RESUMEN
OBJECTIVE: To determine how C reactive protein (CRP), a sensitive marker of inflammation, is related to coronary endothelial function. DESIGN: Changes in quantitative coronary angiographic findings and Doppler flow velocity measurements in response to locally infused acetylcholine were assessed. SETTING: Tertiary cardiology centre. PATIENTS: 46 patients with angiographically normal coronary arteries were divided into groups with normal (< or = 3 mg/l) or increased (> 3 mg/l) CRP concentrations. INTERVENTIONS: Acetylcholine (3 and 30 microg/min) was infused into the left coronary ostium for two minutes. MAIN OUTCOME MEASURES: Percentage change in diameter of epicardial coronary arteries and coronary blood flow (CBF) in response to acetylcholine; and correlations between these parameters and serum CRP concentrations. RESULTS: 15 patients had increased CRP concentrations. The change in coronary artery diameter induced by acetylcholine infusion was similar between the groups but the increase in CBF induced by acetylcholine was smaller in patients with increased CRP concentrations (54.9% v 139.4% with acetylcholine 30 microg/min, p = 0.0030). Multivariate analysis identified increased CRP concentration as independently associated with attenuated CBF response to acetylcholine at 30 microg/min (p = 0.0078, R2 = 0.434). CONCLUSIONS: These findings suggest that inflammation appears to be associated with impaired coronary endothelial function in resistance but not conduit vessels. The data suggest a close relation between chronic vascular inflammation and endothelial dysfunction in atherosclerosis.
Asunto(s)
Acetilcolina/farmacología , Angina de Pecho/fisiopatología , Proteína C-Reactiva/metabolismo , Circulación Coronaria/efectos de los fármacos , Vasos Coronarios/efectos de los fármacos , Vasodilatadores/farmacología , Angina de Pecho/sangre , Vasos Coronarios/fisiopatología , Endotelio Vascular/efectos de los fármacos , Femenino , Humanos , Masculino , Microcirculación/efectos de los fármacos , Persona de Mediana EdadRESUMEN
We present 2 cases of Scimitar syndrome with lung sequestration, 1 of which is infant case and the other is adult. A 3-month-old baby was transferred to our hospital because of severe heart failure. Cardiac ultrasonography and angiography revealed atrial septal defect and lung sequestration. Because of critical condition, she underwent emergency operation. After the operation of abnormal lung resection, her general condition improved. We emphasize that the prognosis is influenced by operation timing. A 20-year-old female was admitted for atrial septal defect. We diagnosed Scimitar syndrome and lung sequestration as the result of close examinations. We carried out definitive operations of the closure of atrial septal defect, the correction of partially anomalous pulmonary venous return and the resection of malformed lung. This adult case is popular one of Scimitar syndrome.
Asunto(s)
Secuestro Broncopulmonar/complicaciones , Secuestro Broncopulmonar/cirugía , Síndrome de Cimitarra/complicaciones , Síndrome de Cimitarra/cirugía , Adulto , Procedimientos Quirúrgicos Cardíacos/métodos , Femenino , Defectos del Tabique Interatrial/complicaciones , Defectos del Tabique Interatrial/cirugía , Humanos , Lactante , MasculinoRESUMEN
Endothelin-1 (ET) is known to stimulate mesangial cell (MC) proliferation, extracellular matrix (ECM) synthesis, and thereby contribute to the progression of glomerulonephritis (GN). To clarify the molecular and cellular mechanisms of how ET is involved in the development of glomerular sclerosis, we investigated the influence of ET on the MC-alpha1beta1 integrin-mediated collagen matrix reorganization using a collagen gel contraction assay. ET enhanced MC-alpha1beta1 integrin-mediated gel contraction in a dose-dependent manner. Addition of the endothelin A (ETA) receptor antagonist, BQ123, into collagen gels abolished ET-induced gel contraction by MC. Cell behavior involved in ET-induced gel contraction was investigated in combination with function-blocking anti-alpha1-integrin antibody. Migration and adhesion assays revealed that ET stimulated alpha1beta1 integrin-mediated MC migration but did not influence cell adhesion to type I collagen (collagen I). Integrin-function blocking studies using anti-alpha1 integrin antibody indicated that MC-alpha1beta1 integrin is required not only for collagen-dependent migration, but also for gel contraction. Zymography showed that ET increased MC matrix metalloproteinase-2 (MMP-2) activity in a dose-dependent manner during MC-induced gel contraction process. Finally, flow cytometry analysis indicated that ET did not affect the cell surface expression of the MC-alpha1beta1 integrin within the collagen gel. These data suggested that ET promotes collagen matrix reorganization through the enhancement of MC-alpha1beta1 integrin-dependent migration and MMP-2 activity. We therefore conclude that ET is a potential molecule inducing pathological collagen matrix remodeling observed in progressive GN.
Asunto(s)
Colágeno/metabolismo , Endotelina-1/farmacología , Mesangio Glomerular/efectos de los fármacos , Integrina alfa1beta1/metabolismo , Animales , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Endotelina-1/metabolismo , Geles/química , Mesangio Glomerular/citología , Mesangio Glomerular/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: Estrogen has been implicated in feeding behavior and adiposity. This study was undertaken to elucidate the mechanism underlying the anti-obesity and anorectic action of estrogen and the role of estrogen receptor (ER) in the central nervous system. METHODS AND RESULTS: Ovariectomy in 8-week-old female Wistar rats induced hyperphagia along with an increase in body weight and abdominal fat accumulation compared to control sham-operated rats. These changes were fully reversed by subcutaneous replacement of estradiol and were abrogated by pair-feeding. Then, the effects of intracerebroventricular infusion of estradiol, alone or in combination with antisense oligodeoxynucleotides (ODN), for ER in ovariectomized rats were examined. The estradiol group showed 10-20% lower daily food intake, and after the 2-week infusion period a 14% reduction in body weight with a similar reduction in abdominal fat compared to the vehicle group. The inhibitory effect of estradiol on food intake and body weight was blocked by co-administration of ER-beta antisense ODN, whereas ER-alpha antisense ODN did not show any influence. CONCLUSION: These results indicate that ER-beta in the central nervous system is involved in the anorectic action of estrogen.
Asunto(s)
Estradiol/farmacología , Obesidad/metabolismo , Receptores de Estrógenos/metabolismo , Tejido Adiposo/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Encéfalo/metabolismo , Esquema de Medicación , Ingestión de Alimentos/efectos de los fármacos , Estradiol/administración & dosificación , Receptor beta de Estrógeno , Femenino , Inyecciones Intraventriculares , Oligodesoxirribonucleótidos Antisentido/administración & dosificación , Oligodesoxirribonucleótidos Antisentido/farmacología , Ovariectomía , Ratas , Ratas WistarRESUMEN
Vascular smooth muscle cell (VSMC) apoptosis has been demonstrated in vascular lesions, such as atherosclerotic and postangioplasty restenotic lesions. Balloon injury also induces VSMC apoptosis. Fas is a death factor that mediates apoptosis when it is activated by its ligand, FasL. Fas-mediated apoptosis was found to be implicated in the pathogenesis of vascular diseases in which Fas/FasL expression was detected. We investigated whether the Fas/FasL interaction mediated acute and chronic VSMC apoptosis and lesion formation in a vascular injury model that may resemble balloon angioplasty. A large spring wire was inserted into the femoral artery of C3H/HeJ (wild-type), C3H-gld (Fas ligand-/-), and C3H-lpr (Fas-/-) mice. The wire was left in place for 1 minute to denude and expand the artery. Massive apoptosis was observed in medial VSMCs from 1 to 7 hours later. There was no difference in the number of apoptotic cells among the 3 groups of mice 4 hours after injury. At 4 weeks, the injured arteries presented signs of concentric neointimal hyperplasia composed exclusively of VSMCs. There was no difference in the degree of neointima hyperplasia (intima/media ratios were as follows: wild type 1.4+/-0.3, gld 1.0+/-0.2, and lpr 1.3+/-0.2) or in the number of apoptotic nuclei among the 3 groups. These findings suggest the existence of other signaling pathways for acute and chronic VSMC apoptosis, at least that induced by mechanical vascular injury.
Asunto(s)
Apoptosis , Arteriopatías Oclusivas/patología , Músculo Liso Vascular/patología , Receptor fas/fisiología , Angioplastia de Balón/efectos adversos , Animales , Arteriopatías Oclusivas/etiología , Proteína Ligando Fas , Cinética , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Transducción de Señal , Estrés Mecánico , Receptor fas/genéticaRESUMEN
NGFI-B is one of the orphan nuclear receptors, and its gene is implicated in the apoptosis of T cells. The aim of this study was to investigate the expression and the role of NGFI-B in vascular smooth muscle cells (VSMCs). Pyrrolidinedithiocarbamate (PDTC) is a modulator of an oxidative state and is reported to induce apoptosis only when the density of VSMCs is low. Under low VSMC density (10 000 cells/cm(2)), addition of PDTC (0.1 to 10 micromol/L) caused apoptosis of VSMCs, which was confirmed by Hoechst 33258 staining under fluorescence microscopy. At low VSMC density, expression of NGFI-B mRNA was induced 1 hour after the addition of PDTC, peaking at 6 hours, and persisted for up to 12 hours. The protein level of NGFI-B was increased 4 hours after PDTC addition and persisted for up to 12 hours. Under low VSMC density, PDTC-induced expression of NGFI-B mRNA was correlated with the magnitude of apoptosis, which was quantified by enzyme immunoassay for histone-associated DNA fragments. In contrast, when the density of VSMCs was high (50 000 cells/cm(2)), PDTC did not induce apoptosis, and the expression of NGFI-B was only transient. This transient expression pattern was also seen when VSMCs were treated with phorbol ester, calcium ionophore, hydrogen peroxide, or angiotensin II, even at low cell density. We next investigated whether the NGFI-B gene may act as a transcription factor under treatment with PDTC by measuring the promoter activity of luciferase reporter plasmids that contained typical NGFI-B-responsive elements. The PDTC-induced transcriptional activity of NGFI-B was 2-fold higher at low cell density than at high cell density. These data demonstrate that NGFI-B can be induced in VSMCs and suggest that NGFI-B may play a role in PDTC-induced VSMC apoptosis.
Asunto(s)
Antioxidantes/farmacología , Apoptosis , Proteínas de Unión al ADN/biosíntesis , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Pirrolidinas/farmacología , Tiocarbamatos/farmacología , Factores de Transcripción/biosíntesis , Animales , Técnicas de Cultivo de Célula/métodos , Núcleo Celular/ultraestructura , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Cinética , Masculino , Músculo Liso Vascular/ultraestructura , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares , Receptores de Esteroides , Elementos de Respuesta , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Activación Transcripcional , TransfecciónRESUMEN
Recent studies suggest that ischemia activates Src and members of the mitogen-activated protein (MAP) kinase superfamily and their downstream effectors, including big MAP kinase 1 (BMK1) and p90 ribosomal S6 kinase (p90RSK). It has also been reported that adenosine is released during ischemia and involved in triggering the protective mechanism of ischemic preconditioning. To assess the roles of Src and adenosine in ischemia-induced MAP kinases activation, we utilized the Src inhibitor PP2 (4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and the adenosine receptor antagonist 8-(p-sulfophenyl) theophylline (SPT) in perfused guinea pig hearts. PP2 (1 microm) inhibited ischemia-induced Src, BMK1 and JNK activation but not JAK2 and p38 activation. SPT inhibited ischemia-mediated p38 and JNK activation. These results demonstrate that Src family kinase and adenosine regulate MAP kinases by parallel pathways. Preconditioning significantly improved both recovery of developed pressure and dp/dt in isolated guinea pig hearts. Since the protective effect of preconditioning was blocked by PP2 (1 microm) and SPT (50 microm), we next investigated the regulation of Src, MAP kinases and p90RSK during preconditioning. The activity and time course of ERK1/2 was not changed, but p90RSK activation by reperfusion was completely inhibited by preconditioning. In contrast, the activation by ischemia of Src, BMK1, p38 and JNK was significantly faster in preconditioned hearts. Maximal BMK1 activation by ischemia was also significantly enhanced by preconditioning. These data suggest important roles for Src family kinases and adenosine in mediating preconditioning, and suggest specific roles for individual MAP kinases in preconditioning.
Asunto(s)
Adenosina/metabolismo , Sistema de Señalización de MAP Quinasas , Isquemia Miocárdica , Miocardio/metabolismo , Miocardio/patología , Familia-src Quinasas/metabolismo , Animales , Western Blotting , Relación Dosis-Respuesta a Droga , Activación Enzimática , Cobayas , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteína Quinasa 7 Activada por Mitógenos , Proteína Quinasa 8 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Perfusión , Pruebas de Precipitina , Daño por Reperfusión/metabolismo , Factores de Tiempo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Hypertension often complicates type 2 diabetes mellitus, and angiotensin converting enzyme inhibitor treatment has been shown to improve insulin resistance in such cases. However, the effect of angiotensin II type-1 (AT(1)) receptor antagonists on insulin resistance is still controversial. To gain further information on this effect, we examined the effect of losartan on insulin resistance in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of type 2 diabetes mellitus. Losartan administration alone lowered systolic blood pressure, but did not improve oral glucose tolerance test or insulin resistance in OLETF rats. However, the administration of losartan with exercise significantly improved both systolic blood pressure and insulin resistance relative to control OLETF rats. On the other hand, losartan treatment, regardless of exercise, increased glucose uptake in excised soleus muscle and fat cells. To explore the beneficial effect of losartan on skeletal muscle glucose uptake, we examined intracellular signaling of soleus muscle. Although Akt activity and glucose transporter type 4 (GLUT4) expressions were not affected by losartan with or without exercise, extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein (MAP) kinase activities were increased by both interventions. These results indicate that angiotensin AT(1) receptor antagonist improved local insulin resistance, but not systemic insulin resistance. These findings may explain the controversy over the effect of angiotensin AT(1) receptor antagonists on insulin resistance in clinical use. The enhancing effect of angiotensin AT(1) receptor antagonist on skeletal muscle glucose uptake may be attributable to MAP kinase activation or other mechanisms rather than phosphatidylinositol 3-kinase activation.
Asunto(s)
Antihipertensivos/farmacología , Resistencia a la Insulina , Losartán/farmacología , Proteínas Musculares , Condicionamiento Físico Animal/fisiología , Proteínas Serina-Treonina Quinasas , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Presión Sanguínea/efectos de los fármacos , Western Blotting , Peso Corporal/efectos de los fármacos , Desoxiglucosa/farmacocinética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Activación Enzimática/efectos de los fármacos , Glucosa/farmacocinética , Glucosa/farmacología , Prueba de Tolerancia a la Glucosa , Transportador de Glucosa de Tipo 4 , Frecuencia Cardíaca/efectos de los fármacos , Insulina/sangre , Proteínas Quinasas JNK Activadas por Mitógenos , Masculino , Proteína Quinasa 1 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Transporte de Monosacáridos/efectos de los fármacos , Proteínas de Transporte de Monosacáridos/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas Endogámicas OLETF , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
A monoclonal antibody, ASH1a/256C (256C), which binds to atherosclerotic lesions in Watanabe heritable hyperlipidemic rabbit (WHHL) aorta in vivo, recognizes complex structures of phosphatidylcholine mixed with neutral lipids. In the present study, a cell culture system is described in which foam cells express 256C-positive lipid droplets. J774.1 macrophages were incubated in the presence of a small volume of WHHL serum for 24 h to produce foam cells, which were then incubated without the WHHL serum for 3 days. Oil red O-positive lipid droplets appeared on day 1, and were present in the cells during the whole incubation period. The lipid droplets in the cells were positively immunostained with antibody 256C on day 4, although they were negative on day 1. Expression of the antigenic lipid droplets was also induced by the addition of acetylated LDL or sera from patients with hyperlipidemia. When foam cells were induced by the addition of WHHL serum, cellular content of cholesteryl ester was greatly increased but then decreased to near basal levels by day 4. Concomitantly, cellular free cholesterol increased during the culture period, indicating that the cholesteryl ester changes to free cholesterol by day 4. The lipid droplets in the foam cells on day 4 were positively stained with filipin, a fluorescent probe for free cholesterol, as well as with 256C antibody, indicating that free cholesterol is enriched in antigenic lipid droplets. These observations suggest that hydrolysis and rearrangement of cellular cholesterol take place in foam cells to form complex structures of phosphatidylcholine and free cholesterol in lipid droplets.
Asunto(s)
Colesterol/análisis , Células Espumosas/química , Células Espumosas/patología , Hiperlipidemias/sangre , Lípidos/análisis , Animales , Anticuerpos Monoclonales , Línea Celular , Colesterol/metabolismo , Ésteres del Colesterol/análisis , Ésteres del Colesterol/metabolismo , Cobre/farmacología , Filipina , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Células Espumosas/metabolismo , Cinética , Metabolismo de los Lípidos , Peroxidación de Lípido , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacología , Macrófagos , Ratones , Fosfatidilcolinas/metabolismo , ConejosRESUMEN
Reversible left ventricular wall motion abnormalities mimicking myocardial infarction have been reported in patients with a noncardiac illness. Their coronary angiograms do not demonstrate organic stenosis or epicardial coronary vasospasm. In this article, two cases of reversible left ventricular contraction abnormality are presented. Electrocardiography showed deep inverted T waves in precordial leads, and the echocardiography revealed diffuse akinesis of the apical region in the acute phase. Coronary angiography showed no significant stenosis or occlusion in either patient. Thallium scintigraphy showed no defect, while the metaiodobenzylguanidine scintigraphy demonstrated significant defects in the apex. The relative coronary flow reserve ratio, measured with an intracoronary Doppler flow wire, was significantly reduced in both patients. Myocardial contrast echocardiography revealed a reversible perfusion defect in the apex in the acute phase in case 2. Transiently impaired coronary microcirculation was thought to be involved in the pathogenesis of the reversible left ventricular dysfunction observed in these patients.
Asunto(s)
Enfermedad Coronaria/fisiopatología , Disfunción Ventricular Izquierda/diagnóstico , Anciano , Anciano de 80 o más Años , Diagnóstico Diferencial , Ecocardiografía , Electrocardiografía , Femenino , Humanos , Masculino , Microcirculación , Infarto del Miocardio/diagnóstico , Aturdimiento Miocárdico/diagnóstico , Sístole/fisiologíaRESUMEN
OBJECTIVE: Durability of the valve seems to be dependent on the remodeling ability of the valve itself, which is controlled by both collagen synthesis and collagenolytic activity of valvular fibroblasts and endothelial cells. However, the balance of collagen synthesis and collagenolysis of the cryopreserved valve has not yet been clearly revealed. Thus, we assessed the collagen synthesis and collagenolysis ability of the cryopreserved valve. METHODS: Twelve valves were divided into 2 groups: freshly harvested valves (n = 6) and cryopreserved valves (n = 6). We measured the collagen content using Sirius red, a dye selective to the collagen. Collagen synthesis was evaluated by means of the tritiated proline incorporation method. Noncollagenase-digestible counts, which represent protein synthesis, and collagenase-digestible counts, which represent collagen synthesis, were estimated. Collagenase activity of the valves was assessed by gelatin zymography. RESULTS: The collagen content of the cryopreserved group was maintained. The noncollagenase-digestible counts of the cryopreserved group decreased from 3862 +/- 1180 counts/mg to 1174 +/- 1362 counts/mg, and the collagenase-digestible counts of the cryopreserved group were 831 +/- 762 counts/mg compared with the value of 1062 +/- 136 counts/mg for the freshly harvested group. The collagenase activity of the cryopreserved group was observed at the same level as that of the freshly harvested group, despite the serious endothelial damage of the cryopreserved valves. CONCLUSIONS: Although the collagen synthesis of cryopreserved valves was relatively maintained, the protein synthesis was highly diminished, and the collagenolysis ability was activated immediately after the thawing process. These results imply that the cryopreservation procedure itself may cause the collagen metabolism to be on the degradable side, which will lead to valve failure.
Asunto(s)
Colágeno/biosíntesis , Colagenasas/metabolismo , Criopreservación , Válvulas Cardíacas/metabolismo , Animales , PorcinosRESUMEN
HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitors, or statins, are prescribed widely to lower cholesterol. Accumulating evidence indicates that statins have various effects on vascular cells, which are independent of their lipid-lowering effect. Here, we tested the hypothesis that statins may augment collateral flow to ischemic tissues. We induced hind-limb ischemia in wild-type mice and treated them with either saline or cerivastatin. Cerivastatin enhanced the blood flow recovery dramatically as determined by Laser Doppler imaging. The mice treated with saline displayed frequent autoamputation of the ischemic toe, which was prevented completely by cerivastatin. Anti-CD31 immunostaining revealed that cerivastatin significantly increased the capillary density. Endothelial nitric oxide synthase (eNOS) activity was enhanced markedly in the mice treated with cerivastatin. The angiogenic effect of cerivastatin was abrogated in eNOS deficient (eNOS-/-) mice. These results indicate that eNOS is essential for cerivastatin to promote collateral growth in response to ischemia.
Asunto(s)
Circulación Colateral/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Isquemia/fisiopatología , Óxido Nítrico Sintasa/metabolismo , Piridinas/farmacología , Animales , Capilares/efectos de los fármacos , Capilares/fisiología , Flujometría por Láser-Doppler , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa/efectos de los fármacos , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Flujo Sanguíneo Regional/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
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
Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) hypertrophy, which results in various cardiovascular diseases. Ang II-induced cellular events have been implicated, in part, in the activation of mitogen-activated protein (MAP) kinases. Although it has been proposed that daily intake of bioflavonoids belonging to polyphenols reduces the incidence of ischemic heart diseases (known as "French paradox"), the precise mechanisms of efficacy have not been elucidated. Thus, we hypothesized that bioflavonoids may affect Ang II-induced MAP kinase activation in cultured rat aortic smooth muscle cells (RASMC). Our findings showed that Ang II stimulated rapid and significant activation of extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK), and p38 in RASMC. Ang II-induced JNK activation was inhibited by 3,3',4',5,7-pentahydroxyflavone (quercetin), a major bioflavonoid in foods of plant origin, whereas ERK1/2 and p38 activation by Ang II were not affected by quercetin. Ang II caused a rapid tyrosine phosphorylation of Src homology and collagen (Shc), which was inhibited by quercetin. Quercetin also inhibited Ang II-induced Shc.p85 association and subsequent activation of phosphatidylinositol 3-kinase (PI3-K)/Akt pathway in RASMC. Furthermore, LY294002, a PI3-K inhibitor and a quercetin derivative, inhibited Ang II-induced JNK activation as well as Akt phosphorylation. Finally, Ang II-induced [(3)H]leucine incorporation was abolished by both quercetin and LY294002. These findings suggest that the preventing effect of quercetin on Ang II-induced VSMC hypertrophy are attributable, in part, to its inhibitory effect on Shc- and PI3-K-dependent JNK activation in VSMC. Thus, inhibition of JNK by quercetin may imply its usefulness for the treatment of cardiovascular diseases relevant to VSMC growth.
Asunto(s)
Angiotensina II/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Quercetina/farmacología , Animales , Cromonas/farmacología , Colágeno/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Leucina/metabolismo , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Morfolinas/farmacología , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Tritio , Tirosina/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos , Dominios Homologos srcRESUMEN
Abnormal mesangial extracellular matrix remodeling by mesangial cells (MCs) is the hallmark of progressive glomerulonephritis (GN). We recently showed, using a type I collagen gel contraction assay, that alpha 1 beta 1 integrin-dependent MC adhesion and migration are necessary cell behaviors for collagen matrix remodeling. To further determine the mechanism of alpha 1 beta 1 integrin-mediated collagen remodeling, we studied the signaling pathways of MCs that participate in the regulation of collagen gel contraction. Immunoprecipitation and phosphotyrosine detection revealed that gel contraction is associated with the enhanced activity and phosphorylation of ERK1/2 by MCs. The tyrosine kinase inhibitors herbimycin and genistein inhibited collagen gel contraction dose dependently. Furthermore, targeting ERK1/2 activity with a MEK inhibitor, PD98059, and antisense ERK1/2 hindered gel contraction in a dose-dependent manner. Similar inhibitory effects on gel contraction and ERK1/2 phosphorylation were observed when MC-mediated gel contraction was performed in the presence of function-blocking anti-alpha1 or anti-beta1 integrin antibodies. However, cell adhesion and migration assays indicated that PD98059 and antisense ERK1/2 blocked alpha 1 beta 1 integrin-dependent MC migration, but did not interfere with collagen adhesion, although there was a marked decrease in ERK1/2 phosphorylation and ERK1/2 protein expression in cell adhesion on type I collagen. None of the above could affect membrane expression of alpha 1 beta 1 integrin. These results suggested that ERK1/2 activation is critical for the alpha 1 beta 1 integrin-dependent MC migration necessary for collagen matrix reorganization. We therefore conclude that ERK1/2 may serve as a possible target for pharmacological inhibition of pathological collagen matrix formation in GN.
Asunto(s)
Colágeno/metabolismo , Matriz Extracelular/fisiología , Mesangio Glomerular/fisiología , Integrinas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Movimiento Celular , Inhibidores Enzimáticos , Flavonoides/farmacología , Mesangio Glomerular/citología , Integrina alfa1beta1 , Proteína Quinasa 1 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/genética , Oligodesoxirribonucleótidos Antisentido/farmacología , Fosforilación , Estimulación Física , Ratas , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
The free radical scavenging and/or generating activity of dopamine-4-O-sulfate was examined and compared with that of dopamine. In humans, dopamine mostly exists in two isomeric forms of sulfate ester conjugates as metabolites; i.e., dopamine-3-O-sulfate and dopamine-4-O-sulfate in the circulation. Dopamine is generally believed to be oxidized by molecular oxygen or another reactive oxygen species under physiological conditions, to form oxidized dopamine derivatives that are cytotoxic. However, it is not known whether dopamine conjugates are generated on interaction with reactive oxygen species or not. In the present study, we measured the susceptibility to oxidization of dopamine-4-O-sulfate by using electron paramagnetic resonance (EPR) spectroscopy and optical absorption spectrometry. Dopamine was easily oxidized and dopamine-derived radicals appeared, whereas dopamine-4-O-sulfate was not oxidized under physiological conditions. Furthermore, dopamine-4-O-sulfate did not react with a strong oxidizing agent, sodium periodate. These results suggest that dopamine-4-O-sulfate has resistance against autoxidation, and seems to be a stable metabolite of dopamine.
Asunto(s)
Dopamina/farmacología , Depuradores de Radicales Libres/farmacología , Dopamina/análogos & derivados , Dopamina/química , Dopamina/efectos de la radiación , Espectroscopía de Resonancia por Spin del Electrón , Depuradores de Radicales Libres/química , Radicales Libres/química , Peroxidasa de Rábano Silvestre/química , Radical Hidroxilo/química , Ácido Peryódico/química , Espectrofotometría Ultravioleta , Superóxidos/química , Rayos Ultravioleta , Xantina Oxidasa/metabolismoRESUMEN
Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) hypertrophy, which results in several cardiovascular diseases. Ang II-induced cellular events have been mediated, in part, by reactive oxygen species (ROS) which also involve activation of mitogen-activated protein (MAP) kinases. Although it has been proposed that the therapeutic administration of antioxidants is useful for vascular diseases, the precise mechanisms which regulate ROS-sensitive signaling events have not been well characterized. Thus, we hypothesized that antioxidants may affect ROS-mediated MAP kinases activation induced by Ang II. The present findings showed that Ang II stimulated rapid and significant activation of ERK 1/2, JNK and p38 MAPK in cultured rat aortic smooth muscle cells (RASMC). Ang II-induced ERK 1/2 activation was not affected by all antioxidants examined, whereas JNK was sensitive to all antioxidants. In contrast, p38 MAPK activation was inhibited by DPI and ascorbic acid concentration-dependently, but by NAC only at high concentration. DETC and Trolox C had no effects on p38 MAPK activation by Ang II. We further examined the effects of antioxidants on Ang II-induced increases in oxygen consumption as an index of ROS generation in RASMC. DPI strongly inhibited Ang II-induced increases in oxygen consumption. DETC also inhibited Ang II-induced oxygen consumption, whereas ascorbic acid markedly augmented it. These findings suggest that the inhibitory effects of antioxidants on MAP kinases activation in VSMC are attributable, in part, to their modulating effects on ROS generation by Ang II in VSMC. Thus, inhibition of MAP kinases by antioxidants may imply their usefulness for relief of cardiovascular diseases.
Asunto(s)
Angiotensina II/farmacología , Antioxidantes/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/enzimología , Vasoconstrictores/farmacología , Acetilcisteína/farmacología , Animales , Aorta Torácica/citología , Ácido Ascórbico/farmacología , Células Cultivadas , Quelantes/farmacología , Cromanos/farmacología , Ditiocarba/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , MAP Quinasa Quinasa 4 , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Compuestos Onio/farmacología , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Adrenomedullin (AM) is a potent vasodilating peptide having a variety of pharmacological properties mainly in respect to vascular pathophysiology. We have previously demonstrated that angiotensin II (Ang II) or natriuretic peptides have influence on the expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) in vascular endothelial cells. The aim of this study was to elucidate the effects of AM on TF and PAI-1 mRNA and protein expression in endothelial cells. As a result, AM inhibited Ang II-induced TF and PAI-1 mRNA expression in a dose- and time-dependent manner. Because the expression of TF and PAI-1 mRNA induced by Ang II was attenuated by the increase of intracellular concentrations of cAMP by forskolin and 8-bromo-cAMP and because AM increased the intracellular level of cAMP in rat aortic endothelial cells, it was indicated that the inhibitory effect of AM on the expressions of TF and PAI-1 was mainly mediated by the cAMP-dependent signal transduction. Furthermore, the inhibitory effect of AM on TF and PAI-1 expression was partly attenuated by an NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester. In conclusion, AM is shown to contribute to the regulation of blood coagulation and fibrinolysis by vascular endothelial cells mainly via the cAMP pathway.