RESUMEN
BACKGROUND: Iodine contrast agents (ICAs) are routinely used by radiologists to help guide intra-articular infiltrations. The aim of this study was to assess the in vitro effects of ICA on platelet function of human autologous Platelet-Rich Plasma (PRP). METHODS: One hundred thirty-seven consecutive patients with symptomatic femoral-patellar osteoarthritis were included. All were addressed to our institution for a fluoroscopy-guided intra-articular PRP infiltration of the pathological femoral-patellar joint. For each patient, 500 µl of PRP were sampled before intra-articular injection. First, PRP samples were mixed with 50 µl of 2 widely used ICA: Visipaque270® (Iodixanol, n = 58) and Iopamiron200® (Iopamidol, n = 69). PRP concentration ([PRP]) was measured at different delays of incubation (t = 0, 5, 10, 15, 20 and 30 min) enabling to calculate PRP ratio (defined as [PRP](t)/[PRP](0mn)) at each delay, for each mixture, in order to quantitatively assess the influence of ICA on PRP ratio. Second, the PRP samples of 10 additional patients were mixed with Visipaque270®, Visipaque270®, Iopamiron200® and phosphate buffer saline (PBS: control solution) in order to qualitatively assess the influence of ICA on platelet aggregation, using ADP, Collagen, Arachidonic acid and TRAP tests. The surface expression of human P-selectin, a marker of α-granule release, in the PRP + Visipaque270® and PRP + Iopamiron200® mixtures was finally compared. Repeated-measures ANOVA, classical 2-way ANOVA and Wilcoxon matched-pairs test were used to study the influence of ICA on PRP quality. RESULTS: There was no significant change in PRP ratio during the first 30mn of incubation (p = 0.991) whatever the ICA (p = 0.926). Whatever the aggregation test, there was no significant difference in the percentage of platelet aggregation between PRP + PBS, PRP + Visipaque270® and PRP + Iopamiron200® (p = 0.998), nor between PRP + PBS and PRP + Visipaque320® (p = 0.470). Finally, there was no significant difference in P-selectin expression between the PRP + Visipaque270® and PRP + Iopamiron200® mixtures (p = 0.500). CONCLUSION: At early delays of incubation, Visipaque® and Iopamiron®, which are two widely used ICA for intra-articular infiltrations, did not influence the in vitro platelet function nor the quality of PRP.
RESUMEN
Whereas hormone replacement/menopause therapy (HRT) in postmenopausal women increases the coronary artery risk, epidemiological studies (protection in premenopaused women) suggest and experimental studies (prevention of the development of fatty streaks in animals) demonstrate a major atheroprotective action of oestradiol (E2). The understanding of the deleterious and beneficial effects of oestrogens is thus required. The immuno-inflammatory system plays a key role in the development of fatty streak deposit as well as in the rupture of the atherosclerotic plaque. Whereas E2 favours an anti-inflammatory effect in vitro (cultured cells), it rather elicits in vivo a proinflammation at the level of several subpopulations of the immuno-inflammatory system, which could contribute to plaque destabilization. Endothelium is another important target for E2, as it potentiates endothelial NO and prostacyclin production, thus promoting the beneficial effects as vasorelaxation and inhibition of platelet aggregation. Prostacyclin, but not NO, appears to be involved in the atheroprotective effect of E2. E2 also accelerates endothelial regrowth, thus favouring vascular healing. Finally, most of these effects of E2 are mediated by oestrogen receptor alpha, and are independent of oestrogen receptor beta. In summary, a better understanding of the mechanisms of oestrogen action not only on the normal and atheromatous arteries, but also on innate and adaptive immune responses is required and should help to optimize the prevention of cardiovascular disease after menopause. These mouse models should help to screen existing and future selective oestrogen receptor modulators.
Asunto(s)
Aterosclerosis/etiología , Estradiol/fisiología , Animales , Aterosclerosis/prevención & control , Vasos Sanguíneos/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Receptor alfa de Estrógeno/fisiología , Femenino , Humanos , Inmunidad Celular/efectos de los fármacos , Inflamación/inducido químicamente , Modelos AnimalesRESUMEN
Osteopontin (OPN), an RGD-containing extracellular matrix protein, is associated with arterial smooth muscle cell (SMC) activation in vitro and in vivo. Many cytokines and growth factors involved in vessel wall remodeling induce OPN overexpression. Moreover, we recently demonstrated that the extracellular nucleotide UTP also induces OPN expression and that OPN is essential for UTP-mediated SMC migration. Thus, we set out to investigate the mechanisms of OPN expression. The aim of this study was to identify transcription factors involved in the regulation of OPN expression in SMCs. First, we explored the contribution of mRNA stabilization and transcription in the increase of UTP-induced OPN mRNA levels. We show that UTP induced OPN mRNA increases via both OPN mRNA stabilization and OPN promoter activation. Then, to identify transcription factors involved in UTP-induced OPN transcription, we located a promoter element activated by UTP within the rat OPN promoter using a gene reporter assay strategy. The -96 to +1 region mediated UTP-induced OPN overexpression (+276+/-60%). Sequence analysis of this region revealed a potential site for AP-1 located at -76. When this AP-1 site was deleted, UTP-induced activation of the -96 to +1 region was totally inhibited. Thus, this AP-1 (-76) site is involved in UTP-induced OPN transcription. A supershift assay revealed that both c-Fos and c-Jun bind to this AP-1 site. Finally, we demonstrate that angiotensin II and platelet-derived growth factor, two main factors involved in vessel wall pathology, also modulated OPN expression via AP-1 activation.
Asunto(s)
Músculo Liso Vascular/efectos de los fármacos , Sialoglicoproteínas/genética , Factor de Transcripción AP-1/metabolismo , Uridina Trifosfato/farmacología , Angiotensina II/farmacología , Animales , Aorta Torácica/citología , Sitios de Unión/genética , Northern Blotting , Células Cultivadas , Ensayo de Cambio de Movilidad Electroforética , Regulación de la Expresión Génica/efectos de los fármacos , Luciferasas/genética , Luciferasas/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Osteopontina , Factor de Crecimiento Derivado de Plaquetas/farmacología , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
Osteopontin (OPN), an RGD containing extracellular matrix protein, is associated with arterial smooth muscle cells (SMC) activation in vitro and in vivo. OPN has been shown to be overexpressed in vascular injury. Its expression can be induced by many factors including growth factors, cytokines, hormones and extracellular nucleotides. We are interested in understanding mechanisms regulating the OPN mRNA steady state level in SMC. We compared the effect of two G-protein coupled receptors agonists (UTP and angiotensin II [AII]) and one tyrosin kinase receptor agonist (PDGF). We explored the effect of these three agonists both on OPN transcription using gene reporter assay and on OPN mRNA stabilisation using actinomycin D. We showed that UTP 100 microM. AII 10 microM and PDGF 50 ng/microL induced OPN transcription. Whereas UTP and AII induced a 366 +/- 81% and 338 +/- 115% activation of transcription respectively, PDGF demonstrated a lower efficiency (195 +/- 59%) inducing the transcription. Moreover, we demonstrated that UTP and AII but not PDGF were able to stabilize OPN mRNA. This effect seems to be specific to G-protein coupled receptor agonists since previous studies demonstrated that intracellular receptor agonists did not stabilise OPN mRNA. Thus, the lower increase of OPN mRNA level in response to PDGF stimulation compared to AII or UTP could be explain by both, the lower activation of the OPN promoter and the effect of UTP and AII on OPN mRNA stabilisation.
Asunto(s)
Angiotensina II/farmacología , Arterias/fisiología , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Sialoglicoproteínas/biosíntesis , Vasoconstrictores/farmacología , Animales , Adhesión Celular , Citocinas , Proteínas de Unión al GTP , Perfilación de la Expresión Génica , Genes Reporteros , Osteopontina , Fosfoproteínas , ARN Mensajero/análisis , Ratas , Ratas Wistar , Receptores de Superficie Celular , Uridina TrifosfatoRESUMEN
Migration and proliferation of arterial smooth muscle cells (SMCs) play a prominent role in the development of atherosclerotic plaques and restenosis lesions. Most of the growth-regulatory molecules potentially involved in these pathological conditions also demonstrate chemotactic properties. Extracellular purine and pyrimidine nucleotides have been shown to induce cell cycle progression and to elicit growth of cultured vascular SMCs. Moreover, the P2Y(2) ATP/UTP receptor was overexpressed in intimal thickening, suggesting a role of these nucleotides in vascular remodeling. Using the Transwell system migration assay, we demonstrate that extracellular ATP, UTP, and UDP exhibit a concentration-dependent chemotactic effect on cultured rat aortic SMCs. UTP, the most powerful nucleotide inducer of migration, elicited significant responses from 10 nmol/L. In parallel, UTP increased osteopontin expression dose-dependently. The blockade of osteopontin or its integrin receptors alpha(v)beta(3)/beta(5) by specific antibodies or antagonists inhibited UTP-induced migration. Moreover, the blockade of ERK-1/ERK-2 MAP kinase or rho protein pathways led to the inhibition of both UTP-induced osteopontin increase and migration, demonstrating the central role of osteopontin in this process. Taken together, these results suggest that extracellular nucleotides, and particularly UTP, can induce arterial SMC migration via the action of osteopontin.
Asunto(s)
Movimiento Celular/fisiología , Espacio Extracelular/metabolismo , Músculo Liso Vascular/metabolismo , Nucleótidos/farmacología , Sialoglicoproteínas/metabolismo , Adenosina Difosfato/farmacología , Adenosina Trifosfato/farmacología , Animales , Aorta , Calcio/metabolismo , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Quimiotaxis/fisiología , Cámaras de Difusión de Cultivos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Oligopéptidos/farmacología , Osteopontina , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Wistar , Uridina Difosfato/farmacología , Uridina Trifosfato/farmacología , Quinasas Asociadas a rho , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Although mineral deposits have long been described to be a prominent feature of atherosclerosis, the mechanisms of arterial calcification are not well understood. However, accumulation of the non-collagenous matrix bone-associated proteins, osteopontin, osteocalcin, and osteonectin, has been demonstrated in atheromatous plaques. The aim of this study was to evaluate the role of these proteins in arterial calcification and, more precisely, during the initiation of this process. A model of rapid aortic calcification was developed in rabbits by an oversized balloon angioplasty. Calcification was followed using von Kossa staining and osteopontin, osteocalcin, and osteonectin were identified using immunohistochemistry. The aortic injury was rapidly followed by calcified deposits that appeared in the media as soon as 2 days after injury and then accumulated in zipper-like structures. Osteonectin was not detected in calcified deposits at any time after injury. In contrast, osteopontin and osteocalcin were detected in 8- and 14-day calcified structures, respectively, but not in the very early 2-day mineral deposits. These results suggest that these matrix proteins, osteopontin, osteocalcin, and osteonectin, are not involved in the initiation step of the aortic calcification process and that the former two might play a role in the regulation of arterial calcification.
Asunto(s)
Arteriosclerosis/metabolismo , Calcinosis/metabolismo , Osteocalcina/metabolismo , Osteonectina/metabolismo , Sialoglicoproteínas/metabolismo , Túnica Media/metabolismo , Angioplastia de Balón/efectos adversos , Animales , Aorta/metabolismo , Enfermedades de la Aorta/etiología , Enfermedades de la Aorta/metabolismo , Calcinosis/etiología , Inmunohistoquímica , Masculino , Microscopía Confocal , Osteopontina , ConejosRESUMEN
In the cardiovascular system, activation of ionotropic (P2X receptors) and metabotropic (P2Y receptors) P2 nucleotide receptors exerts potent and various responses including vasodilation, vasoconstriction, and vascular smooth muscle cell proliferation. Here we examined the involvement of the small GTPase RhoA in P2Y receptor-mediated effects in vascular myocytes. Stimulation of cultured aortic myocytes with P2Y receptor agonists induced an increase in the amount of membrane-bound RhoA and stimulated actin cytoskeleton organization. P2Y receptor agonist-induced actin stress fiber formation was inhibited by C3 exoenzyme and the Rho kinase inhibitor Y-27632. Stimulation of actin cytoskeleton organization by extracellular nucleotides was also abolished in aortic myocytes expressing a dominant negative form of RhoA. Extracellular nucleotides induced contraction and Y-27632-sensitive Ca(2+) sensitization in aortic rings. Transfection of Swiss 3T3 cells with P2Y receptors showed that Rho kinase-dependent actin stress fiber organization was induced in cells expressing P2Y(1), P2Y(2), P2Y(4), or P2Y(6) receptor subtypes. Our data demonstrate that P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptor subtypes are coupled to activation of RhoA and subsequently to Rho-dependent signaling pathways.
Asunto(s)
Músculo Liso Vascular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Purinérgicos P2/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Células 3T3 , Actinas/fisiología , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/fisiología , Membrana Celular/metabolismo , Células Cultivadas , Citoesqueleto/fisiología , Activación Enzimática/fisiología , Fibroblastos/metabolismo , Membranas Intracelulares/fisiología , Péptidos y Proteínas de Señalización Intracelular , Ratones , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Nucleótidos/farmacología , Proteínas Serina-Treonina Quinasas/fisiología , Agonistas del Receptor Purinérgico P2 , Ratas , Ratas Wistar , Transducción de Señal/fisiología , Vasoconstricción/fisiología , Quinasas Asociadas a rhoRESUMEN
Apoptosis of arterial smooth muscle cells (ASMCs) could play an important role in the pathogenesis of atherosclerosis and restenosis. Recent studies have demonstrated that extracellular adenosine induces apoptosis in various cell types. Our aim was to delineate the capacity of this nucleoside to induce ASMC apoptosis in arterial diseases. We demonstrate that adenosine dose-dependently triggers apoptosis of cultured human ASMCs. Apoptotic cell death was quantified by analysis of nuclear chromatin morphology and characterized by DNA laddering. The involvement of adenosine receptors was suggested, because neither an adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, nor an inhibitor of cellular nucleoside transport, dipyridamole, was able to inhibit adenosine-induced ASMC apoptosis. In contrast, an A(1)/A(2)-adenosine receptor antagonist, xanthine amine congener, totally inhibited adenosine-induced apoptosis. Furthermore, among more selective inhibitors of P(1) purinoceptor subtypes, only alloxazine, an antagonist of A(1)- and A(2)-adenosine receptors, completely inhibited adenosine-induced ASMC apoptosis, suggesting that adenosine triggers ASMC apoptosis via either 1 or both of these receptors. However, 8-cyclopentyl-1,3-dipropylxanthine, 8-(3-chlorostyryl) caffeine, and 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(+/-)-dihydropyridine-3,5-dicarboxylate, which are A(1)-, A(2a)-, and A(3)-adenosine receptor antagonists, did not inhibit adenosine-induced apoptosis, suggesting an involvement of the A(2b)-receptor in this process. Moreover, the cAMP increase followed by cAMP-dependent protein kinase activation appears essential to mediate adenosine-induced ASMC apoptosis, thus confirming the previous hypothesis. These results indicate that adenosine-induced apoptosis of ASMCs is essentially mediated via A(2b)-adenosine receptor and involves a cAMP-dependent pathway.
Asunto(s)
Adenosina/fisiología , Apoptosis/fisiología , Arterias/fisiología , Espacio Extracelular/metabolismo , Músculo Liso Vascular/fisiología , Receptores Purinérgicos P1/fisiología , Arterias/citología , Arterias/metabolismo , Células Cultivadas , AMP Cíclico/fisiología , Humanos , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Receptor de Adenosina A2B , Receptores Purinérgicos P1/metabolismoRESUMEN
Extracellular nucleotides, particularly ATP, are involved in the modulation of arterial vasomotricity via P2 purinoceptors present on smooth muscle and endothelial cells. These nucleotides could also be implicated in the smooth muscle cell hyperplasia observed in intimal lesions. In this study, we tried to define the potential role of the P2Y2 (P2u) purinoceptor by studying its expression in normal and balloon-injured rat aortas. The cloning of a rat P2Y2 cDNA from a rat smooth muscle cell cDNA library made it possible to study P2Y2 expression both by Northern blot and in situ hybridization. Northern blot experiments indicated that P2Y2 mRNA was present in rat medial aortic smooth muscle and in cultured rat aortic smooth muscle cells. In situ hybridization indicated that P2Y2 mRNA was present in endothelial cells of the intima and in some smooth muscle cells scattered throughout the media of adult rat aortas, while almost all medial smooth muscle cells of rat embryo aorta expressed this receptor. In contrast with adult aortic media, the majority of neointimal smooth muscle cells found in aortic intimal lesions either 8 or 20 days after balloon injury were positive for P2Y2 mRNA. Moreover, a subpopulation of neointimal cells localized at the luminal surface could be identified by a higher P2Y2 expression than the underlying neointimal smooth muscle cells. These data showing a strong expression of the P2Y2 purinoceptor in the neointima of injured arteries suggest that extracellular nucleotides may be involved, via this receptor, in the intimal hyperplasia and/or chronic constriction observed at the lesion site, and consequently in the restenotic process.
Asunto(s)
Aorta/lesiones , Receptores Purinérgicos P2/metabolismo , Túnica Íntima/metabolismo , Secuencia de Aminoácidos , Angioplastia de Balón/efectos adversos , Animales , Aorta/embriología , Aorta/metabolismo , Secuencia de Bases , Clonación Molecular , ADN Complementario , Expresión Génica , Hibridación in Situ , Masculino , Datos de Secuencia Molecular , Músculo Liso Vascular/metabolismo , Ratas , Ratas Wistar , Receptores Purinérgicos P2Y2RESUMEN
mRNA of the P2u purinoceptor (or nucleotide receptor) is detected both by polymerase chain reaction or Northern blot analyses in cultured aortic smooth muscle cells. When added to the culture medium of these cells, UTP, a specific ligand of the P2u receptor, induces an increased expression of both immediate-early and delayed-early cell cycle-dependent genes. This induction demonstrates similar features (kinetics, concentration dependence) to those obtained after stimulation of aortic smooth cells by exogenous ATP, a common ligand for most P2 purinoceptors. In contrast, 2-methylthioATP, a preferential ligand for P2y purinoceptors, induces only a significant increase of immediate-early genes but not of delayed-early genes. Moreover, the 2-methylthioATP-induced responses (c-fos mRNA increase, free intracellular calcium transient) are lower than those induced by ATP or UTP and are complementary to those of UTP. These results demonstrate that functional P2u receptors are present on cultured aortic smooth muscle cells and suggest that the bulk of responses induced by extracellular ATP on cell cycle progression are mediated via P2u purinoceptors, a hypothesis confirmed by cytofluorometric studies. Since some ATP- or UTP-induced genes code for chemotactic proteins (monocyte chemoattractant protein-1 and osteopontin), this study suggests that these nucleotides may contribute to vascular or blood cell migration and proliferation and consequently to the genesis of arterial diseases.
Asunto(s)
Ciclo Celular/fisiología , Músculo Liso Vascular/citología , Receptores de Superficie Celular/fisiología , Adenosina Trifosfato/fisiología , Animales , Aorta/citología , Secuencia de Bases , Calcio/metabolismo , Expresión Génica/fisiología , Datos de Secuencia Molecular , Músculo Liso Vascular/ultraestructura , Proteínas Proto-Oncogénicas c-fos/genética , ARN Mensajero/metabolismo , Ratas , Uridina Difosfato/metabolismo , Uridina Difosfato/farmacologíaRESUMEN
Because exogenous ATP is suspected to influence the proliferative process, its effects on the cell cycle progression of arterial smooth muscle cells were studied by investigating changes in the mRNA steady-state level of cell cycle-dependent genes. Stimulation of cultured quiescent smooth muscle cells by exogenous ATP induced chronological activation not only of immediate-early but also of delayed-early cell cycle-dependent genes, which were usually expressed after a mitogenic stimulation. In contrast, ATP did not increase late G1 gene mRNA level, demonstrating that this nucleotide induces a limited cell cycle progression of arterial smooth muscle cells through the G1 phase but is not able by itself to induce crossing over the G1-S boundary and consequently DNA synthesis. An increase in c-fos mRNA level was also induced by ADP but not by AMP or adenosine. Moreover, 2-methylthioadenosine 5'-triphosphate but not alpha, beta-methyleneadenosine 5'-triphosphate mediated this kind of response. Taken together, these results demonstrate that extracellular ATP induces the limited progression of arterial smooth muscle cells through the G1 phase via its fixation on P2 gamma receptors.
Asunto(s)
Adenosina Trifosfato/farmacología , Ciclo Celular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/fisiología , Células Cultivadas , Sondas de ADN , Inducción Enzimática , Fase G1/genética , Expresión Génica , Genes fos/efectos de los fármacos , Genes myc/efectos de los fármacos , Histonas/biosíntesis , Histonas/genética , Cinética , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Ornitina Descarboxilasa/biosíntesis , Ornitina Descarboxilasa/genética , Antígeno Nuclear de Célula en Proliferación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Timidina Quinasa/biosíntesis , Timidina Quinasa/genéticaRESUMEN
To isolate the genes involved in the cell cycle G1 phase progression of arterial smooth muscle cells (SMCs), a cDNA clone (M11) was previously selected by differential hybridization screening of a mid-G1 serum-stimulated SMC cDNA library. The delay of induction after mitogenic stimulation, time of expression, and need for new protein synthesis for full expression made it possible to classify this gene in the "delayed early" gene group. Determination of the partial M11 cDNA sequence showed full homology with the osteopontin gene (secreted phosphoprotein 1, 2ar), an Arg-Gly-Asp-containing extracellular matrix protein. Osteopontin mRNA was also detected in the aorta at levels as high as in the kidney but lower than in bone, two tissues in which it has been previously detected. In vitro analysis of osteopontin expression in serum-stimulated quiescent SMCs and asynchronously cycling SMCs demonstrated that osteopontin overexpression was associated with SMC proliferation. In view of our results, the high osteopontin expression observed by others in the injured carotid artery could be explained by the involvement of SMCs in the proliferative process. Taken together, these results suggest that osteopontin may play an important role in pathological processes that are associated with arterial SMC proliferation, such as atherosclerosis or restenosis.
Asunto(s)
Aorta/metabolismo , Músculo Liso Vascular/metabolismo , Sialoglicoproteínas/metabolismo , Animales , Aorta/citología , Ciclo Celular , División Celular , Células Cultivadas , ADN/genética , Músculo Liso Vascular/citología , Osteopontina , ARN Mensajero/metabolismo , Ratas , Sialoglicoproteínas/genética , Factores de TiempoRESUMEN
An increase in cell size and protein content was observed when quiescent arterial smooth muscle cells in culture were incubated with either angiotensin II or III. These effects were inhibited by the specific angiotensin type-1 receptor antagonist losartan (DuP753) but not by CGP42112A. In parallel, a transient and dose-dependent induction of c-fos was demonstrated not only with angiotensins II and III but also with angiotensin I. Both angiotensins II and III exerted their maximal effect at 1 microM, while angiotensin I needed a tenfold-higher concentration to exert an identical effect. As for hypertrophy, losartan also inhibits angiotensin-induced c-fos expression, suggesting that this gene may be involved into the hypertrophic process. Angiotensin-I-mediated c-fos induction is partially inhibited by the angiotensin-converting enzyme inhibitors captopril and trandolaprilate; given that an angiotensin-converting enzyme activity was detected in these smooth muscle cell cultures, these results suggest that angiotensin-I-induced c-fos expression is mediated in part via angiotensin-I conversion to angiotensin II, but also by other unidentified pathway(s). Angiotensin I could essentially induce smooth muscle cell hypertrophy by indirect mechanisms, while angiotensins II and III act directly on smooth muscle cells.
Asunto(s)
Angiotensinas/farmacología , Músculo Liso Vascular/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Angiotensinas/antagonistas & inhibidores , Animales , Compuestos de Bifenilo/farmacología , Captopril/farmacología , Células Cultivadas , ADN/metabolismo , Imidazoles/farmacología , Indoles/farmacología , Losartán , Músculo Liso Vascular/patología , Oligopéptidos/farmacología , ARN/metabolismo , Ratas , Ratas Endogámicas WKY , Tetrazoles/farmacologíaRESUMEN
The expression of a set of cell cycle dependent (CCD) genes (c-fos, c-myc, ornithine decarboxylase (ODC), and thymidine kinase (TK)) was comparatively studied in cultured arterial smooth muscle cells (SMC) during exit from quiescence and exponential proliferation. These genes, which were not expressed in quiescent SMC, were chronologically induced after serum stimulation. c-fos mRNA were rapidly and transiently expressed very early in the G1 phase; c-myc and ODC peaked a few hours after serum stimulation and then remained at an intermediary level throughout the first cell cycle; TK mRNA and activity then appeared at the G1/S boundary and peak in G2/M phases. Except for c-fos, the other genes were also expressed in asynchronously cycling SMC (ACSMC); their expression was studied in elutriated subpopulations representative of cell cycle progression. c-fos mRNA were undetectable in any sorted subpopulations, even in the pure early G1 population. Despite a slight increase as the cell cycle advanced, c-myc and ODC genes were expressed throughout the ACSMC cell cycle. A faint TK activity was found in G1 subpopulations and increased in populations enriched in other phases; in contrast, TK mRNA remained highly expressed in all elutriated subpopulations. This study demonstrates significant modulations in CCD gene expression between quiescent stimulated and asynchronously cycling SMC in culture. This suggests that the events occurring during the emergence of SMC from quiescence are probably different from those in the G1 phase of ACSMC.
Asunto(s)
Ciclo Celular/genética , Regulación de la Expresión Génica , Músculo Liso Vascular/metabolismo , División Celular , Células Cultivadas , Genes fos , Genes myc , Cinética , Músculo Liso Vascular/citología , Ornitina Descarboxilasa/genética , Timidina Quinasa/genéticaRESUMEN
Serum stimulation of arterial smooth muscle cells in culture induces a progression through the cell cycle and cell proliferation. Most genes previously described as cell cycle-dependent in various cell types also demonstrate a cell cycle-dependent expression in arterial smooth muscle cells. As in other cell types, these genes can be classified into three groups according to their mode of expression: "immediate early" genes (c-fos, c-myc, ...), "delayed early" genes (2F1, ...), and "late-G1" genes (proliferating cell nuclear antigen, thymidine kinase, . . .). In addition to these previously described genes, three genes isolated from a cDNA library of stimulated smooth muscle cells have been demonstrated to be cell cycle-dependent: A21, the rat JE gene, and L51 can be classified as "immediate early" genes, while M11 represents a new member of the "delayed early" gene family.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Genes/fisiología , Músculo Liso Vascular/citología , Animales , Ciclo Celular/fisiología , Células Cultivadas , Citometría de Flujo , Fase G1/fisiología , Regulación de la Expresión Génica/genética , Genes/genética , Immunoblotting , Músculo Liso Vascular/fisiología , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/fisiología , ARN/aislamiento & purificación , RatasRESUMEN
8-(N,N-Diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a putative inhibitor of intracellular calcium mobilization, causes a dose-dependent inhibition of serum-induced proliferation of arterial smooth muscle cells in culture. Neither early rise in cytosolic calcium concentration nor induction of early induced cell cycle dependent genes (c-fos, ornithine decarboxylase) are inhibited after serum stimulation in presence of 100 microM TMB-8. In contrast, expression of thymidine kinase, a gene normally induced in late-G1 phase, is entirely inhibited by TMB-8. Taken together with flow cytometry studies, these results indicate that TMB-8 blocks cell cycle progression in mid- or late-G1 phase by a mechanism not directly related to early responses to serum stimulation since TMB-8 is also effective when introduced several hours after serum stimulation.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Ácido Gálico/análogos & derivados , Músculo Liso Vascular/efectos de los fármacos , Animales , Aorta , Calcio/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Citosol/metabolismo , Ácido Gálico/farmacología , Expresión Génica , Interfase/efectos de los fármacos , Masculino , Músculo Liso Vascular/metabolismo , Ornitina Descarboxilasa/análisis , Ornitina Descarboxilasa/genética , ARN Mensajero/biosíntesis , Ratas , Ratas Endogámicas , Timidina Quinasa/análisisRESUMEN
The effect of rifampin on five mollicutes (Spiroplasma citri, Spiroplasma melliferum, Spiroplasma apis, Acholeplasma laidlawii, and Mycoplasma mycoides) was compared with that on Escherichia coli. We found that, in contrast to wild-type E. coli, mollicutes were insensitive to rifampin. DNA-dependent RNA polymerases from S. melliferum and S. apis were purified to the stage where the enzymes were dependent on the addition of exogenous templates for activity. The enzymes were then tested for their sensitivity to rifampin. Spiroplasmal enzymes were at least 1,000 times less sensitive to rifampin than the corresponding E. coli enzyme. This result provides a molecular basis for the resistance of mollicutes to rifampin. The RNA polymerase of S. melliferum was further purified and its subunit composition was investigated. The RNA polymerase has one small and two large subunits. The structure of S. melliferum RNA polymerase therefore resembles that of the eubacterial enzymes in spite of its insensitivity to rifampin.