RESUMEN
Increased cardiovascular morbidity and mortality in individuals with type 2 diabetes (T2DM) is a significant clinical problem. Despite advancements in achieving good glycaemic control, this patient population remains susceptible to macrovascular complications. We previously discovered that vascular smooth muscle cells (SMC) cultured from T2DM patients exhibit persistent phenotypic aberrancies distinct from those of individuals without a diagnosis of T2DM. Notably, persistently elevated expression levels of microRNA-145 co-exist with characteristics consistent with aging, DNA damage and senescence. We hypothesised that increased expression of microRNA-145 plays a functional role in DNA damage signalling and subsequent cellular senescence specifically in SMC cultured from the vasculature of T2DM patients. In this study, markers of DNA damage and senescence were unambiguously and permanently elevated in native T2DM versus non-diabetic (ND)-SMC. Exposure of ND cells to the DNA-damaging agent etoposide inflicted a senescent phenotype, increased expression of apical kinases of the DNA damage pathway and elevated expression levels of microRNA-145. Overexpression of microRNA-145 in ND-SMC revealed evidence of functional links between them; notably increased secretion of senescence-associated cytokines and chronic activation of stress-activated intracellular signalling pathways, particularly the mitogen-activated protein kinase, p38α. Exposure to conditioned media from microRNA-145 overexpressing cells resulted in chronic p38α signalling in naïve cells, evidencing a paracrine induction and reinforcement of cell senescence. We conclude that targeting of microRNA-145 may provide a route to novel interventions to eliminate DNA-damaged and senescent cells in the vasculature and to this end further detailed studies are warranted.
Asunto(s)
Senescencia Celular , Daño del ADN , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , MicroARNs/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Anciano , Efecto Espectador/efectos de los fármacos , Efecto Espectador/genética , Senescencia Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , MicroARNs/genética , Miocitos del Músculo Liso/efectos de los fármacos , Fenotipo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Previous studies of outcomes in people who inject drugs (PWID) with infective endocarditis (IE) have often been retrospective, have had small sample sizes, and the duration of follow-up has been short and limited to patients who were operated on. METHODS: PWID treated for IE between 1 January 2006 and 31 December 2016 were identified from a prospectively collected database. PWID hospitalized with other infections acted as a novel comparison group. Outcomes were all-cause mortality, cause of death, relapse, recurrence, and reoperation. RESULTS: There were 105 episodes of IE in 92 PWID and 112 episodes of other infections in 107 PWID in whom IE was suspected but rejected. Survival at 30 days for the IE group was 85%, and 30-day survival following surgery was 96%. The most common pathogens were Staphylococcus species (60%) and Streptococcus species (30%). The surgical intervention rate was 47%. Survival for the IE group at 1, 3, 5, and 10 years was 74%, 63%, 58%, and 44%, respectively. This was significantly lower compared with the comparator group of other infections in PWID (P = .0002). Mortality was higher in patients who required surgery compared with those who did not (hazard ratio, 1.8 [95% confidence interval, .95-3.3]). The commonest cause of death was infection (66%), usually a further episode of IE (55%). CONCLUSIONS: Although early survival was good, long-term life expectancy was low. This was attributable to ongoing infection risk, rather than other factors known to affect prognosis in PWID. Surgery conferred no long-term survival advantage. More efforts are needed to reduce reinfection risk following an episode of IE in PWID.While early survival for people who inject drugs (PWID) with infective endocarditis is good, long-term survival is poor due to ongoing infection risk. Surgery conferred no long-term survival advantage, so more efforts are needed to reduce reinfection risks for PWID.
Asunto(s)
Consumidores de Drogas , Endocarditis Bacteriana , Endocarditis , Abuso de Sustancias por Vía Intravenosa , Endocarditis/epidemiología , Endocarditis/cirugía , Humanos , Estudios Retrospectivos , Abuso de Sustancias por Vía Intravenosa/complicacionesRESUMEN
Cardiovascular disease is a leading cause of morbidity and mortality. Smooth muscle cells (SMC) comprising the vascular wall can switch phenotypes from contractile to synthetic, which can promote the development of aberrant remodelling and intimal hyperplasia (IH). MicroRNA-21 (miR-21) is a short, non-coding RNA that has been implicated in cardiovascular diseases including proliferative vascular disease and ischaemic heart disease. However, its involvement in the complex development of atherosclerosis has yet to be ascertained. Smooth muscle cells (SMC) were isolated from human saphenous veins (SV). miR-21 was over-expressed and the impact of this on morphology, proliferation, gene and protein expression related to synthetic SMC phenotypes monitored. Over-expression of miR-21 increased the spread cell area and proliferative capacity of SV-SMC and expression of MMP-1, whilst reducing RECK protein, indicating a switch to the synthetic phenotype. Furthermore, platelet-derived growth factor BB (PDGF-BB; a growth factor implicated in vasculoproliferative conditions) was able to induce miR-21 expression via the PI3K and ERK signalling pathways. This study has revealed a mechanism whereby PDGF-BB induces expression of miR-21 in SV-SMC, subsequently driving conversion to a synthetic SMC phenotype, propagating the development of IH. Thus, these signaling pathways may be attractive therapeutic targets to minimise progression of the disease. © 2018 IUBMB Life, 70(7):649-657, 2018.
Asunto(s)
MicroARNs/genética , Músculo Liso Vascular/citología , Vena Safena/citología , Aterosclerosis/genética , Becaplermina/farmacología , Células Cultivadas , Puente de Arteria Coronaria , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Regulación de la Expresión Génica , Humanos , Interleucina-1alfa/genética , Sistema de Señalización de MAP Quinasas , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , MicroARNs/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Fenotipo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Vena Safena/fisiologíaRESUMEN
Type 2 diabetes mellitus prevalence is growing globally, and the leading cause of mortality in these patients is cardiovascular disease. Epigenetic mechanisms such as microRNAs (miRs) and DNA methylation may contribute to complications of type 2 diabetes mellitus. We discovered an aberrant type 2 diabetes mellitus-smooth muscle cell phenotype driven by persistent up-regulation of miR-145. This study aimed to determine whether elevated expression was due to changes in methylation at the miR-145 promoter. Smooth muscle cells were cultured from saphenous veins of 22 non-diabetic and 22 type 2 diabetes mellitus donors. DNA was extracted, bisulphite treated and pyrosequencing used to interrogate methylation at 11 CpG sites within the miR-145 promoter. Inter-patient variation was high irrespective of type 2 diabetes mellitus. Differential methylation trends were apparent between non-diabetic and type 2 diabetes mellitus-smooth muscle cells at most sites but were not statistically significant. Methylation at CpGs -112 and -106 was consistently lower than all other sites explored in non-diabetic and type 2 diabetes mellitus-smooth muscle cells. Finally, miR-145 expression per se was not correlated with methylation levels observed at any site. The persistent up-regulation of miR-145 observed in type 2 diabetes mellitus-smooth muscle cells is not related to methylation at the miR-145 promoter. Crucially, miR-145 methylation is highly variable between patients, serving as a cautionary note for future studies of this region in primary human cell types.
Asunto(s)
Metilación de ADN , Diabetes Mellitus Tipo 2/genética , Epigénesis Genética , MicroARNs/genética , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Regiones Promotoras Genéticas , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Células Cultivadas , Islas de CpG , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , MicroARNs/metabolismo , Persona de Mediana Edad , Fenotipo , Vena Safena/metabolismo , Regulación hacia ArribaRESUMEN
We report a case of spontaneous hemothorax after aortic valve replacement in a 72-year-old female resulting from rupture of a pleural adhesion leading to hemodynamic instability. doi: 10.1111/jocs.12729 (J Card Surg 2016;31:211-213).
Asunto(s)
Implantación de Prótesis de Válvulas Cardíacas , Hemotórax/etiología , Hemotórax/cirugía , Enfermedades Pleurales/complicaciones , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/cirugía , Anciano , Estenosis de la Válvula Aórtica/fisiopatología , Estenosis de la Válvula Aórtica/cirugía , Femenino , Hemodinámica , Hemotórax/diagnóstico por imagen , Humanos , Complicaciones Posoperatorias/diagnóstico por imagen , Rotura Espontánea , Adherencias Tisulares/complicaciones , Resultado del TratamientoRESUMEN
Type 2 diabetes (T2DM) confers increased risk of endothelial dysfunction, coronary heart disease, and vulnerability to vein graft failure after bypass grafting, despite glycaemic control. This study explored the concept that endothelial cells (EC) cultured from T2DM and nondiabetic (ND) patients are phenotypically and functionally distinct. Cultured human saphenous vein- (SV-) EC were compared between T2DM and ND patients in parallel. Proliferation, migration, and in vitro angiogenesis assays were performed; western blotting was used to quantify phosphorylation of Akt, ERK, and eNOS. The ability of diabetic stimuli (hyperglycaemia, TNF-α, and palmitate) to modulate angiogenic potential of ND-EC was also explored. T2DM-EC displayed reduced migration (~30%) and angiogenesis (~40%) compared with ND-EC and a modest, nonsignificant trend to reduced proliferation. Significant inhibition of Akt and eNOS, but not ERK phosphorylation, was observed in T2DM cells. Hyperglycaemia did not modify ND-EC function, but TNF-α and palmitate significantly reduced angiogenic capacity (by 27% and 43%, resp.), effects mimicked by Akt inhibition. Aberrancies of EC function may help to explain the increased risk of SV graft failure in T2DM patients. This study highlights the importance of other potentially contributing factors in addition to hyperglycaemia that may inflict injury and long-term dysfunction to the homeostatic capacity of the endothelium.
Asunto(s)
Diabetes Mellitus Tipo 2/patología , Células Endoteliales/patología , Endotelio Vascular/patología , Vena Safena/patología , Anciano , Anciano de 80 o más Años , Movimiento Celular , Proliferación Celular , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Femenino , Rechazo de Injerto , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Persona de Mediana Edad , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fenotipo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Vena Safena/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Type 2 diabetes (T2DM) promotes premature atherosclerosis and inferior prognosis after arterial reconstruction. Vascular smooth muscle cells (SMC) respond to patho/physiological stimuli, switching between quiescent contractile and activated synthetic phenotypes under the control of microRNAs (miRs) that regulate multiple genes critical to SMC plasticity. The importance of miRs to SMC function specifically in T2DM is unknown. This study was performed to evaluate phenotype and function in SMC cultured from non-diabetic and T2DM patients, to explore any aberrancies and investigate underlying mechanisms. Saphenous vein SMC cultured from T2DM patients (T2DM-SMC) exhibited increased spread cell area, disorganised cytoskeleton and impaired proliferation relative to cells from non-diabetic patients (ND-SMC), accompanied by a persistent, selective up-regulation of miR-143 and miR-145. Transfection of premiR-143/145 into ND-SMC induced morphological and functional characteristics similar to native T2DM-SMC; modulating miR-143/145 targets Kruppel-like factor 4, alpha smooth muscle actin and myosin VI. Conversely, transfection of antimiR-143/145 into T2DM-SMC conferred characteristics of the ND phenotype. Exposure of ND-SMC to transforming growth factor beta (TGFß) induced a diabetes-like phenotype; elevated miR-143/145, increased cell area and reduced proliferation. Furthermore, these effects were dependent on miR-143/145. In conclusion, aberrant expression of miR-143/145 induces a distinct saphenous vein SMC phenotype that may contribute to vascular complications in patients with T2DM, and is potentially amenable to therapeutic manipulation.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , MicroARNs/genética , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Vena Safena/metabolismo , Actinas/genética , Actinas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Femenino , Regulación de la Expresión Génica , Glucosa/metabolismo , Glucosa/farmacología , Humanos , Hipoglucemiantes/uso terapéutico , Interleucina-1alfa/farmacología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , MicroARNs/metabolismo , Persona de Mediana Edad , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/patología , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Fenotipo , Cultivo Primario de Células , Vena Safena/efectos de los fármacos , Vena Safena/patología , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
INTRODUCTION: Type 2 diabetes mellitus (T2DM) promotes adverse myocardial remodeling and increased risk of heart failure; effects that can occur independently of hypertension or coronary artery disease. As cardiac fibroblasts (CFs) are key effectors of myocardial remodeling, we investigated whether inherent phenotypic differences exist in CF derived from T2DM donors compared with cells from nondiabetic (ND) donors. METHODS: Cell morphology (cell area), proliferation (cell counting over 7-day period), insulin signaling [phospho-Akt and phospho-extracellular signal-regulated kinase (ERK) Western blotting], and mRNA expression of key remodeling genes [real-time reverse transcription-polymerase chain reaction (RT-PCR)] were compared in CF cultured from atrial tissue from 14 ND and 12 T2DM donors undergoing elective coronary artery bypass surgery. RESULTS: The major finding was that Type I collagen (COL1A1) mRNA levels were significantly elevated by twofold in cells derived from T2DM donors compared with those from ND donors; changes reflected at the protein level. T2DM cells had similar proliferation rates but a greater variation in cell size and a trend towards increased cell area compared with ND cells. Insulin-induced Akt and ERK phosphorylation were similar in the two cohorts of cells. CONCLUSION: CF from T2DM individuals possess an inherent profibrotic phenotype that may help to explain the augmented cardiac fibrosis observed in diabetic patients. MINI SUMMARY: We investigated whether inherent phenotypic differences exist between CF cultured from donors with or without Type 2 diabetes. Cell morphology, proliferation, insulin signaling, and gene expression were compared between multiple cell populations. The major finding was that Type I collagen levels were elevated in fibroblasts from diabetic donors, which may help explain the augmented cardiac fibrosis observed with diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/patología , Miocardio/patología , Remodelación Atrial/genética , Remodelación Atrial/fisiología , Proliferación Celular , Tamaño de la Célula , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis , Humanos , Interleucina-1alfa/metabolismo , Sistema de Señalización de MAP Quinasas , Miocardio/citología , Miocardio/metabolismo , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor de Insulina/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND/PURPOSE: Coronary heart disease is the leading cause of morbidity in patients with type 2 diabetes mellitus (T2DM), frequently resulting in a requirement for coronary revascularization using the internal mammary artery (IMA) or saphenous vein (SV). Patency rates of SV grafts are inferior to IMA and further impaired by T2DM whilst IMA patencies appear similar in both populations. Smooth muscle cells (SMC) play a pivotal role in graft integration; we therefore examined the phenotype and proliferative function of IMA- and SV-SMC isolated from non-diabetic (ND) patients or those diagnosed with T2DM. METHODS/MATERIALS: SMC were cultured from fragments of SV or IMA. Morphology was analyzed under light microscopy (spread cell area measurements) and confocal microscopy (F-actin staining). Proliferation was analyzed by cell counting. Levels of RhoA mRNA, protein and activity were measured by real-time RT-PCR, western blotting and G-LISA respectively. RESULTS: IMA-SMC from T2DM and ND patients were indistinguishable in both morphology and function. By comparison, SV-SMC from T2DM patients exhibited significantly larger spread cell areas (1.5-fold increase, P<0.05), truncated F-actin fibers and reduced proliferation (33% reduction, P<0.05). Furthermore, lower expression and activity of RhoA were observed in SV-SMC of T2DM patients (37% reduction in expression, P<0.05 and 43% reduction in activity, P<0.01). CONCLUSIONS: IMA-SMC appear impervious to phenotypic modulation by T2DM. In contrast, SV-SMC from T2DM patients exhibit phenotypic and functional changes accompanied by reduced RhoA activity. These aberrancies may be epigenetic in nature, compromising SMC plasticity and SV graft adaptation in T2DM patients. SUMMARY: The internal mammary artery (IMA) is the conduit of choice for bypass grafting and is generally successful in all patients, including those with type 2 diabetes (T2DM). By contrast, saphenous vein (SV) is inferior to IMA and furthermore patients with T2DM suffer strikingly poorer outcomes than their non-diabetic (ND) counterparts. We discovered that SV-SMC from T2DM patients exhibit altered persistent morphology and function compared to ND SV-SMC, with differential expression and activity of the small GTPase RhoA, yet ND and T2DM IMA-SMC were indistinguishable. These data offer an explanation for the superior patency of IMA grafting independent of the presence of diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/complicaciones , Angiopatías Diabéticas/etiología , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Proteína de Unión al GTP rhoA/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Proliferación Celular , Forma de la Célula , Células Cultivadas , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/patología , Angiopatías Diabéticas/enzimología , Angiopatías Diabéticas/patología , Regulación hacia Abajo , Femenino , Humanos , Masculino , Arterias Mamarias/enzimología , Arterias Mamarias/patología , Persona de Mediana Edad , Músculo Liso Vascular/patología , Fenotipo , Vena Safena/enzimología , Vena Safena/patología , Factores de TiempoRESUMEN
Lipoprotein(a) (Lp(a)) is an independent risk factor for the development of cardiovascular disease. Vascular smooth muscle cell (SMC) motility and plasticity, functions that are influenced by environmental cues, are vital to adaptation and remodelling in vascular physiology and pathophysiology. Lp(a) is reportedly damaging to SMC function via unknown molecular mechanisms. Apolipoprotein(a) (apo(a)), a unique glycoprotein moiety of Lp(a), has been demonstrated as its active component. The aims of this study were to determine functional effects of recombinant apo(a) on human vascular SMC motility and explore the underlying mechanism(s). Exposure of SMC to apo(a) in migration assays induced a potent, concentration-dependent chemorepulsion that was RhoA and integrin αVß3-dependent, but transforming growth factor ß-independent. SMC manipulation through RhoA gene silencing, Rho kinase inhibition, statin pre-treatment, αVß3 neutralising antibody and tyrosine kinase inhibition all markedly inhibited apo(a)-mediated SMC migration. Our data reveal unique and potent activities of apo(a) that may negatively influence SMC remodelling in cardiovascular disease. Circulating levels of Lp(a) are resistant to lipid-lowering strategies and hence a greater understanding of the mechanisms underlying its functional effects on SMC may provide alternative therapeutic targets.
Asunto(s)
Apoproteína(a)/farmacología , Quimiotaxis/efectos de los fármacos , Integrina alfaVbeta3/metabolismo , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/enzimología , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Forma de la Célula/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
OBJECTIVES: Transcatheter aortic valve implantation (TAVI) is an established intervention for aortic stenosis. While it is known that the requirement for permanent pacing is higher following CoreValve (Medtronic, Inc., Minneapolis, MN, USA) TAVI than after surgical aortic valve replacement (SAVR), it remains uncertain whether pacing is required in the medium-to-long term. We hypothesized that complete heart block following TAVI is more likely to resolve than that following SAVR. METHODS: A retrospective analysis of prospectively collated data on 528 patients undergoing TAVI or SAVR from May 2008 to December 2010 at a cardiac tertiary referral hospital. Demographic data, timing and indication for pacing post-procedure plus follow-up were recorded. Paced patients were compared and analysed by existing initial indication for pacing. RESULTS: In total, 31 (5.9%) patients received a pacemaker, and there were limited differences between not paced and paced patient characteristics by procedure type. Of these, a greater proportion were implanted post-TAVI compared with SAVR (17 vs 3.2%, P<0.001). The mean time to pacemaker follow-up for TAVI and SAVR was 234 and 188 days, P=0.32, respectively. Fewer patients compared with pacing indication remained in complete heart block at latest follow-up for TAVI (76.5 vs 33.3%, P=0.02) and SAVR (92.9 vs 58.3%, P=0.04). Although, there was a trend towards a greater magnitude of TAVI patients regaining atrioventricular nodal conduction, this did not differ significantly from that seen in SAVR patients. CONCLUSIONS: In keeping with previous reports, this single-centre experience demonstrates that patients undergoing TAVI have higher rates of pacemaker implantation than those following SAVR. However, pacing indication in the short-to-medium term may not persist for all paced patients post-TAVI and -SAVR with the suggestion that a significant proportion recover atrioventricular conduction, which tended to be greatest in TAVI paced patients.
Asunto(s)
Estenosis de la Válvula Aórtica/terapia , Válvula Aórtica/cirugía , Cateterismo Cardíaco , Estimulación Cardíaca Artificial , Bloqueo Cardíaco/terapia , Implantación de Prótesis de Válvulas Cardíacas/métodos , Anciano , Anciano de 80 o más Años , Válvula Aórtica/fisiopatología , Estenosis de la Válvula Aórtica/diagnóstico , Estenosis de la Válvula Aórtica/fisiopatología , Estenosis de la Válvula Aórtica/cirugía , Cateterismo Cardíaco/efectos adversos , Distribución de Chi-Cuadrado , Inglaterra , Femenino , Bloqueo Cardíaco/diagnóstico , Bloqueo Cardíaco/etiología , Bloqueo Cardíaco/fisiopatología , Implantación de Prótesis de Válvulas Cardíacas/efectos adversos , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Marcapaso Artificial , Estudios Retrospectivos , Centros de Atención Terciaria , Factores de Tiempo , Resultado del TratamientoRESUMEN
Cardiac fibroblasts (CF) play a central role in the repair and remodeling of the heart following injury and are important regulators of inflammation and extracellular matrix (ECM) turnover. ECM-regulatory matricellular proteins are synthesized by several myocardial cell types including CF. We investigated the effects of pro-inflammatory cytokines on matricellular protein expression in cultured human CF. cDNA array analysis of matricellular proteins revealed that interleukin-1α (IL-1α, 10ng/ml, 6h) down-regulated connective tissue growth factor (CTGF/CCN2) mRNA by 80% and up-regulated tenascin-C (TNC) mRNA levels by 10-fold in human CF, without affecting expression of thrombospondins 1-3, osteonectin or osteopontin. Western blotting confirmed these changes at the protein level. In contrast, tumor necrosis factor α (TNFα) did not modulate CCN2 expression and had only a modest stimulatory effect on TNC levels. Signaling pathway inhibitor studies suggested an important role for the p38 MAPK pathway in suppressing CCN2 expression in response to IL-1α. In contrast, multiple signaling pathways (p38, JNK, PI3K/Akt and NFκB) contributed to IL-1α-induced TNC expression. In conclusion, IL-1α reduced CCN2 expression and increased TNC expression in human CF. These observations are of potential value for understanding how inflammation and ECM regulation are linked at the level of the CF.
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Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Interleucina-1alfa/fisiología , Miofibroblastos/metabolismo , Tenascina/metabolismo , Células Cultivadas , Factor de Crecimiento del Tejido Conjuntivo/genética , Expresión Génica , Regulación de la Expresión Génica , Humanos , Miocardio/patología , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Tenascina/genética , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
OBJECTIVE: Training in cardiothoracic surgery across Europe remains diverse and variable despite the ever closer integration of European countries at all levels and in all areas of life. Coupled with the increasing ease of movement across Europe, the need for uniform training programmes has arisen to allow for equivalent accreditation and certification. METHODS: We review the current training paradigms within the specialty across the world and in Europe and also explore the concept of competence. RESULTS: There are diverse training systems across the world and in Europe in particular. Competence-based training is the new model of training; however, competence remains difficult to define and measure. We propose a European Training Programme in Cardiothoracic Surgery that aims to standardize training across the European countries. CONCLUSIONS: The difficulties in unifying training across Europe are numerous, but it is time to implement a European Training System in Cardiothoracic Surgery that will deliver a competence-based curriculum.
Asunto(s)
Competencia Clínica/normas , Educación de Postgrado en Medicina/métodos , Cirugía Torácica/educación , Acreditación , Educación Basada en Competencias , Curriculum , Europa (Continente) , Humanos , Enseñanza/métodos , Cirugía Torácica/normasRESUMEN
BACKGROUND AND PURPOSE: The Sigma-1 receptor (Sig1R) impacts on calcium ion signalling and has a plethora of ligands. This study investigated Sig1R and its ligands in relation to endogenous calcium events of endothelial cells and transient receptor potential (TRP) channels. EXPERIMENTAL APPROACH: Intracellular calcium and patch clamp measurements were made from human saphenous vein endothelial cells and HEK 293 cells expressing exogenous human TRPC5, TRPM2 or TRPM3. Sig1R ligands were applied and short interfering RNA was used to deplete Sig1R. TRP channels tagged with fluorescent proteins were used for subcellular localization studies. KEY RESULTS: In endothelial cells, 10-100 µM of the Sig1R antagonist BD1063 inhibited sustained but not transient calcium responses evoked by histamine. The Sig1R agonist 4-IBP and related antagonist BD1047 were also inhibitory. The Sig1R agonist SKF10047 had no effect. Sustained calcium entry evoked by VEGF or hydrogen peroxide was also inhibited by BD1063, BD1047 or 4-IBP, but not SKF10047. 4-IBP, BD1047 and BD1063 inhibited TRPC5 or TRPM3, but not TRPM2. Inhibitory effects of BD1047 were rapid in onset and readily reversed on washout. SKF10047 inhibited TRPC5 but not TRPM3 or TRPM2. Depletion of Sig1R did not prevent the inhibitory actions of BD1063 or BD1047 and Sig1R did not co-localize with TRPC5 or TRPM3. CONCLUSIONS AND IMPLICATIONS: The data suggest that two types of Sig1R ligand (BD1047/BD1063 and 4-IBP) are inhibitors of receptor- or chemically activated calcium entry channels, acting relatively directly and independently of the Sig1R. Chemical foundations for TRP channel inhibitors are suggested.
Asunto(s)
Señalización del Calcio , Endotelio Vascular/metabolismo , Receptores sigma/metabolismo , Canales Catiónicos TRPC/metabolismo , Canales Catiónicos TRPM/metabolismo , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Células HEK293 , Histamina/metabolismo , Humanos , Cinética , Ligandos , Potenciales de la Membrana/efectos de los fármacos , Moduladores del Transporte de Membrana/farmacología , Técnicas de Placa-Clamp , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño , Receptores sigma/agonistas , Receptores sigma/antagonistas & inhibidores , Receptores sigma/genética , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Canales Catiónicos TRPC/antagonistas & inhibidores , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPM/antagonistas & inhibidores , Canales Catiónicos TRPM/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor Sigma-1RESUMEN
Pre-clinical studies suggest that the p38 MAPK signaling pathway plays a detrimental role in cardiac remodeling, but its role in cardiac fibroblast (CF) function is not well defined. We aimed to identify the p38 MAPK subtypes expressed by human CF, study their activation in response to proinflammatory cytokines, and determine which subtypes were important for expression of specific cytokines and matrix metalloproteinases (MMPs). Quantitative real-time RT-PCR analysis of mRNA levels in human CF cultured from multiple patients revealed a consistent pattern of expression with p38α being most abundant, followed by p38γ, then p38δ and only low expression of p38ß (3% of p38α mRNA levels). Immunoblotting confirmed marked protein expression of p38α, γ and δ, with little or no expression of p38ß. Phospho-ELISA and combined immunoprecipitation/immunoblotting techniques demonstrated that the proinflammatory cytokines IL-1α and TNFα selectively activated p38α and p38γ, but not p38δ. Selective p38α siRNA gene silencing reduced IL-1α-induced IL-6 and MMP-3 mRNA expression and protein secretion, without affecting IL-1α-induced IL-1ß and MMP-9 mRNA expression. In conclusion, human CF express the α, γ and δ subtypes of p38 MAPK, and the α subtype is important for IL-1α-induced IL-6 and MMP-3 expression in this cell type.
Asunto(s)
Fibroblastos/metabolismo , Interleucina-6/biosíntesis , Metaloproteinasa 3 de la Matriz/biosíntesis , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Miocardio/citología , Células Cultivadas , Fibroblastos/efectos de los fármacos , Humanos , Interleucina-1alfa/farmacología , Metaloproteinasa 3 de la Matriz/farmacología , Proteína Quinasa 14 Activada por Mitógenos/genéticaRESUMEN
BACKGROUND: Stromelysin (MMP-3) is an important regulator of vascular smooth muscle cell (SMC) invasion, a key contributor to saphenous vein (SV) bypass graft failure. The 5A allele of the common -1612 MMP-3 5A/6A promoter polymorphism reportedly confers increased promoter activity, MMP-3 tissue expression, and susceptibility to a number of vascular pathologies. The aim of this study was to determine whether the MMP-3 5A/6A polymorphism directly influences endogenous MMP-3 expression levels and, consequently, cell invasion, in SV-derived SMC cultured from patients with different genotypes. MATERIAL AND METHODS: Genotyping of 226 patients revealed -1612 MMP-3 5A/6A genotype frequencies of 20.8% 5A/5A, 52.7% 5A/6A, and 26.5% 6A/6A. Using a standardized, controlled protocol, we investigated cytokine- and growth factor-induced MMP-3 expression (real-time polymerase chain reaction [RT-PCR], ELISA) and SV-SMC invasion (Boyden chamber with Matrigel barrier) using cultured SV-SMC from patients with different MMP-3 genotypes. RESULTS: Despite observing a strong correlation between MMP-3 mRNA levels and MMP-3 protein secretion, no significant differences were apparent in MMP-3 expression levels or cell invasion between cells with different MMP-3 5A/6A genotypes. CONCLUSIONS: Our data suggest that the MMP-3 5A/6A promoter polymorphism in isolation does not influence levels of MMP-3 secretion or cellular invasion in human SV-SMC.
Asunto(s)
Movimiento Celular/genética , Genotipo , Metaloproteinasa 3 de la Matriz/genética , Miocitos del Músculo Liso/citología , Polimorfismo Genético/genética , Regiones Promotoras Genéticas/genética , Anciano , Alelos , Células Cultivadas , Estudios de Cohortes , Puente de Arteria Coronaria , Rechazo de Injerto , Humanos , Interleucina-1/farmacología , Masculino , Metaloproteinasa 3 de la Matriz/metabolismo , Persona de Mediana Edad , Miocitos del Músculo Liso/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Estudios Retrospectivos , Vena Safena/trasplante , Regulación hacia Arriba/efectos de los fármacos , Injerto VascularRESUMEN
Transient Receptor Potential Melastatin 3 (TRPM3) is a widely expressed calcium-permeable non-selective cation channel that is stimulated by high concentrations of nifedipine or by physiological steroids that include pregnenolone sulphate. Here we sought to identify steroids that inhibit TRPM3. Channel activity was studied using calcium-measurement and patch-clamp techniques. Progesterone (0.01-10µM) suppressed TRPM3 activity evoked by pregnenolone sulphate. Progesterone metabolites and 17ß-oestradiol were also inhibitory but the effects were relatively small. Dihydrotestosterone was an inhibitor at concentrations higher than 1µM. Corticosteroids lacked effect. Overlay assays indicated that pregnenolone sulphate, progesterone and dihydrotestosterone bound to TRPM3. In contrast to dihydrotestosterone, progesterone inhibited nifedipine-evoked TRPM3 activity or activity in the absence of an exogenous activator, suggesting a pregnenolone sulphate-independent mechanism of action. Dihydrotestosterone, like a non-steroid look-alike compound, acted as a competitive antagonist at the pregnenolone sulphate binding site. Progesterone inhibited endogenous TRPM3 in vascular smooth muscle cells. Relevance of TRPM3 or the progesterone effect to ovarian cells, which have been suggested to express TRPM3, was not identified. The data further define a chemical framework for competition with pregnenolone sulphate at TRPM3 and expand knowledge of steroid interactions with TRPM3, suggesting direct steroid binding and pregnenolone sulphate-independent inhibition by progesterone.
Asunto(s)
Pregnenolona/farmacología , Progesterona/farmacología , Canales Catiónicos TRPM/metabolismo , Corticoesteroides/farmacología , Animales , Señalización del Calcio/efectos de los fármacos , Bovinos , Dihidrotestosterona/farmacología , Estradiol/farmacología , Femenino , Células de la Granulosa/citología , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Células HEK293 , Humanos , Activación del Canal Iónico/efectos de los fármacos , Mifepristona/farmacología , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Nifedipino/farmacología , Pregnenolona/química , Progesterona/química , Unión Proteica/efectos de los fármacos , Receptores de Progesterona/metabolismo , Canales Catiónicos TRPM/agonistasRESUMEN
Neutrophil attraction and adhesion to endothelial cells occurs via well defined mechanisms, yet the ability of other cell types to express neutrophil-binding adhesion molecules is not well studied. Cardiac fibroblasts (CF) are a key cell type involved in repair of the infarcted myocardium, a scenario in which neutrophil recruitment is perceived to be detrimental. Here we determined the effects of proinflammatory cytokines on expression of neutrophil-binding adhesion molecules and neutrophil-attracting chemokines in CF cultured from multiple patients, and explored the underlying regulatory mechanisms. An adhesion molecule focused RT-PCR array identified 5 transcripts that were increased markedly in human CF treated with the proinflammatory cytokine interleukin-1 (IL-1, 10 ng/ml, 6 h); including intercellular cell adhesion molecule (ICAM-1) and E-selectin. Real-time RT-PCR verified the array data and immunoblotting confirmed cytokine-induced ICAM-1 and E-selectin protein expression. Treatment with a panel of pharmacological inhibitors identified the NF-κB pathway as mediating IL-1-induced ICAM-1 and E-selectin mRNA and protein expression. Additionally, E-selectin expression in human CF was markedly potentiated by the JNK inhibitor SP600125, but this was not observed when a more selective inhibitor ((L)-JNKI-1) was used, or in human vascular endothelial cells. IL-1 also stimulated CF to secrete the neutrophil chemoattractant CXCL8 via a p38- and NF-κB-dependent mechanism, as well as inducing CXCL1, CXCL2 and CXCL5 mRNA expression. In conclusion, human CF express neutrophil-binding adhesion molecules and neutrophil chemoattractants in response to proinflammatory cytokines suggesting that, in addition to EC, CF may play an important role in regulating neutrophil recruitment into the infarcted myocardium.