RESUMO
Activation of the peroxisome proliferator-activated receptor (PPAR) gamma, the molecular target for insulin sensitizing thiazolidinediones used in patients with type 2 diabetes, inhibits vascular smooth muscle cell (VSMC) proliferation and prevents atherosclerosis and neointima formation. Emerging evidence indicates that telomerase controls key cellular functions including replicative lifespan, differentiation, and cell proliferation. In the present study, we demonstrate that ligand-induced and constitutive PPARgamma activation inhibits telomerase activity in VSMCs. Telomerase reverse transcriptase (TERT) confers the catalytic activity of telomerase, and PPARgamma ligands inhibit TERT expression through a receptor-dependent suppression of the TERT promoter. 5'-deletion analysis, site-directed mutagenesis, and transactivation studies using overexpression of Ets-1 revealed that suppression of TERT transcription by PPARgamma is mediated through negative cross-talk with Ets-1-dependent transactivation of the TERT promoter. Chromatin immunoprecipitation assays further demonstrated that PPARgamma ligands inhibit Ets-1 binding to the TERT promoter, which is mediated at least in part through an inhibition of Ets-1 expression by PPARgamma ligands. In VSMCs overexpressing TERT, the efficacy of PPARgamma ligands to inhibit cell proliferation is lost, indicating that TERT constitutes an important molecular target for the antiproliferative effects of PPARgamma ligands. Finally, we demonstrate that telomerase activation during the proliferative response after vascular injury is effectively inhibited by PPARgamma ligands. These findings provide a previously unrecognized mechanism for the antiproliferative effects of PPARgamma ligands and support the concept that PPARgamma ligands may constitute a novel therapeutic approach for the treatment of proliferative cardiovascular diseases.
Assuntos
Músculo Liso Vascular/fisiologia , PPAR gama/fisiologia , Telomerase/antagonistas & inibidores , Animais , Aorta , Sequência de Bases , Doenças Cardiovasculares/terapia , Divisão Celular/fisiologia , Células Cultivadas , Primers do DNA , Ativação Enzimática , Microscopia Confocal , Músculo Liso Vascular/citologia , Músculo Liso Vascular/enzimologia , Mutagênese Sítio-Dirigida , PPAR gama/genética , Ratos , Proteínas Recombinantes/metabolismo , Telomerase/metabolismo , TransfecçãoRESUMO
Osteopontin (OPN) is expressed in atherosclerotic lesions, particularly in diabetic patients. To determine the role of OPN in atherogenesis, ApoE-/-OPN+/+, ApoE-/-OPN+/-, and ApoE-/-OPN-/- mice were infused with Ang II, inducing vascular OPN expression and accelerating atherosclerosis. Compared with ApoE-/-OPN+/+ mice, ApoE-/-OPN+/- and ApoE-/-OPN-/- mice developed less Ang II-accelerated atherosclerosis. ApoE-/- mice transplanted with bone marrow derived from ApoE-/-OPN-/- mice had less Ang II-induced atherosclerosis compared with animals receiving ApoE-/-OPN+/+ cells. Aortae from Ang II-infused ApoE-/-OPN-/- mice expressed less CD68, C-C-chemokine receptor 2, and VCAM-1. In response to intraperitoneal thioglycollate, recruitment of leukocytes in OPN-/- mice was impaired, and OPN-/- leukocytes exhibited decreased basal and MCP-1-directed migration. Furthermore, macrophage viability in atherosclerotic lesions from Ang II-infused ApoE-/-OPN-/- mice was decreased. Finally, Ang II-induced abdominal aortic aneurysm formation in ApoE-/-OPN-/- mice was reduced and associated with decreased MMP-2 and MMP-9 activity. These data suggest an important role for leukocyte-derived OPN in mediating Ang II-accelerated atherosclerosis and aneurysm formation.
Assuntos
Angiotensina II/farmacologia , Aneurisma da Aorta Abdominal/etiologia , Arteriosclerose/etiologia , Sialoglicoproteínas/fisiologia , Animais , Aneurisma da Aorta Abdominal/terapia , Apolipoproteínas E/fisiologia , Arteriosclerose/terapia , Movimento Celular , Sobrevivência Celular , Quimiocina CCL2/fisiologia , Citocinas/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Leucócitos/fisiologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Osteopontina , RNA Mensageiro/análise , Receptores CCR2 , Receptores de Quimiocinas/fisiologia , Sialoglicoproteínas/genéticaRESUMO
Osteopontin (OPN) is a proinflammatory cytokine and adhesion molecule implicated in the chemoattraction of monocytes and in cell-mediated immunity. We have recently reported that genetic OPN-deficiency attenuates the development of atherosclerosis in apoE-/- mice identifying OPN as potential target for pharmacological intervention in atherosclerosis. Synthetic agonists for the Liver X Receptor (LXR), members of the nuclear hormone receptor superfamily, prevent the development of atherosclerosis by regulating cholesterol homeostasis and suppressing inflammatory gene expression in macrophages. We demonstrate here that LXR ligands inhibit cytokine-induced OPN expression in macrophages. Two synthetic LXR ligands, T0901317 and GW3965, inhibited TNF-alpha, IL-1beta, INF-gamma and lipopolysaccharide induced OPN mRNA and protein expression in RAW 264.7 macrophages. Transient transfection experiments revealed that LXR ligands suppress cytokine-induced OPN promoter activity. Deletion analysis, heterologous promoter assays, and site-directed mutagenesis identified an activator protein-1 (AP-1) consensus site at -76 relative to the initiation site that supports OPN transcription in macrophages and mediates the effects of LXR ligands to inhibit OPN transcription. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that LXR agonists inhibit cytokine-induced c-Fos and phospho-c-Jun binding to this AP-1 site. Cytokine-induced c-Fos and phospho-c-Jun protein expression was inhibited by LXR ligands and overexpression of c-Fos and c-Jun reversed the inhibitory effect of LXR ligands on OPN promoter activity in transactivation assays. Finally, treatment of C57BL/6J mice with LXR ligands inhibited OPN expression in peritoneal macrophages indicating that the observed effects of LXR ligands to inhibit OPN expression are applicable in vivo. These observations identify the regulation of macrophage OPN expression as a mechanism whereby LXR ligands may impact macrophage inflammatory responses and atherosclerosis. The full text of this article is available online at http://circres.ahajournals.org.
Assuntos
Citocinas/farmacologia , Proteínas de Ligação a DNA/fisiologia , Macrófagos/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Sialoglicoproteínas/genética , Transdução de Sinais/fisiologia , Fator de Transcrição AP-1/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação a DNA/agonistas , Receptores X do Fígado , Camundongos , Camundongos Endogâmicos C57BL , Fator 1 de Transcrição de Octâmero , Receptores Nucleares Órfãos , Osteopontina , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fos/análise , RNA Mensageiro/análise , Receptores Citoplasmáticos e Nucleares/agonistas , Fatores de Transcrição/fisiologia , Fatores Estimuladores UpstreamRESUMO
Angiotensin II (Ang II) is a powerful accelerator of atherosclerosis. Herein, we describe a novel transcription mechanism through which Ang II inhibits macrophage expression of the ATP-binding cassette transporter A1 (ABCA1), a key regulator of reverse cholesterol transport. We demonstrate that chronic Ang II infusion substantially promotes macrophage infiltration, foam cell formation, and atherosclerosis in low-density lipoprotein receptor-deficient mice and significantly reduces ABCA1 expression in peripheral macrophages. Administration of the Ang II type 1 receptor blocker valsartan inhibited Ang II-induced ABCA1 mRNA repression, macrophage cholesterol accumulation, and atherosclerosis. Ang II treatment reduced ABCA1 promoter activity of in vitro cultured mouse peritoneal macrophages, inducing fos-related antigen 2 (Fra2) protein binding to an ABCA1 promoter E-box motif, a site known to negatively regulate macrophage ABCA1 transcription. Valsartan pretreatment blocked Fra2 binding to the ABCA1 promoter, and Fra2 small interfering RNA pretreatment attenuated Ang II-mediated ABCA1 transcriptional inhibition, confirming the role of Fra2 in this process. This new evidence suggests that Ang II, a well-known proinflammatory and pro-oxidative factor, alters macrophage cholesterol homeostasis by repressing ABCA1 to promote foam cell formation and atherosclerosis.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Angiotensina II/farmacologia , Aterosclerose/induzido quimicamente , Macrófagos/metabolismo , Proteínas Repressoras/farmacologia , Transportador 1 de Cassete de Ligação de ATP , Animais , Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/genética , Colesterol/metabolismo , Antígeno 2 Relacionado a Fos/genética , Antígeno 2 Relacionado a Fos/fisiologia , Camundongos , Regiões Promotoras Genéticas , RNA Mensageiro/análise , RNA Interferente Pequeno/farmacologia , Receptores de LDL/fisiologia , Transcrição Gênica/efeitos dos fármacosRESUMO
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily. The 3 PPAR isotypes, PPAR-alpha, PPAR-gamma, and PPAR-delta, play a key role in the regulation of lipid and glucose metabolism. Obesity and the interrelated disorders of the metabolic syndrome have become a major worldwide health problem. In this review, we summarize the critical role of PPARs in regulating inflammation, lipoprotein metabolism, and glucose homeostasis and their potential implications for the treatment of obesity, diabetes, and atherosclerosis.
Assuntos
Aterosclerose/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Obesidade/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Animais , Aterosclerose/complicações , Aterosclerose/terapia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/terapia , Humanos , Obesidade/complicações , Obesidade/terapiaRESUMO
Peroxisome proliferator-activated receptor (PPAR) gamma is activated by thiazolidinediones (TZDs), widely used as insulin-sensitizing agents for the treatment of type 2 diabetes. TZDs have been shown to induce apoptosis in a variety of mammalian cells. In vascular smooth muscle cells (VSMCs), proliferation and apoptosis may be competing processes during the formation of restenotic and atherosclerotic lesions. The precise molecular mechanisms by which TZDs induce apoptosis in VSMCs, however, remain unclear. In the present study, we demonstrate that the TZDs rosiglitazone (RSG), troglitazone (TRO), and a novel non-TZD partial PPARgamma agonist (nTZDpa) induce caspase-mediated apoptosis of human coronary VSMCs. Induction of VSMC apoptosis correlated closely with an upregulation of growth arrest and DNA damage-inducible gene 45 (GADD45) mRNA expression and transcription, a well-recognized modulator of cell cycle arrest and apoptosis. Using adenoviral-mediated overexpression of a constitutively active PPARgamma mutant and the irreversible PPARgamma antagonist GW9662, we provide evidence that PPARgamma ligands induce caspase-mediated apoptosis and GADD45 expression through a receptor-dependent pathway. Deletion analysis of the GADD45 promoter revealed that a 153-bp region between -234 and -81 bp proximal to the transcription start site, containing an Oct-1 element, was crucial for the PPARgamma ligand-mediated induction of the GADD45 promoter. PPARgamma activation induced Oct-1 protein expression and DNA binding and stimulated activity of a reporter plasmid driven by multiple Oct-1 elements. These findings suggest that activation of PPARgamma can lead to apoptosis and growth arrest in VSMCs, at least in part, by inducing Oct-1-mediated transcription of GADD45. The full text of this article is available online at http://www.circresaha.org.
Assuntos
Músculo Liso Vascular/metabolismo , Proteínas/genética , Receptores Citoplasmáticos e Nucleares/fisiologia , Tiazolidinedionas , Fatores de Transcrição/fisiologia , Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Sítios de Ligação/genética , Northern Blotting , Inibidores de Caspase , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Cromanos/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Fator C1 de Célula Hospedeira , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Luciferases/genética , Luciferases/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Mutação , Fator 1 de Transcrição de Octâmero , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/agonistas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Rosiglitazona , Tiazóis/farmacologia , Fatores de Transcrição/agonistas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Troglitazona , Proteínas GADD45RESUMO
Integrins play an important role in vascular smooth muscle cell (VSMC) migration, a crucial event in the development of restenosis and atherosclerosis. Transforming growth factor-beta (TGF-beta) is highly expressed in restenotic and atherosclerotic lesions, and known to induce integrin expression. Peroxisome proliferator-activated receptor alpha (PPARalpha), a member of the nuclear receptor superfamily, regulates gene expression in a variety of vascular cells. We investigated the effects of PPARalpha ligands on TGF-beta-induced beta3 and beta5 integrin expression and potential interaction between PPARalpha and TGF-beta signaling. PPARalpha ligands WY-14643 (100 micromol/L) and 5,8,11,14-eicosatetranoic acid (ETYA, 50 micromol/L) inhibited TGF-beta-induced beta5 integrin protein expression by 72+/-6.8% and 73+/-7.1%, respectively (both P<0.05). TGF-beta-stimulated beta3 integrin expression was not affected by PPARalpha ligands. Both PPARalpha ligands also suppressed TGF-beta-induced beta5 integrin mRNA levels. PPARalpha ligands inhibited TGF-beta-inducible transcription of beta5 integrin by an interaction with a TGF-beta response element between nucleotides -63 and -44, which contains a Sp1/Sp3 transcription factor binding site. Nuclear complexes binding to the TGF-beta response region contained Sp1/Sp3 and TGF-beta-regulated Smad 2, 3, and 4 transcription factors. TGF-beta-stimulated Sp1/Smad4 nuclear complex formation was inhibited by WY-14643 and ETYA with a parallel induction of PPARalpha/Smad4 interactions. However, in vitro pull-down experiments failed to demonstrate direct binding between PPARalpha/Smad4. Both PPARalpha ligands blocked PDGF-directed migration of TGF-beta-pretreated VSMCs, a process mediated, in part, by beta5 integrins. The present study demonstrates that PPARalpha activators inhibit TGF-beta-induced beta5 integrin transcription in VSMCs through a novel indirect interaction between ligand-activated PPARalpha and the TGF-beta-regulated Smad4 transcription factors. The full text of this article is available at http://www.circresaha.org.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Cadeias beta de Integrinas/biossíntese , Músculo Liso Vascular/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/farmacologia , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Ácido 5,8,11,14-Eicosatetrainoico/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Cadeias beta de Integrinas/genética , Integrina beta3/biossíntese , Integrina beta3/genética , Ligantes , Substâncias Macromoleculares , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Pirimidinas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteína Smad4RESUMO
The liver X receptors alpha and beta (LXRalpha and LXRbeta) are important regulators of cholesterol homeostasis in liver and macrophages. Synthetic LXR ligands prevent the development of atherosclerosis in murine models; however, the potential functional relevance of LXRs in vascular smooth muscle cells (VSMCs) has not been investigated. In the present study, we demonstrate that LXRs are expressed and functional in primary human coronary artery VSMCs (CASMCs). LXR ligands inhibited mitogen-induced VSMC proliferation and G1-->S phase progression of the cell cycle. Inhibition of G1 exit by LXR ligands was accompanied by a dose-dependent inhibition of retinoblastoma protein (Rb) phosphorylation, which functions as the key switch for G1-->S cell cycle progression. LXR ligands suppressed mitogen-induced degradation of the cyclin-dependent kinase inhibitor p27Kip1, attenuated cyclin D1 and cyclin A expression, and inhibited the expression of S phase-regulatory minichromosome maintenance protein 6. Stabilization of p27kip1 by LXR ligands was mediated by supressing the transcriptional activation of the S phase kinase-associated protein 2 (Skp2), an F-box protein that targets p27Kip1 for degradation. Inhibition of Rb phosphorylation and G1-->S cell cycle progression by LXR ligands was reversed in VSMCs overexpressing Skp2, indicating that Skp2 as an upstream regulator of p27Kip1 degradation plays a central role in LXR ligand-mediated inhibition of VSMC proliferation. Furthermore, adenovirus-mediated overexpression of the S phase transcription factor E2F, which is released after Rb phosphorylation, reversed the inhibitory effect of LXR ligands on VSMC proliferation and S phase gene expression, suggesting that the primary mechanisms by which LXR ligands inhibit VSMC proliferation occur upstream of Rb phosphorylation. Finally, neointima formation in a model of rat carotid artery balloon injury was significantly attenuated after treatment with the LXR ligand T1317 compared with vehicle-treated animals. These data demonstrate that LXR ligands inhibit VSMC proliferation and neointima formation after balloon injury and suggest that LXR ligands may constitute a novel therapy for proliferative vascular diseases. The full text of this article is available online at http://circres.ahajournals.org.
Assuntos
Lesões das Artérias Carótidas/patologia , Proteínas de Ligação a DNA/fisiologia , Músculo Liso Vascular/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Túnica Íntima/patologia , Túnica Média/patologia , Animais , Anticolesterolemiantes/farmacologia , Benzoatos/farmacologia , Benzilaminas/farmacologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Vasos Coronários/citologia , Inibidor de Quinase Dependente de Ciclina p27 , Proteínas de Ligação a DNA/agonistas , Fase G1/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidrocarbonetos Fluorados , Hiperplasia , Insulina/farmacologia , Ligantes , Receptores X do Fígado , Componente 6 do Complexo de Manutenção de Minicromossomo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Receptores Nucleares Órfãos , Fosforilação/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/agonistas , Proteínas Recombinantes de Fusão/fisiologia , Proteína do Retinoblastoma/metabolismo , Proteínas Quinases Associadas a Fase S/biossíntese , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/fisiologia , Sulfonamidas , Transfecção , Proteínas Supressoras de Tumor/metabolismo , Túnica Íntima/efeitos dos fármacos , Túnica Média/efeitos dos fármacosRESUMO
The incidence of diabetes, now affecting more than 170 million individuals is growing rapidly. Type 2 diabetes, which accounts for 90% of all diabetes cases, is associated with increased cardiovascular morbidity and mortality. Thiazolidinediones (TZDs), used for the treatment of patients with type 2 diabetes improve insulin sensitivity and endothelial dysfunction and exert beneficial effects on the lipid profile by activating the peroxisome proliferator-activated receptor gamma (PPAR-gamma). Moreover, a large body of evidence indicates that TZDs exhibit antiatherogenic effects independent of their antidiabetic and lipid-lowering properties by modulating inflammatory processes. This review will focus on the role of PPAR-gamma agonists in the vessel wall and summarize their effects on C-reactive protein (CRP), plasminogen activator inhibitor type-1 (PAI-1), matrix metalloproteinase-9 (MMP-9), adiponectin and ATP-binding cassette transporter A1 (ABCA1) and their implications for treatment of advanced stages of atherosclerosis, particularly in a setting of type 2 diabetes.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , PPAR gama/agonistas , Tiazolidinedionas/farmacologia , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adiponectina/metabolismo , Animais , Aterosclerose/complicações , Aterosclerose/tratamento farmacológico , Proteína C-Reativa/metabolismo , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Endotélio Vascular/metabolismo , Humanos , Hipoglicemiantes/uso terapêutico , Metaloproteinase 9 da Matriz/metabolismo , Músculo Liso Vascular/metabolismo , PPAR gama/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ensaios Clínicos Controlados Aleatórios como Assunto , Tiazolidinedionas/uso terapêuticoRESUMO
We previously reported that the PPARgamma agonist troglitazone (TRO) inhibits proliferation and induces apoptosis in human MCF-7 breast carcinoma cells. To understand the mechanisms of antiproliferative and pro-apoptotic effects of TRO, we screened a limited DNA array containing 23 genes involved in regulating either the cell cycle and/or apoptosis. Four of the 23 genes screened exhibited regulation by TRO, with growth arrest and DNA damage-inducible gene 45 (GADD45) being the most strongly upregulated. TRO induced GADD45 mRNA expression in a time- and dose-dependent manner. Depletion of GADD45 by siRNA abrogated TRO-induced apoptosis in MCF-7 cells demonstrating the physiological relevance of GADD45 upregulation. Signaling pathways mediating TRO-induced GADD45 were also investigated. Several mitogen-activated protein kinase (MAPK) pathways were involved in the induction of GADD45 by TRO. Inhibition of the c-jun N-terminal kinase MAPK pathway by SP600125 partially abolished TRO-induced GADD45 mRNA, and protein expression and apoptosis. In contrast, inhibition of the p38 MAPK pathway by SB203580, or through overexpression of a dominant-negative mutant of p38 MAPK, augmented GADD45 mRNA induction and GADD45 promoter activation as well as cell apoptosis by TRO. Blockade of the extracellular signal-regulated kinase MAPK pathway by PD98059 also enhanced TRO's effects on GADD45 and apoptosis. Two other PPARgamma agonists pioglitazone and rosiglitazone did not induce GADD45 expression. Our finding of GADD45 induction by TRO may provide a new insight concerning the mechanisms for TRO's antiproliferative and pro-apoptotic effects in breast cancer cells.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Carcinoma/patologia , Cromanos/farmacologia , Proteínas/genética , Transdução de Sinais , Tiazolidinedionas/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Carcinoma/tratamento farmacológico , Carcinoma/genética , Ciclo Celular/genética , Inibidores Enzimáticos/farmacologia , Feminino , Flavonoides/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Imidazóis/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Regiões Promotoras Genéticas , Proteínas/efeitos dos fármacos , Proteínas/metabolismo , Piridinas/farmacologia , RNA Interferente Pequeno , Troglitazona , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por Mitógeno , Proteínas GADD45RESUMO
BACKGROUND: The proliferation of vascular smooth muscle cells (VSMCs) is a known response to arterial injury that is an important part of the process of restenosis and atherosclerosis. People with diabetes have an increased risk of cardiovascular disease resulting from accelerated coronary atherosclerosis. The newest drugs for Type 2 diabetes are thiazolidinediones, which are insulin-sensitizing peroxisome proliferator activating receptor-gamma (PPARgamma) ligands. We investigated the antiproliferative effects of troglitazone, rosiglitazone, and pioglitazone on VSMCs derived from the three vascular beds used for coronary artery by-pass grafting: the internal mammary and radial artery and saphenous veins. METHODS AND RESULTS: The three vessels yielded proliferating cells of slightly differing morphology. Inhibition of cell proliferation was assessed by cell counting and cell cycle studies by Western blotting for phosphorylated retinoblastoma protein. All three thiazolidinediones showed inhibitory potency toward cell proliferation with a potency troglitazone>rosiglitazone approximately pioglitazone, and this potency profile was maintained toward the growth factor and insulin-stimulated phosphorylation of the retinoblastoma protein, which controls cell cycle progression. CONCLUSIONS: The inhibitory potency of clinical thiazolidinediones toward different vascular sources is dependent on the individual thiazolidinedione and very little on the vascular source.
Assuntos
Cromanos/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/metabolismo , Tiazóis/farmacologia , Tiazolidinedionas , Fatores de Transcrição/metabolismo , Becaplermina , Contagem de Células , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Cromanos/metabolismo , Relação Dose-Resposta a Droga , Humanos , Hipoglicemiantes/metabolismo , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Ligantes , Artéria Torácica Interna/citologia , Mitógenos/farmacologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Fosforilação/efeitos dos fármacos , Pioglitazona , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-sis , Artéria Radial/citologia , Proteína do Retinoblastoma/metabolismo , Rosiglitazona , Veia Safena/citologia , Tiazóis/metabolismo , TroglitazonaRESUMO
BACKGROUND: Accumulating evidence suggests that C-reactive protein (CRP), in addition to being a predictor of coronary events, may have direct actions on the vessel wall in the evolution of atherosclerosis. Although accumulation of vascular smooth muscle cells (VSMCs) in the intima is a key event in the development of arterial lesions, apoptosis of VSMCs also plays an important role in progression of atherosclerotic lesions and contributes to increased plaque vulnerability. METHODS AND RESULTS: In the present study we demonstrate that CRP induces caspase-mediated apoptosis of human coronary VSMCs. DNA microarray analysis was used to identify CRP-regulated genes. The growth arrest- and DNA damage-inducible gene 153 (GADD153) mRNA expression was prominently upregulated by CRP. As confirmed by Northern blot analysis, CRP induced a time- and dose-dependent increase of GADD153 mRNA expression. GADD153, a gene involved in growth arrest and apoptosis in vascular and nonvascular cells, is regulated at both transcriptional and posttranscriptional levels. CRP regulation of GADD153 mRNA expression in VSMCs occurs primarily at the posttranscriptional level by mRNA stabilization. Small interfering RNA (siRNA) specifically targeted to GADD153 reduced CRP-induced apoptosis. GADD153 also specifically colocalized to apoptotic VSMCs in human coronary lesions, further supporting a functional role for GADD153 in CRP-induced cell death. CONCLUSIONS: These results demonstrate that GADD153 is a CRP-regulated gene in human VSMCs and plays a causal role in CRP-induced apoptosis. Pharmacological targeting of CRP expression or action may provide a novel therapy for atherosclerosis.
Assuntos
Apoptose/efeitos dos fármacos , Proteína C-Reativa/farmacologia , Vasos Coronários/citologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Humanos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Estimulação QuímicaRESUMO
OBJECTIVES: Osteopontin (OPN) is upregulated in left ventricular hypertrophy and is stimulated by angiotensin II (AngII). Our objective was to determine whether mice deficient in OPN would be protected from AngII-induced cardiac fibrosis. BACKGROUND: Interstitial fibrosis can lead to myocardial dysfunction and ultimately heart failure. Osteopontin activates integrins that regulate cell adhesion, migration, and growth, thus implicating OPN in the process of cardiac fibrosis. METHODS: Osteopontin null (OPN(-/-)) mice (n = 18) and wild-type controls (n = 20) were infused with AngII (2.5 or 3.0 microg/kg/min) for four days or three weeks via osmotic mini-pumps. Hearts were assessed morphometrically and histologically, including quantitative assessment of fibrosis via optical microscopic imaging analysis. Cardiac fibroblasts derived from these mice were evaluated for adhesion and proliferation. Cardiac transcript expression for cytokines, extracellular matrix (ECM), integrin, and atrial natriuretic peptide were assessed. RESULTS: Osteopontin(-/-) mice exhibited less cardiac fibrosis (0.7%) than wild-type mice (8.0%) (p < 0.01) and lowered heart/body weight ratios (0.10% vs. 0.23%) (p < 0.01) after three weeks of AngII infusion. Expression of transforming growth factor-beta, fibronectin, and collagen was not different between OPN(-/-) and wild-type mice, despite the decrease in ECM accumulation in the OPN(-/-) mice. Adhesion to ECM substrates decreased by 30% to 50% in cardiac fibroblasts of OPN(-/-) mice but was restored in OPN(-/-) cells by the addition of recombinant osteopontin. CONCLUSIONS: Osteopontin mediates cardiac fibrosis, probably through the modulation of cellular adhesion and proliferation. Because OPN is increased in cardiac hypertrophy and its lack attenuates fibrosis, understanding of OPN function is essential to extend our knowledge about molecular determinants of cardiac hypertrophy and failure.
Assuntos
Angiotensina II/efeitos adversos , Miocárdio/metabolismo , Miocárdio/patologia , Sialoglicoproteínas/deficiência , Vasoconstritores/efeitos adversos , Angiotensina II/administração & dosagem , Animais , Fator Natriurético Atrial/efeitos dos fármacos , Fator Natriurético Atrial/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Northern Blotting , Cardiomegalia/etiologia , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Camundongos , Camundongos Knockout , Modelos Cardiovasculares , Proteínas Musculares , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Osteopontina , Proteínas Repressoras/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Regulação para Cima/efeitos dos fármacos , Vasoconstritores/administração & dosagemRESUMO
The Liver X Receptors, LXRalpha and LXRbeta are members of the nuclear hormone receptor superfamily which have recently been implicated as novel pharmacological targets for the treatment of cardiovascular diseases. The identification of natural and synthetic ligands for LXRs and the generation of LXR-deficient mice have been crucial for our understanding of the function of these receptors and for the identification of LXR-regulated target genes, particularly with respect to the role of LXRs in regulating cholesterol homeostasis. Synthetic LXRalpha/beta agonists induce cholesterol efflux and reverse cholesterol transport, improve glucose metabolism, inhibit macrophage-derived inflammation, and suppress the proliferation of vascular smooth muscle cells. By regulating the expression of multiple genes involved in these pathways, LXR agonists prevent the development and progression of atherosclerosis and inhibit neointima formation following angioplasty of the arterial wall. In this review, we will summarize the important roles of LXR in metabolism and vascular biology and discuss its implications as potential molecular drug target for the treatment of cardiovascular diseases.
Assuntos
Proteínas de Ligação a DNA/agonistas , Receptores Citoplasmáticos e Nucleares/agonistas , Vasculite/tratamento farmacológico , Animais , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/fisiologia , Progressão da Doença , Humanos , Receptores X do Fígado , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Receptores Nucleares Órfãos , Receptores Citoplasmáticos e Nucleares/fisiologia , Vasculite/fisiopatologia , Vasculite/prevenção & controleRESUMO
Using a cDNA array consisting only of cell cycle genes, we found that a novel nonthiazolidinedione partial peroxisome proliferator-activated receptor gamma (PPARgamma) agonist (nTZDpa) inhibited expression of minichromosome maintenance (MCM) proteins 6 and 7 in vascular smooth muscle cells. MCM proteins are required for the initiation and elongation stages of DNA replication and are regulated by the transcription factor E2F. Mitogen-induced MCM6 and MCM7 mRNA expression was potently inhibited by nTZDpa and to a lesser degree by the full PPARgamma agonist, rosiglitazone. Inhibition of MCM6 and MCM7 expression by nTZDpa and rosiglitazone paralleled their effect to inhibit phosphorylation of the retinoblastoma protein and cell proliferation. Transient transfection experiments revealed that the nTZDpa inhibited mitogen-induced MCM6 and MCM7 promoter activity, implicating a transcriptional mechanism. Adenoviral-mediated E2F overexpression reversed the suppressive effect of nTZDpa on MCM6 and MCM7 expression. Furthermore, activity of a luciferase reporter plasmid driven by multiple E2F elements was inhibited by nTZDpa, indicating that their down-regulation by nTZDpa involves an E2F-dependent mechanism. Overexpression of dominant-negative PPARgamma or addition of a PPARgamma antagonist, GW 9662, blocked nTZDpa inhibition of MCM7 transcription. Adenovirus-mediated overexpression of constitutively active PPARgamma inhibited MCM7 expression in a similar manner as the nTZDpa. These findings provide strong evidence that activation of PPARgamma attenuates MCM7 transcription and support the important role of this nuclear receptor in regulating vascular smooth muscle cell proliferation.
Assuntos
Proteínas de Ciclo Celular/genética , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Indóis/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Proteínas Nucleares/genética , Receptores Citoplasmáticos e Nucleares/agonistas , Sulfetos/farmacologia , Tiazolidinedionas/farmacologia , Fatores de Transcrição/agonistas , Ativação Transcricional/efeitos dos fármacos , Animais , Aorta/citologia , Proteínas de Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Músculo Liso Vascular/metabolismo , Proteínas Nucleares/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Rosiglitazona , Fase S/efeitos dos fármacosRESUMO
Adipose tissue is a dynamic endocrine organ that secretes a number of factors that are increasingly recognized to contribute to systemic and vascular inflammation. Several of these factors, collectively referred to as adipokines, have now been shown regulate, directly or indirectly, a number of the processes that contribute to the development of atherosclerosis, including hypertension, endothelial dysfunction, insulin resistance, and vascular remodeling. Several adipokines are preferentially expressed in visceral adipose tissue, and the secretion of proinflammatory adipokines is elevated with increasing adiposity. Not surprisingly, approaches that reduce adipose tissue depots, including surgical fat removal, exercise, and reduced caloric intake, improve proinflammatory adipokine levels and reduce the severity of their resultant pathologies. Systemic adipokine levels can also be favorably altered by treatment with several of the existing drug classes used to treat insulin resistance, hypertension, and hypercholesterolemia. Greater understanding of adipokine regulation, however, should result in the design of improved treatment strategies to control disease states associated with increase adiposity, an important outcome in view of the growing worldwide epidemic of obesity.
Assuntos
Tecido Adiposo/metabolismo , Arteriosclerose/metabolismo , Obesidade/metabolismo , Vasculite/metabolismo , Tecido Adiposo/imunologia , Animais , Arteriosclerose/imunologia , Humanos , Obesidade/imunologia , Vasculite/imunologiaRESUMO
Atherosclerosis is a complex, chronic disease state that usually arises from the converging action of several pathogenic processes, including hypertension, hyperlidemia, obesity and insulin resistance. Significantly, due to the increasing incidence of type 2 diabetes worldwide, several aspects of the renin-angiotensin system, including the capacity for angiotensin II synthesis and binding are increased in human and animal models of type II diabetes, and potentiate vascular lesion formation. Angiotensin II, an important vasoactive peptide of the renin-angiotensin system, profoundly accelerates atherosclerosis in animal models of diabetes. Conversely, in both human and animal studies, inhibition of angiotensin II synthesis or activity has been shown to significantly reduce atherosclerosis and cardiovascular mortality. Cardiovascular protection is independent of blood pressure and baseline activity of the renin-angiotensin system, suggesting an important and direct role for the vascular renin-angiotensin system in atherosclerotic progression. Angiotensin II appears to accelerate atherosclerosis through activation of several distinct signal transduction pathways, and via these mechanisms can function as a vascular growth and migration factor, a pro-inflammatory cytokine and an oxidative stress agent. Thiazolidinediones, a class of oral insulin-sensitizing agents in broad clinical use for the treatment of type 2 diabetes, have been shown to ameliorate cardiovascular disease in animal trials and clinical studies. Thiazolidinediones also appear to regulate angiotensin II signaling at multiple levels, significantly reducing the expression of the angiotensin II type 1 receptor and repressing signal transduction through this receptor to suppress vascular remodeling, lesion formation, and oxidative stress.
Assuntos
Angiotensina II/fisiologia , Arteriosclerose/fisiopatologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Angiotensina II/metabolismo , Animais , Arteriosclerose/metabolismo , Vasos Sanguíneos/metabolismo , Modelos Animais de Doenças , Humanos , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Vascular smooth muscle cells (VSMCs) in the media of adult arteries are normally quiescent, proliferate at low frequency, and are arrested in the G(0)/G(1) phase of the cell cycle. Proliferation of VSMCs occurs in response to arterial injury and plays a crucial role in the atherosclerotic process and in the pathogenesis of restenosis. Patients with type 2 diabetes mellitus are at increased risk for postangioplasty restenosis, which results from excessive intimal hyperplasia. Insulin sensitizers of the thiazolidinedione (TZD) class inhibit growth of VSMCs by attenuating the activity of important cell-cycle regulators. The TZDs inhibit progression from G(1) to S phase in the cell cycle by blocking growth factor-induced phosphorylation of retinoblastoma tumor suppressor protein (Rb). In animal models of restenosis, TZDs inhibit intimal hyperplasia after mechanical injury in both insulin-sensitive and insulin-resistant vessels. Preliminary clinical studies using troglitazone demonstrate less intimal hyperplasia with this TZD after implantation of coronary stents in individuals with type 2 diabetes. Further large trials are needed to confirm that treatment with a TZD can protect against postangioplasty restenosis.
Assuntos
Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Implante de Prótese Vascular , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/efeitos dos fármacos , Doença da Artéria Coronariana/fisiopatologia , Doença da Artéria Coronariana/terapia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/fisiopatologia , Humanos , Resistência à Insulina/fisiologia , Músculo Liso Vascular/fisiopatologia , Fatores de Risco , StentsRESUMO
Protein kinase C (PKC), a kinase family involved in cell signal transduction, is overexpressed in most pituitary adenoma cells. We studied the effect of tamoxifen, an estrogen receptor antagonist and also a protein kinase inhibitor, on pituitary tumor cell proliferation and the induction of apoptosis; and we compared its effects with those of another PKC inhibitor, staurosporine. Tamoxifen induced growth arrest and apoptosis in a mouse pituitary adenoma cell line, AtT20, and in low-passage human primary pituitary tumor cell cultures. Staurosporine also inhibited pituitary tumor cell growth. PKC activity in AtT20 cells was inhibited by staurosporine and by prolonged treatment with phorbol myristate acetate, which down-regulates PKC activity, but not by tamoxifen, at the dosages used to induce apoptosis. Our findings suggest that tamoxifen induces apoptosis in AtT20 cells independent of a classical PKC isozyme pathway.
Assuntos
Adenoma/patologia , Apoptose/efeitos dos fármacos , Carcinoma/patologia , Proteínas de Neoplasias/fisiologia , Neoplasias Hipofisárias/patologia , Proteína Quinase C/fisiologia , Transdução de Sinais/fisiologia , Tamoxifeno/farmacologia , Adenoma/enzimologia , Adulto , Animais , Carcinoma/enzimologia , Divisão Celular/efeitos dos fármacos , Fragmentação do DNA , Inibidores Enzimáticos/farmacologia , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/fisiologia , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias Hipofisárias/enzimologia , Proteína Quinase C/antagonistas & inibidores , Estaurosporina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologia , Células Tumorais Cultivadas/patologiaRESUMO
Several peroxisome proliferator-activated receptor gamma (PPARgamma) agonists of the thiazolidinedione class inhibit vascular smooth muscle cell proliferation. It is not known whether the antiproliferative activity of PPARgamma agonists is limited to the thiazolidinedione class and/or is directly mediated through PPARgamma-dependent transactivation of target genes. We report here that a novel non-thiazolidinedione partial PPARgamma agonist (nTZDpa) attenuates rat aortic vascular smooth muscle cell proliferation. In a transfection assay for PPARgamma transcriptional activation, the non-thiazolidinedione partial PPARgamma agonist elicited approximately 25% of the maximal efficacy of the full PPARgamma agonist rosiglitazone. In the presence of the non-thiazolidinedione partial PPARgamma agonist, the transcriptional activity of the full agonist, rosiglitazone, was blunted, indicating that the non-thiazolidinedione partial PPARgamma agonist inhibits rosiglitazone-induced PPARgamma activity. The non-thiazolidinedione partial PPARgamma agonist (0.1-10 microM) inhibited vascular smooth muscle cell growth which was accompanied by an inhibition of retinoblastoma protein phosphorylation. Mitogen-induced downregulation of the cyclin-dependent kinase (CDK) inhibitor p27(kip1), and induction of the G1 cyclins cyclin D1, cyclin A, and cyclin E were also attenuated by the non-thiazolidinedione partial PPARgamma agonist. Maximal antiproliferative activity of the non-thiazolidinedione partial PPARgamma agonist required functional PPARgamma as adenovirus-mediated overexpression of a dominant-negative PPARgamma mutant partially reversed its inhibition of vascular smooth muscle cell growth. In contrast, overexpression of dominant-negative PPARgamma did not reverse the inhibitory effect of the non-thiazolidinedione partial PPARgamma agonist on cyclin D1. As the full PPARgamma agonist rosiglitazone exhibited no effect on cyclin D1, inhibition of that G1 cyclin by the non-thiazolidinedione partial PPARgamma agonist likely occurred through a PPARgamma-independent mechanism. These data demonstrate that a non-thiazolidinedione partial PPARgamma agonist may constitute a novel therapeutic for proliferative vascular diseases and could provide additional evidence for the important role of PPARgamma in regulating vascular smooth muscle cell proliferation.