RESUMEN
Hereditary pulmonary veno-occlusive disease (hPVOD) is a severe form of autosomal recessive pulmonary hypertension and is due to biallelic loss of function of the EIF2AK4 gene (alias GCN2) coding for GCN2. GCN2 is a stress kinase that belongs to the integrated stress response pathway (ISR). Three rat lines carrying biallelic Gcn2 mutation were generated and found phenotypically normal and did not spontaneously develop a PVOD-related disease. We submitted these rats to amino acid deprivation to document the molecular and cellular response of the lungs and to identify phenotypic changes that could be involved in PVOD pathophysiology. Gcn2-/- rat lungs were analyzed under basal conditions and 3 days after a single administration of PEG-asparaginase (ASNase). Lung mRNAs were analyzed by RNAseq and single-cell RNAseq (scRNA-seq), flow cytometry, tissue imaging, and Western blots. The ISR was not activated after ASNase treatment in Gcn2-/- rat lungs, and apoptosis was increased. Several proinflammatory and innate immunity genes were overexpressed, and inflammatory cells infiltration was also observed in the perivascular area. Under basal conditions, scRNA-seq analysis of Gcn2-/- rat lungs revealed increases in two T-cell populations, a LAG3+ T-cell population and a proliferative T-cell population. Following ASNase administration, we observed an increase in calprotectin expression involved in TLR pathway activation and neutrophil infiltration. In conclusion, under basal and asparagine and glutamine deprivation induced by asparaginase administration, Gcn2-/- rats display molecular and cellular signatures in the lungs that may indicate a role for Gcn2 in immune homeostasis and provide further clues to the mechanisms of hPVOD development.
Asunto(s)
Hipertensión Pulmonar , Enfermedad Veno-Oclusiva Pulmonar , Animales , Ratas , Pulmón/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Enfermedad Veno-Oclusiva Pulmonar/genética , ARN MensajeroRESUMEN
Background Platelet-derived growth factor is a major regulator of the vascular remodeling associated with pulmonary arterial hypertension. We previously showed that protein widely 1 (PW1+) vascular progenitor cells participate in early vessel neomuscularization during experimental pulmonary hypertension (PH) and we addressed the role of the platelet-derived growth factor receptor type α (PDGFRα) pathway in progenitor cell-dependent vascular remodeling and in PH development. Methods and Results Remodeled pulmonary arteries from patients with idiopathic pulmonary arterial hypertension showed an increased number of perivascular and vascular PW1+ cells expressing PDGFRα. PW1nLacZ reporter mice were used to follow the fate of pulmonary PW1+ progenitor cells in a model of chronic hypoxia-induced PH development. Under chronic hypoxia, PDGFRα inhibition prevented the increase in PW1+ progenitor cell proliferation and differentiation into vascular smooth muscle cells and reduced pulmonary vessel neomuscularization, but did not prevent an increased right ventricular systolic pressure or the development of right ventricular hypertrophy. Conversely, constitutive PDGFRα activation led to neomuscularization via PW1+ progenitor cell differentiation into new smooth muscle cells and to PH development in male mice without fibrosis. In vitro, PW1+ progenitor cell proliferation, but not differentiation, was dependent on PDGFRα activity. Conclusions These results demonstrate a major role of PDGFRα signaling in progenitor cell-dependent lung vessel neomuscularization and vascular remodeling contributing to PH development, including in idiopathic pulmonary arterial hypertension patients. Our findings suggest that PDGFRα blockers may offer a therapeutic add-on strategy to combine with current pulmonary arterial hypertension treatments to reduce vascular remodeling. Furthermore, our study highlights constitutive PDGFRα activation as a novel experimental PH model.
Asunto(s)
Hipertensión Pulmonar , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas , Animales , Proliferación Celular , Células Cultivadas , Humanos , Hipertensión Pulmonar/metabolismo , Hipoxia , Pulmón , Masculino , Ratones , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Remodelación VascularRESUMEN
BACKGROUND: To evaluate the association between donors' and recipients' serum levels of soluble ST2 (sST2) and recipients' outcome after heart transplantation (HT). METHODS: Blood samples were collected in 50 heart donors before organ procurement and in 50 recipients before HT (D0), a week after HT (D7) and at every first year's endomyocardial biopsy (EMB); sST2 levels were evaluated by ELISA. RESULTS: Donors who sustained a cardiac arrest, had significantly higher sST2 levels. Recipients on national high emergency waiting list had significantly higher preoperative sST2 levels compared to recipients who did not. Recipients with postoperative sepsis or continuous renal replacement therapy had significantly higher sST2 levels at D7. Recipients who needed a postoperative ECMO for allograft dysfunction had significantly higher sST2 levels in their corresponding donors. Recipients who died during the hospitalization after the transplantation had significantly higher sST2 levels at D7 compared to recipients who did not. No difference was observed in sST2 levels in recipients who had mild allograft rejection and recipient who did not. CONCLUSIONS: Higher sST2 levels in donors are associated to allograft dysfunction requiring ECMO in recipients; higher postoperative sST2 levels in recipients are associated with in-hospital mortality.
Asunto(s)
Trasplante de Corazón , Proteína 1 Similar al Receptor de Interleucina-1 , Biomarcadores , Humanos , Proteína 1 Similar al Receptor de Interleucina-1/sangre , Donantes de Tejidos , Trasplante HomólogoRESUMEN
Early adaptive cardiac hypertrophy (EACH) is initially a compensatory process to optimize pump function. We reported the emergence of Orai3 activity during EACH. This study aimed to characterize how inflammation regulates store-independent activation of Orai3-calcium influx and to evaluate the functional role of this influx. Isoproterenol infusion or abdominal aortic banding triggered EACH. TNFα or conditioned medium from cardiac CD11b/c cells activated either in vivo [isolated from rats displaying EACH], or in vitro [isolated from normal rats and activated with lipopolysaccharide], were added to adult cardiomyocytes before measuring calcium entry, cell hypertrophy and cell injury. Using intramyocardial injection of siRNA, Orai3 was in vivo knockdown during EACH to evaluate its protective activity in heart failure. Inflammatory CD11b/c cells trigger a store-independent calcium influx in hypertrophied cardiomyocytes, that is mimicked by TNFα. Pharmacological or molecular (siRNA) approaches demonstrate that this calcium influx, depends on TNFR2, is Orai3-driven, and elicits cardiomyocyte hypertrophy and resistance to oxidative stress. Neutralization of Orai3 inhibits protective GSK3ß phosphorylation, impairs EACH and accelerates heart failure. Orai3 exerts a pathophysiological protective impact in EACH promoting hypertrophy and resistance to oxidative stress. We highlight inflammation arising from CD11b/c cells as a potential trigger of TNFR2- and Orai3-dependent signaling pathways.
Asunto(s)
Canales de Calcio/metabolismo , Cardiomegalia/inmunología , Insuficiencia Cardíaca/inmunología , Miocitos Cardíacos/inmunología , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Animales , Antígeno CD11b/metabolismo , Antígeno CD11c/metabolismo , Calcio/metabolismo , Cardiomegalia/inducido químicamente , Cardiomegalia/patología , Cardiomegalia/fisiopatología , Medios de Cultivo Condicionados/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Técnicas de Silenciamiento del Gen , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Humanos , Isoproterenol/toxicidad , Masculino , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/inmunología , Fosforilación/inmunología , ARN Interferente Pequeño/metabolismo , Ratas , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We aimed to analyze the strain-by-strain expression of a large panel of antimicrobial activities counteracting the virulence mechanisms of bacterial vaginosis-associated Prevotella bivia CI-1 and Gardnerella vaginalis 594, pyelonephritis-associated Escherichia coli CFT073, and recurrent cystitis- and preterm labor-associated IH11128 E. coli by Lactobacillus gasseri and Lactobacillus crispatus clinical strains, and L. gasseri ATCC 9857 and KS 120.1, and L. crispatus CTV-05 strains isolated from the cervicovaginal microbiota of healthy women. All L. gasseri and L. crispatus strains exerted antimicrobial activity by secreted lactic acid, which killed the microbial pathogens by direct contact. Potent bactericidal activity was exerted by a very limited number of resident L. gasseri and L. crispatus strains showing the specific ability to a strain to produce and release antibiotic-like compounds. These compounds eradicated the microbial pathogens pre-associated with the surface of cervix epithelial cells, providing efficient protection of the cells against the deleterious effects triggered by toxin-producing G. vaginalis and uropathogenic E. coli. Furthermore, these compounds crossed the cell membrane to kill the pre-internalized microbial pathogens. In addition, all L. gasseri and L. crispatus cells exhibited another non-strain specific activity which inhibited the association of microbial pathogens with cervix epithelial cells with varying efficiency, partially protecting the cells against lysis and detachment triggered by toxin-producing G. vaginalis and uropathogenic E. coli. Our results provide evidence of strain-level specificity for certain antimicrobial properties among cervicovaginal L. gasseri and L. crispatus strains, indicating that the presence of a particular species in the vaginal microbiota is not sufficient to determine its benefit to the host. A full repertory of antimicrobial properties should be evaluated in choosing vaginal microbiota-associated Lactobacillus isolates for the development of live biotherapeutic strategies.
RESUMEN
BACKGROUND: Atrial fibrillation is associated with an atrial cardiomyopathy composed mainly of fibrosis and adipose tissue accumulation. We hypothesized that MRI, when used in an optimal ex vivo setting allowing high spatial resolution without motion artifacts, can help characterizing the complex 3D left atrial (LA) wall composition in human myocardial samples, as compared to histology. METHODS: This prospective case-control study was approved by the institutional review board. 3D MRI acquisitions including saturation-recovery T1 mapping and DIXON imaging was performed at 4.0 T on 9 human LA samples collected from patients who underwent cardiac surgery. Histological quantification of fibrosis and fat was obtained. MRI T1 maps were clustered based on a Gaussian Mixture Model allowing quantification of total, interstitial and fatty fibrosis components. Fat maps were computed from DIXON images and fat fractions were calculated. MRI measurements were performed on the same location as the histological analysis (plane) and on the entire sample volume (3D). RESULTS: High correlations and levels of agreement were observed between MRI and histology for total (r = 0.93), interstitial (r = 0.93) and fatty fibrosis (r = 0.98) and fat (r = 0.96). Native T1 correlated with the amount of fibrosis from MRI and histology. The 3D MRI total, interstitial and fatty fibrosis ranges were between 6% and 23%, 4% and 17.3%; and 1.4% and 19.7% respectively. CONCLUSION: High Field ex vivo MRI was able to quantify different LA myocardial components with high agreement in 2D with histology and moreover to provide 3D quantification of such components whereas in vivo application remains a challenge.
Asunto(s)
Tejido Adiposo/diagnóstico por imagen , Fibrosis/diagnóstico por imagen , Atrios Cardíacos/diagnóstico por imagen , Cardiopatías/diagnóstico por imagen , Imagen por Resonancia Magnética , Miocardio/patología , Tejido Adiposo/patología , Estudios de Casos y Controles , Diagnóstico Diferencial , Fibrosis/patología , Cardiopatías/patología , Cardiopatías/cirugía , Humanos , Interpretación de Imagen Asistida por Computador , Imagenología Tridimensional , Estudios ProspectivosRESUMEN
Purpose To determine whether left atrial (LA) strain quantification with cardiac magnetic resonance (MR) imaging feature tracking is associated with the severity of LA fibrofatty myocardial remodeling at histologic analysis. Materials and Methods This prospective case-control study was approved by the institutional review board. LA strain was evaluated with cardiac MR feature tracking between January 2014 and March 2015 in 13 consecutive patients (mean age, 61 years ± 19; nine male) with mitral regurgitation in the 24 hours before mitral valve surgery and 13 age- and sex-matched healthy control subjects. LA strain parameters were compared first between control subjects and patients and then according to atrial fibrillation and mitral regurgitation status. Associations between LA strain and histology of preoperative biopsies were reported by using receiver operating characteristic curve analysis and Spearman correlation. Results Peak longitudinal atrial strain (PLAS) was significantly lower in patients with mitral regurgitation than in healthy control subjects (P < .001). Increased LA remodeling was significantly related to altered LA strain, and the strongest association was found between PLAS and the degree of fibrofatty myocardial replacement at histologic analysis (r = -0.75, P = .017). LA end-diastolic volume was increased in patients with mitral regurgitation when compared with that in healthy volunteers (P < .001) because of volume overload; however, volume did not correlate with the histologic degree of LA fibrofatty replacement (r = -0.35, P = .330). Conclusion LA strain, especially PLAS, correlates strongly with the degree of fibrofatty replacement at histologic analysis. Such functional imaging biomarker in combination with LA volumetry could help to guide clinical decisions, since myocardial structural remodeling is a known morphologic substrate of LA dysfunction leading to atrial fibrillation with adverse outcome. © RSNA, 2017 Online supplemental material is available for this article.
Asunto(s)
Tejido Adiposo/diagnóstico por imagen , Remodelación Atrial , Atrios Cardíacos/diagnóstico por imagen , Imagen por Resonancia Cinemagnética/métodos , Tejido Adiposo/patología , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Fibrosis/diagnóstico por imagen , Fibrosis/patología , Atrios Cardíacos/patología , Humanos , Masculino , Persona de Mediana Edad , Insuficiencia de la Válvula Mitral/diagnóstico por imagen , Insuficiencia de la Válvula Mitral/patología , Estudios ProspectivosRESUMEN
BACKGROUND: Pw1 gene expression is a marker of adult stem cells in a wide range of tissues. PW1-expressing cells are detected in the heart but are not well characterized. OBJECTIVES: The authors characterized cardiac PW1-expressing cells and their cell fate potentials in normal hearts and during cardiac remodeling following myocardial infarction (MI). METHODS: A human cardiac sample was obtained from a patient presenting with reduced left ventricular (LV) function following a recent MI. The authors used the PW1nLacZ+/- reporter mouse to identify, track, isolate, and characterize PW1-expressing cells in the LV myocardium in normal and ischemic conditions 7 days after complete ligature of the left anterior descending coronary artery. RESULTS: In both human and mouse ischemic hearts, PW1 expression was found in cells that were mainly located in the infarct and border zones. Isolated cardiac resident PW1+ cells form colonies and have the potential to differentiate into multiple cardiac and mesenchymal lineages, with preferential differentiation into fibroblast-like cells but not into cardiomyocytes. Lineage-tracing experiments revealed that PW1+ cells differentiated into fibroblasts post-MI. Although the expression of c-Kit and PW1 showed little overlap in normal hearts, a marked increase in cells coexpressing both markers was observed in ischemic hearts (0.1 ± 0.0% in control vs. 5.7 ± 1.2% in MI; p < 0.001). In contrast to the small proportion of c-Kit+/PW1- cells that showed cardiogenic potential, c-Kit+/PW1+ cells were fibrogenic. CONCLUSIONS: This study demonstrated the existence of a novel population of resident adult cardiac stem cells expressing PW1+ and their involvement in fibrotic remodeling after MI.
Asunto(s)
Factores de Transcripción de Tipo Kruppel/genética , Células Madre Mesenquimatosas/metabolismo , Infarto del Miocardio/genética , Miocardio/metabolismo , ARN/genética , Función Ventricular Izquierda/fisiología , Remodelación Ventricular/genética , Animales , Diferenciación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Citometría de Flujo , Humanos , Factores de Transcripción de Tipo Kruppel/biosíntesis , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Miocardio/patología , Miocitos Cardíacos/metabolismoRESUMEN
We identified murine miR-322, orthologous to human miR-424, as a new regulator of insulin receptor, IGF-1 receptor and sirtuin 4 mRNA in vitro and in vivo in the heart and found that miR-322/424 is highly expressed in the heart of mice. C57Bl/6N mice fed 10weeks of high fat diet (HFD) presented signs of cardiomyopathy and a stable miR-322 cardiac level while cardiac function was slightly affected in 11week-old ob/ob which overexpressed miR-322. We thus hypothesized that mmu-miR-322 could be protective against cardiac consequences of hyperinsulinemia and hyperlipidemia. We overexpressed or knocked-down mmu-miR-322 using AAV9 and monitored cardiac function in wild-type C57Bl/6N mice fed a control diet (CD) or a HFD and in ob/ob mice. The fractional shortening progressively declined while the left ventricle systolic diameter increased in HFD mice infected with an AAVcontrol or with an AAVsponge (decreasing miR-322 bioavailability) but also in ob/ob mice infected with AAVsponge. Similar observations were also found in CD-fed mice infected with AAVsponge. On the contrary over-expressing miR-322 with AAVmiR-322 was efficient in protecting the heart from HFD effects in C57Bl/6N mice. This cardioprotection could be associated with the regulation of identified targets IGF1R, INSR and CD1, a decrease in insulin signaling pathway and an enrichment of genes involved in mitochondrial function and fatty acid oxidation as demonstrated by transcriptome analysis. Altogether, these results emphasize miR-322 as a new potential therapeutic target against cardiac consequences of metabolic syndrome, which represents an increasing burden in the western countries.
Asunto(s)
Cardiopatías/metabolismo , Insulina/metabolismo , Síndrome Metabólico/metabolismo , MicroARNs/biosíntesis , Transducción de Señal , Animales , Dependovirus , Grasas de la Dieta/efectos adversos , Grasas de la Dieta/farmacología , Vectores Genéticos , Cardiopatías/genética , Cardiopatías/patología , Cardiopatías/terapia , Humanos , Hiperinsulinismo/genética , Hiperinsulinismo/metabolismo , Hiperinsulinismo/patología , Hiperinsulinismo/terapia , Hiperlipidemias/genética , Hiperlipidemias/metabolismo , Hiperlipidemias/patología , Hiperlipidemias/terapia , Insulina/genética , Masculino , Síndrome Metabólico/genética , Síndrome Metabólico/patología , Síndrome Metabólico/terapia , Ratones , Ratones Obesos , MicroARNs/genética , Ratas , Ratas Wistar , Transducción GenéticaRESUMEN
UNLABELLED: The ATP-binding cassette transporter MRP4 (encoded by ABCC4) regulates membrane cyclic nucleotides concentrations in arterial cells including smooth muscle cells. MRP4/ABCC4 deficient mice display a reduction in smooth muscle cells proliferation and a prevention of pulmonary hypertension in response to hypoxia. We aimed to study gene transfer of a MRP4/ABCC4 silencing RNA via intratracheal delivery of aerosolized adeno-associated virus 1 (AAV1.shMRP4 or AAV1.control) in a monocrotaline-induced model of pulmonary hypertension in rats. Gene transfer was performed at the time of monocrotaline administration and the effect on the development of pulmonary vascular remodeling was assessed 35 days later. AAV1.shMRP4 dose-dependently reduced right ventricular systolic pressure and hypertrophy with a significant reduction with the higher doses (i.e., >10(11) DRP/animal) as compared to AAV1. CONTROL: The higher dose of AAV1.shMRP4 was also associated with a significant reduction in distal pulmonary arteries remodeling. AAV1.shMRP4 was finally associated with a reduction in the expression of ANF, a marker of cardiac hypertrophy. Collectively, these results support a therapeutic potential for downregulation of MRP4 for the treatment of pulmonary artery hypertension.
RESUMEN
AIMS: Stromal interaction molecule 1 (STIM1) has been shown to control a calcium (Ca(2+)) influx pathway that emerges during the hypertrophic remodelling of cardiomyocytes. Our aim was to determine the interaction of Orai1 and Orai3 with STIM1 and their role in the constitutive store-independent and the store-operated, STIM1-dependent, Ca(2+) influx in cardiomyocytes. METHODS AND RESULTS: We characterized the expression profile of Orai proteins and their interaction with STIM1 in both normal and hypertrophied adult rat ventricular cardiomyocytes. Orai1 and 3 protein levels were unaltered during the hypertrophic process and both proteins co-immunoprecipitated with STIM1. The level of STIM1 and Orai1 were significantly greater in the macromolecular complex precipitated by the Orai3 antibody in hypertrophied cardiomyocytes. We then used a non-viral method to deliver Cy3-tagged siRNAs in vivo to adult ventricular cardiomyocytes and silence Orai channel candidates. Cardiomyocytes were subsequently isolated then the voltage-independent, i.e. store-independent and store-operated Ca(2+) entries were measured on Fura-2 AM loaded Cy3-labelled and control isolated cardiomyocytes. The whole cell patch-clamp technique was used to measure Orai-mediated currents. Specific Orai1 and Orai3 knockdown established Orai3, but not Orai1, as the critical partner of STIM1 carrying these voltage-independent Ca(2+) entries in the adult hypertrophied cardiomyocytes. Orai3 also drove an arachidonic acid-activated inward current. CONCLUSION: Cardiac Orai3 is the essential partner of STIM1 and drives voltage-independent Ca(2+) entries in adult cardiomyocytes. Arachidonic acid-activated currents, which are supported by Orai3, are present in adult cardiomyocytes and increased during hypertrophy.
Asunto(s)
Canales de Calcio/metabolismo , Hipertrofia Ventricular Izquierda/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Ácido Araquidónico/farmacología , Agonistas de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio/genética , Señalización del Calcio , Células Cultivadas , Modelos Animales de Enfermedad , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/patología , Masculino , Glicoproteínas de Membrana/metabolismo , Potenciales de la Membrana , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Proteína ORAI1 , Unión Proteica , Interferencia de ARN , Ratas Wistar , Molécula de Interacción Estromal 1 , Factores de Tiempo , TransfecciónRESUMEN
BACKGROUND: In the senescent heart, the positive inotropic response to ß-adrenoceptor stimulation is reduced, partly by dysregulation of ß1- and ß3-adrenoceptors. The multidrug resistance protein 4 (MRP4) takes part in the control of intracellular cyclic adenosine monophosphate concentration by controlling its efflux but the role of MRP4 in the ß-adrenergic dysfunction of the senescent heart remains unknown. METHODS: The ß-adrenergic responses to isoproterenol were investigated in vivo (stress echocardiography) and in vitro (isolated cardiomyocyte by Ionoptix with sarcomere shortening and calcium transient) in young (3 months old) and senescent (24 months old) rats pretreated or not with MK571, a specific MRP4 inhibitor. MRP4 was quantified in left ventricular homogenates by Western blotting. Data are mean ± SD expressed as percent of baseline value. RESULTS: The positive inotropic effect of isoproterenol was reduced in senescent rats in vivo (left ventricular shortening fraction 120 ± 16% vs. 158 ± 20%, P < 0.001, n = 16 rats) and in vitro (sarcomere shortening 129 ± 37% vs. 148 ± 35%, P = 0.004, n = 41 or 43 cells) as compared to young rats. MRP4 expression increased 3.6-fold in senescent compared to young rat myocardium (P = 0.012, n = 8 rats per group). In senescent rats, inhibition of MRP4 by MK571 restored the positive inotropic effect of isoproterenol in vivo (143 ± 11%, n = 8 rats). In vitro in senescent cardiomyocytes pretreated with MK571, both sarcomere shortening (161 ± 45% vs. 129 ± 37%, P = 0.007, n = 41 cells per group) and calcium transient amplitude (132 ± 25% vs. 113 ± 27%, P = 0.007) increased significantly. CONCLUSION: MRP4 overexpression contributes to the reduction of the positive inotropic response to ß-adrenoceptor stimulation in the senescent heart.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Corazón/efectos de los fármacos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/fisiología , Envejecimiento/fisiología , Animales , Presión Arterial/efectos de los fármacos , Broncodilatadores/farmacología , Calcio/metabolismo , Ecocardiografía de Estrés , Corazón/crecimiento & desarrollo , Isoproterenol/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Propionatos/farmacología , Quinolinas/farmacología , RatasRESUMEN
AIMS: Recent studies have reported a relationship between the abundance of epicardial adipose tissue (EAT) and the risk of cardiovascular diseases including atrial fibrillation (AF). However, the underlying mechanisms are unknown. The aim of this study was to examine the effects of the secretome of human EAT on the histological properties of the myocardium. METHODS AND RESULTS: Samples of EAT and subcutaneous adipose (SAT), obtained from 39 patients undergoing coronary bypass surgery, were analysed and tested in an organo-culture model of rat atria to evaluate the fibrotic properties of human fat depots. The EAT secretome induced global fibrosis (interstitial and peripheral) of rat atria in organo-culture conditions. Activin A was highly expressed in EAT compared with SAT and promoted atrial fibrosis, an effect blocked using neutralizing antibody. In addition, Activin A levels were enhanced in patients with low left-ventricular function. In sections of human atrial and ventricular myocardium, adipose and myocardial tissues were in close contact, together with fibrosis. CONCLUSION: This study provides the first evidence that the secretome from EAT promotes myocardial fibrosis through the secretion of adipo-fibrokines such as Activin A.
Asunto(s)
Adipoquinas/metabolismo , Tejido Adiposo/fisiología , Miocardio/patología , Activinas/metabolismo , Activinas/fisiología , Adipoquinas/fisiología , Animales , Fibrilación Atrial/metabolismo , Fibrilación Atrial/patología , Remodelación Atrial/fisiología , Células Cultivadas , Femenino , Fibrosis/etiología , Fibrosis/patología , Atrios Cardíacos/patología , Humanos , Masculino , Metaloproteinasa 8 de la Matriz/metabolismo , Metaloproteinasa 8 de la Matriz/fisiología , Persona de Mediana Edad , Ratas , Grasa Subcutánea/fisiologíaRESUMEN
BACKGROUND: Phenotypic modulation or switching of vascular smooth muscle cells from a contractile/quiescent to a proliferative/synthetic phenotype plays a key role in vascular proliferative disorders such as atherosclerosis and restenosis. Although several calcium handling proteins that control differentiation of smooth muscle cells have been identified, the role of protein phosphatase inhibitor 1 (I-1) in the acquisition or maintenance of the contractile phenotype modulation remains unknown. METHODS AND RESULTS: In human coronary arteries, I-1 and sarco/endoplasmic reticulum Ca2+ -ATPase expression is specific to contractile vascular smooth muscle cells. In synthetic cultured human coronary artery smooth muscle cells, protein phosphatase inhibitor 1 (I-1 target) is highly expressed, leading to a decrease in phospholamban phosphorylation, sarco/endoplasmic reticulum Ca2+ -ATPase, and cAMP-responsive element binding activity. I-1 knockout mice lack phospholamban phosphorylation and exhibit vascular smooth muscle cell arrest in the synthetic state with excessive neointimal proliferation after carotid injury, as well as significant modifications of contractile properties and relaxant response to acetylcholine of femoral artery in vivo. Constitutively active I-1 gene transfer decreased neointimal formation in an angioplasty rat model by preventing vascular smooth muscle cell contractile to synthetic phenotype change. CONCLUSIONS: I-1 and sarco/endoplasmic reticulum Ca2+ -ATPase synergistically induce the vascular smooth muscle cell contractile phenotype. Gene transfer of constitutively active I-1 is a promising therapeutic strategy for preventing vascular proliferative disorders.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Músculo Liso Vascular/metabolismo , Proteína Fosfatasa 1/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Vasoconstricción/fisiología , Animales , Aorta Torácica/citología , Aorta Torácica/fisiología , Señalización del Calcio/fisiología , Vasos Coronarios/citología , Vasos Coronarios/fisiología , Arteria Femoral/citología , Arteria Femoral/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Arterias Mamarias/citología , Arterias Mamarias/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/citología , Fenotipo , Proteína Fosfatasa 1/genética , Proteínas/genética , Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Enfermedades Vasculares/genética , Enfermedades Vasculares/metabolismoRESUMEN
AIMS: Our aim was to identify new microRNAs (miRNAs) implicated in pathological vascular smooth muscle cells (VSMCs) proliferation and characterize their mechanism of action. METHODS AND RESULTS: MicroRNAs microarray and qRT-PCR results lead us to focus on miR-424 or its rat ortholog miR-322 (miR-424/322). In vitro mir-424/322 level was decreased shortly after the induction of proliferation and increased in a time-dependent manner later on. In vivo its expression increased in the rat carotid artery from Day 4 up to Day 30 after injury. miR-424/322 overexpression in vitro inhibited proliferation and migration without affecting apoptosis and prevented VSMC dedifferentiation. Furthermore, miR-424/322 overexpression resulted in decreased expression of its predicted targets: cyclin D1 and Ca(2+)-regulating proteins calumenin and stromal-interacting molecule 1 (STIM1). Using reporter luciferase assays, we confirmed that cyclin D1 and calumenin mRNAs were direct targets of miR-322, whereas miR-322 effect on STIM1 was indirect. Nevertheless, consistent with the decreased STIM1 level, the store-operated Ca(2+) entry was reduced. We hypothesized that miR-424/322 could be a negative regulator of proliferation overridden in pathological situations. Thus, we overexpressed miR-424/322 in injured rat carotid arteries using an adenovirus, and demonstrated a protective effect against restenosis. CONCLUSION: Our results demonstrate that miR-424/322 is up-regulated after vascular injury. This is likely an adaptive response to counteract proliferation, although this mechanism is overwhelmed in pathological situations such as injury-induced restenosis.
Asunto(s)
Traumatismos de las Arterias Carótidas/metabolismo , MicroARNs/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Neointima , Animales , Apoptosis , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/prevención & control , Arteria Carótida Externa/metabolismo , Arteria Carótida Externa/patología , Desdiferenciación Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , Ciclina D1/metabolismo , Modelos Animales de Enfermedad , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Fenotipo , Ratas , Ratas Wistar , Transducción de Señal , Molécula de Interacción Estromal 1 , Factores de Tiempo , Transfección , Regulación hacia ArribaRESUMEN
Plasticity-related gene-1 (PRG-1) protects neuronal cells from lysophosphatidic acid (LPA) effects. In vascular smooth muscle cells (VSMCs), LPA was shown to induce phenotypic modulation in vitro and vascular remodeling in vivo. Thus we explored the role of PRG-1 in modulating VSMC response to LPA. PCR, Western blot, and immunofluorescence experiments showed that PRG-1 is expressed in rat and human vascular media. PRG-1 expression was strongly inhibited in proliferating compared with quiescent VSMCs both in vitro and in vivo (medial vs. neointimal VSMCs), suggesting that PRG-1 expression is dependent on the cell phenotype. In vitro, adenovirus-mediated overexpression of PRG-1 specifically inhibited LPA-induced rat VSMC proliferation and migration but not platelet-derived growth factor-induced proliferation. This effect was abolished by mutation of a conserved histidine in the lipid phosphate phosphatase family that is essential for interaction with lipid phosphates. In vivo, balloon-induced neointimal formation in rat carotid was significantly decreased in vessels infected with PRG-1 adenovirus compared with ß-galactosidase adenovirus (-71%; P < 0.05). PRG-1 overexpression abolished the activation of the p42/p44 signaling pathway in LPA-stimulated rat VSMCs in culture and in balloon-injured rat carotids. Taken together, these findings provide the first evidence of a protective role of PRG-1 in the vascular media under pathophysiological conditions.
Asunto(s)
Proteínas de Unión a Calmodulina/metabolismo , Movimiento Celular/efectos de los fármacos , Lisofosfolípidos/farmacología , Músculo Liso Vascular/fisiología , Monoéster Fosfórico Hidrolasas/metabolismo , Adenoviridae , Animales , Proteínas de Unión a Calmodulina/genética , Movimiento Celular/fisiología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica/fisiología , Vectores Genéticos , Humanos , Masculino , Músculo Liso Vascular/efectos de los fármacos , Neointima/inducido químicamente , Monoéster Fosfórico Hidrolasas/genética , Ratas , Ratas WistarRESUMEN
Cardiac gene transfer is a powerful molecular tool to improve our understanding of the role of new proteins and mutants in cardiac pathophysiology. There is a need for a simple efficient myocardial gene delivery technique in order to study the physiological role of proteins in their native environment. Here we tested a new method of myocardial nonviral gene delivery, by using the combination of ultrasound energy (USE), liposomes and high pressure injections to the rat heart. Wistar rats were subjected to intra-myocardial injections of liposomes-DNA or siRNA mix. The heart was exposed after an inter-costal incision, and then injections were conducted between two sets of USE heart exposure. Ultrasound application resulted in much higher transfection efficiency (2% of left ventricle) than the liposomes-DNA alone (0.12% of left ventricle) as shown by the beta-galactosidase staining. The ultrasonic based liposomes-DNA delivery resulted in low inflammatory response, as well as in low cardiac fibrosis as shown by total collagen staining. Quantitative real time polymerase chain reaction (PCR) showed that the ultrasonic delivery resulted in cardiac specific transduction. Moreover, 23,906±2197 and 71,883±4065 calcium tolerant transfected cardiac myocytes were isolated following the delivery of a GFP plasmid or tagged siRNA, respectively. This was sufficient to perform single cell physiological measurements and biochemical experiments on homogenates. We developed an interesting safe method for local gene transfer in the heart using ultrasound and liposomes gene delivery. This method is particularly useful to study the effect of gene transfer on cardiac myocytes maintained in their normal environment in animal models.
Asunto(s)
Técnicas de Transferencia de Gen , Miocardio/metabolismo , Ultrasonido/métodos , Animales , Técnicas de Transferencia de Gen/instrumentación , Liposomas , Hígado/metabolismo , Pulmón/metabolismo , Ratones , Contracción Miocárdica , Miocardio/citología , Miocardio/patología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Plásmidos/genética , Ratas , Bazo/metabolismo , Transfección/métodos , Ultrasonido/instrumentación , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
In blood vessels, tone is maintained by agonist-induced cytosolic Ca(2+) oscillations of quiescent/contractile vascular smooth muscle cells (VSMCs). However, in synthetic/proliferative VSMCs, Gq/phosphoinositide receptor-coupled agonists trigger a steady-state increase in cytosolic Ca(2+) followed by a Store Operated Calcium Entry (SOCE) which translates into activation of the proliferation-associated transcription factor NFAT. Here, we report that in human coronary artery smooth muscle cells (hCASMCs), the sarco/endoplasmic reticulum calcium ATPase type 2a (SERCA2a) expressed in the contractile form of the hCASMCs, controls the nature of the agonist-induced Ca(2+) transient and the resulting down-stream signaling pathway. Indeed, restoring SERCA2a expression by gene transfer in synthetic hCASMCs 1) increased Ca(2+) storage capacity; 2) modified agonist-induced IP(3)R Ca(2+) release from steady-state to oscillatory mode (the frequency of agonist-induced IP(3)R Ca(2+) signal was 11.66 ± 1.40/100 s in SERCA2a-expressing cells (n=39) vs 1.37 ± 0.20/100 s in control cells (n=45), p<0.01); 3) suppressed SOCE by preventing interactions between SR calcium sensor STIM1 and pore forming unit ORAI1; 4) inhibited calcium regulated transcription factor NFAT and its down-stream physiological function such as proliferation and migration. This study provides evidence for the first time that oscillatory and steady-state patterns of Ca(2+) transients have different effects on calcium-dependent physiological functions in smooth muscle cells.
Asunto(s)
Señalización del Calcio/fisiología , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Factores de Transcripción NFATC/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Western Blotting , Calcio/metabolismo , Señalización del Calcio/genética , Movimiento Celular/genética , Movimiento Celular/fisiología , Proliferación Celular , Células Cultivadas , Vasos Coronarios/citología , Ciclina D1/genética , Ciclina D1/metabolismo , Humanos , Inmunoprecipitación , Microscopía Confocal , Modelos Biológicos , Factores de Transcripción NFATC/genética , Reacción en Cadena de la Polimerasa , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Aging is the main risk factor for cardiovascular diseases, but the associated molecular mechanisms are poorly understood. The Wnt signaling pathway was shown to be induced during aging in muscle and in the skin, but the regulation and role of Wnt signaling in the aged vessel have not yet been addressed. While screening for age-related changes in gene expression in the intima/media of human mammary arteries, we observed that the expression of frizzled 4 (Fzd4), a Wnt receptor, and of several targets of the Wnt/ß-catenin/TCF signaling pathway [Wnt-inducible secreted protein 1 (WISP1), versican, osteopontin (SPP1), insulin-like growth factor binding protein 2 (IGFBP-2), and p21] were modified with age, suggesting an activation of the Wnt/ß-catenin pathway. In contrast, we did not observe any regulation of forkhead transcription factor (FoxO) target genes. Beta-catenin-activating phosphorylation at position Ser675 was increased in aging mammary arteries, confirming the activation of this pathway. We confirmed in vitro that Wnt3a or Wnt1 treatment of human vascular smooth muscle cells (VSMCs) induced ß-catenin phosphorylation at Ser675 and WISP1, SPP1, and IGFBP-2 expression. In vitro, Wnt treatment induced proliferation and cyclin D1 expression in VSMC from young (6 weeks old) rats but not in cells from older rats (8 months old), even though low-density lipoprotein receptor-related protein 6 and ß-catenin phosphorylation, and ß-catenin nuclear translocation demonstrated ß-catenin activation in both cell types. Beta-catenin silencing demonstrated that Wnt induction of cyclin D1 expression is ß-catenin dependent. Altogether, our data show that the Wnt/ß-catenin/TCF pathway is activated in aging human mammary artery cells, but fails to induce the proliferation of aging vascular cells.
Asunto(s)
Envejecimiento/fisiología , Arterias/fisiología , Transducción de Señal/fisiología , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Arterias/anatomía & histología , Arterias/patología , Proliferación Celular , Células Cultivadas , Ciclina D1/genética , Ciclina D1/metabolismo , Perfilación de la Expresión Génica , Humanos , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/fisiología , Ratas , Proteínas Wnt/genética , beta Catenina/genéticaRESUMEN
Congestive heart failure (HF) is associated with impaired endothelium-dependent nitric oxide-mediated vasodilatation. The aim of this study was to examine the effects of sarco/endoplasmic reticulum (ER) Ca(2+)-ATPase 2a (SERCA2a) gene transfer on endothelial function in a swine HF model. Two months after the creation of mitral regurgitation to induce HF, the animals underwent intracoronary injection of adeno-associated virus (AAV) carrying SERCA2a (n = 7) or saline (n = 6). At 4 months, coronary flow (CF) was measured in the mid-portion of the left anterior descending (LAD) artery. In the failing animals, CF was decreased significantly; SERCA2a gene transfer rescued CF to levels observed in sham-group [ml/min/g, 0.47 +/- 0.064 saline versus 0.89 +/- 0.116, SERCA2a; P < 0.05; 1.00 +/- 0. 185 sham P = NS (nonsignificant)]. In coronary arteries from HF animals, SERCA2a and endothelial isoform of nitric oxide synthase (eNOS) protein expression were decreased, but restored to normal levels by SERCA2a gene transfer. In human coronary artery endothelial cells (HCAECs), SERCA2a overexpression increased eNOS expression, phosphorylation, eNOS promoter activity, Ca(2+) storage capacity, and enhanced histamine-induced calcium oscillations, eNOS activity, and cyclic guanosine monophosphate (cGMP) production. Thus, SERCA2a gene transfer increases eNOS expression and activity by modulating calcium homeostasis to improve CF. These findings suggest that SERCA2a gene transfer improves vascular reactivity in the setting of HF.