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1.
Cardiovasc Res ; 91(3): 492-501, 2011 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-21493704

RESUMO

AIMS: We investigated the molecular mechanism underlying the role of transient receptor potential vanilloid type 1 (TRPV1), a Ca(2+)-permeable non-selective cation channel, in the activation of endothelial nitric oxide (NO) synthase (eNOS) in endothelial cells (ECs) and mice. METHODS AND RESULTS: In ECs, TRPV1 ligands (evodiamine or capsaicin) promoted NO production, eNOS phosphorylation, and the formation of a TRPV1-eNOS complex, which were all abrogated by the TRPV1 antagonist capsazepine. TRPV1 ligands promoted the phosphorylation of Akt, calmodulin-dependent protein kinase II (CaMKII) and TRPV1, and increased the formation of a TRPV1-Akt-CaMKII complex. Removal of extracellular Ca(2+) abolished the ligand-induced increase in the phosphorylation of Akt and CaMKII, formation of a TRPV1-eNOS complex, and eNOS activation. Inhibition of PI3K and CaMKII suppressed the ligand-induced increase in TRPV1 phosphorylation, formation of a TRPV1-eNOS complex, and eNOS activation. TRPV1 activation increased the phosphorylation of Akt, CaMKII, and eNOS in the aortas of wild-type mice but failed to activate eNOS in TRPV1-deficient aortas. Additionally, TRPV1 ligand-induced angiogenesis was diminished in eNOS- or TRPV1-deficient mice. When compared with apolipoprotein E (ApoE)-deficient mice, ApoE/TRPV1-double-knockout mice displayed reduced phosphorylation of eNOS, Akt, and CaMKII in aortas but worsened atherosclerotic lesions. CONCLUSION: TRPV1 activation in ECs may trigger Ca(2+)-dependent PI3K/Akt/CaMKII signalling, which leads to enhanced phosphorylation of TRPV1, increased TRPV1-eNOS complex formation, eNOS activation and, ultimately, NO production.


Assuntos
Células Endoteliais/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/enzimologia , Aterosclerose/genética , Aterosclerose/patologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Bovinos , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico Sintase Tipo III/genética , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinazolinas/farmacologia , Interferência de RNA , Transdução de Sinais , Canais de Cátion TRPV/deficiência , Canais de Cátion TRPV/efeitos dos fármacos , Canais de Cátion TRPV/genética , Fatores de Tempo , Transfecção
2.
J Cell Physiol ; 226(12): 3330-9, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21321940

RESUMO

Erythropoietin (EPO), the key hormone for erythropoiesis, also increases nitric oxide (NO) bioavailability in endothelial cells (ECs), yet the definitive mechanisms are not fully understood. Increasing evidence has demonstrated that ß common receptor (ßCR) plays a crucial role in EPO-mediated non-hematopoietic effects. We investigated the role of ßCR in EPO-induced endothelial NO synthase (eNOS) activation in bovine aortic ECs (BAECs) and the molecular mechanisms involved. Results of confocal microscopy and immunoprecipitation analyses revealed that ßCR was colocalized and interacted with EPO receptor (EPOR) in ECs. Inhibition of ßCR or EPOR by neutralizing antibodies or small interfering RNA abolished the EPO-induced NO production. Additionally, blockage of ßCR abrogated the EPO-induced increase in the phosphorylation of eNOS, Akt, Src, or Janus kinase 2 (JAK2). Immunoprecipitation analysis revealed that treatment with EPO increased the interaction between ßCR and eNOS, which was suppressed by inhibition of Src, JAK2, or Akt signaling with specific pharmacological inhibitors. Furthermore, EPO-induced EC proliferation, migration, and tube formation were blocked by pretreatment with ßCR antibody and Src, JAK2, or PI3K/Akt inhibitors. Moreover, in vivo experiments showed that EPO increased the level of phosphorylated eNOS, Src, JAK2, and Akt, as well as ßCR-eNOS association in aortas and promoted the angiogenesis in Matrigel plug, which was diminished by ßCR or EPOR neutralizing antibodies. Our findings suggest that ßCR may play an integrative role in the EPO signaling-mediated activation of eNOS in ECs.


Assuntos
Subunidade beta Comum dos Receptores de Citocinas/metabolismo , Células Endoteliais/enzimologia , Eritropoetina/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Receptores de Interleucina-3/metabolismo , Transdução de Sinais , Animais , Anticorpos Neutralizantes/farmacologia , Bovinos , Movimento Celular , Proliferação de Células , Células Cultivadas , Subunidade beta Comum dos Receptores de Citocinas/genética , Subunidade beta Comum dos Receptores de Citocinas/imunologia , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática , Eritropoetina/genética , Humanos , Imunoprecipitação , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Neovascularização Fisiológica , Óxido Nítrico/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/imunologia , Receptores da Eritropoetina/metabolismo , Receptores de Interleucina-3/genética , Receptores de Interleucina-3/imunologia , Proteínas Recombinantes , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismo
3.
J Nutr Biochem ; 22(11): 1015-21, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21190831

RESUMO

Wogonin, one component in Scutellaria baicalensis Georgi extracts, has several beneficial properties for cancers and inflammatory diseases. However, the efficacy of wogonin in cholesterol metabolism of macrophages remains unknown. In macrophages, cholesterol uptake is controlled by scavenger receptors (SR-A and CD36) and cholesterol efflux by SR-BI, ATP-binding cassette transporter-A1 (ABCA1) and ABCG1. In the present study, we investigated the effect and underlying molecular mechanism of wogonin on the formation of macrophage foam cells by murine J774.A1 macrophages. Wogonin attenuated oxidized low-density lipoprotein (oxLDL)-induced cholesterol accumulation in macrophages. The binding of oxLDL to macrophages and protein expression of SR-A and CD36 were not affected by wogonin. Wogonin enhanced cholesterol efflux and increased the protein level of ABCA1 without affecting the protein expression of SR-BI or ABCG1. Inhibition of ABCA1 by pharmacological inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt or neutralizing antibody abolished this suppressive effect of wogonin on lipid accumulation. Moreover, the up-regulation of ABCA1 protein by wogonin resulted from a decrease in degradation rate of ABCA1 protein, with no effect on ABCA1 mRNA expression. This reduction in ABCA1 degradation was due to increased protein phosphatase 2B (PP2B)-mediated ABCA1 dephosphorylation, as evidenced by increased interaction between ABCA1 and PP2B; pharmacological inhibition of PP2B would prevent wogonin-induced ABCA1 protein expression, dephosphorylation and attenuation of lipid accumulation. Collectively, wogonin increases the protein stability of ABCA1 via PP2B-mediated dephosphorylation, thus leading to reduced cholesterol accumulation in macrophage foam cells.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Calcineurina/metabolismo , Colesterol/metabolismo , Flavanonas/farmacologia , Macrófagos/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Transportador 1 de Cassete de Ligação de ATP , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Células Cultivadas , Células Espumosas/efeitos dos fármacos , Lipoproteínas/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Receptores Depuradores Classe A/metabolismo , Receptores Depuradores Classe B/metabolismo
4.
Free Radic Biol Med ; 50(1): 47-54, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21034810

RESUMO

α-Lipoic acid (α-LA), a key cofactor in cellular energy metabolism, has protective activities in atherosclerosis, yet the detailed mechanisms are not fully understood. In this study, we examined whether α-LA affects foam cell formation and its underlying molecular mechanisms in murine macrophages. Treatment with α-LA markedly attenuated oxidized low-density lipoprotein (oxLDL)-mediated cholesterol accumulation in macrophages, which was due to increased cholesterol efflux. Additionally, α-LA treatment dose-dependently increased protein levels of ATP-binding cassette transporter A1 (ABCA1) and ABCG1 but had no effect on the protein expression of SR-A, CD36, or SR-BI involved in cholesterol homeostasis. Furthermore, α-LA increased the mRNA expression of ABCA1 and ABCG1. The upregulation of ABCA1 and ABCG1 by α-LA depended on liver X receptor α (LXRα), as evidenced by an increase in the nuclear levels of LXRα and LXRE-mediated luciferase activity and its prevention of the expression of ABCA1 and ABCG1 after inhibition of LXRα activity by the pharmacological inhibitor geranylgeranyl pyrophosphate (GGPP) or knockdown of LXRα expression with small interfering RNA (siRNA). Consistently, α-LA-mediated suppression of oxLDL-induced lipid accumulation was abolished by GGPP or LXRα siRNA treatment. In conclusion, LXRα-dependent upregulation of ABCA1 and ABCG1 may mediate the beneficial effect of α-LA on foam cell formation.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Células Espumosas/efeitos dos fármacos , Lipoproteínas/genética , Receptores Nucleares Órfãos/fisiologia , Ácido Tióctico/farmacologia , Transportador 1 de Cassete de Ligação de ATP , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Células Cultivadas , Células Espumosas/metabolismo , Células Espumosas/patologia , Humanos , Lipoproteínas/metabolismo , Lipoproteínas LDL/metabolismo , Receptores X do Fígado , Camundongos , Receptores Nucleares Órfãos/antagonistas & inibidores , Receptores Nucleares Órfãos/metabolismo , Fosfatos de Poli-Isoprenil/farmacologia , RNA Interferente Pequeno/farmacologia , Elementos de Resposta/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia , Transfecção , Regulação para Cima/efeitos dos fármacos
5.
J Cell Biochem ; 111(1): 104-10, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20506155

RESUMO

Berberine, a botanical alkaloid purified from Cortidis rhizoma, has effects in cardiovascular diseases, yet the mechanism is not fully understood. Foam cells play a critical role in the progression of atherosclerosis. This study aimed to investigate the effect of berberine on the formation of foam cells by macrophages and the underlying mechanism. Treatment with berberine markedly suppressed oxidized low-density lipoprotein (oxLDL)-mediated lipid accumulation, which was due to an increase in cholesterol efflux. Berberine enhanced the mRNA and protein expression of ATP-binding membrane cassette transport protein A1 (ABCA1) but did not alter the protein level of ABCG1 or other scavenger receptors. Additionally, functional inhibition of ABCA1 with a pharmacological inhibitor or neutralizing antibody abrogated the effects of berberine on cholesterol efflux and lipid accumulation. Moreover, berberine induced the nuclear translocation and activation of liver X receptor alpha (LXRalpha) but not its protein expression. Knockdown of LXRalpha mRNA expression by small interfering RNA abolished the berberine-mediated protective effects on ABCA1 protein expression and oxLDL-induced lipid accumulation in macrophages. These data suggest that berberine abrogates the formation of foam cells by macrophages by enhancing LXRalpha-ABCA1-dependent cholesterol efflux.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Berberina/farmacologia , Colesterol/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Receptores Nucleares Órfãos/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Animais , Linhagem Celular , Humanos , Lipoproteínas LDL/metabolismo , Receptores X do Fígado , Macrófagos/citologia , Camundongos
6.
Circulation ; 121(16): 1828-37, 2010 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-20385932

RESUMO

BACKGROUND: In addition to the hematopoietic effect of erythropoietin, increasing evidence suggests that erythropoietin also exerts protective effects for cardiovascular diseases. However, the role of erythropoietin and its underlying mechanism in macrophage foam cell formation are poorly understood. METHODS AND RESULTS: Compared with wild-type specimens, erythropoietin was increased in atherosclerotic aortas of apolipoprotein E-deficient (apoE(-/-)) mice, mainly in the macrophage foam cells of the lesions. Erythropoietin levels in culture medium and macrophages were significantly elevated in response to oxidized low-density lipoprotein in a dose-dependent manner. Furthermore, erythropoietin markedly attenuated lipid accumulation in oxidized low-density lipoprotein-treated macrophages, a result that was due to an increase in cholesterol efflux. Erythropoietin treatment significantly increased ATP-binding cassette transporters (ABC) A1 and ABCG1 mRNA and protein levels without affecting protein expression of scavenger receptors, including scavenger receptor-A, CD36, and scavenger receptor-BI. The upregulation of ABCA1 and ABCG1 by erythropoietin resulted from liver X receptor alpha activation, which was confirmed by its prevention on expression of ABCA1 and ABCG1 after pharmacological or small interfering RNA inhibition of liver X receptor alpha. Moreover, the erythropoietin-mediated attenuation on lipid accumulation was abolished by such inhibition. Finally, reduced lipid accumulation and marked increase in ABCA1 and ABCG1 were demonstrated in erythropoietin-overexpressed macrophages. CONCLUSIONS: Our data suggest that erythropoietin suppresses foam cell formation via the liver X receptor alpha-dependent upregulation of ABCA1 and ABCG1.


Assuntos
Aterosclerose/tratamento farmacológico , Cardiotônicos/farmacologia , Eritropoetina/farmacologia , Células Espumosas/efeitos dos fármacos , Receptores Nucleares Órfãos/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Doenças da Aorta/tratamento farmacológico , Doenças da Aorta/imunologia , Doenças da Aorta/patologia , Apolipoproteínas E/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Antígenos CD36/genética , Células Cultivadas , Células Espumosas/metabolismo , Células Espumosas/patologia , Lipídeos/biossíntese , Lipoproteínas/genética , Lipoproteínas/metabolismo , Lipoproteínas LDL/farmacologia , Receptores X do Fígado , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/metabolismo , Receptores Depuradores Classe A/genética , Receptores Depuradores Classe B/genética
7.
Cardiovasc Res ; 82(3): 468-75, 2009 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19307231

RESUMO

AIMS: Valsartan, a selective angiotensin II type 1 receptor (AT1R) blocker, has beneficial effects in the cardiovascular system in part by its increase of nitric oxide (NO) bioavailability, yet the mechanisms are unclear. We investigated the molecular mechanisms underlying this effect in endothelial cells (ECs). METHODS AND RESULTS: NO production was examined by Griess reagent assay, DAF-2 DA fluorescence staining and cGMP ELISA kits. Protein interaction was determined by western blotting and immunoprecipitation. Treating bovine or human aortic ECs with valsartan increased NO production, as evidenced by elevated level of stable NO metabolites and intracellular cGMP. Valsartan increased the phosphorylation but not the protein level of endothelial NO synthase (eNOS). Inhibition of phosphoinositide-3 kinase (PI3K)/Akt and Src pathways by specific inhibitors suppressed valsartan-induced NO release. In addition, valsartan increased the tyrosine residue phosphorylation of AT1R, which was attenuated by inhibition of Src but not PI3K activities. Valsartan also suppressed the interaction of eNOS and AT1R, which was blocked by Src or PI3K inhibition. CONCLUSION: Valsartan-induced NO production in ECs is mediated through Src/PI3K/Akt-dependent phosphorylation of eNOS. Valsartan-induced AT1R phosphorylation depends on Src but not PI3K, whereas valsartan-induced suppression of AT1R-eNOS interaction depends on Src/PI3K/Akt signalling. These results indicate a novel vasoprotective mechanism of valsartan in upregulating NO production in ECs.


Assuntos
Anti-Hipertensivos/farmacologia , Células Endoteliais/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Tetrazóis/farmacologia , Valina/análogos & derivados , Animais , Aorta/citologia , Bovinos , Células Cultivadas , Humanos , Óxido Nítrico/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Valina/farmacologia , Valsartana , Quinases da Família src/metabolismo
8.
Life Sci ; 84(3-4): 97-104, 2009 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-19041881

RESUMO

AIMS: Resistin promotes macrophage-foam cell formation, but the mechanisms are unclear. In macrophages, lipid uptake is regulated by scavenger receptors (SR-A and CD36), while the cholesterol efflux is regulated by SR-BI, ATP-binding cassette transporter-A1 (ABCA1) and ABCG1. We investigated the mechanisms underlying the dysregulation by resistin of these regulators leading to promotion of lipid accumulation in bone marrow-derived macrophages. MAIN METHODS: Western blotting, real-time PCR and oil red O staining were performed. KEY FINDINGS: Resistin exacerbated lipid accumulation in oxLDL-treated macrophages. Resistin treatment of oxLDL-untreated macrophages showed increased SR-A and CD36 mRNA and protein levels, and decreased ABCA1 protein level, while having no effect on SR-BI or ABCG1 expression. Up-regulation of SR-A and CD36 by resistin resulted from activation of AP-1 and PPARgamma, respectively, and this was confirmed by the lack of activation of either after AP-1 inhibition using curcumin or SP600125, or PPARgamma inhibition using GW9662, respectively. The down-regulation of ABCA1 by resistin was not accompanied by a reduced mRNA level or an activation of LXRalpha/RXR, but resulted from enhanced protein degradation as revealed by the abolition of the down-regulation after inhibition of the proteasome pathway using ALLN or MG-132. A combined inhibition by SP600125, GW9662 and ALLN prevented resistin-induced exacerbation of lipid accumulation in oxLDL-treated macrophages. SIGNIFICANCE: Resistin promotes foam cell formation via dysregulation of SR-A, CD36 and ABCA1. SR-A and CD36 are transcriptionally up-regulated by resistin through AP-1 and PPARgamma, respectively, whereas ABCA1 is down-regulated by resistin through proteasome-mediated enhancement of protein degradation.


Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Antígenos CD36/fisiologia , Metabolismo dos Lipídeos , Macrófagos/metabolismo , Resistina/fisiologia , Receptores Depuradores Classe A/fisiologia , Transportador 1 de Cassete de Ligação de ATP , Animais , Colesterol/metabolismo , Lipoproteínas LDL/fisiologia , PPAR gama/fisiologia , Complexo de Endopeptidases do Proteassoma/fisiologia , Ratos , Ratos Sprague-Dawley , Fator de Transcrição AP-1/fisiologia
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