RESUMEN
AIMS: Peroxisome proliferator-activated receptor-alpha (PPARalpha) is a nuclear receptor regulating cardiac metabolism that also has anti-inflammatory properties. Since the activation of inflammatory signalling pathways is considered to be important in cardiac hypertrophy and fibrosis, it is anticipated that PPARalpha modulates cardiac remodelling. Accordingly, in this study the hypothesis was tested that the absence of PPARalpha aggravates the cardiac hypertrophic response to pressure overload. METHODS AND RESULTS: Male PPARalpha-/- and wild-type mice were subjected to transverse aortic constriction (TAC) for 28 days. TAC resulted in a more pronounced increase in ventricular weight and left ventricular (LV) wall thickness in PPARalpha-/- than in wild-type mice. Compared with sham-operated mice, TAC did not affect cardiac function in wild-type mice, but significantly depressed LV ejection fraction and LV contractility in PPARalpha-/- mice. Moreover, after TAC mRNA levels of hypertrophic (atrial natriuretic factor, alpha-skeletal actin), fibrotic (collagen 1, matrix metalloproteinase-2), and inflammatory (interleukin-6, tumour necrosis factor-alpha, cyclo-oxygenase-2) marker genes were higher in PPARalpha-/- than in wild-type mice. The mRNA levels of genes involved in fatty acid metabolism (long-chain acyl-CoA synthetase, hydroxyacyl-CoA dehydrogenase) were decreased in PPARalpha-/- mice, but were not further compromised by TAC. CONCLUSION: The present findings show that the absence of PPARalpha results in a more pronounced hypertrophic growth response and cardiac dysfunction that are associated with an enhanced expression of markers of inflammation and extracellular matrix remodelling. These findings indicate that PPARalpha exerts salutary effects during cardiac hypertrophy.
Asunto(s)
Hipertrofia Ventricular Izquierda/metabolismo , Miocardio/metabolismo , PPAR alfa/metabolismo , Remodelación Ventricular , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Actinas/metabolismo , Animales , Aorta Torácica/cirugía , Factor Natriurético Atrial/metabolismo , Coenzima A Ligasas/metabolismo , Colágeno Tipo I/metabolismo , Ciclooxigenasa 2/metabolismo , Modelos Animales de Enfermedad , Fibrosis , Hipertrofia Ventricular Izquierda/diagnóstico por imagen , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/fisiopatología , Interleucina-6/metabolismo , Ligadura , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Contracción Miocárdica , Miocardio/enzimología , Miocardio/patología , PPAR alfa/deficiencia , PPAR alfa/genética , ARN Mensajero/metabolismo , Volumen Sistólico , Factor de Necrosis Tumoral alfa/metabolismo , Ultrasonografía , Función Ventricular Izquierda , Remodelación Ventricular/genéticaRESUMEN
OBJECTIVE: The development of heart failure is invariably associated with extensive fibrosis. Treatment with Peroxisome Proliferator-Activated Receptor (PPAR) ligands has been shown to attenuate cardiac fibrosis, but the molecular mechanism underlying this protective effect has remained largely unknown. In this study the potential of each PPAR isoform (PPARalpha, delta, and gamma) to attenuate cardiac fibroblast proliferation, fibroblast (CF) to myofibroblast (CMF) transdifferentiation, and collagen synthesis was investigated. METHODS AND RESULTS: PPARdelta was found to be the most abundant isoform in both CF and CMF. Only the PPARdelta ligand GW501516, but not PPARalpha ligand Wy-14,643 or PPARgamma ligand rosiglitazone, significantly increased PPAR-dependent promoter activity and expression of the PPAR-responsive gene UCP2 ( approximately 5-fold). GW501516 reduced the proliferation rate of CF (-38%) and CMF (-26%), which was associated with increased expression of the cell cycle inhibitor gene G0/G1 switch gene 2 (G0S2). Exposure of CF to the PPARdelta ligand or adenoviral overexpression of PPARdelta significantly decreased alpha-smooth muscle actin (alpha-SMA) levels, indicating a reduced CF to CMF transition. The inhibition of transdifferentiation by PPARdelta correlated with an increase in PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome ten) expression. (3)H-Proline incorporation assays demonstrated a GW501516 induced decline in collagen synthesis (-36%) in CF. CONCLUSION: Cardiac fibroblast proliferation, fibroblast to myofibroblast differentiation and collagen synthesis were reduced after activation of PPARdelta, suggesting that PPARdelta represents an attractive molecular target for attenuating cardiac fibrosis.
Asunto(s)
Fibroblastos/metabolismo , Miocitos Cardíacos/metabolismo , PPAR delta/metabolismo , Animales , Animales Recién Nacidos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colágeno/biosíntesis , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibrosis , Humanos , Inmunohistoquímica , Ligandos , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , PPAR alfa/análisis , PPAR alfa/genética , PPAR alfa/metabolismo , PPAR delta/análisis , PPAR delta/genética , PPAR gamma/análisis , PPAR gamma/genética , PPAR gamma/metabolismo , Proliferadores de Peroxisomas/farmacología , Pirimidinas/farmacología , Ratas , Ratas Endogámicas Lew , Rosiglitazona , Tiazoles/farmacología , Tiazolidinedionas/farmacología , Transducción Genética/métodosRESUMEN
Expression of the tissue-specific gap junction protein connexin(Cx)40 is regulated by the interaction of ubiquitous and tissue-specific factors such as Sp1 and GATA4. Cardiac Cx40 expression is altered under pathological conditions such as atrial fibrillation. A human promoter polymorphism, a G-->A change at position -44 that has been associated with atrial-specific arrhythmias, is located between the TBE-NKE-Sp and GATA consensus transcription factor binding sites important for the regulation of the mouse Cx40 gene. The presence of the A-allele at position -44 in promoter-reporter constructs significantly reduces promoter activity. Using electrophoretic mobility shift assays and luciferase reporter assays in various cell types, we show that Sp1 and GATA4 are important regulators of human Cx40 gene transcription and that the -44 G-->A polymorphism negatively affects the promoter regulation by the transcription factors Sp1 and GATA4.
Asunto(s)
Conexinas/genética , Factor de Transcripción GATA4/fisiología , Regulación de la Expresión Génica/fisiología , Polimorfismo Genético , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/fisiología , Animales , Secuencia de Bases , Cartilla de ADN , Humanos , Ratones , Proteína alfa-5 de Unión ComunicanteRESUMEN
In the mammalian heart, the gap junction protein connexin45 (Cx45) has a characteristic spatiotemporal expression pattern and is involved in mediating the rapid spreading of the electrical impulse that precedes coordinated contraction. The aim of this study was to isolate and characterize the rat Cx45 gene and to investigate its expression pattern in various tissues and cell lines. The gene consists of four exons (termed E1a, E1b, E2, and E3), of which the complete protein-coding sequence as well as a small part of the 5' -untranslated region (5'-UTR) reside on E3. 5' -Rapid amplification of cDNA ends (5' -RACE) analysis demonstrated the existence of four transcripts, which all contained the same coding region (derived from E3) but differed in the composition of their 5'-UTR. Analysis of Cx45 RNA expression in various rat tissues and cultured cell lines revealed that the transcripts composed of either E1a, E2, and E3 (i.e., E1a/2/3) or of E1b, E2, and E3 (E1b/2/3) sequences are both ubiquitously expressed. Comparison of the rat Cx45 gene structure with its murine ortholog indicated both similarities and species-specific differences in Cx45 gene organization. These findings will allow for the mapping and characterization of the rat Cx45 gene regulatory regions.
Asunto(s)
Regiones no Traducidas 5'/genética , Conexinas/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Conexinas/genética , ADN Complementario/genética , Exones , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Ratas , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Worldwide, millions of patients are affected annually by healthcare-associated infection (HCAI), impacting up to 80,000 patients in European Hospitals on any given day. This represents not only public health risk, but also an economic burden. Complementing routine hand hygiene practices, cleaning and disinfection, antimicrobial coatings hold promise based, in essence, on the application of materials and chemicals with persistent bactericidal or -static properties onto surfaces or in textiles used in healthcare environments. The focus of considerable commercial investment and academic research energies, such antimicrobial coating-based approaches are widely believed to have potential in reduction of microbial numbers on surfaces in clinical settings. This belief exists despite definitive evidence as to their efficacy and is based somewhat on positive studies involving, for example, copper, silver or gold ions, titanium or organosilane, albeit under laboratory conditions. The literature describes successful delay and/or prevention of recontamination following conventional cleaning and disinfection by problematic microbes such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE), among others. However, there is a scarcity of studies assessing antimicrobial surfaces other than copper in the clinical environment, and a complete lack of published data regarding the successful implementation of these materials on clinically significant outcomes (including HCAI). Through its Cooperation in Science and Technology program (COST), the European Commission has funded a 4-year initiative to establish a network of stakeholders involved in development, regulation and use of novel anti-microbial coatings for prevention of HCAI. The network (AMiCI) comprises participants of more than 60 universities, research institutes and companies across 29 European countries and, to-date, represents the most comprehensive consortium targeting use of these emergent technologies in healthcare settings. More specifically, the network will prioritise coordinated research on the effects (both positive and negative) of antimicrobial coatings in healthcare sectors; know-how regarding availability and mechanisms of action of (nano)-coatings; possible adverse effects of such materials (e.g., potential emergence of microbial resistance or emission of toxic agents into the environment); standardised performance assessments for antimicrobial coatings; identification and dissemination of best practices by hospitals, other clinical facilities, regulators and manufacturers.
Asunto(s)
Infección Hospitalaria/prevención & control , Antiinfecciosos/química , Antiinfecciosos/uso terapéutico , Humanos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Enterococos Resistentes a la Vancomicina/efectos de los fármacosRESUMEN
Rapid spreading of the electrical impulse throughout the heart is essential for coordinated contraction and is mediated by electrical coupling of cardiomyocytes through gap junction channels composed of connexin40 (Cx40), connexin43 (Cx43) or connexin45 (Cx45). Each of these connexin proteins has a characteristic developmental and regional expression pattern in the heart. Alterations in this pattern may result in abnormal cellular coupling and consequently contribute to irregularities in cardiac rhythm. Indeed, alterations in cardiac connexin expression have been correlated with cardiovascular disease for which the molecular mechanisms, however, are largely unknown. Transcription factors and their target elements in the genome regulate the expression of genes during development and in response to extracellular signals in a cell type-specific and quantitative manner. Altered transcriptional regulation of gene expression is a characteristic feature in the development of cardiac disease which may influence the connexin expression pattern as well. In this review, we will summarize what is known on transcriptional regulation of the Cx40, Cx43 and Cx45 genes in general, with an emphasis on the heart.
Asunto(s)
Conexinas/genética , Regulación del Desarrollo de la Expresión Génica , Miocardio/metabolismo , Transcripción Genética , Animales , Conexina 43/genética , Uniones Comunicantes/metabolismo , Humanos , Ratones , Contracción Miocárdica/genética , Proteína alfa-5 de Unión ComunicanteRESUMEN
Altered transcriptional control is likely to contribute to the down-regulation of connexin 43 (Cx43) expression observed in many forms of heart disease. However, little is known about the factors regulating Cx43 transcription in the heart under (patho)physiological conditions. Therefore, a systematic study of rat Cx43 (rCx43) proximal promoter regulation in rat primary neonatal ventricular cardiomyocytes (NCM) and, for comparison, different cell types was initiated. Luciferase assays revealed that, in NCM, the proximal promoter is preserved in a conserved region extending from 148 nucleotides upstream towards 281 nucleotides downstream relative to the transcription initiation site (TIS). Further deletional analysis suggested the involvement of four putative Sp- and two AP1-binding sites. The binding of both Sp1 and Sp3 to the Sp-binding elements and AP1 to the AP1-binding elements was demonstrated by electrophoretic mobility shift assays (EMSA). Promoter-luciferase assays using the natural rCx43 proximal promoter and mutated derivatives in NCM, HL-1 and A7r5 cells revealed that all sites contribute to basal promoter activity. Trans-activation of the Cx43 proximal promoter with Sp1 and Sp3 in Drosophila Schneider line 2 (SL2) cells demonstrated that Sp1 and, to a lesser extent, Sp3 determine rCx43 promoter activation. Thus Sp1, Sp3 and AP1 determine basal Cx43 expression. In addition, we studied the effect of the cardiac transcription factor Nkx2.5 on Cx43 regulation. NCM were infected with adenovirus encoding either beta-galactosidase (control) or Nkx2.5. Cx43 protein and mRNA were significantly decreased after Nkx2.5 infection as shown by Western and Northern blot analyses. Promoter-reporter assays demonstrated that the rCx43 promoter was down-regulated approximately twofold upon Nkx2.5 overexpression. Therefore, in NCM, Nkx2.5 appears to play a role in the regulation of Cx43 expression.
Asunto(s)
Conexina 43/genética , Miocitos Cardíacos/metabolismo , Regiones Promotoras Genéticas/genética , Animales , Animales Recién Nacidos , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Expresión Génica , Regulación de la Expresión Génica , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Miocitos Cardíacos/citología , Unión Proteica , Ratas , Ratas Wistar , Elementos de Respuesta/genética , Alineación de Secuencia , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3 , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismo , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
BACKGROUND: Bacterial resistance against the classic antibiotics is posing an increasing challenge for the prevention and treatment of infections in health care environments. The introduction of antimicrobial nanocoatings with active ingredients provides alternative measures for active killing of microorganisms, through a preventive hygiene approach. PURPOSE: The purpose of this study was to investigate the antimicrobial activity of a panel of antimicrobial coatings available on the European market. METHODS: A comparative, biased selection of commercially available antimicrobial coatings was tested for antimicrobial efficiency. Suppliers were contacted to deliver their coatings on glass and/or stainless steel substrates. In total, 23 coatings from eleven suppliers were received, which were investigated for their effect on the growth of Escherichia coli, using the International Organization for Standardization (ISO) 22196 protocol. RESULTS: The majority of nanomaterial-containing coatings (n=13) contained nanosilver (n=12), while only one had photocatalytic TiO2 as the active particle. The differences in antimicrobial activity among all of the coatings, expressed as log reduction values, varied between 1.3 and 6.6, while the variation within the nanomaterial-based group was between 2.0 and 6.2. Although nanosilver coatings were on average very effective in reducing the number of viable bacteria after challenge, the strongest log reduction (6.6) was seen with a coating that has immobilized, covalently bound quaternary ammonium salt in its matrix. Besides these two compounds, coatings containing TiO2, poly(dimethylsiloxane), triclosan, or zinc pyrithione evoked 100% killing of E. coli. CONCLUSION: Our findings indicate that nanosilver dominates the nanoparticle-based coatings and performs adequately. However, considering the unknowns in relation to ecotoxicological emission and effects, it needs further consideration before widespread application into different environments.
RESUMEN
Accumulating evidence indicates an important role for inflammation in cardiac hypertrophy and failure. Peroxisome proliferator-activated receptors (PPARs) have been reported to attenuate inflammatory signaling pathways and, as such, may interfere with cardiac remodeling. Accordingly, the objectives of the present study were to explore the relationship between cardiomyocyte hypertrophy and inflammation and to investigate whether PPARalpha and PPARdelta are able to inhibit NF-kappaB activation and, consequently, the hypertrophic growth response of neonatal rat cardiomyocytes (NCM). mRNA levels of markers of both hypertrophy and inflammation were increased following treatment with the pro-hypertrophic factor phenylephrine (PE) or the chemokine TNF-alpha. Induction of inflammatory genes was found to be fast (within 2 h after stimulation) and transient, while induction of hypertrophic marker genes was more gradual (peaking at 24-48 h). Inflammatory and hypertrophic pathways appeared to converge on NF-kappaB as both PE and TNF-alpha increased NF-kappaB binding activity as measured by electrophoretic mobility shift assay. Following transient transfection, the p65-induced transcriptional activation of a NF-kappaB reporter construct was significantly blunted after co-transfection of PPARalpha or PPARdelta in the presence of their respective ligands. Finally, adenoviral overexpression of PPARalpha and PPARdelta markedly attenuated cell enlargement and the expression of hypertrophic marker genes in PE-stimulated NCM. The collective findings reveal a close relationship between hypertrophic and inflammatory signaling pathways in the cardiomyocyte. It was shown that both PPARalpha and PPARdelta are able to mitigate cardiomyocyte hypertrophy in vitro by inhibiting NF-kappaB activation.
Asunto(s)
Cardiomegalia/metabolismo , Regulación de la Expresión Génica , Inflamación , Miocitos Cardíacos/metabolismo , PPAR alfa/metabolismo , PPAR delta/metabolismo , Adenoviridae/metabolismo , Animales , Animales Recién Nacidos , Modelos Biológicos , FN-kappa B/metabolismo , Ratas , Ratas Endogámicas Lew , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Cardiac hypertrophic remodelling, initiated by signalling cascades in response to increased workload, injury or intrinsic disease, is initially adaptive. However, prolonged hypertrophy as a consequence of pathological stress leads to maladaptive changes that increase the risk for fatal ventricular arrhythmias. One of these changes is the remodelling of myocardial gap junctions, which provide for electrical coupling of adjacent cardiomyocytes. Myocardial gap junctions are composed of three connexin isotypes, connexin40 (Cx40), -43 (Cx43), and -45 (Cx45) and each display a characteristic developmental and regional expression pattern. Alterations in the distribution and expression of Cx43, the predominant isoform in the adult ventricles, has been the main focus of examination in humans, experimental animal models and cultured cardiomyocytes in response to hypertrophy. The molecular mechanisms and signalling pathways underlying these changes have been studied less thoroughly. In this review we summarize what is known about the remodelling of myocardial gap junctions during hypertrophy, the putative underlying mechanisms and functional consequences thereof.
Asunto(s)
Conexinas/metabolismo , Hipertrofia Ventricular Izquierda/metabolismo , Miocardio/metabolismo , Animales , Comunicación Celular , Insuficiencia Cardíaca/metabolismo , Humanos , Ratones , Ratones Transgénicos , Modelos Biológicos , Miocitos Cardíacos/metabolismo , Estrés MecánicoRESUMEN
The rat gap junction protein connexin40 (rCx40) has a characteristic developmental and regional expression pattern, for which the exact regulatory mechanisms are not known. To identify the molecular factors controlling Cx40 expression, its proximal promoter was characterized. The proximal rCx40 promoter is the most conserved noncoding region within the Cx40-gene known thus far and contains five potential binding sites for Sp-family transcription factors. The binding of both Sp1 and Sp3 to each of these DNA elements was demonstrated by EMSA. Luciferase assays of the natural rCx40 proximal promoter or mutated derivatives in Cx40-expressing (NCM, primary rat neonatal cardiomyocytes and A7r5, rat smooth muscle embryonic thoracic aorta cells) and -nonexpressing cells (N2A, mouse neuroblastoma cells) revealed that all sites are contributing to basal promoter activity. Trans-activation assays in Drosophila Schneider line 2 cells demonstrated that Sp1 and Sp3 activate the rCx40 proximal promoter in a dose-dependent and additive manner.
Asunto(s)
Conexinas/genética , Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Células Cultivadas , Ensayo de Cambio de Movilidad Electroforética , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Ratas , Ratas Wistar , Elementos de Respuesta , Alineación de Secuencia , Factor de Transcripción Sp3 , Proteína alfa-5 de Unión ComunicanteRESUMEN
The two major isoforms of smoothelin (A and B) contain a calponin homology (CH) domain, colocalize with alpha-smooth muscle actin (alpha-SMA) in stress fibers and are only expressed in contractile smooth muscle cells (SMCs). Based on these findings, we hypothesized that smoothelins are involved in smooth muscle cell contraction, presumably via interaction with actin. The interaction between smoothelins and three different actin isoforms (alpha- and gamma-smooth muscle and alpha-skeletal actin [alpha-SKA]) was investigated using several in vitro assays. Smoothelin-B co-immunoprecipitated with alpha-smooth muscle actin from pig aorta extracts. In rat embryonic fibroblasts, transfected smoothelins-A and -B associated with stress fibers. In vitro dot blot assays, in which immobilized actin was overlaid with radio-labeled smoothelin, showed binding of smoothelin-A to actin filaments, but not to monomeric G-actin. A truncated smoothelin, containing the calponin homology domain, associated with stress fibers when transfected and bound to actin filaments in overlay, but to a lesser extent. ELISA results showed that the binding of smoothelin to actin has no significant isoform specificity. Our results indicate an interaction between smoothelin and actin filaments. Moreover, the calponin homology domain and its surrounding sequences appear to be sufficient to accomplish this interaction, although the presence of other domains is apparently necessary to facilitate and/or strengthen the binding to actin.