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1.
Transgenic Res ; 20(6): 1203-16, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21360304

RESUMO

In order to study the effects of Hepatocyte Growth Factor (HGF) in the heart, two transgenic mice were developed, one carrying a bidirectional HGF-TetO-GFP responder construct and the other carrying a α-MHC-tTA transactivator construct. Crosses were carried out between heterozygotes, so that litters contained bitransgenic α-MHC-tTA/HGF-TetO-GFP+, thus expressing HGF and GFP exclusively in the heart and only in the absence of Doxycycline. Our data show that the expression of HGF was indeed restricted to the heart and that the expression was limited to the timeframe of the absence of Doxycycline. Surprisingly the expression was variable even between bitransgenic littermates. In the setting of a model of ischemia-reperfusion, the expression of HGF ameliorates cardiac functionality, enhances proliferation and diminishes the scarred area, proving that this is a good model to study the beneficial influences and functional roles of HGF in the heart.


Assuntos
Doxiciclina/farmacologia , Coração/fisiopatologia , Fator de Crescimento de Hepatócito/metabolismo , Animais , Western Blotting , Linhagem Celular , Proliferação de Células , Colágeno/metabolismo , Cruzamentos Genéticos , Meios de Cultivo Condicionados/metabolismo , Cães , Ecocardiografia , Feminino , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Coração/efeitos dos fármacos , Fator de Crescimento de Hepatócito/genética , Heterozigoto , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Modelos Animais , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Gravidez , Reação em Cadeia da Polimerase em Tempo Real
2.
J Cell Biochem ; 110(1): 70-9, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20213742

RESUMO

Accumulating evidences point to a significant role for the chromogranin A (CgA)-derived peptide vasostatin 1 (VS-1) in the protective modulation of the cardiovascular activity, because of its ability to counteract the adrenergic signal. We have recently shown that VS-1 induces a PI3K-dependent-nitric oxide (NO) release by endothelial cells, contributing to explain the mechanism of its cardio-suppressive and vasodilator properties. However, the cellular processes upstream the eNOS activation exerted by this peptide are still unknown, as typical high-affinity receptors have not been identified. Here we hypothesize that in endothelial cells VS-1 acts, on the basis of its cationic and amphipathic properties, as a cell penetrating peptide, binding to heparan sulfate proteoglycans (HSPGs) and activating eNOS phosphorylation (Ser1179) through a PI3K-dependent, endocytosis-coupled mechanism. In bovine aortic endothelial cells (BAE-1 cells) endocytotic vesicles trafficking was quantified by confocal microscopy with a water-soluble membrane dye; caveolin 1 (Cav1) shift from plasma membrane was studied by immunofluorescence staining; VS-1-dependent eNOS phosphorylation was assessed by immunofluorescence and immunoblot analysis. Our experiments demonstrate that VS-1 induces a marked increase in the caveolae-dependent endocytosis, (115 +/- 23% endocytotic spots/cell/field in VS-1-treated cells with respect to control cells), that is significantly reduced by both heparinase III (HEP, 17 +/- 15% above control) and Wortmannin (Wm, 7 +/- 22% above control). Heparinase, Wortmannin, and methyl-beta-cyclodextrin (MbetaCD) abolish the VS-1-dependent eNOS phosphorylation (P(Ser1179)eNOS). These results suggest a novel signal transduction pathway for endogenous cationic and amphipathic peptides in endothelial cells: HSPGs interaction and caveolae endocytosis, coupled with a PI3K-dependent eNOS phosphorylation.


Assuntos
Cromogranina A/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Fragmentos de Peptídeos/farmacologia , Proteoglicanas/metabolismo , Androstadienos/farmacologia , Animais , Bovinos , Cavéolas/efeitos dos fármacos , Cavéolas/metabolismo , Caveolina 1/metabolismo , Endocitose/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Heparina Liase/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Vesículas Transportadoras/efeitos dos fármacos , Vesículas Transportadoras/metabolismo , Wortmanina
3.
Cell Mol Biol Lett ; 14(1): 100-12, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-18839068

RESUMO

Ischemic diseases are characterized by the presence of pro-apoptotic stimuli, which initiate a cascade of processes that lead to cell injury and death. Several molecules and events represent detectable indicators of the different stages of apoptosis. Among these indicators is phosphatidylserine (PS) translocation from the inner to the outer leaflet of the plasma membrane, which can be detected by annexinV (ANXA5) conjugation. This is a widely used in vivo and in vitro assay marking the early stages of apoptosis. We report here on an original method that employs PS-ANXA5 conjugation to target stem cells to apoptotic cells. Mesenchymal stem cells (MSCs) from GFP-positive transgenic rats were biotinylated on membrane surfaces with sulfosuccinimidyl-6-(biotinamido) hexanoate (sulfo-NHS-LC-biot) and then bound to avidin. The avidin-biotinylated MSCs were labeled with biotin conjugated ANXA5. Bovine aortic endothelial cells (BAE-1 cells) were exposed to UVC to induce caspasedependent apoptosis. Finally, we tested the ability of ANXA5-labeled MSCs to bind BAE-1 apoptotic cells: suspended ANXA5-labeled MSCs were seeded for 1 hour on a monolayer of UV-treated or control BAE-1 cells. After washing, the number of MSCs bound to BAE-1 cells was evaluated by confocal microscopy. Statistical analysis demonstrated a significant increase in the number of MSCs tagged to apoptotic BAE-1 cells. Therefore, stem cell ANXA5 tagging via biotin-avidin bridges could be a straightforward method of improving homing to apoptotic tissues.


Assuntos
Anexina A5/metabolismo , Apoptose , Movimento Celular , Células Endoteliais/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Coloração e Rotulagem , Animais , Biotinilação , Bovinos , Linhagem Celular , Células Endoteliais/metabolismo , Cetonas/metabolismo , Ratos
4.
Am J Physiol Heart Circ Physiol ; 292(6): H2906-12, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17293489

RESUMO

Vasostatins (VSs) are vasoactive peptides derived from chromogranin A (CgA), a protein contained in secretory granules of chromaffin and other cells. The negative inotropic effect and the reduction of isoproterenol (Iso)-dependent inotropism induced by VSs in the heart suggest that they have an antiadrenergic function. However, further investigation of the mechanisms of action of VSs is needed. The aim of the present study was to define the signaling pathways activated by VS-1 in mammalian ventricular myocardium and cultured endothelial cells that lead to the modulation of cardiac contractility. Ca(2+) and nitric oxide (NO) fluorometric confocal imaging was used to study the effects induced by recombinant human VS-1 [STA-CgA-(1-76)] on contractile force, L-type Ca(2+) current, and Ca(2+) transients under basal conditions and after beta-adrenergic stimulation in rat papillary muscles and ventricular cells and the effects on intracellular Ca(2+) concentration and NO production in cultured bovine aortic endothelial (BAE-1) cells. VS-1 had no effect on basal contractility of papillary muscle, but the effect of Iso stimulation was reduced by 27%. Removal of endocardial endothelium and inhibition of NO synthesis and phosphatidylinositol 3-kinase (PI3K) activity abolished the antiadrenergic effect of VS-1 on papillary muscle. In cardiomyocytes, 10 nM VS-1 was ineffective on basal and Iso (1 microM)-stimulated L-type Ca(2+) current and Ca(2+) transients. In BAE-1 cells, VS-1 induced a Ca(2+)-independent increase in NO production that was blocked by the PI3K inhibitor wortmannin. Our results suggest that the antiadrenergic effect of VS-1 is mainly due to a PI3K-dependent NO release by endothelial cells, rather than a direct action on cardiomyocytes.


Assuntos
Antagonistas Adrenérgicos beta/metabolismo , Sinalização do Cálcio , Cromogranina A/metabolismo , Células Endoteliais/metabolismo , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Óxido Nítrico/metabolismo , Músculos Papilares/metabolismo , Fragmentos de Peptídeos/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Androstadienos/farmacologia , Animais , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Bovinos , Células Cultivadas , Cromogranina A/farmacologia , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Feminino , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Humanos , Técnicas In Vitro , Isoproterenol/antagonistas & inibidores , Isoproterenol/farmacologia , Força Muscular , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Músculos Papilares/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Ratos , Proteínas Recombinantes/metabolismo , Wortmanina
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