RESUMO
The cytokine resistin and the chemokine fractalkine (FKN) were found at increased levels in human atherosclerotic plaque, in the subendothelium, but their role in this location still needs to be characterized. Recently, high local resistin in the arterial vessel wall was shown to contribute to an enhanced accumulation of macrophages by mechanisms that need to be clarified. Our recent data showed that resistin activated smooth muscle cells (SMC) by up-regulating FKN and MCP-1 expression and monocyte chemotaxis by activating toll-like receptor 4 (TLR4) and Gi/o proteins. Since in the vessel wall both endothelial cells (EC) and SMC respond to cytokines and promote atherosclerosis, we questioned whether subendothelial resistin (sR) has a role in vascular cells cross-talk leading to enhanced monocyte transmigration and we investigated the mechanisms involved. To this purpose we used an in vitro system of co-cultured SMC and EC activated by sR and we analyzed monocyte transmigration. Our results indicated that: (1) sR enhanced monocyte transmigration in EC/SMC system compared to EC cultured alone; (2) sR activated TLR4 and Gi/o signaling in EC/SMC system and induced the secretion of more FKN and MCP-1 compared to EC cultured alone and used both chemokines to specifically recruit monocytes by CX3CR1 and CCR2 receptors. Moreover, FKN produced by resistin in EC/SMC system, by acting on CX3CR1 on EC/SMC specifically contributes to MCP-1 secretion in the system and to the enhanced monocyte transmigration. Our study indicates new possible targets for therapy to reduce resistin-dependent enhanced macrophage infiltration in the atherosclerotic arterial wall.
Assuntos
Quimiocina CCL2/metabolismo , Quimiocina CX3CL1/metabolismo , Células Endoteliais/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Miócitos de Músculo Liso/metabolismo , Resistina/metabolismo , Receptor 4 Toll-Like/metabolismo , Humanos , Monócitos/metabolismo , Transdução de SinaisRESUMO
During the early phase of atherosclerosis, monocytes attach to and migrate through the vessel wall where they activate and communicate with smooth muscle cells (SMC) affecting plaque progression by largely unknown mechanisms. Activation of STAT3 transcription factor is suggested to be critically involved in dedifferentiation, migration, and proliferation of SMC in the neointima formation after vascular injury. Monocytes-SMC cross-talk induces an inflammatory phenotype of the resident SMC, but the involvement of STAT3 in phenotype switching is not known. Resistin is a cytokine found in human atheroma associated to monocytes/macrophages with role in inflammation associated with cardiovascular disease. The aim of this study was to follow the effect of activated monocytes-SMC cross-talk on STAT3 activation and subsequent resistin and reactive oxygen species (ROS) production. Our results showed that the interaction of activated monocytes with SMC determines: (i) phosphorylation of STAT3 and reduction of SOCS3 expression in both cell types; (ii) intracellular ROS production dependent on NADPH oxidase (by increased Nox1 expression) and STAT3 activation in SMC; (iii) up-regulation of resistin expression in monocytes dependent on STAT3 activation. Furthermore, exposure of SMC to resistin induces ROS by increasing NADPH oxidase activity and the p22phox and Nox1 expression. In conclusion, the cross-talk between SMC and monocytes activates STAT3 transcription factor and lead to resistin up-regulation in monocytes and ROS production in SMC. Moreover, resistin increases the ROS levels in SMC. These data indicate that monocyte-SMC communication may represent an important factor for progression of the atherosclerotic lesion.
Assuntos
Monócitos/citologia , Monócitos/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Resistina/metabolismo , Fator de Transcrição STAT3/metabolismo , Western Blotting , Separação Celular , Células Cultivadas , Humanos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Resistin is a significant local and systemic regulatory cytokine involved in inflammation. Suppressors of cytokine signaling (SOCS) proteins are intracellular regulators of receptor signal transduction induced by several cytokines in a cytokine and cell specific manner. Resistin up-regulates SOCS3 expression in mice adipocytes but it is not known whether this is a common occurrence in other cells. We questioned whether resistin-induces SOCS3 in human endothelial cells and if signal transducer and activator of transcription (STAT) proteins are involved in the process. The Real-Time PCR and Western blot analysis showed that in resistin-activated HEC the gene and protein expression of SOCS3 were significantly increased. Furthermore, resistin induced activation of STAT3 as characterized by increased tyrosine phosphorylation. Resistin-induced SOCS3 expression was blocked by specific inhibitors of STAT3 signaling and by the transfection of siRNA specific for STAT3. Silencing of SOCS3 gene expression by transfection with SOCS3 siRNA reduced the expression of resistin induced-P-selectin and fractalkine in HEC. Together, our results demonstrate that in HEC (1) resistin up-regulates SOCS3 expression and activates STAT3 transcription factor; (2) the increase in SOCS3 mRNA and protein expression as well as STAT3 activation have a long-lasting effect (up to 18h); (3) inhibition of SOCS3 function prevents resistin-induced expression of cell adhesion molecules P-selectin and fractalkine and thus activation of endothelial cells. The data uncover a new resistin-mediated mechanism in human endothelial cells and designate SOCS3 as a novel therapeutic target to modulate resistin-dependent inflammation in vessel wall diseases.
Assuntos
Células Endoteliais/metabolismo , Inflamação/genética , Resistina/fisiologia , Fator de Transcrição STAT3/metabolismo , Proteínas Supressoras da Sinalização de Citocina/genética , Ativação Transcricional , Células Endoteliais/efeitos dos fármacos , Humanos , Resistina/farmacologia , Fator de Transcrição STAT3/agonistas , Proteína 3 Supressora da Sinalização de Citocinas , Regulação para CimaRESUMO
OBJECTIVE: In atherosclerotic lesions, fractalkine (CX3CL1) and its receptor (CX3CR1) expressed by smooth muscle cells (SMC) and monocytes/macrophages, mediate the heterotypic anchorage and chemotaxis of these cells. We questioned whether, during the close interaction of monocytes with SMC, the CX3CL1/CX3CR1 pair modulates the expression of pro-atherogenic molecules in these cells. METHODS AND RESULTS: SMC were co-cultured with monocytes or LPS-activated monocytes (18h) and then the cells were separated and individually investigated for the gene and protein expression of TNFα, IL-1ß, IL-6, CX3CR1 and metalloproteinases (MMP-2, MMP-9). We found that SMC-monocyte interaction induced, in each cell type, an increased mRNA and protein expression of TNFα, IL-1ß, IL-6, CX3CR1, MMP-2 and MMP-9. Blocking the binding of fractalkine to CX3CR1 (by pre-incubation of monocytes with anti-CX3CR1 or by CX3CR1 siRNA transfection) before cell co-culture decreased the production of TNFα, CX3CR1 and MMP-9. Monocyte-SMC interaction induced the phosphorylation of p38MAPK and activation of AP-1 transcription factor. Silencing the p65 (NF-kB subunit) inhibited the IL-1ß and IL-6 and silencing c-jun inhibited the TNFα, CX3CR1 and MMP-9 induced by SMC-monocyte interaction. CONCLUSIONS: The cross-talk between SMC and monocytes augments the inflammatory response in both cell types as revealed by the increased expression of TNFα, IL-1ß, IL-6, CX3CR1 and MMPs. Up-regulation of TNFα, CX3CR1 and MMP-9 is further increased upon interaction of SMC with activated monocytes and is dependent on fractalkine/CXRCR1 pair. These data imply that the fractalkine/CX3RCR1 axis may represent a therapeutic target to impede the inflammatory process associated with atherosclerosis.
Assuntos
Aorta/metabolismo , Quimiocina CX3CL1/metabolismo , Monócitos/metabolismo , Músculo Liso Vascular/metabolismo , Receptores de Quimiocinas/metabolismo , Aorta/citologia , Aorta/efeitos dos fármacos , Western Blotting , Receptor 1 de Quimiocina CX3C , Células Cultivadas , Quimiocina CX3CL1/antagonistas & inibidores , Quimiocina CX3CL1/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Monócitos/citologia , Monócitos/efeitos dos fármacos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Quimiocinas/antagonistas & inibidores , Receptores de Quimiocinas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
Resistin is a cytokine which plays an important role in cardiovascular disease by influencing systemic inflammation and endothelial activation. In human endothelial cells (HEC) it increases the expression of P-selectin and fractalkine, and enhances monocyte adhesion by antioxidant mechanisms. This study investigated whether the natural antioxidants curcumin (CC) and an extract of Morus alba leaves (MA) have protective effects in resistin-activated HEC. HEC were exposed to 100 ng/mL resistin for 6 and 18 h in the absence or presence of MA or CC and the expression of fractalkine and P-selectin was determined by RT-PCR and western blot. Intracellular accumulation of reactive oxygen species (ROS) was monitored by fluorimetry and NADPH oxidase activity by a lucigenin-enhanced chemiluminescence assay. In addition, adhesion assays using the monocytic U937 cells were performed. The results showed that treatment of HEC exposed to resistin with MA and CC: (1) inhibited significantly P-selectin and fractalkine expression, (2) inhibited the increase in the intracellular ROS level, (3) reduced NADPH activation and (4) reduced monocytes adhesion to HEC. The results indicate that MA and curcumin target resistin-induced human endothelial activation partly via antioxidant mechanisms and suggest that they may represent therapeutic agents in vascular disease mediated by resistin.
Assuntos
Curcumina/química , Células Endoteliais/efeitos dos fármacos , Morus/química , Extratos Vegetais/farmacologia , Resistina/farmacologia , Antioxidantes/farmacologia , Adesão Celular , Linhagem Celular , Quimiocina CX3CL1/metabolismo , Células Endoteliais/metabolismo , Humanos , NADPH Oxidases/metabolismo , Selectina-P/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Resistin has emerged as a significant local and systemic regulatory cytokine involved in inflammation. In diabetic patients, the serum resistin level is increased, monocytes/macrophages being an important source of resistin production. We therefore hypothesize that high glucose concentrations (HG) regulate resistin expression in human monocytes. Our aim has been to uncover the potential signalling pathways involved in this process. We have also questioned whether insulin has an effect on the regulation of resistin expression induced by HG. Human monocytes (U937 cell line) were exposed to 25 mM glucose for 24 h and then resistin gene expression and protein levels were determined by reverse transcription with the polymerase chain reaction and Western blot assays. We found that (1) the gene expression and protein level of resistin were up-regulated by HG; (2) the inhibitors of the mitogen-activated protein kinases (MAPKs) p38 (SB203580), extracellular signal-regulated kinases 1/2 (ERK1/2; PD98059) and c-Jun N-terminal kinase (SP600125) and of the transcription factor nuclear factor kappa-B (PDTC) inhibited HG-induced resistin protein production and (3) insulin reduced HG-induced resistin expression via a mechanism independent of phosphatidylinositol 3-kinase (PI3K) or p38 and ERK1/2. Therefore, HG significantly increases resistin gene expression and protein production in the U937 cell line by mechanisms involving MAPKs and the transcription factor NF-kB, whereas insulin reduces its expression. This study adds new data concerning the molecular mechanisms involved in the pro-inflammatory effects of HG on human monocytes.
Assuntos
Glucose/farmacologia , Insulina/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monócitos/metabolismo , NF-kappa B/metabolismo , Resistina/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Flavonoides/farmacologia , Humanos , Imidazóis/farmacologia , Insulina/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases , Piridinas/farmacologia , Resistina/genética , Células U937 , Regulação para CimaRESUMO
Resistin and high glucose (HG) are concomitantly present at elevated concentration in diabetic's plasma; both are pro-inflammatory agents acting on vascular cells by mechanisms that are not fully understood. We questioned whether resistin and HG affect the expression of major adhesion molecules, P-selectin and fractalkine in human endothelial cells (HEC). The results showed that in HEC (i) resistin increased P-selectin expression; (ii) HG up-regulated Fk expression; (iii) P-selectin and fractalkine were functional increasing monocyte adhesion to activated cells. Co-stimulation with resistin and HG increased P-selectin and fractalkine mRNA and protein and induced monocyte adhesion, generated an increase in NADPH oxidase activity and of the intracellular reactive oxygen species and activated the NF-kB and AP-1 transcription factors at similar values as those of each activator. In conclusion in HEC, resistin and HG induce the up-regulation of P-selectin and fractalkine and the ensuing increased monocyte adhesion by a mechanism involving oxidative stress and NF-kB and AP-1 activation.
Assuntos
Quimiocina CX3CL1/biossíntese , Células Endoteliais/fisiologia , Hiperglicemia/metabolismo , Monócitos/fisiologia , Selectina-P/biossíntese , Resistina/metabolismo , Glicemia/metabolismo , Adesão Celular , Linhagem Celular , Quimiocina CX3CL1/genética , Diabetes Mellitus/sangue , Diabetes Mellitus/metabolismo , Regulação da Expressão Gênica , Glucose/farmacologia , Humanos , Monócitos/efeitos dos fármacos , NADPH Oxidases/metabolismo , NF-kappa B/metabolismo , Selectina-P/genética , Espécies Reativas de Oxigênio , Resistina/farmacologia , Fator de Transcrição AP-1/metabolismo , Regulação para CimaRESUMO
Resistin is a cytokine and fractalkine (Fk) a cell adhesion molecule and chemokine that contribute to human vascular inflammation by mechanisms not clearly defined. We questioned whether resistin induces Fk expression in human endothelial cells (HEC), compared the effect with that of the pro-inflammatory cytokine, TNF-alpha, and evaluated the consequences of co-stimulating HEC with both activators on Fk induction and on the signalling molecules involved. We found that resistin up-regulated Fk expression at comparable level to that of TNF-alpha by a mechanism involving P38 and JNK MAPK and NF-kappaB. Co-stimulation of cells with resistin and TNF-alpha did not increase Fk expression induced by every single inducer. Moreover resistin reduced the expression induced by TNF-alpha in HEC. The new data uncover Fk as a novel molecular link between resistin and inflammation and show that resistin and TNF-alpha have no additive effect in Fk up-regulation or on the signalling molecules implicated.