RESUMEN
Nonalcoholic steatohepatitis (NASH) is an emerging health crisis with no approved therapies. Obeticholic acid (OCA), a farnesoid X receptor (FXR) agonist, shows promise in NASH trials. However, the precise mechanisms mediating OCA effects and impact on cholesterol metabolism are not fully understood. We explored the pharmaco-toxicological effects of OCA on patho-physiological pathways in hepatocytes using a previously described perfused organotypic liver system that allows culture in near-physiological insulin/glucose milieus, and exhibits drug responses at clinically-relevant concentrations. Primary hepatocytes experienced 48-hour exposure to OCA at concentrations approximating therapeutic (0.5µM) and supratherapeutic (10µM) levels. Global transcriptomics by RNAseq was complimented by cellular viability (MTT), CYP activity assays, and secreted FGF19 levels in the media. Dose-dependent, transcriptional effects suggested suppression of bile acid synthesis (↓CYP7A1, ↓CYP27A1) and increased bile efflux (↑ABCB4, ↑ABCB11, ↑OSTA, ↑OSTB). Pleiotropic effects included suppression of TGFß and IL-6 signaling pathways, and signatures suggestive of HDL suppression (↑SCARB1, ↓ApoAI, ↓LCAT) and LDL elevation (↑ApoB, ↓CYP7A1). OCA exhibited direct FXR-mediated effects with increased FGF19 secretion. Transcriptomics revealed regulation of metabolic, anti-inflammatory, and anti-fibrotic pathways beneficial in NASH, and predicted cholesterol profiles consistent with clinical findings. Follow-up studies under lipotoxic/inflammatory conditions would corroborate these effects in a disease-relevant environment.
Asunto(s)
Ácido Quenodesoxicólico/análogos & derivados , Hepatocitos/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ácido Quenodesoxicólico/farmacología , Ácido Quenodesoxicólico/toxicidad , Colesterol/metabolismo , Hepatocitos/metabolismo , Humanos , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Transcriptoma/efectos de los fármacosRESUMEN
In vitro primary hepatocyte systems typically elicit drug induction and toxicity responses at concentrations much higher than corresponding in vivo or clinical plasma C(max) levels, contributing to poor in vitro-in vivo correlations. This may be partly due to the absence of physiological parameters that maintain metabolic phenotype in vivo. We hypothesized that restoring hemodynamics and media transport would improve hepatocyte architecture and metabolic function in vitro compared with nonflow cultures. Rat hepatocytes were cultured for 2 wk either in nonflow collagen gel sandwiches with 48-h media changes or under controlled hemodynamics mimicking sinusoidal circulation within a perfused Transwell device. Phenotypic, functional, and metabolic parameters were assessed at multiple times. Hepatocytes in the devices exhibited polarized morphology, retention of differentiation markers [E-cadherin and hepatocyte nuclear factor-4α (HNF-4α)], the canalicular transporter [multidrug-resistant protein-2 (Mrp-2)], and significantly higher levels of liver function compared with nonflow cultures over 2 wk (albumin ~4-fold and urea ~5-fold). Gene expression of cytochrome P450 (CYP) enzymes was significantly higher (fold increase over nonflow: CYP1A1: 53.5 ± 10.3; CYP1A2: 64.0 ± 15.1; CYP2B1: 15.2 ± 2.9; CYP2B2: 2.7 ± 0.8; CYP3A2: 4.0 ± 1.4) and translated to significantly higher basal enzyme activity (device vs. nonflow: CYP1A: 6.26 ± 2.41 vs. 0.42 ± 0.015; CYP1B: 3.47 ± 1.66 vs. 0.4 ± 0.09; CYP3A: 11.65 ± 4.70 vs. 2.43 ± 0.56) while retaining inducibility by 3-methylcholanthrene and dexamethasone (fold increase over DMSO: CYP1A = 27.33 and CYP3A = 4.94). These responses were observed at concentrations closer to plasma levels documented in vivo in rats. The retention of in vivo-like hepatocyte phenotype and metabolic function coupled with drug response at more physiological concentrations emphasizes the importance of restoring in vivo physiological transport parameters in vitro.
Asunto(s)
Hemodinámica/fisiología , Hepatocitos/metabolismo , Hepatocitos/ultraestructura , Circulación Hepática/fisiología , Hígado/irrigación sanguínea , Animales , Western Blotting , Sistema Enzimático del Citocromo P-450/metabolismo , Inmunohistoquímica , Hígado/citología , Masculino , Microscopía Electrónica de Transmisión , Ratas , Ratas Endogámicas F344 , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: We previously demonstrated that upregulation of intermediate-conductance Ca(2+)-activated K(+) channels (K(Ca)3.1) is necessary for mitogen-induced phenotypic modulation in isolated porcine coronary smooth muscle cells (SMCs). The objective of the present study was to determine the role of K(Ca)3.1 in the regulation of coronary SMC phenotypic modulation in vivo using a swine model of postangioplasty restenosis. METHODS AND RESULTS: Balloon angioplasty was performed on coronary arteries of swine using either noncoated or balloons coated with the specific K(Ca)3.1 blocker TRAM-34. Expression of K(Ca)3.1, c-jun, c-fos, repressor element-1 silencing transcription factor (REST), smooth muscle myosin heavy chain (SMMHC), and myocardin was measured using qRT-PCR in isolated medial cells 2 hours and 2 days postangioplasty. K(Ca)3.1, c-jun, and c-fos mRNA levels were increased 2 hours postangioplasty, whereas REST expression decreased. SMMHC expression was unchanged at 2 hours, but decreased 2 days postangioplasty. Use of TRAM-34 coated balloons prevented K(Ca)3.1 upregulation and REST downregulation at 2 hours, SMMHC and myocardin downregulation at 2 days, and attenuated subsequent restenosis 14 and 28 days postangioplasty. Immunohistochemical analysis demonstrated corresponding changes at the protein level. CONCLUSIONS: Blockade of K(Ca)3.1 by delivery of TRAM-34 via balloon catheter prevented smooth muscle phenotypic modulation and limited subsequent restenosis.
Asunto(s)
Estenosis Coronaria/prevención & control , Vasos Coronarios/efectos de los fármacos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Músculo Liso Vascular/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Pirazoles/farmacología , Angioplastia Coronaria con Balón/efectos adversos , Animales , Diferenciación Celular/efectos de los fármacos , Estenosis Coronaria/patología , Vasos Coronarios/metabolismo , Vasos Coronarios/patología , Modelos Animales de Enfermedad , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Masculino , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Fenotipo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Porcinos , Factores de Transcripción/metabolismoRESUMEN
This chapter will review conditional mouse model systems that have been developed to study gene function in skeletal, cardiac, and vascular smooth muscle cells in vivo with an emphasis on the utility of these models for investigating developmental and pathophysiological gene function in muscle. In general, these systems have utilized muscle-specific/selective promoter-enhancers in conjunction with site-specific DNA recombinases, e.g., Cre-loxP, and fusion proteins with these recombinases that confer temporal control, such as tamoxifen-inducible CreER systems. A major focus of this chapter will be to discuss unique challenges of studying Cre-mediated mutagenesis/gene targeting in these muscle types during development and in the adult animal, some of which are inherent of the muscle cell type being studied. For example, unlike cardiac and skeletal muscle cells, the vascular SMC is extremely plastic and able to undergo rapid phenotypic modulation to various environmental cues in vivo. Thus, employing SMC marker gene promoter enhancers for conditional gene targeting in SMCs must take into account the possibility and/or certainty that the particular SMC promoter enhancers used may or may not be transcriptionally active in SMCs of a vessel wall under normal and some pathophysiological conditions. Moreover, individual floxed loci within the same muscle cell type and tissue have different degrees of sensitivity to Cre, most likely dependent on the chromatin state of that particular gene, i.e., closed/condensed state or open/active state. Thus, Cre recombination may be ineffective for specific floxed gene DNA. Lastly, rigorous efforts must be made to confirm the degree of recombination in a tissue, taking into full account the multicellularity of the tissue, to understand the extent of the physiological effect in that organ.
Asunto(s)
Músculos/embriología , Músculos/metabolismo , Animales , Marcación de Gen , Ratones , Modelos Animales , Mutagénesis , Recombinasas/metabolismoRESUMEN
A hallmark of smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and restenosis is suppression of SMC differentiation marker genes, proliferation, and migration. Blockade of intermediate-conductance Ca(2+)-activated K(+) channels (IKCa1) has been shown to inhibit restenosis after carotid balloon injury in the rat; however, whether IKCa1 plays a role in SMC phenotypic modulation is unknown. Our objective was to determine the role of IKCa1 channels in regulating coronary SMC phenotypic modulation and migration. In cultured porcine coronary SMCs, platelet-derived growth factor-BB (PDGF-BB) increased TRAM-34 (a specific IKCa1 inhibitor)-sensitive K(+) current 20-fold; increased IKCa1 promoter histone acetylation and c-jun binding; increased IKCa1 mRNA approximately 4-fold; and potently decreased expression of the smooth muscle differentiation marker genes smooth muscle myosin heavy chain (SMMHC), smooth muscle alpha-actin (SMalphaA), and smoothelin-B, as well as myocardin. Importantly, TRAM-34 completely blocked PDGF-BB-induced suppression of SMMHC, SMalphaA, smoothelin-B, and myocardin and inhibited PDGF-BB-stimulated migration by approximately 50%. Similar to TRAM-34, knockdown of endogenous IKCa1 with siRNA also prevented the PDGF-BB-induced increase in IKCa1 and decrease in SMMHC mRNA. In coronary arteries from high fat/high cholesterol-fed swine demonstrating signs of early atherosclerosis, IKCa1 expression was 22-fold higher and SMMHC, smoothelin-B, and myocardin expression significantly reduced in proliferating vs. nonproliferating medial cells. Our findings demonstrate that functional upregulation of IKCa1 is required for PDGF-BB-induced coronary SMC phenotypic modulation and migration and support a similar role for IKCa1 in coronary SMC during early coronary atherosclerosis.
Asunto(s)
Vasos Coronarios/citología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/fisiología , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/fisiología , Regulación hacia Arriba , Actinas/genética , Animales , Becaplermina , Biomarcadores , Técnicas de Cultivo de Célula , Diferenciación Celular , División Celular , Movimiento Celular , Células Cultivadas , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Modelos Biológicos , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/citología , Cadenas Pesadas de Miosina/genética , Fenotipo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas c-sis , Pirazoles/farmacología , ARN Mensajero/metabolismo , Porcinos , Porcinos Enanos , Túnica Media/citologíaRESUMEN
A hallmark of smooth muscle cell (SMC) phenotypic switching in atherosclerotic lesions is suppression of SMC differentiation marker gene expression. Yet little is known regarding the molecular mechanisms that control this process. Here we show that transcription of the SMC differentiation marker gene SM22alpha is reduced in atherosclerotic lesions and identify a cis regulatory element in the SM22alpha promoter required for this process. Transgenic mice carrying the SM22alpha promoter-beta-galactosidase (beta-gal) reporter transgene were crossed to apolipoprotein E (ApoE)-/- mice. Cells of the fibrous cap, intima, and underlying media showed complete loss of beta-gal activity in advanced atherosclerotic lesions. Of major significance, mutation of a G/C-rich cis element in the SM22alpha promoter prevented the decrease in SM22alpha promoter-beta-gal reporter transgene expression, including in cells that compose the fibrous cap of the lesion and in medial cells in proximity to the lesion. To begin to assess mechanisms whereby the G/C repressor element mediates suppression of SM22alpha in atherosclerosis, we tested the hypothesis that effects may be mediated by platelet-derived growth factor (PDGF)-BB-induced increases in the G/C binding transcription factor Sp1. Consistent with this hypothesis, results of studies in cultured SMCs showed that: (1) PDGF-BB increased expression of Sp1; (2) PDGF-BB and Sp1 profoundly suppressed SM22alpha promoter activity as well as smooth muscle myosin heavy chain promoter activity through mechanisms that were at least partially dependent on the G/C cis element; and (3) a short interfering RNA to Sp1 increased basal expression and attenuated PDGF-BB induced suppression of SM22alpha. Together, these results support a model whereby a G/C repressor element within the SM22alpha promoter mediates transcriptional repression of this gene within phenotypically modulated SMCs in experimental atherosclerosis and provide indirect evidence implicating PDGF-BB and Sp1 as possible mediators of these effects.
Asunto(s)
Arteriosclerosis/genética , Silenciador del Gen/fisiología , Proteínas de Microfilamentos/genética , Proteínas Musculares/genética , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Regiones Promotoras Genéticas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Aorta/citología , Apolipoproteínas E/genética , Arteriosclerosis/etiología , Arteriosclerosis/patología , Becaplermina , Diferenciación Celular/genética , Células Cultivadas/metabolismo , Células Cultivadas/patología , Cruzamientos Genéticos , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/genética , Genes Reporteros , Hipercolesterolemia/complicaciones , Hipercolesterolemia/genética , Operón Lac , Ratones , Ratones Endogámicos CBA , Ratones Transgénicos , Proteínas Musculares/biosíntesis , Miocitos del Músculo Liso/patología , Fenotipo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Factor de Crecimiento Derivado de Plaquetas/fisiología , Unión Proteica , Proteínas Proto-Oncogénicas c-sis , Ratas , Proteínas Recombinantes de Fusión/fisiología , Elemento de Respuesta al Suero , Factor de Transcripción Sp1/fisiologíaRESUMEN
Vascular smooth muscle cell (SMC) contraction is mediated in part by calcium influx through L-type voltage-gated Ca2+ channels (VGCC) and activation of the RhoA/Rho kinase (ROK) signaling cascade. We tested the hypothesis that Ca2+ influx through VGCCs regulates SMC differentiation marker expression and that these effects are dependent on RhoA/ROK signaling. Depolarization-induced activation of VGCCs resulted in a nifedipine-sensitive increase in endogenous smooth muscle myosin heavy chain (SMMHC) and SM alpha-actin expression and CArG-dependent promoter activity, as well as c-fos promoter activity. The ROK inhibitor, Y-27632, prevented depolarization-induced increase in SMMHC/SM alpha-actin but had no effect on c-fos expression. Conversely, the Ca2+/calmodulin-dependent kinase inhibitor, KN93, prevented depolarization-induced increases in c-fos expression with no effect on SMMHC/SM alpha-actin. Depolarization increased expression of myocardin, a coactivator of SRF that mediates CArG-dependent transcription of SMC marker gene promoters containing paired CArG cis regulatory elements (SMMHC/SM alpha-actin). Both nifedipine and Y-27632 prevented the depolarization-induced increase in myocardin expression. Moreover, short interfering RNA (siRNA) specific for myocardin attenuated depolarization-induced SMMHC/SM alpha-actin transcription. Chromatin immunoprecipitation (ChIP) assays revealed that depolarization increased SRF enrichment of the CArG regions in the SMMHC, SM alpha-actin, and c-fos promoters in intact chromatin. Whereas Y-27632 decreased basal and depolarization-induced SRF enrichment in the SMMHC/SM alpha-actin promoter regions, it had no effect of SRF enrichment of c-fos. Taken together, these results provide evidence for a novel mechanism whereby Ca2+ influx via VGCCs stimulates expression of SMC differentiation marker genes through mechanisms that are dependent on ROK, myocardin, and increased binding of SRF to CArG cis regulatory elements.
Asunto(s)
Canales de Calcio Tipo L/fisiología , Regulación del Desarrollo de la Expresión Génica , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Proteínas Serina-Treonina Quinasas/fisiología , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Actinas/fisiología , Animales , Aorta , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas/citología , Células Cultivadas/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes fos , Péptidos y Proteínas de Señalización Intracelular , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Cadenas Pesadas de Miosina/fisiología , Nifedipino/farmacología , Proteínas Nucleares/fisiología , Organoides/citología , Técnicas de Placa-Clamp , Cloruro de Potasio/farmacología , Transporte de Proteínas , ARN Interferente Pequeño/farmacología , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Elemento de Respuesta al Suero/genética , Factor de Respuesta Sérica/fisiología , Transactivadores/fisiología , Transfección , Quinasas Asociadas a rho , Proteína de Unión al GTP rhoA/fisiologíaRESUMEN
Substantial evidence exists supporting the role of chronic exercise in reducing the incidence and severity of coronary vascular disease. Physical inactivity is an independent risk factor for coronary heart disease suggesting that the cardioprotective effect of exercise is due, in part, to an intrinsic adaptation within the coronary vasculature. Surprisingly, a paucity of information exists regarding the intrinsic cellular changes within the coronary vasculature associated with exercise training and even less is known regarding the effect of physical activity on long-term phenotypic modulation of coronary smooth muscle (CSM). The purpose of this symposium is to provide a concise update on the current knowledge regarding CSM adaptation to exercise training and the potential for these adaptations to contribute to exercise-induced cardioprotection. The potential role of CSM in exercise-induced cardioprotection will be approached from two perspectives. First, endurance exercise training effects on the regulation of coronary vasomotor tone via changes in CSM calcium regulation will be reviewed, i.e. short-term functional adaptation. Secondly, we will discuss potential long-term consequences of this altered calcium regulation, i.e. exercise-induced phenotypic modulation of CSM. We propose that exercise training alters CSM intracellular calcium regulation to reduce Ca2+-dependent activation of the contractile apparatus and Ca2+-dependent gene transcription and increase activation of sarcolemmal potassium channels. The overall effect is to increase the gain of the vasomotor system and maintain a stable, contractile CSM phenotype.
Asunto(s)
Enfermedad Coronaria/prevención & control , Ejercicio Físico/fisiología , Músculo Liso Vascular/fisiopatología , Adaptación Fisiológica , Calcio/metabolismo , Canales de Calcio/metabolismo , Circulación Coronaria/fisiología , Enfermedad Coronaria/fisiopatología , Vasos Coronarios/fisiopatología , Endotelinas/metabolismo , Regulación de la Expresión Génica/fisiología , Humanos , Microcirculación/fisiopatología , Mitógenos/fisiología , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Fenotipo , Canales de Potasio/metabolismo , Vasoconstrictores/metabolismo , Sistema Vasomotor/fisiopatologíaRESUMEN
Endothelin-1 (ET-1) signaling mechanisms have been implicated in the pathogenesis of excess coronary artery disease in diabetic dyslipidemia. We hypothesized that in diabetic dyslipidemia ET-1-induced coronary smooth muscle calcium (Ca2+m) and tyrosine phosphorylation would be increased, and the lipid lowering agent, atorvastatin, would inhibit these increases. Male Yucatan miniature swine groups were treated for 20 weeks: normal low-fat fed control, high-fat/cholesterol fed (hyperlipidemic), hyperlipidemic made diabetic with alloxan (diabetic dyslipidemic), and diabetic dyslipidemic treated with atorvastatin (atorvastatin-treated). Blood glucose values were 5-fold greater in diabetic dyslipidemic and atorvastatin-treated versus control and hyperlipidemic. Total and low-density lipoprotein (LDL) plasma cholesterol in hyperlipidemic, diabetic dyslipidemic, and atorvastatin-treated were approximately 5-fold greater than control. Intravascular ultrasound detectable coronary disease and hypertriglyceridemia were only observed in diabetic dyslipidemic and were abolished by atorvastatin. In freshly isolated cells, the Ca2+m response to ET-1 in diabetic dyslipidemic was greater than in control, hyperlipidemic, and atorvastatin-treated groups. Selective ET-1 receptor antagonists showed in the control group that the ETB subtype inhibits ETA regulation of Ca2+m. There was almost a complete switch of receptor subtype regulation of Ca2+m from largely ETA in control to an increased inhibitory interaction between ETA and ETB in hyperlipidemic and diabetic dyslipidemic groups, such that neither ETA nor ETB antagonist alone could block the ET-1-induced Ca2+m response. The inhibitory interaction was attenuated in the atorvastatin-treated group. In single cells, basal and ET-1-induced tyrosine phosphorylation in diabetic dyslipidemic were more than 3- and 6-fold greater, respectively, than in control, hyperlipidemic, and atorvastatin-treated. Attenuation by atorvastatin of coronary disease and ET-1-induced Ca2+m and tyrosine phosphorylation signaling with no change in cholesterol provides strong evidence for direct actions of atorvastatin and/or triglycerides on the vascular wall.
Asunto(s)
Anticolesterolemiantes/uso terapéutico , Señalización del Calcio/fisiología , Enfermedad de la Arteria Coronaria/prevención & control , Complicaciones de la Diabetes , Ácidos Heptanoicos/uso terapéutico , Hiperlipidemias/complicaciones , Pirroles/uso terapéutico , Animales , Atorvastatina , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Enfermedad de la Arteria Coronaria/etiología , Dieta , Modelos Animales de Enfermedad , Endotelina-1/farmacología , Endotelinas/farmacología , Masculino , Fosforilación , Receptor de Endotelina A , Receptores de Endotelina/metabolismo , Porcinos , Tirosina/metabolismoRESUMEN
Physical inactivity is an independent risk factor for coronary heart disease, yet the mechanism(s) of exercise-related cardioprotection remains unknown. We tested the hypothesis that coronary smooth muscle after exercise training would have decreased mitogen-induced phenotypic modulation and enhanced regulation of nuclear Ca(2+). Yucatan swine were endurance exercise trained (EX) on a treadmill for 16-20 wk. EX reduced endothelin-1-induced DNA content by 40% compared with sedentary (SED) swine (P < 0.01). EX decreased single cell peak endothelin-1-induced cytosolic Ca(2+) responses compared with SED by 16% and peak nuclear Ca(2+) responses by 33% (P < 0.05), as determined by confocal microscopy. On the basis of these results, we hypothesized that sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and intracellular Ca(2+) stores in native smooth muscle are spatially localized to dissociate cytosolic Ca(2+) and nuclear Ca(2+). Subcellular localization of SERCA in living and fixed cells revealed a distribution of SERCA near the sarcolemma and on the nuclear envelope. These results show that EX enhances nuclear Ca(2+) regulation, possibly via SERCA, which may be one mechanism by which coronary smooth muscle cells from EX are less responsive to mitogen-induced phenotypic modulation.
Asunto(s)
Señalización del Calcio/fisiología , Núcleo Celular/metabolismo , Vasos Coronarios/metabolismo , Músculo Liso Vascular/metabolismo , Condicionamiento Físico Animal , Animales , Bromodesoxiuridina , Cafeína/farmacología , Calcio/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Núcleo Celular/ultraestructura , Separación Celular , Vasos Coronarios/citología , Vasos Coronarios/efectos de los fármacos , ADN/metabolismo , Endotelina-1/farmacología , Femenino , Colorantes Fluorescentes , Técnicas In Vitro , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/ultraestructura , Fenotipo , Esfuerzo Físico/fisiología , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Porcinos EnanosRESUMEN
Atorvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, alters bulk myoplasmic Ca2+ regulation and inhibits phenotypic modulation and proliferation of vascular smooth muscle in culture. Nuclear Ca2+ (Ca(n)) signaling is tightly coupled to transcriptional events and cell growth. Therefore, we hypothesized that in vivo treatment with atorvastatin would attenuate alterations in mitogen-induced Ca(n) signaling associated with coronary atherosclerosis. Three groups of male Yucatan pigs were treated for 20 weeks: controls, alloxan-induced diabetics fed an atherogenic diet and diabetics fed an atherogenic diet plus atorvastatin (80 mg/day). Right coronary artery single-cell cytosolic Ca2+ (Ca(c)) and Ca(n) responses to the mitogen endothelin-1 (5 x 10(-8) M) were measured by laser confocal microscopy using the calcium indicator Fluo-4. We observed a 39% increase in Ca(c) and a 52% increase in Ca(n) responses to endothelin-1 in cells from diabetic dyslipidemic arteries compared to control. These alterations were prevented in animals treated with atorvastatin. We show that during proliferation, the nucleus of a smooth muscle cell becomes rounded and loses the characteristic multilobular shape, clefts and invaginations. Consistent with this, a redistribution of Ca2+ stores from a transnuclear morphology in controls to a more perinuclear morphology occurred in cells from diabetic dyslipidemic arteries and was prevented by atorvastatin. In addition, the peak Ca(n) responses to endothelin-1 were inversely correlated (r = 0.712) with the extent of the transnuclear distribution of Ca2+ stores and directly correlated (r = 0.874) with the extent of atherosclerosis, as assessed in vivo by intravascular ultrasound. These findings indicate that chronic treatment with atorvastatin directly decreases mitogen-induced Ca(n) mobilization, which we suggest is related to the spatial localization of Ca(n) stores.
Asunto(s)
Anticolesterolemiantes/uso terapéutico , Señalización del Calcio , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Ácidos Heptanoicos/uso terapéutico , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Hipercolesterolemia/tratamiento farmacológico , Músculo Liso Vascular/efectos de los fármacos , Pirroles/uso terapéutico , Aloxano/efectos adversos , Animales , Anticolesterolemiantes/administración & dosificación , Anticolesterolemiantes/farmacología , Arterias/efectos de los fármacos , Arterias/metabolismo , Atorvastatina , Glucemia/análisis , Calcio/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Núcleo Celular/patología , Núcleo Celular/ultraestructura , Células Cultivadas , Colesterol/sangre , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/complicaciones , Enfermedad de la Arteria Coronaria/metabolismo , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/metabolismo , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/complicaciones , Endotelina-1/farmacología , Ácidos Heptanoicos/administración & dosificación , Ácidos Heptanoicos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Hipercolesterolemia/sangre , Hipercolesterolemia/complicaciones , Hipercolesterolemia/metabolismo , Masculino , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Pirroles/administración & dosificación , Pirroles/farmacología , PorcinosRESUMEN
The phenotypic dedifferentiation of vascular smooth muscle cells (SMCs) is an early event associated with cell culturing and vascular injury. The purpose of this study was to evaluate the SMC phenotype underlying the functional responsiveness of SMCs to nucleotides in organ culture. Porcine coronary arteries were either used fresh, cold stored (5 degrees C) 4 days, or organ cultured (37 degrees C) 4 days. Fura-2 digital imaging of single SMCs was used to measure the myoplasmic calcium (Ca(m)) response to 10 microM of the following nucleotide receptor agonists: UTP, UDP, ATP, ADP, and 2-MeSATP. In contrast to the nucleotides UDP, ATP, ADP, and 2-MeSATP, the Ca(m) response increased 10-fold and the number of cells that responded to UTP increased 5-fold in SMCs from organ culture compared to SMCs from fresh or cold-stored arteries. Simultaneous imaging of Ca(m), DNA content, and SR distribution in SMCs from organ culture indicated that the UTP-induced Ca(m) increase occurred exclusively in SMCs that had a dedifferentiated cell phenotype. Three-dimensional image reconstruction of the nucleus and sarcoplasmic reticulum (SR) revealed a novel transnuclear SR distribution that intertwined with the nucleus in fresh SMCs, while in SMCs from organ culture the SR was predominantly perinuclear and cytoplasmic. This study demonstrates that the functional up-regulation of UTP-sensitive receptors and the disappearance of the transnuclear SR distribution are novel features of dedifferentiated coronary SMCs.
Asunto(s)
Vasos Coronarios , Músculo Liso Vascular/metabolismo , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/fisiología , Retículo Sarcoplasmático/ultraestructura , Animales , Cafeína/farmacología , Calcio/metabolismo , Carbocianinas , Diferenciación Celular , ADN/análisis , Colorantes Fluorescentes , Regulación de la Expresión Génica/efectos de los fármacos , Indoles , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/ultraestructura , Nucleótidos/farmacología , Técnicas de Cultivo de Órganos , Cloruro de Potasio/farmacología , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2Y1 , Porcinos , Uridina Trifosfato/farmacologíaRESUMEN
Arterial injury models for coronary artery disease have demonstrated an enhanced expression and function of either the endothelin(A) or endothelin(B) (ET(A) or ET(B)) receptor subtype. We hypothesized that organ culture would enhance the physiological function of ET receptors in the porcine right coronary artery. Arteries were either cold stored (4 degrees C) or organ cultured (37 degrees C) for 4 days. After 4 days, the artery was either 1) sectioned into rings to measure the ET-1-induced isometric tension response (3 x 10(-10)-3 x 10(-7) M), or 2) enzymatically dispersed and the isolated smooth muscle cells imaged using fura-2 to measure the myoplasmic calcium (Ca(m)) response to 3 x 10(-8) M ET-1 ( approximately EC(50)). Isometric tension and Ca(m) to ET-1 were measured in the absence and presence of bosentan (nonselective ET(A) or ET(B) receptor antagonist), BQ788 (ET(B)-selective antagonist), and BQ123 (ET(A)-selective antagonist). Compared with cold storage, organ culture induced a 2-fold increase in tension development (3 x 10(-7) M ET-1) and Ca(m) (3 x 10(-8) M ET-1), which was inhibited with bosentan, thus confirming the enhanced responses to ET-1 were due to ET receptor activation. BQ123 also inhibited the enhanced contraction and Ca(m) responses to ET-1. In contrast, BQ788 failed to inhibit tension development and Ca(m) responses to ET-1 in organ culture and cold storage. Sarafotoxin 6C (ET(B) agonist) failed to elicit an increased Ca(m) response in organ culture compared with cold storage. Our results indicate the increased tension development and Ca(m) responses to ET-1 in organ culture are attributable to ET(A) receptors, and not ET(B) receptors.