RESUMO
CXCL10, the chemokine with potent chemotactic activity on immune cells and other non-immune cells expressing its receptor CXCR3, has been demonstrated to involve in myocardial infarction, which was resulted from hypoxia/ischemia. The cardiac microvascular endothelial cells (CMECs) are the first cell type which is implicated by hypoxia/ischemia. However, the potential molecular mechanism by which hypoxia/ischemia regulates the expression of CXCL10 in CMECs remains unclear. In the present study, the expression of CXCL10 was firstly examined by real-time PCR and ELISA analysis. Several potential binding sites (BS) for transcription factors including NF-kappaB (NFkB), HIF1 alpha (HIF1α) and FoxO3a were identified in the promoter region of CXCL10 gene from -2000 bp to -1 bp using bioinformatics software. Luciferase reporter gene vectors for CXCL10 promoter and for activation of above transcription factors were constructed. The activation of NFkB, hypoxia-inducible transcription factor-1 alpha (HIF-1α) and FoxO3a was also analyzed by Western blotting. It was shown that the production of CXCL10 in CMECs was significantly increased by hypoxia/ischemia treatment, in parallel with the activation of CXCL10 promoter examined by reporter gene vector system. Furthermore, transcription factors including NFkB, HIF1α and FoxO3a were activated by hypoxia/ischemia in CMECs. However, over-expression of NFkB, but not that of HIF1α or FoxO3a, significantly promoted the activation of CXCL10 promoter reporter gene. These findings indicated that CXCL10 production in CMECs was significantly increased by hypoxia/ischemia, at least in part, through activation of NFkB pathway and subsequently binding to CXCL10 promoter, finally promoted the transcription of CXCL10 gene.
Assuntos
Quimiocina CXCL10/metabolismo , Vasos Coronários/citologia , Células Endoteliais/metabolismo , NF-kappa B/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Western Blotting , Hipóxia Celular , Células Cultivadas , Quimiocina CXCL10/genética , Ensaio de Imunoadsorção Enzimática , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia , NF-kappa B/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
CXCL10 is a chemokine with potent chemotactic activity for immune and non-immune cells expressing its receptor CXCR3. Previous studies have demonstrated that CXCL10 is involved in myocardial infarction. However, the role of CXCL10 in cardiac microvascular endothelial cell (CMEC) regulation and related mechanisms remains unclear. In this study, we investigated the effects of CXCL10 on the CMEC migration and explored its potential molecular mechanism by wound healing, cell proliferation and viability analysis. Furthermore, migration-related signaling pathways, including FAK, Erk, p38 and Smad, were examined by Western blotting. We found that CXCL10 significantly promotes CMEC migration under normal conditions and during hypoxia/ischemia. However, no significant differences in CMEC proliferation and viability were observed with or without CXCL10 treatment. CXCL10-mediated CMEC migration was greatly blocked by treatment with an anti-CXCR3 antibody. Although CXCL10 treatment promoted phosphorylation and activation of the FAK, Erk, and p38 pathways during hypoxia/ischemia, CXCL10-mediated CMEC migration was significantly blocked by p38 and FAK inhibitors, but not by an Erk inhibitor. Furthermore, CXCL10-mediated FAK activation was suppressed by the p38 inhibitor. These findings indicated that the CXCL10/CXCR3 pathway promotes the migration of CMECs under normal conditions and during hypoxia/ischemia in a proliferation-independent manner, at least in part, through regulation of the p38/FAK pathways.
Assuntos
Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL10/farmacologia , Células Endoteliais/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Receptores CXCR3/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Western Blotting , Hipóxia Celular , Células Cultivadas , Vasos Coronários/citologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Quinase 1 de Adesão Focal/antagonistas & inibidores , Expressão Gênica/efeitos dos fármacos , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Ratos Sprague-Dawley , Receptores CXCR3/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
FoxO3a plays an important role in the aging process and decreases with age. However, the potential regulatory roles of FoxO3a in processes involved in cardiac microvascular endothelial cell (CMEC) senescence, and its underlying molecular mechanisms have not been elucidated. This study demonstrates that FoxO3a is deactivated in senescent CMECs together with the inhibition of proliferation and tube formation. Furthermore, the activation of the antioxidant enzymes catalase and SOD, downstream FoxO3a targets, was significantly decreased, thereby leading to cell cycle arrest in G1-phase by increased ROS generation and subsequently the activation of the p27(Kip1) pathway. However, FoxO3a overexpression in primary low-passage CMECs not only significantly suppressed the senescence process by increasing the activation of catalase and SOD but also markedly inhibited ROS generation and p27(Kip1) activation, although it failed to reverse cellular senescence. Moreover, both cell viability and tube formation were greatly increased by FoxO3a overexpression in primary CMECs during continuous passage. In addition, FoxO3a, deficiency in low-passage CMECs, accelerated the senescence process. Collectively, our data suggest that FoxO3a suppresses the senescence process in CMECs by regulating the antioxidant/ROS/p27(Kip1) pathways, although it fails to reverse the cellular senescent phenotype.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/genética , Células Endoteliais/metabolismo , Fatores de Transcrição Forkhead/genética , Miocárdio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Sequência de Bases , Catalase/genética , Catalase/metabolismo , Sobrevivência Celular , Senescência Celular , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Células Endoteliais/patologia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Regulação da Expressão Gênica , Genes Reporter , Lentivirus/genética , Luciferases/genética , Luciferases/metabolismo , Dados de Sequência Molecular , Miocárdio/patologia , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
FoxO3a, a member of the forkhead transcription factors, has been demonstrated to be involved in myocardial ischemia/reperfusion (I/R) injury. Cardiac microvascular endothelial cells (CMECs) are some of the predominant cells damaged immediately after myocardial I/R injury. Despite the importance of injured CMECs in an ischemic heart, little is known about the involvement of FoxO3a in regulating CMECs injury. Thus, we used rat CMECs following simulated I/R to examine FoxO3a activation and signaling in relation to survival, the cell cycle and apoptosis in CMECs. We found that Akt negatively regulates activation of the FoxO3a pathway by phosphorylating FoxO3a in CMECs as demonstrated with an Akt inhibitor and activator. Upon I/R injury, the FoxO3a pathway was significantly activated in CMECs, which was accompanied by Akt deactivation. In parallel, the I/R of CMECs induced G1-phase arrest through p27(Kip1) up-regulation and significant activation of caspase-3. Accordingly, inhibition of the FoxO3a pathway by IGF-1, an Akt activator, could significantly block the I/R-enhanced activation of p27(Kip1) and caspase-3 in CMECs. Collectively, our results indicate that the FoxO3a pathway is involved in the I/R injury of CMECs at least in part through the regulation of cell cycle arrest and apoptosis, suggesting that the FoxO3a pathway may be a novel therapeutic target that protects against microvascular endothelial damage in ischemic hearts.