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1.
Am J Respir Cell Mol Biol ; 62(6): 732-746, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32048876

RESUMO

Pulmonary vasoconstriction resulting from intermittent hypoxia (IH) contributes to pulmonary hypertension (pHTN) in patients with sleep apnea (SA), although the mechanisms involved remain poorly understood. Based on prior studies in patients with SA and animal models of SA, the objective of this study was to evaluate the role of PKCß and mitochondrial reactive oxygen species (mitoROS) in mediating enhanced pulmonary vasoconstrictor reactivity after IH. We hypothesized that PKCß mediates vasoconstriction through interaction with the scaffolding protein PICK1 (protein interacting with C kinase 1), activation of mitochondrial ATP-sensitive potassium channels (mitoKATP), and stimulated production of mitoROS. We further hypothesized that this signaling axis mediates enhanced vasoconstriction and pHTN after IH. Rats were exposed to IH or sham conditions (7 h/d, 4 wk). Chronic oral administration of the antioxidant Tempol or the PKCß inhibitor LY-333531 abolished IH-induced increases in right ventricular systolic pressure and right ventricular hypertrophy. Furthermore, scavengers of O2- or mitoROS prevented enhanced PKCß-dependent vasoconstrictor reactivity to endothelin-1 in pulmonary arteries from IH rats. In addition, this PKCß/mitoROS signaling pathway could be stimulated by the PKC activator PMA in pulmonary arteries from control rats, and in both rat and human pulmonary arterial smooth muscle cells. These responses to PMA were attenuated by inhibition of mitoKATP or PICK1. Subcellular fractionation and proximity ligation assays further demonstrated that PKCß acutely translocates to mitochondria upon stimulation and associates with PICK1. We conclude that a PKCß/mitoROS signaling axis contributes to enhanced vasoconstriction and pHTN after IH. Furthermore, PKCß mediates pulmonary vasoconstriction through interaction with PICK1, activation of mitoKATP, and subsequent mitoROS generation.


Assuntos
Hipertensão Pulmonar/fisiopatologia , Hipóxia/fisiopatologia , Mitocôndrias/fisiologia , Proteína Quinase C beta/fisiologia , Artéria Pulmonar/fisiopatologia , Vasoconstrição/fisiologia , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Células Cultivadas , Óxidos N-Cíclicos/farmacologia , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/metabolismo , Sequestradores de Radicais Livres/farmacologia , Humanos , Hipertensão Pulmonar/etiologia , Hipóxia/complicações , Hipóxia/enzimologia , Indóis/farmacologia , Masculino , Maleimidas/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiopatologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Canais de Potássio/metabolismo , Mapeamento de Interação de Proteínas , Artéria Pulmonar/enzimologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Síndromes da Apneia do Sono/fisiopatologia , Marcadores de Spin , Acetato de Tetradecanoilforbol/farmacologia
2.
Am J Physiol Heart Circ Physiol ; 318(2): H470-H483, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31922892

RESUMO

Reactive oxygen species (ROS), mitochondrial dysfunction, and excessive vasoconstriction are important contributors to chronic hypoxia (CH)-induced neonatal pulmonary hypertension. On the basis of evidence that PKCß and mitochondrial oxidative stress are involved in several cardiovascular and metabolic disorders, we hypothesized that PKCß and mitochondrial ROS (mitoROS) signaling contribute to enhanced pulmonary vasoconstriction in neonatal rats exposed to CH. To test this hypothesis, we examined effects of the PKCß inhibitor LY-333,531, the ROS scavenger 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (TEMPOL), and the mitochondrial antioxidants mitoquinone mesylate (MitoQ) and (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) on vasoconstrictor responses in saline-perfused lungs (in situ) or pressurized pulmonary arteries from 2-wk-old control and CH (12-day exposure, 0.5 atm) rats. Lungs from CH rats exhibited greater basal tone and vasoconstrictor sensitivity to 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U-46619). LY-333,531 and TEMPOL attenuated these effects of CH, while having no effect in lungs from control animals. Basal tone was similarly elevated in isolated pulmonary arteries from neonatal CH rats compared with control rats, which was inhibited by both LY-333,531 and mitochondria-targeted antioxidants. Additional experiments assessing mitoROS generation with the mitochondria-targeted ROS indicator MitoSOX revealed that a PKCß-mitochondrial oxidant signaling pathway can be pharmacologically stimulated by the PKC activator phorbol 12-myristate 13-acetate in primary cultures of pulmonary artery smooth muscle cells (PASMCs) from control neonates. Finally, we found that neonatal CH increased mitochondrially localized PKCß in pulmonary arteries as assessed by Western blotting of subcellular fractions. We conclude that PKCß activation leads to mitoROS production in PASMCs from neonatal rats. Furthermore, this signaling axis may account for enhanced pulmonary vasoconstrictor sensitivity following CH exposure.NEW & NOTEWORTHY This research demonstrates a novel contribution of PKCß and mitochondrial reactive oxygen species signaling to increased pulmonary vasoconstrictor reactivity in chronically hypoxic neonates. The results provide a potential mechanism by which chronic hypoxia increases both basal and agonist-induced pulmonary arterial smooth muscle tone, which may contribute to neonatal pulmonary hypertension.


Assuntos
Hipóxia/metabolismo , Proteína Quinase C beta/metabolismo , Animais , Animais Recém-Nascidos , Doença Crônica , Óxidos N-Cíclicos/farmacologia , Inibidores Enzimáticos , Feminino , Sequestradores de Radicais Livres , Indóis/farmacologia , Maleimidas/farmacologia , Compostos Organofosforados/farmacologia , Estresse Oxidativo , Gravidez , Proteína Quinase C beta/antagonistas & inibidores , Artéria Pulmonar/efeitos dos fármacos , Circulação Pulmonar , Ratos , Espécies Reativas de Oxigênio , Marcadores de Spin , Ubiquinona/análogos & derivados , Ubiquinona/farmacologia , Vasoconstrição , Vasoconstritores/farmacologia
3.
Am J Respir Cell Mol Biol ; 62(6): 709-718, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31945301

RESUMO

Chronic hypoxia (CH) augments depolarization-induced pulmonary vasoconstriction through superoxide-dependent, Rho kinase-mediated Ca2+ sensitization. Nicotinamide adenine dinucleotide phosphate oxidase and EGFR (epidermal growth factor receptor) signaling contributes to this response. Caveolin-1 regulates the activity of a variety of proteins, including EGFR and nicotinamide adenine dinucleotide phosphate oxidase, and membrane cholesterol is an important regulator of caveolin-1 protein interactions. We hypothesized that derangement of these membrane lipid domain components augments depolarization-induced Ca2+ sensitization and resultant vasoconstriction after CH. Although exposure of rats to CH (4 wk, ∼380 mm Hg) did not alter caveolin-1 expression in intrapulmonary arteries or the incidence of caveolae in arterial smooth muscle, CH markedly reduced smooth muscle membrane cholesterol content as assessed by filipin fluorescence. Effects of CH on vasoreactivity and superoxide generation were examined using pressurized, Ca2+-permeabilized, endothelium-disrupted pulmonary arteries (∼150 µm inner diameter) from CH and control rats. Depolarizing concentrations of KCl evoked greater constriction in arteries from CH rats than in those obtained from control rats, and increased superoxide production as assessed by dihydroethidium fluorescence only in arteries from CH rats. Both cholesterol supplementation and the caveolin-1 scaffolding domain peptide antennapedia-Cav prevented these effects of CH, with each treatment restoring membrane cholesterol in CH arteries to control levels. Enhanced EGF-dependent vasoconstriction after CH similarly required reduced membrane cholesterol. However, these responses to CH were not associated with changes in EGFR expression or activity, suggesting that cholesterol regulates this signaling pathway downstream of EGFR. We conclude that alterations in membrane lipid domain signaling resulting from reduced cholesterol content facilitate enhanced depolarization- and EGF-induced pulmonary vasoconstriction after CH.


Assuntos
Cálcio/fisiologia , Caveolina 1/biossíntese , Colesterol/fisiologia , Hipóxia/fisiopatologia , Lipídeos de Membrana/fisiologia , Músculo Liso Vascular/metabolismo , Artéria Pulmonar/fisiopatologia , Vasoconstrição/fisiologia , Animais , Caveolina 1/genética , Doença Crônica , Receptores ErbB/fisiologia , Hipóxia/metabolismo , Masculino , Potenciais da Membrana , Artéria Pulmonar/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Superóxidos/metabolismo
4.
Am J Respir Cell Mol Biol ; 62(1): 61-73, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31264901

RESUMO

Chronic hypoxia augments pressure- and agonist-induced pulmonary vasoconstriction through myofilament calcium sensitization. NADPH oxidases contribute to the development of pulmonary hypertension, and both epidermal growth factor receptor and Src kinases can regulate NADPH oxidase. We tested the hypothesis that Src-epidermal growth factor receptor (EGFR) signaling mediates enhanced vasoconstrictor sensitivity after chronic hypoxia through NADPH oxidase-derived superoxide generation. Protocols employed pharmacological inhibitors in isolated, pressurized rat pulmonary arteries to examine the contribution of a variety of signaling moieties to enhanced vascular tone after chronic hypoxia. Superoxide generation in pulmonary arterial smooth muscle cells was assessed using the fluorescent indicator dihydroethidium. Indices of pulmonary hypertension were measured in rats treated with the EGFR inhibitor gefitinib. Inhibition of NADPH oxidase, Rac1 (Ras-related C3 botulinum toxin substrate 1), and EGFR abolished pressure-induced pulmonary arterial tone and endothelin-1 (ET-1)-dependent calcium sensitization and vasoconstriction after chronic hypoxia. Consistently, chronic hypoxia augmented ET-1-induced superoxide production through EGFR signaling, and rats treated chronically with gefitinib displayed reduced right ventricular pressure and diminished arterial remodeling. Src kinases were also activated by ET-1 after chronic hypoxia and contributed to enhanced basal arterial tone and vasoconstriction in response to ET-1. A role for matrix metalloproteinase 2 to mediate Src-dependent EGFR activation is further supported by our findings. Our studies support a novel role for an Src kinase-EGFR-NADPH oxidase signaling axis to mediate enhanced pulmonary vascular smooth muscle Ca2+ sensitization, vasoconstriction, and pulmonary hypertension after chronic hypoxia.


Assuntos
Receptores ErbB/metabolismo , Hipóxia/tratamento farmacológico , Pulmão/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacocinética , Quinases da Família src/metabolismo , Animais , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/metabolismo , Hipóxia/metabolismo , Pulmão/metabolismo , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Ratos , Ratos Sprague-Dawley
5.
Antioxid Redox Signal ; 31(12): 898-915, 2019 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-30569735

RESUMO

Significance: Pulmonary hypertension (PH) is characterized by elevated vascular resistance due to vasoconstriction and remodeling of the normally low-pressure pulmonary vasculature. Redox stress contributes to the pathophysiology of this disease by altering the regulation and activity of membrane receptors, K+ channels, and intracellular Ca2+ homeostasis. Recent Advances: Antioxidant therapies have had limited success in treating PH, leading to a growing appreciation that reductive stress, in addition to oxidative stress, plays a role in metabolic and cell signaling dysfunction in pulmonary vascular cells. Reactive oxygen species generation from mitochondria and NADPH oxidases has substantial effects on K+ conductance and membrane potential, and both receptor-operated and store-operated Ca2+ entry. Critical Issues: Some specific redox changes resulting from oxidation, S-nitrosylation, and S-glutathionylation are known to modulate membrane receptor and ion channel activity in PH. However, many sites of regulation that have been elucidated in nonpulmonary cell types have not been tested in the pulmonary vasculature, and context-specific molecular mechanisms are lacking. Future Directions: Here, we review what is known about redox regulation of membrane receptors and ion channels in PH. Further investigation of the mechanisms involved is needed to better understand the etiology of PH and develop better targeted treatment strategies.


Assuntos
Hipertensão Pulmonar/metabolismo , Canais Iônicos/metabolismo , Oxirredução , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Cálcio/metabolismo , Humanos , Potássio/metabolismo , Resistência Vascular
6.
Am J Physiol Heart Circ Physiol ; 314(5): H1011-H1021, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29373038

RESUMO

Chronic hypoxia (CH) augments basal and endothelin-1 (ET-1)-induced pulmonary vasoconstrictor reactivity through reactive oxygen species (ROS) generation and RhoA/Rho kinase (ROCK)-dependent myofilament Ca2+ sensitization. Because ROCK promotes actin polymerization and the actin cytoskeleton regulates smooth muscle tension, we hypothesized that actin polymerization is required for enhanced basal and ET-1-dependent vasoconstriction after CH. To test this hypothesis, both end points were monitored in pressurized, endothelium-disrupted pulmonary arteries (fourth-fifth order) from control and CH (4 wk at 0.5 atm) rats. The actin polymerization inhibitors cytochalasin and latrunculin attenuated both basal and ET-1-induced vasoconstriction only in CH vessels. To test whether CH directly alters the arterial actin profile, we measured filamentous actin (F-actin)-to-globular actin (G-actin) ratios by fluorescent labeling of F-actin and G-actin in fixed pulmonary arteries and actin sedimentation assays using homogenized pulmonary artery lysates. We observed no difference in actin polymerization between groups under baseline conditions, but ET-1 enhanced actin polymerization in pulmonary arteries from CH rats. This response was blunted by the ROS scavenger tiron, the ROCK inhibitor fasudil, and the mDia (RhoA effector) inhibitor small-molecule inhibitor of formin homology domain 2. Immunoblot analysis revealed an effect of CH to increase both phosphorylated (inactive) and total levels of the actin disassembly factor cofilin but not phosphorylated cofilin-to-total cofilin ratios. We conclude that actin polymerization contributes to increased basal pulmonary arterial constriction and ET-1-induced vasoconstrictor reactivity after CH in a ROS- and ROCK-dependent manner. Our results further suggest that enhanced ET-1-mediated actin polymerization after CH is dependent on mDia but independent of changes in the phosphorylated cofilin-to-total cofilin ratio. NEW & NOTEWORTHY This research is the first to demonstrate a role for actin polymerization in chronic hypoxia-induced basal pulmonary arterial constriction and enhanced agonist-induced vasoconstrictor activity. These results suggest that a reactive oxygen species-Rho kinase-actin polymerization signaling pathway mediates this response and may provide a mechanistic basis for the vasoconstrictor component of pulmonary hypertension.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Hipertensão Pulmonar/etiologia , Hipóxia/complicações , Artéria Pulmonar/metabolismo , Remodelação Vascular , Vasoconstrição , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/patologia , Fatores de Despolimerização de Actina/metabolismo , Animais , Doença Crônica , Modelos Animais de Doenças , Endotelina-1/farmacologia , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/fisiopatologia , Hipóxia/metabolismo , Hipóxia/patologia , Hipóxia/fisiopatologia , Masculino , Estresse Oxidativo , Fosforilação , Polimerização , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , Ratos Sprague-Dawley , Remodelação Vascular/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo
7.
PLoS One ; 12(6): e0180455, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28666030

RESUMO

Pulmonary arterial hypertension is associated with a decreased antioxidant capacity. However, neither the contribution of reactive oxygen species to pulmonary vasoconstrictor sensitivity, nor the therapeutic efficacy of antioxidant strategies in this setting are known. We hypothesized that reactive oxygen species play a central role in mediating both vasoconstrictor and arterial remodeling components of severe pulmonary arterial hypertension. We examined the effect of the chemical antioxidant, TEMPOL, on right ventricular systolic pressure, vascular remodeling, and enhanced vasoconstrictor reactivity in both chronic hypoxia and hypoxia/SU5416 rat models of pulmonary hypertension. SU5416 is a vascular endothelial growth factor receptor antagonist and the combination of chronic hypoxia/SU5416 produces a model of severe pulmonary arterial hypertension with vascular plexiform lesions/fibrosis that is not present with chronic hypoxia alone. The major findings from this study are: 1) compared to hypoxia alone, hypoxia/SU5416 exposure caused more severe pulmonary hypertension, right ventricular hypertrophy, adventitial lesion formation, and greater vasoconstrictor sensitivity through a superoxide and Rho kinase-dependent Ca2+ sensitization mechanism. 2) Chronic hypoxia increased medial muscularization and superoxide levels, however there was no effect of SU5416 to augment these responses. 3) Treatment with TEMPOL decreased right ventricular systolic pressure in both hypoxia and hypoxia/SU5416 groups. 4) This effect of TEMPOL was associated with normalization of vasoconstrictor responses, but not arterial remodeling. Rather, medial hypertrophy and adventitial fibrotic lesion formation were more pronounced following chronic TEMPOL treatment in hypoxia/SU5416 rats. Our findings support a major role for reactive oxygen species in mediating enhanced vasoconstrictor reactivity and pulmonary hypertension in both chronic hypoxia and hypoxia/SU5416 rat models, despite a paradoxical effect of antioxidant therapy to exacerbate arterial remodeling in animals with severe pulmonary arterial hypertension in the hypoxia/SU5416 model.


Assuntos
Modelos Animais de Doenças , Hipertensão Pulmonar/metabolismo , Artéria Pulmonar/patologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Óxidos N-Cíclicos/administração & dosagem , Endotelina-1/metabolismo , Hipertensão Pulmonar/prevenção & controle , Hipertrofia Ventricular Direita/patologia , Masculino , Artéria Pulmonar/metabolismo , Artéria Pulmonar/fisiopatologia , Ratos , Ratos Sprague-Dawley , Marcadores de Spin , Superóxidos/metabolismo , Vasoconstrição
8.
Am J Physiol Heart Circ Physiol ; 313(4): H828-H838, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28733445

RESUMO

Augmented vasoconstrictor reactivity is thought to play an important role in the development of chronic hypoxia (CH)-induced neonatal pulmonary hypertension. However, whether this response to CH results from pulmonary endothelial dysfunction and reduced nitric oxide (NO)-mediated vasodilation is not well understood. We hypothesized that neonatal CH enhances basal tone and pulmonary vasoconstrictor sensitivity by limiting NO-dependent pulmonary vasodilation. To test this hypothesis, we assessed the effects of the NO synthase (NOS) inhibitor Nω-nitro-l-arginine (l-NNA) on baseline pulmonary vascular resistance (PVR) and vasoconstrictor sensitivity to the thromboxane mimetic U-46619 in saline-perfused lungs (in situ) from 2-wk-old control and CH (12-day exposure, 0.5 atm) Sprague-Dawley rats. Basal tone was defined as that reversed by exogenous NO (spermine NONOate). CH neonates displayed elevated right ventricular systolic pressure (in vivo) and right ventricular hypertrophy, indicative of pulmonary hypertension. Perfused lungs from CH rats demonstrated greater baseline PVR, basal tone, and U-46619-mediated vasoconstriction compared with control rats in the absence of l-NNA. l-NNA markedly increased baseline PVR and reactivity to U-46619 in lungs from CH neonates, further augmenting vasoconstrictor sensitivity compared with control lungs. Exposure to CH also enhanced NO-dependent vasodilation to arginine vasopressin, pulmonary expression of NOS III [endothelial NOS (eNOS)], and eNOS phosphorylation at activation residue Ser1177 However, CH did not alter lung nitrotyrosine levels, a posttranslational modification reflecting [Formula: see text] scavenging of NO. We conclude that, in contrast to our hypothesis, enhanced basal tone and agonist-induced vasoconstriction after neonatal CH is limited by increased NO-dependent pulmonary vasodilation resulting from greater eNOS expression and phosphorylation at activation residue Ser1177NEW & NOTEWORTHY This research is the first to demonstrate enhanced nitric oxide-dependent vasodilation that limits increased vasoconstrictor reactivity in neonatal pulmonary hypertension. These results suggest that augmented vasoconstriction in this setting reflects changes in smooth muscle reactivity rather than a reduction in nitric oxide-dependent pulmonary vasodilation.


Assuntos
Hipóxia/fisiopatologia , Óxido Nítrico , Circulação Pulmonar , Vasoconstritores/farmacologia , Vasodilatação , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Animais , Animais Recém-Nascidos , Doença Crônica , Inibidores Enzimáticos/farmacologia , Sequestradores de Radicais Livres/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Nitroarginina/farmacologia , Ratos , Ratos Sprague-Dawley , Tirosina/análogos & derivados , Tirosina/metabolismo , Resistência Vascular/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos
9.
Arterioscler Thromb Vasc Biol ; 35(11): 2374-83, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26381868

RESUMO

OBJECTIVE: We and others have previously shown that RhoA-dependent stimulation of myocardin-related transcription factor nuclear localization promotes smooth muscle cell (SMC) marker gene expression. The goal of this study was to provide direct in vivo evidence that actin polymerization by the diaphanous-related formins contributes to the regulation of SMC differentiation and phenotype. APPROACH AND RESULTS: Conditional Cre-based genetic approaches were used to overexpress a well-characterized dominant-negative variant of mDia1 (DNmDia) in SMC. DNmDia expression in SM22-expressing cells resulted in embryonic and perinatal lethality in ≈20% of mice because of defects in myocardial development and SMC investment of peripheral vessels. Although most DNmDia(+)/SM22Cre(+) mice exhibited no overt phenotype, the re-expression of SMC differentiation marker gene expression that occurs after carotid artery ligation was delayed, and this effect was accompanied by a significant decrease in myocardin-related transcription factor-A nuclear localization. Interestingly, neointima growth was inhibited by expression of DNmDia in SMC and this was likely because of a defect in directional SMC migration and not to defects in SMC proliferation or survival. Finally, by using the tamoxifen-inducible SM MHC-CreER(T2) line, we showed that SMC-specific induction of DNmDia in adult mice decreased SMC marker gene expression. CONCLUSIONS: Our demonstration that diaphanous-related formin signaling plays a role in heart and vascular development and the maintenance of SMC phenotype provides important new evidence that Rho/actin/myocardin-related transcription factor signaling plays a critical role in cardiovascular function.


Assuntos
Proteínas de Transporte/metabolismo , Cardiopatias Congênitas/metabolismo , Músculo Liso Vascular/metabolismo , Miocárdio/metabolismo , Miócitos de Músculo Liso/metabolismo , Transdução de Sinais , Actinas/metabolismo , Animais , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Artérias Carótidas/cirurgia , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Proteínas de Transporte/genética , Diferenciação Celular , Linhagem Celular , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Forminas , Coração/embriologia , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/patologia , Ligadura , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Desenvolvimento Muscular , Proteínas Musculares/metabolismo , Músculo Liso Vascular/embriologia , Músculo Liso Vascular/patologia , Músculo Liso Vascular/cirurgia , Miocárdio/patologia , Miócitos de Músculo Liso/patologia , Neointima , Fenótipo , Polimerização , Fatores de Tempo , Transfecção , Lesões do Sistema Vascular
10.
Am J Physiol Heart Circ Physiol ; 307(3): H379-90, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24906914

RESUMO

We have previously shown that RhoA-mediated actin polymerization stimulates smooth muscle cell (SMC)-specific transcription by regulating the nuclear localization of the myocardin-related transcription factors (MRTFs). On the basis of the recent demonstration that nuclear G-actin regulates MRTF nuclear export and observations from our laboratory and others that the RhoA effector, mDia2, shuttles between the nucleus and cytoplasm, we investigated whether nuclear RhoA signaling plays a role in regulating MRTF activity. We identified sequences that control mDia2 nuclear-cytoplasmic shuttling and used mDia2 variants to demonstrate that the ability of mDia2 to fully stimulate MRTF nuclear accumulation and SMC-specific gene transcription was dependent on its localization to the nucleus. To test whether RhoA signaling promotes nuclear actin polymerization, we established a fluorescence recovery after photobleaching (FRAP)-based assay to measure green fluorescent protein-actin diffusion in the nuclear compartment. Nuclear actin FRAP was delayed in cells expressing nuclear-targeted constitutively active mDia1 and mDia2 variants and in cells treated with the polymerization inducer, jasplakinolide. In contrast, FRAP was enhanced in cells expressing a nuclear-targeted variant of mDia that inhibits both mDia1 and mDia2. Treatment of 10T1/2 cells with sphingosine 1-phosphate induced RhoA activity in the nucleus and forced nuclear localization of RhoA or the Rho-specific guanine nucleotide exchange factor (GEF), leukemia-associated RhoGEF, enhanced the ability of these proteins to stimulate MRTF activity. Taken together, these data support the emerging idea that RhoA-dependent nuclear actin polymerization has important effects on transcription and nuclear structure.


Assuntos
Núcleo Celular/enzimologia , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/genética , Actinas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Aorta Torácica/enzimologia , Células COS , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diferenciação Celular , Chlorocebus aethiops , Recuperação de Fluorescência Após Fotodegradação , Forminas , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , NADPH Desidrogenase/genética , NADPH Desidrogenase/metabolismo , Interferência de RNA , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Fatores de Tempo , Transativadores/genética , Fatores de Transcrição/genética , Transfecção , Proteína rhoA de Ligação ao GTP
11.
Biophys J ; 106(8): 1741-50, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24739173

RESUMO

A method for fitting sedimentation velocity experiments using whole boundary Lamm equation solutions is presented. The method, termed parametrically constrained spectrum analysis (PCSA), provides an optimized approach for simultaneously modeling heterogeneity in size and anisotropy of macromolecular mixtures. The solutions produced by PCSA are particularly useful for modeling polymerizing systems, where a single-valued relationship exists between the molar mass of the growing polymer chain and its corresponding anisotropy. The PCSA uses functional constraints to identify this relationship, and unlike other multidimensional grid methods, assures that only a single molar mass can be associated with a given anisotropy measurement. A description of the PCSA algorithm is presented, as well as several experimental and simulated examples that illustrate its utility and capabilities. The performance advantages of the PCSA method in comparison to other methods are documented. The method has been added to the UltraScan-III software suite, which is available for free download from http://www.ultrascan.uthscsa.edu.


Assuntos
Algoritmos , Ultracentrifugação/métodos , Animais , Bovinos , Clatrina/química , Clatrina/metabolismo , DNA/química , Método de Monte Carlo , Polimerização
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