RESUMEN
Calcification of the bicuspid aortic valve (BAV) involves differential expression of various RNA genes, which is achieved through complex regulatory networks that are controlled in part by transcription factors and microRNAs. We previously found that miR-195-5p regulates the osteogenic differentiation of valvular interstitial cells (VICs) by targeting the TGF-ß pathway. However, the transcriptional regulation of miR-195-5p in calcified BAV patients is not yet clear. In this study, stenotic aortic valve tissues from patients with BAVs and tricuspid aortic valves (TAVs) were collected. Candidate transcription factors of miR-195-5p were predicted by bioinformatics analysis and tested in diseased valves and in male porcine VICs. SP2 gene expression and the corresponding protein levels in BAV were significantly lower than those in TAV, and a low SP2 expression level environment in VICs resulted in remarkable increases in RNA expression levels of RUNX2, BMP2, collagen 1, MMP2, and MMP9 and the corresponding proteins. ChIP assays revealed that SP2 directly bound to the transcription promoter region of miR-195-5p. Cotransfection of SP2 shRNA and a miR-195-5p mimic in porcine VICs demonstrated that SP2 repressed SMAD7 expression via miR-195-5p, while knockdown of SP2 increased the mRNA expression of SMAD7 and the corresponding protein and attenuated Smad 2/3 expression. Immunofluorescence staining of diseased valves confirmed that the functional proteins of osteogenesis differentiation, including RUNX2, BMP2, collagen 1, and osteocalcin, were overexpressed in BAVs. In Conclusion, the transcription factor Sp2 is expressed at low levels in VICs from BAV patients, which has a negative impact on miR-195-5p expression by binding its promoter region and partially promotes calcification through a SMAD-dependent pathway.
Asunto(s)
Enfermedad de la Válvula Aórtica Bicúspide/patología , Calcinosis/patología , Osteoblastos/patología , Proteína smad7/metabolismo , Factor de Transcripción Sp2/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Válvula Tricúspide/patología , Animales , Enfermedad de la Válvula Aórtica Bicúspide/genética , Enfermedad de la Válvula Aórtica Bicúspide/metabolismo , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Calcinosis/genética , Calcinosis/metabolismo , Diferenciación Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Femenino , Humanos , Masculino , MicroARNs , Persona de Mediana Edad , Osteoblastos/metabolismo , Osteogénesis , Proteína smad7/genética , Factor de Transcripción Sp2/genética , Porcinos , Factor de Crecimiento Transformador beta1/genética , Válvula Tricúspide/metabolismoRESUMEN
BACKGROUND/AIMS: Aortic stenosis caused by leaflet calcification in the bicuspid aortic valve (BAV) is more accelerative than that in the tricuspid aortic valve (TAV). MicroRNA-195 (miR-195) is downregulated more in stenotic than in insufficient BAVs, but its expression in BAVs compared with TAVs is unclear. We aimed to investigate the roles of miR-195 and its calcification-related target SMAD7 in stenotic BAVs compared with those in TAVs. METHODS: Twenty-one stenotic BAV and 29 TAV samples were collected from surgical patients and examined for the expression of miR-195 and SMAD7 by RT-PCR. The samples were also assessed by western blotting and immunohistochemistry for the functional protein alteration associated with calcification. Dual-luciferase assay was performed to determine the putative target of miR-195 before the effects of miR-195 expression on osteogenic progression was demonstrated in cultured porcine valve interstitial cells (VICs). RESULTS: Compared with TAV, the expression of miR-195 was remarkably lower in the BAV leaflet with higher expression of SMAD7, which was then validated as a direct target of miR-195. Their negative correlation was then confirmed in cultured VICs. Under an osteogenic environment, the cellular calcification was promoted in miR-195-repressed VICs expressing higher BMP-2 and Runx2 and higher activity of MMP-2 compared with the controls. Finally, higher MMP-2 and MMP-9 expression and more collagen distribution were observed in BAV than TAV samples. CONCLUSIONS: miR-195 is downregulated more in stenotic BAV than TAV in this study. The downregulation of miR-195 is associated with valvular calcification via targeting SMAD7, which promotes the remodeling of the extracellular matrix.
Asunto(s)
Válvula Aórtica/anomalías , Enfermedades de las Válvulas Cardíacas/metabolismo , MicroARNs/metabolismo , Proteína smad7/metabolismo , Regiones no Traducidas 3' , Adulto , Anciano , Animales , Antagomirs/metabolismo , Válvula Aórtica/metabolismo , Estenosis de la Válvula Aórtica/metabolismo , Estenosis de la Válvula Aórtica/patología , Secuencia de Bases , Enfermedad de la Válvula Aórtica Bicúspide , Proteína Morfogenética Ósea 2/metabolismo , Línea Celular , Colágeno/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Femenino , Células HEK293 , Humanos , Inmunohistoquímica , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Alineación de Secuencia , Proteína smad7/antagonistas & inhibidores , Proteína smad7/genética , Porcinos , Válvula Tricúspide/metabolismoRESUMEN
Dysfunction of macroautophagy/autophagy in macrophages contributes to atherosclerosis. Impaired autophagy-lysosomal degradation system leads to lipid accumulation, facilitating atherosclerotic plaque. ATG14 is an essential regulator for the fusion of autophagosomes with lysosomes. Whether ATG14 plays a role in macrophage autophagy dysfunction in atherosclerosis is unknown. To investigate the effects of ATG14 on macrophage autophagy, human atherosclerotic plaque, apoe-/- mice and cultured mouse macrophages were evaluated. Overexpression of ATG14 by adenovirus was used to reveal its function in autophagy, inflammation and atherosclerotic plaque formation. Results showed that impaired autophagy function with reduction of ATG14 expression existed in macrophages of human and mouse atherosclerotic plaques. Ox-LDL impaired autophagosome-lysosome fusion with reduction of ATG14 expression in macrophages. Overexpression of ATG14 in macrophages enhanced fusion of autophagosomes with lysosomes and promoted lipid degradation, decreasing Ox-LDL-induced apoptosis and inflammatory response. Augmenting ATG14 expression reversed the autophagy dysfunction in macrophages of apoe-/- mice plaque, blunted SQSTM1/p62 accumulation, inhibited inflammation, and upregulated the population of Treg cells, resulting in alleviating atherosclerotic lesions.Abbreviations: ABCC1: ATP-binding cassette, sub-family C (CFTR/MRP), member 1; ABCA1: ATP-binding cassette, sub-family A (ABC1), member 1; Ad-Atg14: adenovirus vector carrying the mouse Atg14 gene; Ad-LacZ: adenovirus vector carrying the gene for bacterial ß-galactosidase; apoe-/-: apolipoprotein E knockout; ATG14: autophagy-related 14; CD68: CD68 antigen; DAPI: 4',6-diamidino-2-phenylindole; Dil-ox-LDL: Dil-oxidized low density lipoprotein; ELISA: enzyme-linked immunosorbent assay; HFD: high-fat diet (an atherogenic diet); IL: interleukin; LAMP2: lysosomal-associated membrane protein 2; LDL-C: low density lipoprotrein cholesterol; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; ND: normal diet; Ox-LDL: oxidized low density lipoprotein; PBMC: peripheral blood mononuclear cells; SQSTM1/p62: sequestosome 1; SREBF1/SREBP1c: sterol regulatory element binding transcription factor 1; SREBF2/SREBP2: sterol regulatory element binding factor 2; STX17: syntaxin 17; TC: serum total cholesterol; TG: triglyceride; TNF: tumor necrosis factor; IFN: interferon; Treg cell: regulatory T cell.
Asunto(s)
Aterosclerosis , Autofagosomas , Animales , Aterosclerosis/patología , Autofagosomas/metabolismo , Autofagia , Inflamación/metabolismo , Leucocitos Mononucleares/metabolismo , Lisosomas/metabolismo , Macroautofagia , Macrófagos/metabolismo , RatonesRESUMEN
One key risk factor of aortic valve stenosis in clinical practice is bicuspid aortic valve (BAV). Increasing evidence indicates that numerous microRNAs (miRs/miRNAs) are involved in BAV calcification via their target genes. miR3303p was found to be involved in the deterioration of BAV calcification by miR profiling in human calcified BAV and tricuspid aortic valve (TAV) tissues in the present study and the underlying mechanism was investigated. RNA sequencing was performed on four BAV and four TAV tissues from patients with aortic stenosis before these leaflets were examined for the expression levels of miR3303p and CREBbinding protein (CREBBP) by reverse transcriptionPCR. The alteration of functional factors associated with calcification was also assessed by Western blotting and immunohistochemistry in human aortic tissue samples. The putative target of miR3303p was detected by dualluciferase assay in 293 cells. Furthermore, the influence of miR3303p expression on osteogenic progression was explored in cultured porcine valve interstitial cells (VICs). Rescue experiments of CRBBP were performed to confirm the influence of the miR3303pCREBBP pathway in the calcification progress in porcine VICs. RNA sequencing indicated distinct expression of miR3303p in human BAV tissues compared with TAV, which was then confirmed by PCR. CREBBP expression levels in human BAV and TAV leaflets also demonstrated the opposite alterations. This negative correlation was then confirmed in cultured porcine VICs. Under an osteogenic environment, cellular calcification was promoted in miR3303poverexpressed porcine VICs expressing higher bone morphogenetic protein 2, Runtrelated transcription factor 2, matrix metalloproteinase (MMP)2, MMP9 and collagen I compared with controls. Rescue experiments further confirmed that miR3303p played its role via targeting CREBBP in porcine VICs. Collectively, miR3303p was upregulated in calcified BAV compared with TAV. The upregulation of miR3303p promotes the calcification progress partially via targeting CREBBP.
Asunto(s)
Estenosis de la Válvula Aórtica/genética , Enfermedad de la Válvula Aórtica Bicúspide/genética , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , MicroARNs/genética , Regiones no Traducidas 3' , Adulto , Anciano , Animales , Estenosis de la Válvula Aórtica/metabolismo , Enfermedad de la Válvula Aórtica Bicúspide/metabolismo , Células Cultivadas , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ARN , Porcinos , Válvula Tricúspide/metabolismo , Regulación hacia ArribaRESUMEN
The SWI/SNF complex is a multimeric chromatin remodeling complex that has vital roles in regulating gene expression and cancer development. However, to date few studies have deeply explored the mechanism of SMARCA2 inactivation. We applied multi-omics analysis to explore the mechanism of SMARCA2 inactivation in The Cancer Genome Atlas (TCGA) database and performed the dCas9-DNMT3a system to evaluate the role of promoter methylation in SMARCA2 transcriptional regulation. We also assessed the tumor suppressing roles of SMARCA2 in lung cancer development by in vitro experiments. SMARCA2 promoter hypermethylation was significantly associated with decreased expression of SMARCA2. This result was further confirmed in the results of our own tissues. In addition, we observed that the mRNA level decreased by about 3 folds while the CpG island of promoter is nearly 30% hypermethylated by dCas9-DNMT3a system in H1299 cells. We identified SMARCA2 as a tumor suppressor gene whose expression was downregulated in lung cancers. Its inactivation was significantly associated with the poor survival of lung cancer patients [hazard ratio, HRâ¯=â¯0.35 (0.27-0.45)]. Besides, we found that SMARCA2 was a tumor suppressor and can significantly inhibit lung cancer cell vitality. We found that promoter hypermethylation contribute to the inactivation of SMARCA2. We also verified its oncogenetic roles of BRM inactivation in lung adenocarcinoma, which may provide a potential target for the clinical treatment.
Asunto(s)
Adenocarcinoma/patología , Biomarcadores de Tumor/genética , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/patología , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Adenocarcinoma/genética , Apoptosis , Carcinogénesis , Movimiento Celular , Proliferación Celular , Ensamble y Desensamble de Cromatina , Humanos , Neoplasias Pulmonares/genética , Mutación , Pronóstico , Células Tumorales CultivadasRESUMEN
BACKGROUND: Despite the successful creation of complex lesion sets during hybrid ablation (HA), reoccurrence of atrial fibrillation (AF), and/or atrial arrhythmia and procedural complications still occur. The main objective of this study was to compare the efficacy and safety between HA and transcatheter ablation (TA). METHODS: We searched Pubmed, Embase, and the Cochrane Central Register of Controlled Trials (CENTRAL) database up to October 2017. Studies that satisfied our predefined inclusion criteria were included. Of the 894 records, 4 studies encompassing 331 patients were included in our study. We assessed pooled data using random-effect or fixed-effect model. The main endpoint was freedom of atrial arrhythmia after follow-up duration, secondary results were procedure time and intraoperative and postoperative adverse events. Similarly, tertiary outcomes were endocardial time, fluoroscopy time, and postoperative hospitalization. RESULTS: Compared with TA, HA treatment through mini-thoracotomy access improved superiority in freedom of atrial arrhythmia after follow-up duration (odds ratio [OR]â=â6.67, 95% confidence interval [CI]: 2.63-16.90), but HA increased the incidence of intraoperative and postoperative adverse events for AF patients (ORâ=â2.98, 95% CI: 1.30-6.83). HA through either mini-thoracotomy or transdiaphragmatic/subxiphoid access had longer procedure time and postoperative hospitalization than TA. However, endocardial time was shorter than TA. CONCLUSIONS: For AF patients, HA possessed of an overall superior outcome using mini-thoracotomy way to TA. Although HA had longer procedure time, it yielded a reduction in endocardial time. Meanwhile, we should pay attention to the significantly high risk of intraoperative and postoperative adverse events that the HA generated.
Asunto(s)
Técnicas de Ablación/métodos , Fibrilación Atrial/cirugía , Ablación por Catéter/métodos , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Toracotomía/métodos , Resultado del TratamientoRESUMEN
Autophagy regulates the metabolism, survival and function of numerous types of cell, including cells that comprise the cardiovascular system. The dysfunction of autophagy has been demonstrated in atherosclerosis, restenotic lesions and hypertensive vessels. As a member of the Ras GTPase superfamily, Rab7 serves a significant role in the regulation of autophagy. The present study evaluated how Rab7 affects the proliferation and invasion, and phenotypic transformations of aortic dissection (AD) smooth muscle cells (SMCs) via autophagy. Rab7 was overexpressed in AD tissues and the percentage of synthetic human aortic SMCs (HASMCs) was higher in AD tissues compared with NAD tissues. Downregulation of Rab7 decreased cell growth, reduced the number of invasive cells and decreased the percentage cells in the G1 phase. Autophagy of HASMCs was inhibited following Rab7 knockdown. Inhibition of autophagy with 3methyladenine or Rab7 knockdown suppressed the phenotypic conversion of contractile to synthetic HASMCs. The action of Rab7 may be mediated by inhibiting the Ras/Raf/mitogenactivated protein kinase (MAPK) kinase (MEK)/extracellular signal related kinase (ERK) signaling pathway. In conclusion, the results revealed that Rab7mediated autophagy regulated the behavior of SMCs and the phenotypic transformations in AD via activation of the Ras/Raf/MEK/ERK signaling pathway. The findings of the present study may improve understanding of the role Rab7 in the molecular etiology of AD and suggests the application of Rab7 as a novel therapeutic target in the treatment of human AD.
Asunto(s)
Disección Aórtica/etiología , Disección Aórtica/metabolismo , Autofagia , Miocitos del Músculo Liso/metabolismo , Fenotipo , Transducción de Señal , Proteínas de Unión al GTP rab/metabolismo , Ciclo Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas de Unión a GTP rab7 , Quinasas raf/metabolismo , Proteínas ras/metabolismoRESUMEN
BACKGROUND: Marfan syndrome (MFS) is an inherited connective tissue disorder affecting the ocular, skeletal and cardiovascular systems. Previous studies of MFS have demonstrated the association between genetic defects and clinical manifestations. Our purpose was to investigate the role of novel genetic variants in determining MFS clinical phenotypes. METHODS: We sequenced the whole exome of 19 individuals derived from three Han Chinese families. The sequencing data were analyzed by a standard pipeline. Variants were further filtered against the public database and an in-house database. Then, we performed pedigree analysis under different inheritance patterns according to American College of Medical Genetics guidelines. Results were confirmed by Sanger sequencing. RESULTS: Two novel loss-of-function indels (c.5027_5028insTGTCCTCC, p.D1677Vfs*8; c.5856delG, p.S1953Lfs*27) and one nonsense variant (c.8034C>A, p.Y2678*) of FBN1 were identified in Family 1, Family 2 and Family 3, respectively. All affected members carried pathogenic mutations, whereas other unaffected family members or control individuals did not. These different kinds of loss of function (LOF) variants of FBN1 were located in the cbEGF region and a conserved domain across species and were not reported previously. CONCLUSIONS: Our study extended and strengthened the vital role of FBN1 LOF mutations in the pathogenesis of MFS with an autosomal dominant inheritance pattern. We confirm that genetic testing by next-generation sequencing of blood DNA can be fundamental in helping clinicians conduct mutation-based pre- and postnatal screening, genetic diagnosis and clinical management for MFS.