RESUMEN
BACKGROUND: The human Bcr gene was originally identified by its presence in the chimeric Bcr/Abl oncogene, which is causative for chronic myeloblastic leukemia. Because Bcr encodes a serine/threonine protein kinase, we studied its kinase activity and determined the role of Bcr in the PDGF signaling pathway to ERK1/2 activation and DNA synthesis in rat aortic smooth muscle cells (RASMCs). METHODS AND RESULTS: In RASMCs, platelet-derived growth factor-BB (PDGF) stimulated Bcr kinase activity, with a maximum at 1 minute. Because phosphatidylinositol 3'-kinase (PI3-K) is essential for Bcr/Abl leukemogenesis, we evaluated the role of mouse PDGF-beta-receptor binding sites for PI3-K (Y708, Y719) and for phospholipase C-gamma (Y977, Y989) in PDGF-mediated Bcr kinase activation. The mutant PDGF receptor Y708F/Y719F but not Y977F/Y989F showed significantly reduced Bcr kinase activity. To determine the role of Bcr in PDGF-mediated signal transduction events leading to ERK1/2 and its downstream Elk1 transcription activation, wild-type (WT) and kinase-negative (KN) Bcr were transiently expressed in RASMCs. Bcr WT enhanced, whereas Bcr KN inhibited, PDGF-stimulated ERK1/2 and Elk1 transcriptional activity. Overexpression of Bcr also enhanced PDGF-induced Ras/Raf-1 activity and DNA synthesis, but this regulation is independent of the kinase activity of Bcr. Finally, we found that Bcr expression was increased in the neointimal layer after balloon injury of rat carotid artery. CONCLUSIONS: These results demonstrated the importance of Bcr in PDGF-mediated events, such as activation of Ras, Raf-1, ERK1/2, and Elk1, and stimulation of DNA synthesis.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Proteínas Oncogénicas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas , Animales , Aorta , Células CHO , Arterias Carótidas/metabolismo , Células Cultivadas , Cricetinae , ADN/biosíntesis , Activación Enzimática/efectos de los fármacos , Expresión Génica , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/citología , Proteínas Oncogénicas/genética , Estructura Terciaria de Proteína/fisiología , Proteínas Proto-Oncogénicas c-bcr , Proteínas Proto-Oncogénicas c-raf/metabolismo , Ratas , Receptores del Factor de Crecimiento Derivado de Plaquetas/genética , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transfección , Túnica Íntima/metabolismo , Proteínas ras/metabolismoRESUMEN
M. Zhang, D. Methot, V. Poppa, Y. Fujio, K. Walsh and C. E. Murry. Cardiomyocyte Grafting for Cardiac Repair: Graft Cell Death and Anti-Death Strategies. Journal of Molecular and Cellular Cardiology (2001) 33, 907-921. Recent studies indicate that cardiomyocyte grafting forms new myocardium in injured hearts. It is unknown, however, whether physiologically significant amounts of new myocardium can be generated. Pilot experiments showed that death of grafted rat neonatal cardiomyocytes limited formation of new myocardium after acute cryoinjury. Time-course studies showed that, at 30 min after grafting, only 1.8(+/-0.4)% of graft cells were TUNEL-positive. At 1 day, however, TUNEL indices increased to 32.1(+/-3.5)% and remained high at 4 days, averaging 9.8(+/-3.8)%. By 7 days, TUNEL decreased to 1.0(+/-0.2)%. Electron microscopy revealed that dead cells had features of both irreversible ischemic injury and apoptosis. To test whether ischemia contributed to poor graft survival, grafts were placed into vascularized 2-week-old cardiac granulation tissue or normal myocardium. TUNEL indices were reduced by 53% and 86%, respectively. Adenoviral infection of graft cells with the cytoprotective kinase Akt, or constitutively active Akt, reduced TUNEL indices by 31% and 40%, respectively, compared to beta -gal-transfected controls. Neither treatment reached statistical significance compared to untreated controls, however. Heat shock reduced cardiomyocyte death in vitro in response to serum deprivation, glucose depletion, and viral activation of the Fas death pathway. When cardiomyocytes were heat shocked prior to grafting, graft cell death in vivo was reduced by 54% at day 1. Therefore, high levels of cardiomyocyte death occur for at least 4 days after grafting into injured hearts, in large part due to ischemia. Death can be limited by activating the Akt pathway and even more effectively by heat shock prior to transplantation.
Asunto(s)
Miocardio/citología , Miocardio/metabolismo , Proteínas Serina-Treonina Quinasas , Trasplante de Tejidos/métodos , Adenoviridae/genética , Animales , Western Blotting , Muerte Celular , Supervivencia Celular , Células Cultivadas , Medio de Cultivo Libre de Suero/metabolismo , Calor , Etiquetado Corte-Fin in Situ , Isquemia , Masculino , Microscopía Electrónica , Miocardio/ultraestructura , Necrosis , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas Endogámicas F344 , Factores de TiempoRESUMEN
Synthesis of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) is critical for normal vascular homeostasis. eNOS function is rapidly regulated by agonists and blood flow and chronically by factors that regulate mRNA stability and gene transcription. Recently, localization of eNOS to specialized plasma membrane invaginations termed caveolae has been proposed to be required for maximal eNOS activity. Because caveolae are highly enriched in cholesterol, and hypercholesterolemia is associated with increased NO production, we first studied the effects of cholesterol loading on eNOS localization and NO production in cultured bovine aortic endothelial cells (BAECs). Caveolae-enriched fractions were prepared by OptiPrep gradient density centrifugation. Treatment of BAECs with 30 microgram/mL cholesterol for 24 hours stimulated significant increases in total eNOS protein expression (1.50-fold), eNOS associated with caveolae-enriched membranes (2.23-fold), and calcium ionophore-stimulated NO production (1.56-fold). Because reactive oxygen species (ROS) contribute to endothelial dysfunction in hypercholesterolemia, we next studied the effects of ROS on eNOS localization and caveolae number. Treatment of BAECs for 24 hours with 1 micromol/L LY83583, a superoxide-generating napthoquinolinedione, decreased caveolae number measured by electron microscopy and prevented the cholesterol-mediated increases in eNOS expression. In vitro exposure of caveolae-enriched membranes to ROS (xanthine plus xanthine oxidase) dissociated caveolin more readily than eNOS from the membranes. These results show that cholesterol treatment increases eNOS expression, whereas ROS treatment decreases eNOS expression and the association of eNOS with caveolin in caveolae-enriched membranes. Our data suggest that oxidative stress modulates endothelial function by regulating caveolae formation, eNOS expression, and eNOS-caveolin interactions.
Asunto(s)
Caveolinas , Colesterol/farmacología , Endotelio Vascular/enzimología , Óxido Nítrico Sintasa/metabolismo , Especies Reactivas de Oxígeno/fisiología , Animales , Aorta/citología , Aorta/enzimología , Aorta/metabolismo , Aorta/ultraestructura , Bovinos , Caveolina 1 , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Endotelio Vascular/ultraestructura , Proteínas de la Membrana/metabolismo , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo III , Fracciones Subcelulares/metabolismoRESUMEN
Axl is a receptor tyrosine kinase originally identified as a transforming gene product in human myeloid leukemia cells. Cultured rat vascular smooth muscle cells also express Axl, where it has been proposed that Axl may play a role in cell proliferation. In the current study, we tested the hypotheses that Axl expression would parallel neointima formation in balloon-injured rat carotid, and that Axl expression would be regulated by growth factors present at sites of vascular injury. Ribonuclease protection assay showed dynamic increases in Axl mRNA in vessels, with peak expression 7 and 14 days after injury. Immunohistochemical analysis confirmed these results and demonstrated that Axl protein expression was localized primarily to cells of the neointima after injury. Northern blot analysis indicated increased mRNA expression for the secreted Axl ligand, Gas6, in injured carotids, with a time course paralleling that of Axl upregulation. Axl and Gas6 expression were temporally correlated with neointima formation, suggesting a role for Axl signaling in this process. Other studies, performed in cultured rat vascular smooth muscle cells, revealed positive regulation of Axl mRNA expression by thrombin or angiotensin II but not by basic fibroblast growth factor, platelet-derived growth factor-BB, or transforming growth factor-ss1. Western blot analysis confirmed these results, showing that Axl protein expression was specifically increased by thrombin or angiotensin II. Our results implicate Axl as a potential mediator of vascular smooth muscle migration and proliferation caused by vascular injury and G protein-coupled receptor agonists.
Asunto(s)
Traumatismos de las Arterias Carótidas , Cateterismo/efectos adversos , Proteínas de Unión al GTP/metabolismo , Proteínas Oncogénicas/biosíntesis , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Receptores de Superficie Celular/agonistas , Angiotensina II/farmacología , Animales , Factor 2 de Crecimiento de Fibroblastos/farmacología , Masculino , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas , Ratas , Ratas Sprague-Dawley , Receptores de Superficie Celular/metabolismo , Trombina/farmacología , Factor de Crecimiento Transformador beta/farmacología , Tirosina Quinasa del Receptor AxlRESUMEN
Endothelial nitric oxide synthase (eNOS) has been shown to be regulated both transcriptionally and posttranslationally in cultured endothelial cells, but eNOS regulatory mechanisms in vivo have not been elucidated. Because one of the strongest stimuli for eNOS expression in tissue culture is cell proliferation and because increased NO production would be beneficial in the setting of arterial injury, we hypothesized that eNOS expression should be increased in regenerating endothelium after a denuding injury. Rat aortas underwent partial endothelial denudation by passage of a deflated balloon catheter, and eNOS expression was studied 48 hours after injury. Immunohistochemistry with eNOS monoclonal antibody, NADPH diaphorase activity assay under conditions specific for eNOS, and mRNA hybridization were performed in situ on perfusion-fixed rat aortic segments. The vessels were studied en face to enhance visualization compared with cross sections. eNOS protein and mRNA expression were significantly increased in regenerating and migrating endothelial cells at the wound edge, with translocation of eNOS to the plasma membrane at the leading edge. Similar results were obtained when endothelial cells were studied in a tissue culture wound model. An important role for transforming growth factor (TGF)-beta1 in regulating eNOS expression was suggested by the ability of a TGF-beta1-neutralizing antibody to limit induction of eNOS at the wound edge. Increased eNOS expression after wounding appears to be related to signal events associated with cell migration as well as proliferation, because eNOS expression in vivo increased in nonproliferating cells and TGF-beta1-neutralizing antibody inhibited eNOS expression but stimulated proliferation. The current study is the first to suggest an important role in vivo for increased eNOS, and perhaps NO production, in the process of endothelial regeneration and wound repair.
Asunto(s)
Aorta/enzimología , Endotelio Vascular/enzimología , Óxido Nítrico Sintasa/metabolismo , Regeneración/fisiología , Análisis de Varianza , Animales , Aorta/fisiología , Técnicas de Cultivo , Endotelio Vascular/fisiología , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Hibridación in Situ , Masculino , NADPH Deshidrogenasa/metabolismo , Óxido Nítrico Sintasa de Tipo III , Ratas , Estadísticas no ParamétricasRESUMEN
Vascular remodeling is regulated by a combination of hemodynamic, environmental, and genetic factors and may be influenced by age. To evaluate age-dependent remodeling in rats, we developed and used a quantitative highly reproducible model of carotid flow alteration. Fourteen juvenile (99+/-3 g) and 9 adult (199+/-5 g) male inbred Fischer rats underwent ligation of the left internal and external carotid arteries under anesthesia. Left common carotid blood flow immediately decreased by approximately 93%, whereas flow in the contralateral carotid increased by approximately 46%. After 4 weeks, the left carotid outer diameter (OD) significantly decreased in both juvenile and adult rats (as measured in vivo and by histological morphometry) compared with sham-operated rats. Changes in shear stress acutely mirrored the changes in blood flow. OD increased and shear stress returned to initial values after chronic exposure to increased flow in juvenile but not adult rats. To develop a simple quantitative index of remodeling that would not require killing the animals, we measured the OD in vivo and compared the ratio of right to left OD (OD ratio [ODR]) between groups. The initial ODR for all groups was approximately 1.0. After 4 weeks of altered flow, the ODR was significantly greater in juvenile than in adult rats (1.48+/-0.05 versus 1.29+/-0.04, respectively; P=.030), indicating that juvenile rats experienced more extensive remodeling than did the adult rats. We also found that unilateral carotid ligation caused a left versus right difference in endothelial NO synthase protein levels after 4 weeks that was not present in the sham-operated animals. Thus, the model described here shows that flow-induced vascular remodeling is dependent on age and supports the hypothesis that the driving force for remodeling involves shear stress and possibly NO. Because the model is quantitative, it allows dissection of the genetic factors that regulate remodeling in inbred rat strains.