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1.
Am J Physiol Heart Circ Physiol ; 280(6): H2779-88, 2001 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-11356636

RESUMEN

The cellular mechanisms that contribute to the acceleration of atherosclerosis in aging populations are poorly understood, although it is hypothesized that changes in the proliferative capacity of vascular smooth muscle cells is contributory. We addressed the relationship among aging, generation of reactive oxygen species (ROS), and proliferation in primary culture smooth muscle cells (SMC) derived from the aortas of young (4 mo old) and aged (16 mo old) mice to understand the phenotypic modulation of these cells as aging occurs. SMC from aged mice had decreased proliferative capacity in response to alpha-thrombin stimulation, yet generated higher levels of ROS and had constitutively increased mitogen-activated protein kinase activity, in comparison with cells from younger mice. These effects may be explained by dysregulation of cell cycle-associated proteins such as cyclin D1 and p27Kip1 in SMC from aged mice. Increased ROS generation was associated with decreased endogenous antioxidant activity, increased lipid peroxidation, and mitochondrial DNA damage. Accrual of oxidant-induced damage and decreased proliferative capacity in SMC may explain, in part, the age-associated transition to plaque instability in humans with atherosclerosis.


Asunto(s)
Envejecimiento/metabolismo , Aorta/metabolismo , Proteínas de Ciclo Celular , Músculo Liso Vascular/metabolismo , Proteínas Supresoras de Tumor , Animales , Aorta/citología , División Celular/fisiología , Células Cultivadas , Ciclina D1/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Daño del ADN , ADN Mitocondrial/metabolismo , Glutatión/metabolismo , Peróxido de Hidrógeno/metabolismo , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mitosis/fisiología , Modelos Cardiovasculares , Músculo Liso Vascular/citología , Oxidación-Reducción , Reacción en Cadena de la Polimerasa , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo
2.
Circulation ; 100(6): 659-65, 1999 Aug 10.
Artículo en Inglés | MEDLINE | ID: mdl-10441105

RESUMEN

BACKGROUND: Smooth muscle cell (SMC) proliferation is a critical component of neointimal formation in many models of vascular injury and in human lesions as well. Cell-cycle inhibition by gene transfer techniques can block SMC proliferation and lesion formation in animal models, although these methods are not yet applicable to the treatment of human disease. Flavopiridol is a recently identified, potent, orally available cyclin-dependent kinase inhibitor. METHODS AND RESULTS: Using human aortic SMCs, we found that flavopiridol in concentrations as low as 75 nmol/L resulted in nearly complete inhibition of basic fibroblast growth factor-induced and thrombin-induced proliferation. At this dose, flavopiridol inhibited cyclin-dependent kinase activity, as measured by histone H1 phosphorylation, but had no effect on mitogen-activated protein kinase activation. Induction of the cell cycle-related proteins cyclin D1, proliferating cell nuclear antigen, and phosphorylated retinoblastoma protein was also blocked by flavopiridol. Flavopiridol had no effect on cellular viability. To test whether flavopiridol had a similar activity in vivo when administered orally, we examined neointimal formation in rat carotid arteries after balloon injury. Flavopiridol 5 mg/kg reduced neointimal area by 35% and 39% at 7 and 14 days, respectively, after injury. CONCLUSIONS: Flavopiridol inhibits SMC growth in vitro and in vivo. Its oral availability and selectivity for cyclin-dependent kinases make it a potential therapeutic tool in the treatment of SMC-rich vascular lesions.


Asunto(s)
Traumatismos de las Arterias Carótidas , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Inhibidores de Crecimiento/farmacología , Proteínas Musculares/antagonistas & inhibidores , Músculo Liso Vascular/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Piperidinas/farmacología , Cicatrización de Heridas/efectos de los fármacos , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/enzimología , Arterias Carótidas/patología , Cateterismo/efectos adversos , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Hiperplasia , Masculino , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/patología , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Trombina/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Túnica Íntima/efectos de los fármacos , Túnica Íntima/enzimología , Túnica Íntima/lesiones , Túnica Íntima/patología
3.
J Biol Chem ; 274(28): 19814-22, 1999 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-10391925

RESUMEN

Thrombin is a potent vascular smooth muscle cell (VSMC) mitogen. Because recent evidence implicates reactive oxygen intermediates (ROI) in VSMC proliferation in general and atherogenesis in particular, we investigated whether ROI generation is necessary for thrombin-induced mitogenesis. Treatment of human aortic smooth muscle cells with thrombin increased DNA synthesis, an effect that was antagonized by diphenyleneiodonium but not by other inhibitors of cellular oxidase systems. This effect of thrombin was accompanied by increased O-2 and H2O2 generation and NADH/NADPH consumption. ROI generation in response to thrombin pretreatment could also be blocked by diphenyleneiodonium, suggesting that the NAD(P)H oxidase was necessary for ROI generation and thrombin-induced mitogenesis. Because of observed differences between the VSMC and neutrophil oxidase, we examined whether the cytosolic components of the phagocytic NAD(P)H oxidase were present in VSMC. p47(phox) and Rac2 were present in VSMC. Furthermore, thrombin increased expression of p47(phox) and Rac2 and stimulated their translocation to the cell membrane. We examined whether p47(phox) might be similarly regulated in vivo in a rat aorta balloon injury model and found that p47(phox) protein was increased after injury. Immunocytochemistry localized expression of p47(phox) to the neointima and media of injured arteries. Our data demonstrate that generation of O-2 and H2O2 is required for thrombin-mediated mitogenesis in VSMC and that p47(phox) is regulated by thrombin in vitro and is associated with vascular lesion formation in vivo.


Asunto(s)
Músculo Liso Vascular/enzimología , NADH NADPH Oxidorreductasas/metabolismo , Fosfoproteínas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Trombina/farmacología , Animales , Aorta , División Celular/efectos de los fármacos , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Proteínas de Unión al GTP/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Mitógenos/farmacología , NAD/metabolismo , NADP/metabolismo , NADPH Oxidasas , Ratas , Superóxidos/metabolismo , Proteínas de Unión al GTP rac
4.
J Biol Chem ; 271(42): 26320-8, 1996 Oct 18.
Artículo en Inglés | MEDLINE | ID: mdl-8824285

RESUMEN

Thrombin, via activation of vascular endothelial and smooth muscle cell thrombin receptors, modulates vascular wall healing. To understand the mechanisms that regulate human thrombin receptor (HTR) expression, we cloned and characterized the HTR gene. The HTR gene consists of Exon I, which contains the 5'-regulatory region and 85 nucleotides of coding sequence; a approximately 15-kb intron; and Exon II, which contains the remainder of the coding sequence and the entire 3'-untranslated region. Multiple transcription initiation sites were identified by S1 mapping and ribonuclease protection assay. DNA sequence analysis indicated the presence of two SP-1-AP-2 consensus binding sequences, near or within the transcription initiation sites, and consensus binding sequences for numerous regulatory proteins that potentially modulate HTR expression. Functional analysis of the HTR promoter was performed by transfecting human microvascular endothelial cells with HTR promoter region-luciferase constructs. The highest level of expression was obtained with a 0.7-kb promoter sequence and was progressively less with fragments of 0.54, 1.16, 1.6, and approximately3.2 kb. The data presented in this report provide a foundation for further characterization of the HTR gene and the mechanisms that regulate its expression within the blood vessel wall.


Asunto(s)
Regiones Promotoras Genéticas , Receptores de Trombina/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Mapeo Cromosómico , Clonación Molecular , ADN , Exones , Eliminación de Gen , Humanos , Intrones , Microscopía Electrónica , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo
5.
Circ Res ; 75(6): 1029-38, 1994 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-7955141

RESUMEN

Blood vessels respond to injury by initiating cell proliferation and migration that result in vascular lesion formation. To determine the roles of thrombin and the thrombin receptor in this process, we characterized thrombin receptor expression in normal and injured arteries, thrombin receptor-mediated smooth muscle cell mitogenesis, and the regulation of thrombin receptor mRNA expression in vitro. Thrombin receptor mRNA was not detected in normal rat or baboon arteries by in situ hybridization. Immunohistochemistry using an antithrombin receptor antibody (TR-R9), directed against the thrombin cleavage site of the rat aortic smooth muscle cell thrombin receptor, revealed low-level staining for thrombin receptor protein in endothelial cells and smooth muscle cells of normal arteries. In contrast, balloon catheter injury increased thrombin mRNA expression in medial smooth muscle cells within 6 hours. This increased thrombin receptor expression continued within the media and in neointimal cells throughout vascular lesion formation, predominantly in areas of active cell proliferation. In vitro, alpha-thrombin stimulates rat aortic smooth muscle cell proliferation in a concentration-dependent manner. That thrombin receptor activation is required for the mitogenic response was confirmed by demonstrating that the polyclonal antibody TR-R9 inhibits thrombin-induced cell proliferation. Thrombin receptor mRNA synthesis was induced by both basic fibroblast growth factor (maximal stimulation of 1.8-fold at 1 hour) and platelet-derived growth factor (maximal stimulation of 2.4-fold at 8 and 24 hours) in quiesced cultured rat aortic smooth muscle cells. In summary, upregulation of smooth muscle cell thrombin receptor expression occurs very early after vascular injury and continues throughout neointimal development.(ABSTRACT TRUNCATED AT 250 WORDS)


Asunto(s)
Angioplastia , Arteriosclerosis/patología , Vasos Sanguíneos/lesiones , Receptores de Trombina/genética , Regulación hacia Arriba , Animales , Especificidad de Anticuerpos , Northern Blotting , Células Cultivadas , Endarterectomía , Factor 2 de Crecimiento de Fibroblastos/farmacología , Inmunohistoquímica , Hibridación in Situ , Músculo Liso Vascular/lesiones , Músculo Liso Vascular/patología , Factor de Crecimiento Derivado de Plaquetas/farmacología , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Receptores de Trombina/efectos de los fármacos , Receptores de Trombina/inmunología , Trombina/aislamiento & purificación
6.
Am J Physiol ; 264(6 Pt 2): F989-95, 1993 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-7686719

RESUMEN

To study receptors for angiotensin II, polyclonal rabbit anti-peptide antisera were prepared against the peptide QDDCPKAGRHC corresponding to amino acids 15-24 of the rat AT1A and AT1B receptors. Western analysis of rat tissues showed a major band of approximately 43 kDa. The antisera immunoprecipitated AT1-receptor protein produced in vitro. Immunohistochemical analysis of rat tissues showed intense staining of arterial and arteriolar smooth muscle. Other tissues that contained AT1-receptor protein included hepatocytes, the zona glomerulosa of the adrenal gland, and the smooth muscle of the bronchus, gut, ureter, and epididymis. In the kidney, intense staining was observed in all small arteries and arterioles. Both afferent and efferent arterioles contain approximately equal intensities of immunoreactive AT1 protein. The inner stripe of the outer medulla has a moderate level of receptors within thick ascending limb epithelium. Proximal tubular epithelium also expresses receptor protein. Glomerular immunoreactive AT1 protein is found within mesangial cells and varies in intensity among different rat strains. Lewis and Wistar rats demonstrated moderate glomerular staining, whereas the CD and Sprague-Dawley strains showed lesser levels of reactivity. The fact that glomerular mesangial cells are the primary locus of angiotensin II action within the glomerulus.


Asunto(s)
Receptores de Angiotensina/metabolismo , Secuencia de Aminoácidos , Animales , Sueros Inmunes/química , Inmunohistoquímica/métodos , Masculino , Datos de Secuencia Molecular , Pruebas de Precipitina , Ratas , Ratas Sprague-Dawley , Coloración y Etiquetado , Distribución Tisular
7.
Artículo en Inglés | MEDLINE | ID: mdl-1690284

RESUMEN

Natural infection of sooty mangabey monkeys with simian immunodeficiency virus, designated SIV/SMM, results in long-term persistent infections with little or no disease. In contrast, experimental infection of macaques with isolates of SIV/SMM induces chronic and progressive disease that terminates in an AIDS-like illness and death in most animals. To determine whether antibodies might be important in preventing the development of disease in mangabeys or progression of disease in macaques, humoral immune responses to SIV/SMM were compared in 13 macaques infected for up to 43 months and in infected and uninfected mangabeys selected at random from among a breeding colony. Total SIV/SMM-specific antibody titers, profiles of antibodies to specific viral proteins, neutralizing antibodies that inhibited infectivity of cell-free virus or syncytia formation, antibodies that inhibited reverse transcriptase activity, and antibodies to lymphocyte cell-surface antigens were assessed. The results indicated that in macaques the magnitude of the SIV/SMM-specific antibody response and progression of disease were functions of virus load. Surprisingly, asymptomatic mangabeys also had high virus loads with, on average, lower antibody titers than macaques. In both species, the presence of neutralizing antibodies or antibodies that inhibited SIV/SMM reverse transcriptase activity did not correlate with protection from clinical disease. A correlation was observed, however, between the development of disease and the presence of antibodies to an 18-kDa protein that is found on the surface of activated lymphocytes and appears to be related to histone H2B. A similar correlation has been observed in association with HIV infection in humans, suggesting that some manifestations of both human and simian AIDS may result from autoimmune reactions.


Asunto(s)
Anticuerpos Antivirales/biosíntesis , Enfermedades de los Monos/inmunología , Infecciones por Retroviridae/veterinaria , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Autoanticuerpos/análisis , Cercopithecidae/inmunología , Macaca/inmunología , Enfermedades de los Monos/microbiología , Pruebas de Neutralización , ADN Polimerasa Dirigida por ARN/metabolismo , Infecciones por Retroviridae/enzimología , Infecciones por Retroviridae/inmunología , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación
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