ nuclear export to the cytosol. CONCLUSIONS: This study provides the first evidence that uPA interferes with PPARγ transcriptional activity in hepatocytes, resulting in downregulation of PON1 expression and its secretion to the medium. This may explain, at least in part, the prooxidative effect of uPA in the vascular wall.
Assuntos
Arildialquilfosfatase/metabolismo , Núcleo Celular/metabolismo , Regulação para Baixo/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , PPAR gama/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Arildialquilfosfatase/genética , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Hepatócitos/citologia , Humanos , MAP Quinase Quinase Quinases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Endogâmicos Lew , Receptores de Ativador de Plasminogênio Tipo Uroquinase/deficiência , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Atherogenesis refers to the development of atheromatous plaques in the inner lining of the arteries. These atherosclerotic lesions are characterized by accumulation of monocyte-derived macrophage-foam cells loaded with cholesterol, which eventually undergo apoptotic death, leading finally to formation of the necrotic core of the plaque. Atheroma formation also involves the recruitment of smooth muscle cells (SMC) from the media into the intima, where they proliferate and form the neointima in a process called "remodeling". Cells in the advanced atherosclerotic plaques express high levels of the serine protease urokinase-type plasminogen activator (uPA) and its receptor (uPAR). uPA is a multi-functional multi-domain protein that is not only a regulator of fibrinolysis, but it is also associated with several acute and chronic pathologic conditions. uPA mediate the extracellular matrix (ECM) degradation, and plays a pivotal role in cell adhesion, migration and proliferation, during tissue remodeling. On cell surface uPA binds to the high affinity urokinase receptor, providing a strictly localized proteolysis of ECM proteins. The uPA/uPAR complex also activates intracellular signaling, thus regulating cellular function. An imbalance in the uPA/uPAR system leads to dis-orders in tissue structure and function. This review summarizes recent progress in understanding the role and mechanisms of the uPA/uPAR system in atherogenesis.
Assuntos
Aterosclerose/etiologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Aterosclerose/patologia , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células , Humanos , Macrófagos/fisiologia , Monócitos/fisiologia , Estresse Oxidativo , Remodelação Ventricular/fisiologiaRESUMO
BACKGROUND: Pomegranate extract (POMx) consumption has been shown to reduce the incidence and severity of collagen-induced arthritis in mice. OBJECTIVES: To investigate whether pomegranate consumption affects disease activity in patients with rheumatoid arthritis (RA), in relation to their serum oxidative status. METHODS: In this pilot 12 week open-labeled study eight patients with active RA consumed POMx (10 ml/day) for 12 weeks. Patients' joint status and serum oxidative status (lipid peroxidation, total thiols group, paraoxonase 1 activity) were evaluated at baseline and at week 12. RESULTS: Six patients completed the study. POMx consumption significantly (P < 0.02) reduced the composite Disease Activity Index (DAS28) by 17%, which could be related mostly to a significant (P < 0.005) reduction in the tender joint count (by 62%). These results were associated with a significant (P < 0.02) reduction in serum oxidative status and a moderate but significant (P < 0.02) increase in serum high density lipoprotein-associated paraoxonase 1 (PON1) activity. The addition of POMx to serum from RA patients reduced free radical-induced lipid peroxidation by up to 25%. CONCLUSIONS: The pomegranate consumption reduced DAS28 in RA patients, and this effect could be related to the antioxidative property of pomegranates. Dietary supplementation with pomegranates may be a useful complementary strategy to attenuate clinical symptoms in RA patients.
Assuntos
Artrite Reumatoide/terapia , Lythraceae , Estresse Oxidativo , Fitoterapia , Índice de Gravidade de Doença , Arildialquilfosfatase/sangue , Sedimentação Sanguínea , Feminino , Humanos , Peroxidação de Lipídeos , Peróxidos Lipídicos/sangue , Pessoa de Meia-Idade , Projetos Piloto , Extratos Vegetais/uso terapêuticoRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Arildialquilfosfatase/deficiência , Receptores Depuradores Classe B/biossíntese , Animais , Arildialquilfosfatase/genética , Linhagem Celular , Humanos , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/farmacologia , Lisofosfatidilcolinas/farmacologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Regulação para CimaRESUMO
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.
Assuntos
Arildialquilfosfatase/biossíntese , Ácido Elágico/farmacologia , Ácido Gálico/farmacologia , Hepatócitos/enzimologia , Taninos Hidrolisáveis/farmacologia , Lythraceae , PPAR gama/fisiologia , Regulação para Cima , Células Cultivadas , HumanosRESUMO
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.
Assuntos
HDL-Colesterol/metabolismo , Diabetes Mellitus Experimental/metabolismo , Glucose/metabolismo , Hiperglicemia/metabolismo , Macrófagos Peritoneais/metabolismo , Receptores Depuradores Classe B/biossíntese , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Animais , Aterosclerose/etiologia , Ciclopentanos/farmacologia , Diabetes Mellitus Experimental/complicações , Glucose/farmacologia , Hiperglicemia/complicações , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Receptores Depuradores Classe B/antagonistas & inibidores , Tiossemicarbazonas/farmacologiaRESUMO
OBJECTIVE: The high-density lipoprotein (HDL)-associated paraoxonase-1 (PON1)/free PON1 ratio is lower in diabetic patients in comparison with healthy controls. Because diabetes is associated with increased oxidative stress, we hypothesized that a labeled recombinant PON1 (rePON1) would detect differences in HDL capacity to bind PON1 under specific experimental conditions, such as oxidation, addition of polyphenols, or in vivo dosing of diabetic patients with polyphenols. METHODS: In the present study we determined labeled rePON1 binding to HDL under various oxidative conditions by using polyacrylamide gel electrophoresis for the separation of free labeled rePON1 from HDL-bound labeled rePON1. RESULTS: The HDL-rePON1/free rePON1 ratio gradually decreased as the extent of HDL oxidation increased, and the antioxidants vitamin E or pomegranate juice (PJ) inhibited the redistribution of rePON1. PJ or its purified polyphenols, punicalagin, gallic acid, or ellagic acid, increased rePON1 binding also to non-oxidized HDL. Further, rePON1 associated more efficiently with HDLs isolated from diabetic patients after PJ consumption versus HDLs isolated before PJ consumption. CONCLUSIONS: We conclude that 1) oxidative stress impairs binding of fluorescein isothiocyanate-labeled rePON1 to HDL and 2) PJ polyphenols directly increase the HDL-rePON1 association beyond their antioxidative effect.
Assuntos
Arildialquilfosfatase/sangue , Bebidas , Diabetes Mellitus/sangue , Flavonoides/farmacologia , Lipoproteínas HDL/sangue , Lythraceae , Fenóis/farmacologia , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Eletroforese em Gel de Poliacrilamida/métodos , Ativação Enzimática , Flavonoides/administração & dosagem , Flavonoides/sangue , Ácido Gálico/sangue , Ácido Gálico/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Fenóis/administração & dosagem , Fenóis/sangue , Extratos Vegetais/sangue , Extratos Vegetais/farmacologia , PolifenóisRESUMO
BACKGROUND: Members of the signal transducer and activator of transcription (Stat) family of transcription factors traverse the nuclear membrane through a specialized structure, called the nuclear pore complex (NPC), which represents a selective filter for the import of proteins. Karyophilic molecules can bind directly to a subset of proteins of the NPC, collectively called nucleoporins. Alternatively, the transport is mediated via a carrier molecule belonging to the importin/karyopherin superfamily, which transmits the import into the nucleus through the NPC. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we provide evidence for an alternative Stat1 nuclear import mechanism, which is mediated by the shuttle protein nucleolin. We observed Stat1-nucleolin association, nuclear translocation and specific binding to the regulatory DNA element GAS. Using expression of nucleolin transgenes, we found that the nuclear localization signal (NLS) of nucleolin is responsible for Stat1 nuclear translocation. We show that this mechanism is utilized upon differentiation of myeloid cells and is specific for the differentiation step from monocytes to macrophages. CONCLUSIONS/SIGNIFICANCE: Our data add the nucleolin-Stat1 complex as a novel functional partner for the cell differentiation program, which is uniquely poised to regulate the transcription machinery via Stat1 and nuclear metabolism via nucleolin.
Assuntos
Diferenciação Celular , Núcleo Celular/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator de Transcrição STAT1/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Sequência de Bases , Antígenos CD36/metabolismo , Linhagem Celular , Inativação Gênica , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Sinais de Localização Nuclear/metabolismo , Fosfoproteínas/química , Ligação Proteica , Proteínas de Ligação a RNA/química , Relação Estrutura-Atividade , Fatores de Tempo , NucleolinaRESUMO
Changes in the abundance and properties of blood lipoproteins are generally considered major causes for varied pathological conditions and diseases. Using novel chromatic biomimetic vesicle and cell assays, we present here for the first time evidence for significant changes in lipoproteins' interactions with artificial membranes. Specifically, we demonstrate significant differences in membrane binding between lipoproteins (both low-density lipoprotein [LDL] and high-density lipoprotein [HDL]) harvested from diabetic patients vs. healthy controls as well as between oxidized and native lipoproteins. The chromatic assays, complemented by biophysical techniques and electron microscopy, point to significant reduction of surface membrane binding of the lipoproteins as a consequence of diabetes or oxidation. Overall, our results indicate that the substantial modulation of membrane interactions revealed by the chromatic assays may be used as a new and potentially powerful marker for screening and prediction of diseases associated with oxidative stress.
Assuntos
Biomarcadores/metabolismo , Diabetes Mellitus Tipo 2/diagnóstico , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Membranas/metabolismo , Estresse Oxidativo , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/metabolismo , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Humanos , Bicamadas Lipídicas , Masculino , Lipídeos de Membrana/metabolismo , Pessoa de Meia-Idade , OxirreduçãoRESUMO
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.
Assuntos
Arildialquilfosfatase/genética , Macrófagos/enzimologia , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/fisiologia , Transdução de Sinais/fisiologia , Proteína de Ligação a Elemento Regulador de Esterol 2/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Regulação da Expressão Gênica , Humanos , NADPH Oxidases/fisiologia , Ativador de Plasminogênio Tecidual/farmacologia , Transcrição GênicaRESUMO
BACKGROUND: Low density lipoprotein oxidation is a major pathogenic pathway in atherosclerosis. Previous studies suggested that aspirin, a commonly prescribed drug in patients with atherosclerosis, when given in a dose of 300 mg/day may decrease LDL susceptibility to oxidative modification. However, the effect of the more common lower dose aspirin on LDL oxidation is not known. OBJECTIVES: To examine the effect of aspirin administration (low dosage) on the susceptibility of LDL to oxidative modification in healthy volunteers. METHODS: Aspirin 75 mg was administered daily for 2 weeks to 10 healthy volunteers selected from the medical staff and students at the faculty of medicine. The main outcome measure was ex vivo oxidation of LDL by ultraviolet C irradiation or by peroxyl free radicals generated by AAPH (2,2'-azobis 2-amidinopropane hydrochloride). The extent of LDL oxidation was determined by measuring the formed amounts of thiobarbituric acid-reactive substances, lipid peroxides and conjugated dienes. RESULTS: Following exposure to UVc irradiation there was a significant (P 0.01) increase (10.8%) in TBARS concentrations and a significant (P < 0.05) increase (5.4%) in PD concentrations in LDL withdrawn after aspirin treatment as compared to LDL withdrawn before aspirin treatment. Following incubation with AAPH there was a significant (P < 0.05) increase (15%) in PD concentrations and a significant (P < 0.05) reduction (10%) of the LDL oxidation lag time in LDL withdrawn after aspirin intake as compared to LDL withdrawn before aspirin treatment. CONCLUSIONS: Aspirin treatment given to healthy volunteers at a dose of 75 mg/day increased the susceptibility of their plasma LDL to oxidative modification ex vivo. Our study provides, for the first time, in vivo evidence of pro-oxidative properties of aspirin already suggested by previous in vitro trials.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Aspirina/farmacologia , Lipoproteínas LDL/metabolismo , Aspirina/administração & dosagem , Humanos , Lipoproteínas LDL/efeitos da radiação , Masculino , Oxirredução , Raios UltravioletaRESUMO
BACKGROUND: Low plasma concentrations of high-density lipoprotein (HDL) are associated with increased risk of cardiovascular disease. However, recently several studies have questioned the protective role of high plasma HDL levels. OBJECTIVE: This study was designed to evaluate HDL functions in women with high plasma HDL cholesterol and very low risk profile with relation to subclinical carotid atherosclerosis (ATS). METHODS: Included were 158 middle-aged women with plasma HDL >60 mg/dL and Framingham risk score <7% who had B-mode ultrasound of the carotid arteries. Subclinical ATS was determined by the presence of plaques and/or intima-media thickness (IMT) >1.0 mm. RESULTS: ATS was observed in 51 women, with the majority (n=41) having carotid plaques, some with advanced morphology. In a multivariable model analysis, each, HDL or age, were independently associated with increased prevalence of ATS. Odds ratios for ATS were 3.1 and 2.5 greater for age>60 years and HDL >70 mg/dL, respectively. None of HDL functions determined by its antioxidative properties, reverse-cholesterol transport, or activities of HDL-associated enzyme were different between -ATS and +ATS. C-reactive protein was similar in both groups. CONCLUSION: Subclinical carotid ATS is present in one-third of middle-aged women independently of conventional risk factors. A greater ATS prevalence was associated with very high HDL values. We could not find association between ATS and HDL dysfunction.
RESUMO
Up-regulation of heme oxygenase-1 (HO-1) and altered cholesterol (CH) metabolism are characteristic of Alzheimer-diseased neural tissues. The liver X receptor (LXR) is a molecular sensor of CH homeostasis. In the current study, we determined the effects of HO-1 over-expression and its byproducts iron (Fe(2+)), carbon monoxide (CO) and bilirubin on CH biosynthesis, CH efflux and oxysterol formation in cultured astroglia. HO-1/LXR interactions were also investigated in the context of CH efflux. hHO-1 over-expression for 3 days ( approximately 2-3-fold increase) resulted in a 30% increase in CH biosynthesis and a two-fold rise in CH efflux. Both effects were abrogated by the competitive HO inhibitor, tin mesoporphyrin. CO, released from administered CORM-3, significantly enhanced CH biosynthesis; a combination of CO and iron stimulated CH efflux. Free iron increased oxysterol formation three-fold. Co-treatment with LXR antagonists implicated LXR activation in the modulation of CH homeostasis by heme degradation products. In Alzheimer's disease and other neuropathological states, glial HO-1 induction may transduce ambient noxious stimuli (e.g. beta-amyloid) into altered patterns of glial CH homeostasis. As the latter may impact synaptic plasticity and neuronal repair, modulation of glial HO-1 expression (by pharmacological or other means) may confer neuroprotection in patients with degenerative brain disorders.