RESUMEN
BACKGROUND: C/EBP homologous protein-10 (CHOP-10) is a novel developmentally regulated nuclear protein that emerges as a critical transcriptional integrator among pathways regulating differentiation, proliferation, and survival. In the present study, we analyzed the role of CHOP-10 in postnatal neovascularization. METHODS AND RESULTS: Ischemia was induced by right femoral artery ligation in wild-type and CHOP-10(-/-) mice. In capillary structure of skeletal muscle, CHOP-10 mRNA and protein levels were upregulated by ischemia and diabetes mellitus. Angiographic score, capillary density, and foot perfusion were increased in CHOP-10(-/-) mice compared with wild-type mice. This effect was associated with a reduction in apoptosis and an upregulation of endothelial nitric oxide synthase (eNOS) levels in ischemic legs of CHOP-10(-/-) mice compared with wild-type mice. In agreement with these results, eNOS mRNA and protein levels were significantly upregulated in CHOP-10 short interfering RNA-transfected human endothelial cells, whereas overexpression of CHOP-10 inhibited basal transcriptional activation of the eNOS promoter. Using a chromatin immunoprecipitation assay, we also showed that CHOP-10 was bound to the eNOS promoter. Interestingly, enhanced postischemic neovascularization in CHOP-10(-/-) mice was fully blunted in CHOP-10/eNOS double-knockout animals. Finally, we showed that induction of diabetes mellitus is associated with a marked upregulation of CHOP-10 that substantially inhibited postischemic neovascularization. CONCLUSIONS: This study identifies CHOP-10 as an important transcription factor modulating vessel formation and maturation.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Neovascularización Patológica/enzimología , Óxido Nítrico Sintasa de Tipo III/genética , Factor de Transcripción CHOP/genética , Animales , Animales Recién Nacidos , Células Cultivadas , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/genética , Arteria Femoral/enzimología , Arteria Femoral/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica/genética , Óxido Nítrico Sintasa de Tipo III/biosíntesis , Unión Proteica/genética , Factor de Transcripción CHOP/biosíntesis , Factor de Transcripción CHOP/deficiencia , Activación Transcripcional/genética , Regulación hacia Arriba/genéticaRESUMEN
OBJECTIVE: Catecholamines have been shown to control bone marrow (BM)-derived cell egress, yet the cellular and molecular mechanisms involved in this effect and their subsequent participation to postischemic vessel growth are poorly understood. METHODS AND RESULTS: Tyrosine hydroxylase mRNA levels, as well as dopamine (DA) and norepinephrine (NE) contents, were increased in the ischemic BM of mice with right femoral artery ligation. Angiographic score, capillary density, and arteriole number were markedly increased by treatments with DA (IP, 50 mg/kg, 5 days) or NE (IP, 2.5 mg/kg, 5 days). Using chimeric mice lethally irradiated and transplanted with BM-derived cells from green fluorescent protein mice, we showed that DA and NE enhanced by 70% (P<0.01) and 62% (P<0.001), respectively, the number of green fluorescent protein-positive BM-derived cells in ischemic tissue and promoted their ability to differentiate into cells with endothelial and inflammatory phenotypes. Similarly, both DA and NE increased the in vitro differentiation of cultured BM-derived cells into cells with endothelial phenotype. This increase was blunted by the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester. DA and NE also upregulated the number of CD45-positive cells in blood 3 days after ischemia and that of macrophages in ischemic tissue 21 days after ischemia. Of interest, DA and NE increased BM endothelial nitric oxide synthase (eNOS) mRNA levels and were unable to promote BM-derived cell mobilization in chimeric eNOS-deficient mice lethally irradiated and transplanted with BM-derived cells from wild-type animals. Furthermore, administration of a ß2 adrenergic agonist (clenbuterol, IP, 2 mg/kg, 5 days) and that of a dopaminergic D1/D5 receptor agonist (SKF-38393, IP, 2.5 mg/kg, 5 days) also enhanced BM-derived cell mobilization and subsequently postischemic vessel growth. CONCLUSION These results unravel, for the first time, a major role for the sympathetic nervous system in BM-derived cell egress through stromal eNOS activation.
Asunto(s)
Células de la Médula Ósea/enzimología , Médula Ósea/enzimología , Diferenciación Celular , Movimiento Celular , Células Endoteliales/metabolismo , Isquemia/enzimología , Músculo Esquelético/irrigación sanguínea , Óxido Nítrico Sintasa de Tipo III/metabolismo , Sistema Nervioso Simpático/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/inervación , Células de la Médula Ósea/efectos de los fármacos , Trasplante de Médula Ósea , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Dopamina/metabolismo , Agonistas de Dopamina/farmacología , Células Endoteliales/efectos de los fármacos , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Arteria Femoral/cirugía , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Miembro Posterior , Isquemia/fisiopatología , Ligadura , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neovascularización Fisiológica , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/genética , Norepinefrina/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal , Células del Estroma/enzimología , Sistema Nervioso Simpático/efectos de los fármacos , Sistema Nervioso Simpático/fisiopatología , Factores de Tiempo , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismo , Regulación hacia ArribaRESUMEN
OBJECTIVE: Leukocyte infiltration in ischemic areas is a hallmark of myocardial infarction, and overwhelming infiltration of innate immune cells has been shown to promote adverse remodeling and cardiac rupture. Recruitment of inflammatory cells in the ischemic heart depends highly on the family of CC-chemokines and their receptors. Here, we hypothesized that the chemokine decoy receptor D6, which specifically binds and scavenges inflammatory CC-chemokines, might limit inflammation and adverse cardiac remodeling after infarction. METHODS AND RESULTS: D6 was expressed in human and murine infarcted myocardium. In a murine model of myocardial infarction, D6 deficiency led to increased chemokine (C-C motif) ligand 2 and chemokine (C-C motif) ligand 3 levels in the ischemic heart. D6-deficient (D6(-/-)) infarcts displayed increased infiltration of pathogenic neutrophils and Ly6Chi monocytes, associated with strong matrix metalloproteinase-9 and matrix metalloproteinase-2 activities in the ischemic heart. D6(-/-) mice were cardiac rupture prone after myocardial infarction, and functional analysis revealed that D6(-/-) hearts had features of adverse remodeling with left ventricle dilation and reduced ejection fraction. Bone marrow chimera experiments showed that leukocyte-borne D6 had no role in this setting, and that leukocyte-specific chemokine (C-C motif) receptor 2 deficiency rescued the adverse phenotype observed in D6(-/-) mice. CONCLUSIONS: We show for the first time that the chemokine decoy receptor D6 limits CC-chemokine-dependent pathogenic inflammation and is required for adequate cardiac remodeling after myocardial infarction.
Asunto(s)
Inflamación/prevención & control , Infarto del Miocardio/inmunología , Miocardio/inmunología , Receptores CCR10/metabolismo , Receptores de Quimiocina/metabolismo , Remodelación Ventricular , Animales , Antígenos Ly/metabolismo , Trasplante de Médula Ósea , Quimiocina CCL2/metabolismo , Quimiocina CCL3/metabolismo , Quimiotaxis , Modelos Animales de Enfermedad , Genotipo , Rotura Cardíaca Posinfarto/inmunología , Rotura Cardíaca Posinfarto/patología , Humanos , Hipertrofia Ventricular Izquierda/inmunología , Hipertrofia Ventricular Izquierda/patología , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Mediadores de Inflamación/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/inmunología , Infarto del Miocardio/complicaciones , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Miocardio/metabolismo , Miocardio/patología , Infiltración Neutrófila , Neutrófilos/inmunología , Fenotipo , Receptores CCR2/deficiencia , Receptores CCR2/genética , Receptores de Quimiocina/deficiencia , Receptores de Quimiocina/genética , Transducción de Señal , Volumen Sistólico , Ultrasonografía , Función Ventricular Izquierda , Receptor de Quimiocina D6RESUMEN
BACKGROUND: The hypoxia-inducible transcription factor (HIF) subunits are destabilized via the O(2)-dependent prolyl hydroxylase domain proteins (PHD1, PHD2, and PHD3). We investigated whether inhibition of PHDs via upregulating HIF might promote postischemic neovascularization. METHODS AND RESULTS: Mice with right femoral artery ligation were treated, by in vivo electrotransfer, with plasmids encoding for an irrelevant short hairpin RNA (shRNA) (shCON [control]) or specific shRNAs directed against HIF-1alpha (shHIF-1alpha), PHD1 (shPHD1), PHD2 (shPHD2), and PHD3 (shPHD3). The silencing of PHDs induced a specific and transient downregulation of their respective mRNA and protein levels at day 2 after ischemia and, as expected, upregulated HIF-1alpha. As a consequence, 2 key hypoxia-inducible proangiogenic actors, vascular endothelial growth factor-A and endothelial nitric oxide synthase, were upregulated at the mRNA and protein levels. In addition, monocyte chemotactic protein-1 mRNA levels and infiltration of Mac-3-positive macrophages were enhanced in ischemic leg of mice treated with shPHD2 and shPHD3. Furthermore, activation of HIF-1alpha-related pathways was associated with changes in postischemic neovascularization. At day 14, silencing of PHD2 and PHD3 increased vessel density by 2.2- and 2.6-fold, capillary density by 1.8- and 2.1-fold, and foot perfusion by 1.2- and 1.4-fold, respectively, compared with shCON (P<0.001). shPHD1 displayed a lower proangiogenic effect. Of interest, coadministration of shHIF-1alpha with shPHD3 abrogated shPHD3-related effects, suggesting that activation of endogenous HIF-1-dependent pathways mediated the proangiogenic effects of PHD silencing. CONCLUSIONS: We demonstrated that a direct inhibition of PHDs, and more particularly PHD3, promoted therapeutic revascularization. Furthermore, we showed that activation of the HIF-1 signaling pathway is required to promote this revascularization.
Asunto(s)
Terapia Genética/métodos , Isquemia/terapia , Neovascularización Fisiológica/fisiología , Procolágeno-Prolina Dioxigenasa/genética , Transducción de Señal/fisiología , Animales , Quimiocinas/metabolismo , Arteria Femoral , Silenciador del Gen , Miembro Posterior/irrigación sanguínea , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Inflamación/metabolismo , Isquemia/metabolismo , Isquemia/fisiopatología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo III/metabolismo , Plásmidos/farmacología , Procolágeno-Prolina Dioxigenasa/metabolismo , ARN Mensajero/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: CD4+ and CD8+ T lymphocytes are key regulators of postischemic neovascularization. T-cell activation is promoted by 2 major costimulatory signalings, the B7/CD28 and CD40-CD40 ligand pathways. Interestingly, CD28 interactions with the structurally related ligands B7-1 and B7-2 are also required for the generation and homeostasis of CD4+CD25+ regulatory T cells (Treg cells), which play a critical role in the suppression of immune responses and the control of T-cell homeostasis. We hypothesized that Treg cell activation may modulate the immunoinflammatory response to ischemic injury, leading to alteration of postischemic vessel growth. METHODS AND RESULTS: Ischemia was induced by right femoral artery ligation in CD28-, B7-1/2-, or CD40-deficient mice (n=10 per group). CD40 deficiency led to a significant reduction in the postischemic inflammatory response and vessel growth. In contrast, at day 21 after ischemia, angiographic score, foot perfusion, and capillary density were increased by 2.0-, 1.2-, and 1.8-fold, respectively, in CD28-deficient mice, which showed a profound reduction in the number of Treg cells compared with controls. Similarly, disruption of B7-1/2 signaling or anti-CD25 treatment and subsequent Treg deletion significantly enhanced postischemic neovascularization. These effects were associated with enhanced accumulation of CD3-positive T cells and Mac-3-positive macrophages in the ischemic leg. Conversely, treatment of CD28(-/-) mice with the nonmitogenic anti-CD3 monoclonal antibody enhanced the number of endogenous Treg cells and led to a significant reduction of the postischemic inflammatory response and neovascularization. Finally, coadministration of Treg cells and CD28(-/-) splenocytes in Rag1(-/-) mice with hindlimb ischemia abrogated the CD28(-/-) splenocyte-induced activation of the inflammatory response and neovascularization. CONCLUSIONS: Treg cell response modulates postischemic neovascularization.
Asunto(s)
Isquemia Miocárdica/inmunología , Linfocitos T Reguladores/inmunología , Animales , Antígeno B7-1/genética , Antígeno B7-1/inmunología , Antígeno B7-2/genética , Antígeno B7-2/inmunología , Antígenos CD28/genética , Antígenos CD28/inmunología , Linfocitos T CD4-Positivos/inmunología , Antígenos CD40/deficiencia , Antígenos CD40/genética , Antígenos CD40/inmunología , Linfocitos T CD8-positivos/inmunología , Capilares/inmunología , Citometría de Flujo , Miembro Posterior , Inmunohistoquímica , Isquemia/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/inmunología , Neovascularización Fisiológica , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Acute myocardial infarction is a severe ischemic disease responsible for heart failure and sudden death. Here, we show that after acute myocardial infarction in mice, mature B lymphocytes selectively produce Ccl7 and induce Ly6C(hi) monocyte mobilization and recruitment to the heart, leading to enhanced tissue injury and deterioration of myocardial function. Genetic (Baff receptor deficiency) or antibody-mediated (CD20- or Baff-specific antibody) depletion of mature B lymphocytes impeded Ccl7 production and monocyte mobilization, limited myocardial injury and improved heart function. These effects were recapitulated in mice with B cell-selective Ccl7 deficiency. We also show that high circulating concentrations of CCL7 and BAFF in patients with acute myocardial infarction predict increased risk of death or recurrent myocardial infarction. This work identifies a crucial interaction between mature B lymphocytes and monocytes after acute myocardial ischemia and identifies new therapeutic targets for acute myocardial infarction.
Asunto(s)
Linfocitos B/fisiología , Corazón/fisiopatología , Monocitos/fisiología , Infarto del Miocardio/fisiopatología , Animales , Factor Activador de Células B/sangre , Linfocitos B/metabolismo , Quimiocina CCL7/biosíntesis , Quimiocina CCL7/sangre , Humanos , Depleción Linfocítica , Ratones , Infarto del Miocardio/metabolismo , Transducción de SeñalRESUMEN
AIMS: Monocyte systemic levels are known to be a major determinant of ischaemic tissue revascularization, but the mechanisms mediating mobilization of different monocyte subsets-Ly6C(hi) and Ly6C(lo)-to the blood and their respective role in post-ischaemic neovascularization are not clearly understood. Here, we hypothesized that distinct chemokine/chemokine receptor pathways, namely CCL2/CCR2, CX3CL1/CX3CR1, and CCL5/CCR5, differentially control monocyte subset systemic levels, and might thus impact post-ischaemic vessel growth. METHODS AND RESULTS: In a model of murine hindlimb ischaemia, both Ly6C(hi) and Ly6C(lo) monocyte circulating levels were increased after femoral artery ligation. CCL2/CCR2 activation enhanced blood Ly6C(hi) and Ly6C(lo) monocyte counts, although the opposite effect was seen in mice with CCL2 or CCR2 deficiency. CX3CL1/CX3CR1 strongly impacted Ly6C(lo) monocyte levels, whereas CCL5/CCR5 had no role. Only CCL2/CCR2 signalling influenced neovascularization, which was increased in mice overexpressing CCL2, whereas it markedly decreased in CCL2-/- mice. Moreover, adoptive transfer of Ly6C(hi)-but not Ly6C(lo)-monocytes enhanced vessel growth and blood flow recovery. CONCLUSION: Altogether, our data demonstrate that regulation of proangiogenic Ly6C(hi) monocytes systemic levels by CCL2/CCR2 controls post-ischaemic vessel growth, whereas Ly6C(lo) monocytes have no major role in this setting.