RESUMO
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.
Assuntos
Arginina/análogos & derivados , Pressão Sanguínea/fisiologia , ATPases Transportadoras de Cálcio/metabolismo , Músculo Liso Vascular/enzimologia , Sistema Vasomotor/fisiologia , Animais , Aorta/enzimologia , Arginina/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/genética , Proteínas de Transporte de Cátions , Linhagem Celular , Células Cultivadas , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Doxiciclina/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genótipo , Humanos , Técnicas In Vitro , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiologia , Camundongos , Camundongos Transgênicos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I , Fenilefrina/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática , Cloreto de Potássio/farmacologia , Vasoconstrição/efeitos dos fármacos , Sistema Vasomotor/efeitos dos fármacosRESUMO
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.
Assuntos
Estenose das Carótidas/prevenção & controle , Genes Dominantes , Músculo Liso Vascular/metabolismo , Proteínas Proto-Oncogênicas c-myb/biossíntese , Fatores de Transcrição , Animais , Bromodesoxiuridina , Estenose das Carótidas/patologia , Divisão Celular/genética , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/farmacologia , Tetraciclina/farmacologia , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/metabolismo , Túnica Íntima/patologiaRESUMO
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.