RESUMEN
OBJECTIVES: Whether the severity of left main coronary artery (LMCA) disease reflects LMCA and overall coronary atherosclerotic burden is not known. We aimed to assess nonculprit LMCA disease characteristics and the relationship with atherosclerosis in the rest of the coronary arteries as well as patient outcomes. PATIENTS AND METHODS: In the PROSPECT study, 697 patients with acute coronary syndromes underwent three-vessel gray-scale and radiofrequency intravascular ultrasound after percutaneous coronary intervention. RESULTS: Overall, 552 patients with adequate LMCA imaging were compared according to LMCA plaque burden. The tertile with the highest plaque burden in the LMCA had the smallest LMCA minimum lumen area (17.4, 14.2, 10.5, lowest through highest tertiles, respectively, P<0.0001) and the greatest percent necrotic core volume (2.8, 5.6, 9.5%, lowest through highest tertiles, respectively, P<0.0001). Furthermore, the tertile with the highest plaque burden was also significantly associated with the highest overall non-LMCA percent atheroma volume within the major epicardial arteries (48.3, 49.2, 50.8%, lowest through highest tertiles, respectively, P<0.0001). After adjusting for patient background, the LMCA plaque burden was independently associated with non-LMCA percent atheroma volume (P=0.003). Of the three PROSPECT predictors of future nonculprit major adverse cardiac events (MACE) (minimum lumen area≤4 mm, plaque burden≥70%, and virtual histology thin-cap fibroatheroma), the tertile with the highest LMCA plaque burden had the highest number of patients with at least one of three PROSPECT predictors (P=0.03). In multivariable model, though total atheroma volume (per 1%) was an independent predictor of all MACE [hazard ratio (95% confidence interval)=1.06 (1.01-1.11), P=0.02] and strong trend for non-culprit-related MACE [hazard ratio (95% confidence interval)=1.06 (1.00-1.13), P=0.06], plaque burden at LMCA was not (all MACE, P=0.90, non-culprit-related MACE, P=0.85). CONCLUSION: The severity of atherosclerosis in LMCA predicted the overall atherosclerotic plaque burden as well as the presence of high-risk plaques in the three major epicardial coronary arteries.
Asunto(s)
Síndrome Coronario Agudo/diagnóstico por imagen , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Vasos Coronarios/diagnóstico por imagen , Placa Aterosclerótica , Ultrasonografía Intervencional , Síndrome Coronario Agudo/patología , Síndrome Coronario Agudo/cirugía , Anciano , Enfermedad de la Arteria Coronaria/patología , Enfermedad de la Arteria Coronaria/cirugía , Vasos Coronarios/patología , Vasos Coronarios/cirugía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Intervención Coronaria Percutánea/efectos adversos , Valor Predictivo de las Pruebas , Factores de Riesgo , Índice de Severidad de la Enfermedad , Factores de Tiempo , Resultado del TratamientoRESUMEN
OBJECTIVES: This study evaluated the relationship between platelet reactivity and plaque morphology using grayscale and radiofrequency intravascular ultrasound (IVUS) virtual histology (VH). BACKGROUND: Recent studies have reported that high on-treatment platelet reactivity (HPR) is associated with higher plaque volume and the presence of multivessel disease; however, the association between HPR and plaque morphology has not been evaluated. METHODS: The ADAPT-DES (Dual AntiPlatelet Therapy With Drug Eluting Stents) intravascular ultrasound substudy was a prospective, multicenter, observational study of 8,582 patients undergoing percutaneous coronary intervention with drug-eluting stents in whom platelet reactivity on clopidogrel was assessed routinely. The current analysis included 909 culprit lesions from 773 patients with pre-intervention grayscale IVUS and IVUS-VH. HPR was defined as platelet reactivity >208 P2Y12 reaction unit in point-of-care P2Y12 testing by the VerifyNow assay, measured during steady-state platelet inhibition in patients receiving an antiplatelet agent. RESULTS: HPR was associated with 3-vessel coronary artery disease (31.0% vs. 24.4%; p = 0.04). The incidence of fibroatheroma was higher in patients with HPR than those without HPR (77.1% vs. 68.9%; p = 0.01). The HPR group had larger percent plaque and media volume (plaque and media/external elastic membrane volume: 58.1% [95% confidence interval (CI): 57.1% to 59.0%] vs. 56.6% [95% CI: 55.8% to 57.5%]; p = 0.03) and plaque burden at the minimum lumen site (76.7% [95% CI: 75.7% to 77.8%] vs. 75.0% [95% CI: 74.0% to 76.0%]; p = 0.02). Despite a similar prevalence of attenuated plaque, patients with HPR had longer culprit lesion attenuated plaque length (8.0 [95% CI: 7.0 to 9.1] mm vs. 6.5 [95% CI: 5.9 to 7.1] mm; p = 0.01). On multivariate analysis, the presence of angiographic calcium (odds ratio [OR]: 1.85: 95% CI: 1.33 to 2.56; p = 0.0002) and HPR (OR: 1.45; 95% CI: 1.05 to 2.01; p = 0.02) were independent predictors for a culprit lesion fibroatheroma. CONCLUSIONS: HPR was associated with increased culprit lesion atherosclerotic burden and adverse plaque morphology among patients undergoing percutaneous coronary intervention. Platelet reactivity might be associated with not only blood clot formation, but also severity of atherosclerosis. (Assessment of Dual AntiPlatelet Therapy With Drug Eluting Stents [ADAPT-DES]; NCT00638794).
Asunto(s)
Enfermedad de la Arteria Coronaria/terapia , Vasos Coronarios/diagnóstico por imagen , Intervención Coronaria Percutánea , Inhibidores de Agregación Plaquetaria/uso terapéutico , Agregación Plaquetaria/efectos de los fármacos , Ticlopidina/análogos & derivados , Ultrasonografía Intervencional , Anciano , Distribución de Chi-Cuadrado , Clopidogrel , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Resistencia a Medicamentos , Stents Liberadores de Fármacos , Femenino , Fibrosis , Alemania , Humanos , Análisis de los Mínimos Cuadrados , Modelos Logísticos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Intervención Coronaria Percutánea/efectos adversos , Intervención Coronaria Percutánea/instrumentación , Placa Aterosclerótica , Inhibidores de Agregación Plaquetaria/efectos adversos , Pruebas de Función Plaquetaria , Valor Predictivo de las Pruebas , Estudios Prospectivos , Factores de Riesgo , Ticlopidina/efectos adversos , Ticlopidina/uso terapéutico , Factores de Tiempo , Resultado del Tratamiento , Estados UnidosRESUMEN
AIMS: The aim of this study was to investigate the reproducibility of intravascular optical coherence tomography (IVOCT) assessments, including a comparison to intravascular ultrasound (IVUS). Intra-observer and inter-observer variabilities of IVOCT have been previously described, whereas inter-institute reliability in multiple laboratories has never been systematically studied. METHODS AND RESULTS: In 2 independent laboratories with intravascular imaging expertise, 100 randomized matched data sets of IVOCT and IVUS images were analysed by 4 independent observers according to published consensus document definitions. Intra-observer, inter-observer, and inter-institute variabilities of IVOCT qualitative and quantitative measurements vs. IVUS measurements were assessed. Minor inter- and intra-observer variability of both imaging techniques was observed for detailed qualitative and geometric analysis, except for inter-observer mixed plaque identification on IVUS (κ = 0.70) and for inter-observer fibrous cap thickness measurement reproducibility on IVOCT (ICC = 0.48). The magnitude of inter-institute measurement differences for IVOCT was statistically significantly less than that for IVUS concerning lumen cross-sectional area (CSA), maximum and minimum lumen diameters, stent CSA, and maximum and minimum stent diameters (P < 0.001, P < 0.001, P < 0.001, P = 0.02, P < 0.001, and P = 0.01, respectively). Minor inter-institute measurement variabilities using both techniques were also found for plaque identification. CONCLUSION: In the measurement of lumen CSA, maximum and minimum lumen diameters, stent CSA, and maximum and minimum stent diameters by analysts from two different laboratories, reproducibility of IVOCT was more consistent than that of IVUS.
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Angioplastia Coronaria con Balón/métodos , Enfermedad Coronaria/diagnóstico por imagen , Enfermedad Coronaria/terapia , Tomografía de Coherencia Óptica/métodos , Ultrasonografía Intervencional/métodos , Angioplastia Coronaria con Balón/mortalidad , Cateterismo Cardíaco/métodos , Consenso , Enfermedad Coronaria/mortalidad , Procedimientos Quirúrgicos Electivos/métodos , Femenino , Humanos , Masculino , Variaciones Dependientes del Observador , Reproducibilidad de los Resultados , Stents , Tasa de Supervivencia , Resultado del Tratamiento , Grado de Desobstrucción Vascular/fisiologíaRESUMEN
BACKGROUND: Although smoking is a risk factor for coronary atherosclerosis, the age-related impact on lesion characteristics and plaque instability remains unclear. PATIENTS AND METHODS: In ADAPT-DES, 780 patients with 916 culprit lesions were evaluated by preprocedural grayscale and virtual histology-intravascular ultrasound. RESULTS: Current smokers (smoking within 1 month) more often presented with acute coronary syndrome (67 vs. 51 vs. 51%, P<0.05) compared with former smokers (no smoking for >1 month) or nonsmokers. In patients 65 or more years of age, current smokers (vs. nonsmokers) showed larger normalized volumes of plaque and media [8.6 (7.8-9.4) vs. 7.2 (6.8-7.7) mm/mm, P=0.016] and external elastic membrane [14.4 (13.2-15.5) vs. 12.8 (12.2-13.4) mm/mm, P=0.05]. At the minimal lumen area site, despite a greater plaque burden, the larger external elastic membrane area [14.4 (13.1-15.7) vs. 12.0 (11.3-12.7) mm, P=0.003] contributed toward preserving the minimal lumen area [2.6 (2.4-2.7) vs. 2.6 (2.5-2.7) mm, P=0.91] in current smokers (vs. nonsmokers) 65 or more years of age. Moreover, current smokers (vs. nonsmokers) 65 or more years of age showed a greater normalized necrotic core volume [1.19 (0.96-1.46) vs. 0.75 (0.66-0.85) mm/mm, P=0.0007], more thin-cap fibroatheromas (61 vs. 48%, P=0.04), and plaque ruptures (38 vs. 26%, P=0.051). Conversely, in patients younger than 65 years of age, there was no significant difference in culprit lesion morphology among current, former, and nonsmokers. CONCLUSION: In patients 65 or more years (not in patients<65 years), smoking increased culprit lesion plaque instability (greater plaque with more necrotic core, thin-cap fibroatheromas, positive remodeling, and plaque ruptures).
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Síndrome Coronario Agudo/diagnóstico por imagen , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Vasos Coronarios/diagnóstico por imagen , Placa Aterosclerótica , Fumar/efectos adversos , Ultrasonografía Intervencional , Síndrome Coronario Agudo/etiología , Síndrome Coronario Agudo/terapia , Factores de Edad , Anciano , Enfermedad de la Arteria Coronaria/etiología , Enfermedad de la Arteria Coronaria/terapia , Estudios Transversales , Femenino , Fibrosis , Humanos , Masculino , Persona de Mediana Edad , Necrosis , Valor Predictivo de las Pruebas , Medición de Riesgo , Factores de Riesgo , Rotura Espontánea , Cese del Hábito de Fumar , Prevención del Hábito de Fumar , Factores de Tiempo , Remodelación VascularRESUMEN
BACKGROUND: Obesity is a cardiovascular risk factor, but the obesity paradox in patients undergoing percutaneous coronary intervention is poorly understood. METHODS AND RESULTS: Assessment of Dual Antiplatelet Therapy With Drug-Eluting Stents (ADAPT-DES) was a prospective, multicenter study of patients undergoing drug-eluting stent implantation. Overall, 780 patients (916 culprit lesions) were evaluated by grayscale and virtual histology-intravascular ultrasound pre-percutaneous coronary intervention. Poststenting intravascular ultrasound was done in 780 patients (894 treated lesions). Patients were divided into body mass index (BMI) tertiles. The high-BMI group had more diabetes mellitus, hypertension, and hyperlipidemia and more frequent plaque ruptures compared with the low-BMI group. At the minimal lumen area site, the high-BMI group had a larger plaque area (11.7 [11.0-12.4] versus 9.8 [9.3-10.4] mm(2)) and a greater plaque burden (77.3% [76.1%-78.5%] versus 74.4% [73.1%-75.8%]) compared with the low-BMI group; however, a larger external elastic membrane area (14.6 [13.8-15.3] versus 12.7 [12.1-13.3] mm(2)) resulted in a similar minimal lumen area compared with the low-BMI group. Post stenting, the high-BMI group had a significantly larger stent area versus the lower-BMI group. At 1-year follow-up, the high-BMI group was associated with less clinically driven target lesion revascularization compared with the low-BMI group in both the overall and the propensity-matched cohorts. CONCLUSIONS: A high BMI was associated with a greater plaque burden; however, a larger external elastic membrane preserved lumen dimensions and was associated with a larger stent area during intravascular ultrasound-guided stent implantation. Thus, despite more comorbidities, greater plaque burden, and more plaque rupture, a high BMI was not associated with worse outcomes after drug-eluting stent implantation. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT00638794.
Asunto(s)
Implantación de Prótesis Vascular , Enfermedad de la Arteria Coronaria/epidemiología , Trombosis Coronaria/prevención & control , Vasos Coronarios/cirugía , Obesidad/epidemiología , Intervención Coronaria Percutánea , Inhibidores de Agregación Plaquetaria/administración & dosificación , Complicaciones Posoperatorias/prevención & control , Anciano , Índice de Masa Corporal , Comorbilidad , Enfermedad de la Arteria Coronaria/mortalidad , Enfermedad de la Arteria Coronaria/terapia , Trombosis Coronaria/etiología , Vasos Coronarios/patología , Stents Liberadores de Fármacos/estadística & datos numéricos , Femenino , Estudios de Seguimiento , Humanos , Imagenología Tridimensional , Masculino , Persona de Mediana Edad , Obesidad/mortalidad , Obesidad/terapia , Paclitaxel , Estudios Prospectivos , Análisis de Supervivencia , Resultado del Tratamiento , Ultrasonografía Intervencional/métodos , Estados UnidosRESUMEN
AIMS: Mechanical complications contribute to bare metal and first-generation drug-eluting stent (DES) failure. However, the importance of the mechanical complications of second-generation DES remains unclear. We report mechanical complications associated with everolimus-eluting stent (EES) failures. METHODS AND RESULTS: We retrospectively analysed 177 consecutive EES-treated lesions in 136 patients who underwent intravascular ultrasound (IVUS) at follow-up. Mechanical complications were identified in 17 patients (five stable angina, 10 unstable angina, two non-ST-elevation myocardial infarction [NSTEMI] without angiographic thrombus). Fifteen (88.2%) were treated with repeat revascularisation. By IVUS, there were 16 focal (94.1%) and one diffuse (5.9%) in-stent restenoses. Complete stent fracture with separation was seen in only one, partial stent fracture with separation was seen in three, and in 13 there was longitudinal deformation (n=2) or stent strut fracture (n=11) with overlapping of the proximal and distal stent fragments. In 13 EES with evidence of overlapping in the setting of either fracture or deformation, there was a 35.5±12.2% smaller stent area compared to the adjacent proximal and distal stent fragments, and >50% neointimal hyperplasia in 12 (92.3%). CONCLUSIONS: We found EES mechanical complications, often followed by longitudinal deformation or fracture leading to excessive neointimal hyperplasia, in-stent restenosis, and repeat revascularisation.
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Stents Liberadores de Fármacos/efectos adversos , Sirolimus/análogos & derivados , Ultrasonografía Intervencional/métodos , Everolimus , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Sirolimus/administración & dosificaciónRESUMEN
OBJECTIVES: Glagov proposed that remodeling delayed development of significant coronary artery stenoses until plaque occupied, on average, 40% of arterial area (40% plaque burden). The aim of the current study was to confirm the previously proposed concept of coronary remodeling as first described by Glagov who studied postmortem left main coronary arteries (LMCAs). METHODS: Using the in-vivo intravascular ultrasound data from the Providing Regional Observations to Study Predictors of Events in the Coronary Tree (PROSPECT) study, we evaluated 552 LMCAs in 552 patients. RESULTS: External elastic membrane cross-sectional areas (CSAs) increased in proportion to the increase in plaque and media CSAs (r=0.61, P<0.0001), especially when the plaque burden was 20% or lower (r=0.88, P<0.0001). For more advanced atherosclerosis (>40% plaque burden), there was an inverse relationship between lumen CSA and plaque burden (r=-0.57, P<0.0001), whereas this relationship was weak in the presence of less than 40% plaque burden. The frequency of virtual histology derived thin-cap fibroatheroma increased with increasing plaque burden. In contrast, the frequency of pathological intimal thickening decreased. CONCLUSION: The previously proposed remodeling concept of Glagov was validated in vivo in the PROSPECT study patients. In addition, the present study suggested that plaque phenotype worsened with increasing LMCA plaque growth.
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Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Vasos Coronarios/diagnóstico por imagen , Placa Aterosclerótica , Anciano , Enfermedad de la Arteria Coronaria/terapia , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Intervención Coronaria Percutánea , Fenotipo , Valor Predictivo de las Pruebas , Resultado del Tratamiento , Ultrasonografía IntervencionalRESUMEN
AIMS: This study aimed to use intravascular ultrasound (IVUS) data to reveal the mechanism of lesion progression in the native coronary circulation proximal to bypass grafts after coronary artery bypass grafting (CABG). METHODS AND RESULTS: We reviewed IVUS images in 86 patients with an angiographically significant left main coronary artery (LMCA) stenosis. Overall, 41 patients underwent CABG more than 6 months (mean 8.2 ± 6.1 years) previously and had at least one patent graft to the left coronary artery system. The number of patent grafts to the left coronary artery was 1.4 ± 0.7. Comparing patent graft vs. non-CABG groups, external elastic membrane and lumen areas and remodeling index at the minimum lumen area (MLA) site trended smaller with no difference in the plaque & media area. In addition, patients in the patent graft group had more LMCA calcium whether defined by cross-sectional (arc at the MLA site of 141 ± 109° vs. 88 ± 108°, P = 0.025) or longitudinal measurements (calcium length index, calculated as LMCA calcium length divided by total LMCA length, 0.69 ± 0.38 vs. 0.50 ± 0.42, P = 0.035). CONCLUSIONS: Negative remodeling may be the main mechanism of lesion progression proximal to a patent bypass graft, and more calcium was found in LMCA after CABG compared with non-CABG patients.
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Calcinosis/diagnóstico por imagen , Puente de Arteria Coronaria/métodos , Estenosis Coronaria/patología , Vasos Coronarios/patología , Anciano , Anciano de 80 o más Años , Circulación Coronaria , Estenosis Coronaria/diagnóstico por imagen , Estenosis Coronaria/cirugía , Vasos Coronarios/diagnóstico por imagen , Vasos Coronarios/cirugía , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Tiempo , UltrasonografíaRESUMEN
Vascular calcification is an active and regulated process that is similar to bone formation. While calcium channel blockers (CCBs) have been shown to improve outcomes in atherosclerotic vascular disease, it remains unknown whether CCBs have an effect on the process of vascular calcification. Here we investigated whether CCBs inhibit osteogenic differentiation and matrix mineralization of vascular smooth muscle cells induced by Msx2, a key factor of vascular calcification. Human aortic smooth muscle cells (HASMCs) were transduced with adenovirus expressing MSX2 and were treated with 3 distinct CCBs. Azelnidipine, a dihydropyridine subclass of CCBs, significantly decreased alkaline phosphatase (ALP) activity of Msx2-overexpressed HASMCs, whereas verapamil and diltiazem had no effect. Furthermore, azelnidipine, but not verapamil and diltiazem, significantly decreased matrix mineralization of Msx2-overexpressing HASMCs. Azelnidipine significantly attenuated the induction of ALP gene expression by Msx2, a key transcription factor in osteogenesis, while it did not reduce enzymatic activity of ALP. Furthermore, azelnidipine inhibited the ability of Msx2 to activate the ALP gene, but had no effect on Notch-induced Msx2 expression. Given that L-type calcium channels are equally blocked by these CCBs, our results suggest that azelnidipine inhibits the Msx2-dependent process of vascular calcification by mechanisms other than inhibition of calcium channel activity.
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Ácido Azetidinocarboxílico/análogos & derivados , Calcificación Fisiológica/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Dihidropiridinas/farmacología , Proteínas de Homeodominio/antagonistas & inhibidores , Músculo Liso Vascular/fisiología , Osteogénesis/efectos de los fármacos , Fosfatasa Alcalina/efectos de los fármacos , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Ácido Azetidinocarboxílico/farmacología , Diferenciación Celular , Células Cultivadas , Cartilla de ADN/química , Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Transfección , Calcificación Vascular/metabolismo , Calcificación Vascular/fisiopatologíaRESUMEN
AIM: Connective tissue growth factor (CTGF), a direct target gene of transforming growth factor-ß (TGF-ß) signaling, plays an important role in the development of atherosclerosis. We previously showed that Runx2, a key transcription factor in osteoblast differentiation, regulates osteogenic conversion and dedifferentiation of vascular smooth muscle cells (VSMCs). In this study, we investigated the hypothesis that Runx2 modulates CTGF gene expression via the regulation of TGF-ß signaling. METHODS AND RESULTS: Expression of the Runx2 gene was decreased, and CTGF mRNA levels were reciprocally increased by TGF-ß in a time-dependent manner in cultured human aortic smooth muscle cells (HASMCs) and C3H10T1/2 cells. Forced expression of Runx2 decreased and the reduction of Runx2 expression by small interfering RNA enhanced both basal and TGF-ß-stimulated CTGF gene expression in HASMCs. Site-directed mutation analysis of the CTGF promoter indicated that transcriptional repression by Runx2 was mediated by the Smad-binding element (SBE) under basal and TGF-ß-stimulated conditions. Data obtained from immunoblots of Runx2-, Smad3- or Smad4-transfected cells and chromatin immunoprecipitation analysis indicated that Runx2 interacts with Smad3 at the SBE. Immunohistochemistry revealed that the expression of Runx2 and CTGF was distinct and almost mutually exclusive in human atherosclerotic plaque. CONCLUSIONS: These results for the first time demonstrate that Runx2/Smad3 complex negatively regulates endogenous and TGF-ß-induced CTGF gene expression in VSMCs. Thus, the induction of Runx2 expression contributes to the phenotypic modulation of VSMCs, in which the TGF-ß/Smad pathway plays a major role.
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Factor de Crecimiento del Tejido Conjuntivo/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Regulación de la Expresión Génica , Músculo Liso Vascular/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Aorta/citología , Aorta/metabolismo , Secuencia de Bases , Western Blotting , Células COS , Células Cultivadas , Chlorocebus aethiops , Inmunoprecipitación de Cromatina , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Ensayo de Inmunoadsorción Enzimática , Humanos , Técnicas para Inmunoenzimas , Luciferasas/metabolismo , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Músculo Liso Vascular/citología , Fosforilación , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico , Transducción de Señal , Proteína smad3/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Notch is an ancient cell-signaling system that regulates the specification of cell fate. This study examined the role of Notch in the epithelial-mesenchymal transition (EMT) and myofibroblast differentiation of cultured RLE-6TN cells (i.e., rat alveolar epithelial cells). The activation of Notch, either by ectopic expression of the Notch intracellular domain or by the co-culture of RLE-6TN cells with L-Jagged1 cells, induces the expression of smooth muscle α-actin (SMA) and other mesenchymal marker genes (collagen I and vimentin), and reduces the expression of epithelial marker genes (E-cadherin, occludin, and zonula occludens-1). The pharmacologic inhibition of the endogenous Notch signal significantly inhibited the transforming growth factor-ß (TGF-ß)-induced expression of SMA. Cell migratory capacity was increased by Notch. Luciferase assays revealed that the CC(A/T)(6)GG (CArG) box and the TGF-ß control element (TCE) are required for Notch-induced SMA gene transcription. DNA microarray analysis revealed that members of the TGF-ß family as well as Jagged1 were induced in RLE-6TN cells by Notch. Western blot analysis showed that Notch induced the phosphorylation of Smad3, and the TGF-ß receptor type I/activin receptor-like kinase 5 (ALK5) kinase inhibitor SB431542 markedly reduced the Notch-induced expression of SMA. Enzyme-linked immunosorbent assays confirmed the production of TGF-ß1 from RLE-6TN cells by Notch. Immunohistochemistry of a bleomycin-induced model of pulmonary fibrosis and lung specimens from patients with idiopathic interstitial pneumonias showed that Notch was strongly expressed in myofibroblasts, identified as SMA-positive cells. These data indicate that Notch induces myofibroblast differentiation through a TGF-ß-Smad3 pathway that activates SMA gene transcription in a CArG-dependent and TCE-dependent manner in alveolar epithelial cells. Our data also imply that Notch induces the EMT phenotype, with increased migratory behavior in pulmonary fibrosis.
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Fibroblastos/metabolismo , Mioblastos/metabolismo , Alveolos Pulmonares/metabolismo , Receptores Notch/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Antígenos de Diferenciación/biosíntesis , Línea Celular , Movimiento Celular , Técnicas de Cocultivo , Transición Epitelial-Mesenquimal , Fibroblastos/patología , Regulación de la Expresión Génica , Masculino , Mioblastos/patología , Fosforilación , Alveolos Pulmonares/patología , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Ratas , Ratas Wistar , Elementos de Respuesta , Transducción de SeñalRESUMEN
AIM: Vascular calcification is prevalent in patients with diabetes and chronic kidney disease. Receptor for advanced glycation end products (RAGE) and its multiple ligands have been implicated in the pathogenesis of accelerated atherosclerosis; however, little is known about the effects of RAGE activation on vascular calcification. METHODS AND RESULTS: Cultured rat and human aortic smooth muscle cells (HASMC) were transduced with adenovirus expressing RAGE. Expression of myocardin and the SMC-marker genes was significantly repressed in these cells. RAGE activation inhibited myocardin-induced expression of the SMC genes in mouse embryonic mesenchymal C3H10T1/2 cells. Interestingly, RAGE activation induced alkaline phosphatase (ALP) expression, calcium deposition, and Msx2 expression, a crucial transcription factor for osteogenic differentiation, in HASMC. RAGE-induced osteogenic differentiation was significantly inhibited by endogenous secretory RAGE. RAGE-induced ALP and Msx2 expression was completely abrogated by DAPT, an inhibitor of the Notch signaling pathway. PD98059 (MEK inhibitor) effectively blunted RAGE-induced Notch1 and Msx2 gene expression. Simultaneous stimulation with bone morphogenetic protein 2 (BMP2) and RAGE signaling synergistically induced expressions of Msx2 and ALP in HASMC. Immunohistochemistry revealed that the human calcifying atherosclerotic plaque expressed RAGE, Notch components and Msx2. The ALP activity induced in RAGE-overexpressing HASMCs by human serum was positively correlated with the serum creatinine level, but not with phosphate and hemoglobin A1c levels. CONCLUSIONS: These results indicate that activation of RAGE not only inhibits myocardin-dependent SMC gene expression, but also induces osteogenic differentiation of vascular SMC through Notch/Msx2 induction. These results provide a novel insight into the role of RAGE axis in vascular calcification.
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Músculo Liso Vascular/citología , Osteogénesis/fisiología , Receptores Inmunológicos/metabolismo , Adenoviridae/genética , Fosfatasa Alcalina/metabolismo , Animales , Aterosclerosis , Proteína Morfogenética Ósea 2/metabolismo , Calcio/metabolismo , Hemoglobina Glucada/química , Humanos , Inmunohistoquímica , Ligandos , Ratones , Ratones Endogámicos C3H , Ratas , Receptor para Productos Finales de Glicación Avanzada , Receptores Notch/metabolismo , Transducción de Señal , Calcificación VascularRESUMEN
Vascular calcification is regulated in a process similar to bone formation. BMP2 (bone morphogenetic protein 2) is essential for osteoblastic differentiation of mesenchymal progenitor cells and thus has been implicated in the development of vascular calcification. Here we examined whether Notch signaling interacts with BMP2 signaling to regulate osteogenic differentiation and mineralization of vascular smooth muscle cells (SMCs). BMP2 alone scarcely induced the expression of alkaline phosphatase (ALP), an ectoenzyme crucially required for active biomineralization, in human aortic SMCs (HASMCs), despite its strong induction in osteoblast precursor MC3T3-E1 cells. Notably, overexpression of the Notch1 intracellular domain (N1-ICD) markedly enhanced BMP2-mediated induction of ALP activity and mineralization of HASMCs. In HASMCs, expression of Msx2 gene, a well documented BMP2 target gene in osteoblasts, was barely induced by BMP2 alone, and N1-ICD clearly enhanced the BMP2-driven Msx2 gene expression. Deletion and site-directed mutation analysis of Msx2 gene promoter revealed that the RBPJk-binding site was necessary for BMP2 responsiveness. Using the RBPJk-deficient cells and siRNA for RBPJk, we showed that RBPJk was required for BMP2 induction of Msx2 gene expression and ALP activity. Moreover, we showed that Smad1, a transcription factor downstream of BMP2 signaling, interacted with N1-ICD to form a complex within the Msx2 promoter. Immunohistochemistry of human calcifying atherosclerotic plaques revealed colocalized expression of Notch1, BMP2, and Msx2. These results indicate that the Notch intracellular domain·RBPJk complex enhances the BMP2-induced Msx2 gene expression by cooperating with Smad1 and suggest that Notch signaling makes vascular SMC responsive to BMP2 and promotes vascular calcification.
Asunto(s)
Proteína Morfogenética Ósea 2/metabolismo , Calcinosis/metabolismo , Diferenciación Celular , Proteínas de Homeodominio/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Osteoblastos/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal , Animales , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Proteína Morfogenética Ósea 2/genética , Calcinosis/genética , Calcinosis/patología , Línea Celular , Proteínas de Homeodominio/genética , Humanos , Ratones , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Osteoblastos/patología , Receptor Notch1/genéticaRESUMEN
BACKGROUND: Fibroblast growth factor 23 (FGF-23) is a novel bone-derived phosphate-regulating hormone, and serum FGF-23 levels are associated with mortality among hemodialysis (HD) patients. However, the pathophysiological role of FGF-23 in those patients remains unclear, so the association between serum FGF-23 levels and known cardiac biomarkers or echocardiographic measurements were investigated in long-term HD patients without cardiac symptoms. METHODS AND RESULTS: The 87 consecutive patients treated in a single HD center (51 males, 36 females; mean age 64 years, mean HD duration 5.8 years) were included in this study. Comprehensive echocardiography was performed after HD. Blood samples were obtained before HD. Serum FGF-23 levels in dialysis patients were 1,171±553pg/ml. In univariate analysis, serum phosphate (r=0.443, P<0.001) and calcium levels (r=0.256, P=0.04), left ventricular mass index (LVMI) (r=0.268, P=0.039) were significantly associated with FGF-23 levels. Neither the B-type natriuretic peptide (BNP) nor the cardiac troponin T level was correlated with FGF-23. In multivariate regression analysis, only LVMI (ß=0.287, P=0.031, confidence interval (CI) 0.390-8.040) and phosphate levels (ß=0.419, P=0.001, CI 57.12-207.7) and calcium levels (ß=0.277, P=0.025, CI 24.95-360.1) remained significantly correlated with FGF-23. CONCLUSIONS: Beside BNP, FGF-23 was identified as a factor that is significantly associated with LVMI. FGF-23 could be a novel biomarker of left ventricular overload, which is closely associated with the increased risk of death in HD patients.
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Factores de Crecimiento de Fibroblastos/sangre , Hipertrofia Ventricular Izquierda/sangre , Hipertrofia Ventricular Izquierda/terapia , Péptido Natriurético Encefálico/sangre , Diálisis Renal , Troponina T/sangre , Anciano , Biomarcadores/sangre , Femenino , Factor-23 de Crecimiento de Fibroblastos , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de RiesgoRESUMEN
Expression of bone-associated proteins and osteoblastic transcription factor Runx2 in arterial cells has been implicated in the development of vascular calcification. However, the signaling upstream of the Runx2-mediated activation of osteoblastic program in vascular smooth muscle cells (VSMC) is poorly understood. We examined the effects of fibroblast growth factor-2 (FGF-2), an important regulator of bone formation, on osteoblastic differentiation of VSMC. Stimulation of cultured rat aortic SMC (RASMC) with FGF-2 induced the expression of the osteoblastic markers osteopontin (OPN) and osteocalcin. Luciferase assays showed that FGF-2 induced osteocyte-specific element (OSE)-dependent transcription. Downregulation of Runx2 by siRNA repressed the basal and FGF-2-stimulated expression of the OPN gene in RASMC. FGF-2 produced hydrogen peroxide in RASMC, as evaluated by fluorescent probe. Induction of OPN expression by FGF-2 was inhibited not only by PD98059 (MEK1 inhibitor) and PP1 (c-Src inhibitor), but also by an antioxidant, N-acetyl cysteine. Nuclear extracts from FGF-2-treated RASMC exhibited increased DNA-binding of Runx2 to its target sequence. Immunohistochemistry of human coronary atherectomy specimens and calcified aortic tissues showed that expression of FGF receptor-1 and Runx2 was colocalized. In conclusion, these results suggest that FGF-2 plays a role in inducing osteoblastic differentiation of VSMC by activating Runx2 through mitogen-activated protein kinase (MAPK)-dependent- and oxidative stress-sensitive-signaling pathways.
Asunto(s)
Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Factor 2 de Crecimiento de Fibroblastos/fisiología , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Osteoblastos/metabolismo , Osteogénesis , Animales , Aterosclerosis/genética , Aterosclerosis/metabolismo , Proteína Tirosina Quinasa CSK , Diferenciación Celular , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Factor 2 de Crecimiento de Fibroblastos/farmacología , Expresión Génica , Técnicas de Silenciamiento del Gen , Marcadores Genéticos , Humanos , Peróxido de Hidrógeno/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Osteopontina/genética , Proteínas Tirosina Quinasas/metabolismo , Ratas , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Activación Transcripcional , Familia-src QuinasasRESUMEN
OBJECTIVE: Vascular calcification is closely correlated with cardiovascular morbidity and mortality. Here, we demonstrate the role of Notch signaling in osteogenic differentiation and mineralization of vascular smooth muscle cells (SMCs). METHODS AND RESULTS: The Msx2 gene, a key regulator of osteogenesis, was highly induced by coculture with Notch ligand-expressing cells or overexpression of Notch intracellular domains (NICDs) in human aortic SMCs (HASMCs). Furthermore, the Notch1 intracellular domain (N1-ICD) overexpression markedly upregulated alkaline phosphatase (ALP) activity and matrix mineralization of HASMCs. A knockdown experiment with a small interfering RNA confirmed that Msx2 mediated N1-ICD-induced osteogenic conversion of HASMCs. Interestingly, Msx2 induction by N1-ICD was independent of bone morphogenetic protein-2 (BMP-2), an osteogenic morphogen upstream of Msx2. The transcriptional activity of the Msx2 promoter was significantly enhanced by N1-ICD overexpression. The RBP-Jk binding element within the Msx2 promoter was critical to Notch-induced Msx2 gene expression. Correspondingly, N1-ICD overexpression did not induce the Msx2 expression in RBP-Jk-deficient fibroblasts. Immunohistochemistry of human carotid artery specimens revealed localization of Notch1, Jagged1 and Msx2 to fibrocalcific atherosclerotic plaques. CONCLUSIONS: These results imply a new mechanism for osteogenic differentiation of vascular SMCs in which Notch/RBP-Jk signaling directly induces Msx2 gene expression and suggest its crucial role in mediating vascular calcification.
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Diferenciación Celular/fisiología , Proteínas de Homeodominio/genética , Miocitos del Músculo Liso/citología , Activación Transcripcional , Aorta/citología , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/fisiología , Receptores Notch/fisiología , Transducción de SeñalRESUMEN
OBJECTIVE: TGFbeta and proliferation/phenotypic switching of smooth muscle cells (SMCs) play a pivotal role in pathogenesis of atherosclerotic and restenotic lesions after angioplasty. We have previously shown that the protein inhibitor of activated STAT (PIAS)1 activates expression of SMC differentiation marker genes including smooth muscle (SM) alpha-actin by interacting with serum response factor (SRF) and class I bHLH proteins. Here, we tested the hypothesis that TGFbeta activates SM alpha-actin through PIAS1. METHODS AND RESULTS: An siRNA specific for PIAS1 and ubc9, an E2-ligase for sumoylation, inhibited TGFbeta-induced expression of SM alpha-actin in cultured SMCs as determined by real-time RT-PCR. Overexpression of PIAS1 increased SM alpha-actin promoter activity in a TGFbeta control element (TCE)-dependent manner. Because the TCE within the SM alpha-actin promoter could mediate repression through interaction with KLF4, we tested whether PIAS1 regulates the function of KLF4 for SMC gene expression. PIAS1 interacted with KLF4 in mammalian two-hybrid and coimmunoprecipitation assays, and overexpression of PIAS1 inhibited KLF4-repression of SM alpha-actin promoter activity. Moreover, PIAS1 promoted degradation of KLF4 through sumoylation. CONCLUSIONS: These results provide evidence that PIAS1 promotes TGFbeta-induced activation of SM alpha-actin gene expression at least in part by promoting sumoylation and degradation of the TCE repressor protein, KLF4.
Asunto(s)
Actinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Factores de Transcripción de Tipo Kruppel/antagonistas & inhibidores , Proteínas Inhibidoras de STAT Activados/fisiología , Factor de Crecimiento Transformador beta/farmacología , Animales , Aorta/fisiología , Células COS/citología , Células COS/fisiología , Chlorocebus aethiops , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/fisiología , Ratones , Ratones Endogámicos BALB C , Músculo Liso Vascular/fisiología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN/genética , ARN/aislamiento & purificación , ARN Interferente Pequeño/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Phenotypic plasticity and the switching of vascular smooth muscle cells (SMCs) play a critical role in atherosclerosis. Although Runx2, a key osteogenic transcription factor, is expressed in atherosclerotic plaques, the molecular mechanisms by which Runx2 regulates SMC differentiation remain unclear. Here we demonstrated that Runx2 repressed SMC differentiation induced by myocardin, which acts as a coactivator for serum response factor (SRF). Myocardin-mediated induction of SMC gene expression was enhanced in mouse embryonic fibroblasts derived from Runx2 null mice compared to wild-type mice. Forced expression of Runx2 decreased the expression of SMC genes and promoted osteogenic gene expression, whereas the reduction of Runx2 expression by small interfering RNA enhanced SMC differentiation in human aortic SMCs. Runx2 interacted with SRF and interfered with the formation of the SRF/myocardin ternary complex. Thus, this study provides the first evidence that Runx2 inhibits SRF-dependent transcription, as a corepressor independent of its DNA binding. We propose that Runx2 plays a pivotal role in osteogenic conversion tightly coupled with repression of the SMC phenotype in atherosclerotic lesions.