RESUMEN
Mechanisms mediating the cardioprotective actions of glucagon-like peptide 1 (GLP-1) were unknown. Here, we show in both ex vivo and in vivo models of ischemic injury that treatment with GLP-1(28-36), a neutral endopeptidase-generated (NEP-generated) metabolite of GLP-1, was as cardioprotective as GLP-1 and was abolished by scrambling its amino acid sequence. GLP-1(28-36) enters human coronary artery endothelial cells (caECs) through macropinocytosis and acts directly on mouse and human coronary artery smooth muscle cells (caSMCs) and caECs, resulting in soluble adenylyl cyclase Adcy10-dependent (sAC-dependent) increases in cAMP, activation of protein kinase A, and cytoprotection from oxidative injury. GLP-1(28-36) modulates sAC by increasing intracellular ATP levels, with accompanying cAMP accumulation lost in sAC-/- cells. We identify mitochondrial trifunctional protein-α (MTPα) as a binding partner of GLP-1(28-36) and demonstrate that the ability of GLP-1(28-36) to shift substrate utilization from oxygen-consuming fatty acid metabolism toward oxygen-sparing glycolysis and glucose oxidation and to increase cAMP levels is dependent on MTPα. NEP inhibition with sacubitril blunted the ability of GLP-1 to increase cAMP levels in coronary vascular cells in vitro. GLP-1(28-36) is a small peptide that targets novel molecular (MTPα and sAC) and cellular (caSMC and caEC) mechanisms in myocardial ischemic injury.
Asunto(s)
Cardiotónicos/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Mitocondrias Cardíacas/metabolismo , Subunidad alfa de la Proteína Trifuncional Mitocondrial/metabolismo , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/prevención & control , Animales , Vasos Coronarios/metabolismo , Vasos Coronarios/patología , AMP Cíclico/genética , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Péptido 1 Similar al Glucagón/genética , Humanos , Ratones , Ratones Noqueados , Mitocondrias Cardíacas/genética , Mitocondrias Cardíacas/patología , Subunidad alfa de la Proteína Trifuncional Mitocondrial/genética , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Isquemia Miocárdica/genética , Isquemia Miocárdica/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Sistemas de Mensajero Secundario/genéticaRESUMEN
The proto-oncogene c-myb (and corresponding nuclear transcription factor, c-Myb) regulates the proliferation and differentiation of hematologic and vascular smooth muscle cells; however, the role of c-Myb in blood pressure regulation is unknown. Here, we show that mice homozygous for a hypomorphic c-myb allele ( c-myb h/h) conferring reduced c-Myb activity manifest reduced peripheral blood and kidney B220+ B-cells and have decreased systolic (104±2 versus 120±1 mm Hg; P<0.0001) and diastolic blood pressure (71±2 versus 83±1 mm Hg; P<0.0001) compared with WT (wild type) mice. Additionally, c-myb h/h mice had lower susceptibility to deoxycorticosterone acetate-salt experimental hypertension. Although cardiac (echocardiography) and resistance artery (perfusion myography) functions were normal, metabolic cage studies revealed that c-myb h/h mice had increased 24-hour urine output and sodium excretion versus WT. Reconstitution of WT mice with c-myb h/h bone marrow transplant and chimeric bone marrow transplant using mice lacking B-cells ( J H T; h/h>WT and h/h:J H T>WT, respectively) decreased blood pressure and increased 24-hour urine output compared with controls ( WT>WT; WT:J H T>WT). J H T mice also had decreased systolic (103±2 versus 115±1 mm Hg; P<0.0001) and diastolic blood pressure (71±2 versus 79±1; P<0.01) and increased 24-hour urine output versus WT. Real-time quantitative reverse transcription polymerase chain reaction of kidney medulla revealed reduced V2R (vasopressin receptor 2) expression in c-myb h/h and J H T mice. These data implicate B-cells in the regulation of V2R and its associated effects on salt and water handling and blood pressure homeostasis.
Asunto(s)
Linfocitos B/metabolismo , Presión Sanguínea/fisiología , Hipertensión/inmunología , Miocitos del Músculo Liso/metabolismo , Animales , Linfocitos B/patología , Diferenciación Celular , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Hipertensión/metabolismo , Hipertensión/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/patología , Proteínas Proto-Oncogénicas c-myb/biosíntesis , Proteínas Proto-Oncogénicas c-myb/genética , ARN/genéticaRESUMEN
BACKGROUND: MicroRNAs (miR) are small non-coding RNAs that regulate diverse biological functions. The bicistronic gene miR-143/145 determines cell fate and phenotype of vascular smooth muscle cells (VSMC), in part, by destabilizing Elk-1 mRNA. The transcription factor c-Myb also regulates differentiation and proliferation of VSMC, and here we test whether these effects may be mediated by miR-143/145. METHODS & RESULTS: Flow cytometry of cardiovascular-directed d3.75 embryoid bodies (EBs) isolated smooth muscle progenitors with specific cell surface markers. In c-myb knockout (c-myb -/-) EB, these progenitors manifest low levels of miR-143 (19%; p<0.05) and miR-145 (6%; p<0.01) expression as compared to wild-type (wt) EB. Primary VSMC isolated from transgenic mice with diminished expression (c-myblx/lx) or reduced activity (c-mybh/h) of c-Myb also manifest low levels of miR-143 (c-myblx/lx: 50%; c-mybh/h: 41%), and miR-145 (c-myblx/lx: 49%; c-mybh/h: 56%), as compared to wt (P<0.05). Sequence alignment identified four putative c-Myb binding sites (MBS1-4) in the proximal promoter (PP) of the miR-143/145 gene. PP-reporter constructs revealed that point mutations in MBS1 and MBS4 abrogated c-Myb-dependent transcription from the miR-143/145 PP (P<0.01). Chromatin immunoprecipitation (ChIP) revealed preferential c-Myb binding at MBS4 (p<0.001). By conjugating Elk-1 3'-untranslated region (UTR) to a reporter and co-transducing wt VSMC with this plus a miR-143-antagomir, and co-transducing c-myblx/lx VSMC with this plus a miR-143-mimic, we demonstrate that c-Myb's ability to repress Elk-1 is mediated by miR-143. CONCLUSION: c-Myb regulates VSMC gene expression by transcriptional activation of miR-143/145.
Asunto(s)
MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-myb/metabolismo , Regiones no Traducidas 3' , Animales , Antagomirs/metabolismo , Sitios de Unión , Células Cultivadas , Inmunoprecipitación de Cromatina , Ratones , Ratones Noqueados , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Mutación Puntual , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myb/genética , Activación Transcripcional/genética , Proteína Elk-1 con Dominio ets/genética , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
Background: Heart failure (HF) is associated with reduced expression of plasma membrane Ca2+-ATPase 4 (PMCA4). Cardiac-specific overexpression of human PMCA4b in mice inhibited nNOS activity and reduced cardiac hypertrophy by inhibiting calcineurin. Here we examine temporally regulated cardiac-specific overexpression of hPMCA4b in mouse models of myocardial ischemia reperfusion injury (IRI) ex vivo, and HF following experimental myocardial infarction (MI) in vivoMethods and results: Doxycycline-regulated cardiomyocyte-specific overexpression and activity of hPMCA4b produced adaptive changes in expression levels of Ca2+-regulatory genes, and induced hypertrophy without significant differences in Ca2+ transients or diastolic Ca2+ concentrations. Total cardiac NOS and nNOS-specific activities were reduced in mice with cardiac overexpression of hPMCA4b while nNOS, eNOS and iNOS protein levels did not differ. hMPCA4b-overexpressing mice also exhibited elevated systolic blood pressure vs. controls, with increased contractility and lusitropy in vivo In isolated hearts undergoing IRI, hPMCA4b overexpression was cardioprotective. NO donor-treated hearts overexpressing hPMCA4b showed reduced LVDP and larger infarct size versus vehicle-treated hearts undergoing IRI, demonstrating that the cardioprotective benefits of hPMCA4b-repressed nNOS are lost by restoring NO availability. Finally, both pre-existing and post-MI induction of hPMCA4b overexpression reduced infarct expansion and improved survival from HF.Conclusions: Cardiac PMCA4b regulates nNOS activity, cardiac mass and contractility, such that PMCA4b overexpression preserves cardiac function following IRI, heightens cardiac performance and limits infarct progression, cardiac hypertrophy and HF, even when induced late post-MI. These data identify PMCA4b as a novel therapeutic target for IRI and HF.
Asunto(s)
Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/prevención & control , Miocitos Cardíacos/enzimología , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Animales , Señalización del Calcio , Modelos Animales de Enfermedad , Insuficiencia Cardíaca/enzimología , Insuficiencia Cardíaca/fisiopatología , Insuficiencia Cardíaca/prevención & control , Humanos , Hipertrofia Ventricular Izquierda/enzimología , Hipertrofia Ventricular Izquierda/fisiopatología , Hipertrofia Ventricular Izquierda/prevención & control , Preparación de Corazón Aislado , Ratones Transgénicos , Contracción Miocárdica , Infarto del Miocardio/enzimología , Infarto del Miocardio/genética , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/enzimología , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/fisiopatología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo I/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/genética , Regulación hacia Arriba , Función Ventricular Izquierda , Presión VentricularRESUMEN
Short-term studies in subjects with diabetes receiving glucagon-like peptide 1 (GLP-1)-targeted therapies have suggested a reduced number of cardiovascular events. The mechanisms underlying this unexpectedly rapid effect are not known. We cloned full-length GLP-1 receptor (GLP-1R) mRNA from a human megakaryocyte cell line (MEG-01), and found expression levels of GLP-1Rs in MEG-01 cells to be higher than those in the human lung but lower than in the human pancreas. Incubation with GLP-1 and the GLP-1R agonist exenatide elicited a cAMP response in MEG-01 cells, and exenatide significantly inhibited thrombin-, ADP-, and collagen-induced platelet aggregation. Incubation with exenatide also inhibited thrombus formation under flow conditions in ex vivo perfusion chambers using human and mouse whole blood. In a mouse cremaster artery laser injury model, a single intravenous injection of exenatide inhibited thrombus formation in normoglycemic and hyperglycemic mice in vivo. Thrombus formation was greater in mice transplanted with bone marrow lacking a functional GLP-1R (Glp1r(-/-)), compared with those receiving wild-type bone marrow. Although antithrombotic effects of exenatide were partly lost in mice transplanted with bone marrow from Glp1r(-/-) mice, they were undetectable in mice with a genetic deficiency of endothelial nitric oxide synthase. The inhibition of platelet function and the prevention of thrombus formation by GLP-1R agonists represent potential mechanisms for reduced atherothrombotic events.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/metabolismo , Péptidos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Trombosis/prevención & control , Ponzoñas/farmacología , Animales , Línea Celular , Exenatida , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/agonistas , Humanos , Pulmón/metabolismo , Ratones , Óxido Nítrico Sintasa de Tipo III/deficiencia , Páncreas/metabolismoRESUMEN
OBJECTIVE: To understand the molecular pathways underlying the cardiac preconditioning effect of short-term caloric restriction (CR). BACKGROUND: Lifelong CR has been suggested to reduce the incidence of cardiovascular disease through a variety of mechanisms. However, prolonged adherence to a CR life-style is difficult. Here we reveal the pathways that are modulated by short-term CR, which are associated with protection of the mouse heart from ischemia. METHODS: Male 10-12 wk old C57bl/6 mice were randomly assigned to an ad libitum (AL) diet with free access to regular chow, or CR, receiving 30% less food for 7 days (d), prior to myocardial infarction (MI) via permanent coronary ligation. At d8, the left ventricles (LV) of AL and CR mice were collected for Western blot, mRNA and microRNA (miR) analyses to identify cardioprotective gene expression signatures. In separate groups, infarct size, cardiac hemodynamics and protein abundance of caspase 3 was measured at d2 post-MI. RESULTS: This short-term model of CR was associated with cardio-protection, as evidenced by decreased infarct size (18.5±2.4% vs. 26.6±1.7%, N=10/group; P=0.01). mRNA and miR profiles pre-MI (N=5/group) identified genes modulated by short-term CR to be associated with circadian clock, oxidative stress, immune function, apoptosis, metabolism, angiogenesis, cytoskeleton and extracellular matrix (ECM). Western blots pre-MI revealed CR-associated increases in phosphorylated Akt and GSK3ß, reduced levels of phosphorylated AMPK and mitochondrial related proteins PGC-1α, cytochrome C and cyclooxygenase (COX) IV, with no differences in the levels of phosphorylated eNOS or MAPK (ERK1/2; p38). CR regimen was also associated with reduced protein abundance of cleaved caspase 3 in the infarcted heart and improved cardiac function.
Asunto(s)
Restricción Calórica , Daño por Reperfusión Miocárdica/metabolismo , Transcriptoma , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Citocromos c/genética , Citocromos c/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandina-Endoperóxido Sintasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
CONTEXT: Regulator of G-protein signaling-2 (RGS2) inhibits Gq-mediated regulation of Ca(2+) signalling in vascular smooth muscle cells (VSMC). OBJECTIVE: RGS2 knockout (RGS2KO) mice are hypertensive and show arteriolar remodeling. VSMC proliferation modulates intracellular Ca(2+) concentration [Ca(2+)]i. RGS2 involvement in VSMC proliferation had not been examined. METHODS: Thymidine incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion assays measured cell proliferation. Fura-2 ratiometric imaging quantified [Ca(2+)]i before and after UTP and thapsigargin. [(3)H]-labeled inositol was used for phosphoinositide hydrolysis. Quantitative RT-PCR and confocal immunofluorescence of select Ca(2+) transporters was performed in primary aortic VSMC. RESULTS AND DISCUSSION: Platelet-derived growth factor (PDGF) increased S-phase entry and proliferation in VSMC from RGS2KO mice to a greater extent than in VSMC from wild-type (WT) controls. Consistent with differential PDGF-induced changes in Ca(2+) homeostasis, RGS2KO VSMC showed lower resting [Ca(2+)]i but higher thapsigargin-induced [Ca(2+)]i as compared with WT. RGS2KO VSMC expressed lower mRNA levels of plasma membrane Ca(2+) ATPase-4 (PMCA4) and Na(+) Ca(2+) Exchanger (NCX), but higher levels of sarco-endoplasmic reticulum Ca(2+) ATPase-2 (SERCA2). Western blot and immunofluorescence revealed similar differences in PMCA4 and SERCA2 protein, while levels of NCX protein were not reduced in RGS2KO VSMC. Consistent with decreased Ca(2+) efflux activity, (45)Ca-extrusion rates were lower in RGS2KO VSMC. These differences were reversed by the PMCA inhibitor La(3+), but not by replacing extracellular Na(+) with choline, implicating differences in the activity of PMCA and not NCX. CONCLUSION: RGS2-deficient VSMC exhibit higher rates of proliferation and coordinate plasticity of Ca(2+)-handling mechanisms in response to PDGF stimulation.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/fisiología , Proteínas RGS/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Células Cultivadas , Ratones , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas RGS/genéticaRESUMEN
We explored the role played by plasma membrane calcium ATPase-4 (PMCA4) and its alternative splice variants in the cell cycle of vascular smooth muscle cells (VSMC). A novel variant (PMCA4e) was discovered. Quantitative real-time-PCR-quantified PMCA4 splice variant proportions differed in specific organs. The PMCA4a:4b ratio in uninjured carotid arteries (â¼1:1) was significantly reduced by wire denudation injury (to â¼1:3) by modulation of alternative splicing, as confirmed by novel antibodies against PMCA4a/e and PMCA4b. Laser capture microdissection localized this shift to the media and adventitia. Primary carotid VSMC from PMCA4 knock-out (P4KO) mice showed impaired [(3)H]thymidine incorporation and G1 phase arrest as compared with wild type (P4WT). Electroporation of expression constructs encoding PMCA4a, PMCA4b, and a PMCA4b mutant lacking PDZ binding rescued this phenotype of P4KO cells, whereas a mutant with only 10% of normal Ca(2+) efflux activity could not. Microarray of early G1-synchronized VSMC showed 39-fold higher Rgs16 (NFAT (nuclear factor of activated T-cells) target; MAPK inhibitor) and 69-fold higher Decorin (G1 arrest marker) expression in P4KO versus P4WT. Validation by Western blot also revealed decreased levels of Cyclin D1 and NFATc3 in P4KO. Microarrays of P4KO VSMC rescued by PMCA4a or PMCA4b expression showed reversal of perturbed Rgs16, Decorin, and NFATc3 expression levels. However, PMCA4a rescue caused a 44-fold reduction in AP-2ß, a known anti-proliferative transcription factor, whereas PMCA4b rescue resulted in a 50-fold reduction in p15 (Cyclin D1/Cdk4 inhibitor). We conclude that Ca(2+) efflux activity of PMCA4 underlies G1 progression in VSMC and that PMCA4a and PMCA4b differentially regulate specific downstream mediators.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Calcio/metabolismo , Ciclo Celular , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Animales , ATPasas Transportadoras de Calcio/genética , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Células Cultivadas , Clonación Molecular , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/citología , ATPasas Transportadoras de Calcio de la Membrana Plasmática/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Distribución TisularRESUMEN
OBJECTIVE: The IP3 receptor-1 (IP3R1) mediates Ca2+ signals critical to vascular smooth muscle cell (VSMC) proliferation. The cell cycle-associated transcription factor c-Myb increases Ca2+ at the G1/S transition. Here we show the mechanism through which c-Myb regulates expression of IP3R1. METHODS & RESULTS: Ribonuclease protection confirmed transcriptional start (TS), and qRT-PCR revealed a 6-fold increase in IP3R1 mRNA as immortalized VSMC progress from G0 to G1/S. A c-Myb neutralizing antibody decreased IP3R1 mRNA expression 3-fold, and abolished the 3.4-fold increase in IP3R1 protein observed at G1/S. Primary aortic VSMCs in culture and proliferating carotid VSMCs in vivo showed similar regulation of IP3R1 mRNA and protein. Sequence analysis of a 3.1-Kb mouse IP3R1 promoter revealed 17 putative c-Myb binding sites. Reporter assays demonstrated a 2-fold increase in promoter activity in G1/S- versus G0-synchronized VSMCs, which was abolished by functional c-Myb knockdown or deletion of promoter sequences upstream and downstream of TS. Point mutations in Myb sites-13 or -15 significantly blunted G1/S-specific promoter induction in both immortalized and primary VSMCs. Gel shift and ChIP confirmed binding of c-Myb to sites-13 and -15 in G1/S stage VSMCs. CONCLUSION: c-Myb regulates cell cycle-associated IP3R1 transcription in VSMCs via specific highly conserved Myb-binding sites in the IP3R1 promoter.
Asunto(s)
Canales de Calcio/metabolismo , Ciclo Celular/fisiología , Glicoproteínas de Membrana/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myb/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transcripción Genética , Animales , Calcio/metabolismo , Canales de Calcio/genética , Arterias Carótidas/cirugía , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/fisiopatología , Línea Celular , Proliferación Celular , Inmunoprecipitación de Cromatina , Secuencia Conservada , ADN/metabolismo , Modelos Animales de Enfermedad , Ensayo de Cambio de Movilidad Electroforética , Genes Reporteros , Receptores de Inositol 1,4,5-Trifosfato , Luciferasas , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/citología , Mutación , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Análisis de Secuencia de ADN , Transfección , Regulación hacia ArribaRESUMEN
In vascular smooth muscle cells (SMCs), several mechanisms act in concert to regulate the intracellular calcium concentration [Ca2+]i, which may in turn affect vascular tone. One such mechanism is the extrusion of Ca2+ by the plasma membrane calcium ATPase (PMCA). To address, in particular, the role of the neuronal nitric oxide synthase (nNOS)-associating isoform PMCA4b in regulating vascular tone, a doxycycline-responsive transgene for human PMCA4b was overexpressed in arterial SMCs of mice. Overexpression of hPMCA4b resulted in a 2-fold increase in total aortic PMCA4 protein expression and significant real-time RT-PCR-documented differences in the levels of endogenous mouse PMCA1, PMCA4, SERCA2, and IP3R1 gene expression in arterial SMCs. Whereas no significant difference in basal [Ca2+]i or Ca2+ sensitivity was observed in vascular SMCs or mesenteric arteries, respectively, from hPMCA4b-overexpressing versus control mice, hPMCA4b-overexpressing mice revealed a reduced set-point and increased extent of myogenic response and heightened sensitivity to vasoconstrictors. Treatment of arteries with an nNOS inhibitor resulted in a reduced set-point and increased extent of the myogenic response in control but not hPMCA4b-overexpressing mice. Moreover, aortic SMCs from hPMCA4b-overexpressing mice exhibited reduced levels of cGMP under both basal and phenylephrine-stimulated conditions. These changes were associated with significant doxycycline-reversible elevations in blood pressure. Taken together, these data show that overexpression of hPMCA4b in arterial SMCs increases vascular reactivity and blood pressure, an effect that may be mediated in part by negative regulation of nNOS.
Asunto(s)
Arginina/análogos & derivados , Presión Sanguínea/fisiología , ATPasas Transportadoras de Calcio/metabolismo , Músculo Liso Vascular/enzimología , Sistema Vasomotor/fisiología , Animales , Aorta/enzimología , Arginina/farmacología , Presión Sanguínea/efectos de los fármacos , Calcio/metabolismo , ATPasas Transportadoras de Calcio/genética , Proteínas de Transporte de Catión , Línea Celular , Células Cultivadas , GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Doxiciclina/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Genotipo , Humanos , Técnicas In Vitro , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/fisiología , Ratones , Ratones Transgénicos , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo I , Fenilefrina/farmacología , ATPasas Transportadoras de Calcio de la Membrana Plasmática , Cloruro de Potasio/farmacología , Vasoconstricción/efectos de los fármacos , Sistema Vasomotor/efectos de los fármacosRESUMEN
Calcineurin mediates repression of plasma membrane Ca2+-ATPase-4 (PMCA4) expression in neurons, whereas c-Myb is known to repress PMCA1 expression in vascular smooth muscle cells (VSMC). Here, we describe a novel mouse VSMC line (MOVAS) in which 45Ca efflux rates decreased 50%, fura 2-AM-based intracellular Ca2+ concentrations ([Ca2+]i) increased twofold, and real-time RT-PCR and Western blot revealed a approximately 40% decrease in PMCA4 expression levels from G0 to G1/S in the cell cycle, where PMCA4 constituted approximately 20% of total PMCA protein. Although calcineurin activity increased fivefold as MOVAS progressed from G0 to G1/S, inhibition of this increase with either BAPTA or retroviral transduction with peptide inhibitors of calcineurin (CAIN), or its downstream target nuclear factor of activated T cells (NFAT) (VIVIT), had no effect on the repression of PMCA4 mRNA expression at G1/S. By contrast, Ca2+-independent activity of the calmodulin-dependent protein kinase-II (CaMK-II) increased eightfold as MOVAS progressed from G0 to G1/S, and treatment with an inhibitor of CaMK-II (KN-93) or transduction of a c-Myb-neutralizing antibody significantly alleviated the G1/S-associated repression of PMCA4. These data show that G1/S-specific PMCA4 repression in proliferating VSMC is brought about by c-Myb and CaMK-II and that calcineurin may regulate cell cycle-associated [Ca2+]i through alternate targets.
Asunto(s)
Calcineurina/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Ácido Egtácico/análogos & derivados , Músculo Liso Vascular/citología , Músculo Liso Vascular/enzimología , Animales , Anticuerpos/farmacología , Bencilaminas/farmacología , Inhibidores de la Calcineurina , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , ATPasas Transportadoras de Calcio/genética , Proteínas Portadoras/genética , Proteínas de Transporte de Catión , Línea Celular , Membrana Celular/enzimología , Tamaño de la Célula/fisiología , Quelantes/farmacología , Ácido Egtácico/farmacología , Inhibidores Enzimáticos/farmacología , Fase G1/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Ratones , Ratones Endogámicos C57BL , Oligopéptidos/genética , Fenotipo , ATPasas Transportadoras de Calcio de la Membrana Plasmática , Proteínas Proto-Oncogénicas c-myb/inmunología , Proteínas Proto-Oncogénicas c-myb/metabolismo , Fase S/fisiología , Sulfonamidas/farmacología , TransfecciónRESUMEN
Inhibiting activity of the c-Myb transcription factor attenuates G1 to S phase cell cycle transitions in vascular smooth muscle cells (SMCs) in vitro. To determine the effects of arterial SMC-specific expression of a dominant-negative c-Myb molecule (Myb-Engrailed) on vascular remodeling in vivo, we performed carotid artery wire-denudation in 2 independent lines of binary transgenic mice with SM22alpha promoter-defined Doxycycline-suppressible expression of Myb-Engrailed. Adult mice with arterial SMC-specific expression of Myb-Engrailed were overtly normal in appearance and did not display any changes in cardiovascular structure or physiology. However, bromodeoxyuridine-defined arterial SMC proliferation, neointima formation, medial hyperplasia, and arterial remodeling were markedly decreased in mice expressing arterial SMC-restricted Myb-Engrailed after arterial injury. These data suggest that c-Myb activity in arterial SMCs is not essential for arterial structure or function during development, but is involved in the proliferation of arterial SMCs as occurs in vascular pathology, and that the expression of a dominant-negative c-Myb can dramatically reduce adverse arterial remodeling in an in vivo model of restenosis. As such, this model represents a novel tissue-specific strategy for the potential gene therapy of diseases characterized by arterial SMC proliferation.