RESUMO
Heart failure (HF) is a leading cause of death and disability globally. Heritable factors and the extent and pattern of myocardial fibrosis are important determinants of outcomes in patients with HF. In a genome-wide association study of mortality in HF, we recently identified a genetic polymorphism on chromosome 5q22 associated with HF mortality. Here, we sought to study the mechanisms by which this variant may influence myocardial disease processes. We find that the risk allele is located in an enhancer motif upstream of the TSLP gene (encoding thymic stromal lymphopoietin), conferring increased binding of the transcription factor nescient helix-loop helix 1 (NHLH1) and increased TSLP expression in human heart. Further, we find that increased strain of primary human myocardial fibroblasts results in increased TSLP expression and that the TSLP receptor is expressed in myocardial mast cells in human single nuclei RNA sequence data. Finally, we show that TSLP overexpression induces increased transforming growth factor ß expression in myocardial mast cells and tissue fibrosis. Collectively, our findings based on follow-up of a human genetic finding implicate a novel pathway in myocardial tissue homeostasis and remodeling.
Assuntos
Insuficiência Cardíaca , Linfopoietina do Estroma do Timo , Humanos , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Citocinas/genética , Fibroblastos , Estudo de Associação Genômica Ampla , Insuficiência Cardíaca/genética , Mastócitos , Miócitos CardíacosRESUMO
Acute cellular rejection (ACR) is a leading cause of graft loss and death after heart transplantation despite effective immunosuppressive therapies. The identification of factors that impair graft vascular barrier function or promote immune cell recruitment during ACR could provide new therapeutic opportunities for the treatment of patients who receive transplants. In 2 ACR cohorts, we found the extracellular vesicle-associated cytokine TWEAK to be elevated during ACR. Vesicular TWEAK promoted expression of proinflammatory genes and the release of chemoattractant cytokines from human cardiac endothelial cells. We conclude that vesicular TWEAK is a novel target with potential therapeutic implications in ACR.
RESUMO
Inadequate adaption to mechanical forces, including blood pressure, contributes to development of arterial aneurysms. Recent studies have pointed to a mechanoprotective role of YAP and TAZ in vascular smooth muscle cells (SMCs). Here, we identified reduced expression of YAP1 in human aortic aneurysms. Vascular SMC-specific knockouts (KOs) of YAP/TAZ were thus generated using the integrin α8-Cre (Itga8-Cre) mouse model (i8-YT-KO). i8-YT-KO mice spontaneously developed aneurysms in the abdominal aorta within 2 weeks of KO induction and in smaller arteries at later times. The vascular specificity of Itga8-Cre circumvented gastrointestinal effects. Aortic aneurysms were characterized by elastin disarray, SMC apoptosis, and accumulation of proteoglycans and immune cell populations. RNA sequencing, proteomics, and myography demonstrated decreased contractile differentiation of SMCs and impaired vascular contractility. This associated with partial loss of myocardin expression, reduced blood pressure, and edema. Mediators in the inflammatory cGAS/STING pathway were increased. A sizeable increase in SOX9, along with several direct target genes, including aggrecan (Acan), contributed to proteoglycan accumulation. This was the earliest detectable change, occurring 3 days after KO induction and before the proinflammatory transition. In conclusion, Itga8-Cre deletion of YAP and TAZ represents a rapid and spontaneous aneurysm model that recapitulates features of human abdominal aortic aneurysms.
Assuntos
Aneurisma da Aorta Abdominal , Aneurisma Aórtico , Animais , Humanos , Camundongos , Aorta Abdominal , Aneurisma Aórtico/genética , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/metabolismo , Modelos Animais de Doenças , Músculo Liso Vascular/metabolismoRESUMO
While restoration of coronary blood flow to the ischemic heart is the most effective strategy for reducing infarct size, reperfusion injury represents a significant limiting factor on clinical outcomes in myocardial infarction patients. Ischemic preconditioning (IPC) has been shown to inhibit reperfusion injury and represents an attractive model for studying cardioprotective signal transduction pathways. Long non-coding RNAs (lncRNAs) are a structurally and functionally heterogenous class of RNA transcripts with unknown roles in IPC-induced cardioprotection. Through microarray-based expression profiling of 31,423 lncRNAs in cardiac tissue from IPC mice, we identified the nuclear transcript Neat1 to be rapidly and robustly decreased in response to IPC. siRNA-mediated knock down of Neat1 reduced apoptosis and necrosis in murine cardiomyocytes (CM) and human iPS-derived CMs in response to prolonged hypoxia and hypoxia-reoxygenation, assessed with Annexin V/propidium iodide-staining, a Caspase 3/7 activity assay, LDH release, and western blot for cleaved Caspase 3. Mechanistically, Neat1 was shown to regulate processing of pro-apoptotic microRNA-22 (miR-22) in murine and human CM nuclei using a luciferase reporter assay. Hypoxia-induced downregulation of Neat1 was shown to result in accumulation of unprocessed pri-miRNA and decreased availability of biologically active miRNA, including miR-22. Addition of exogenous synthetic miR-22 reversed the protective effect of Neat1 knock down in human iPS-CM. In conclusion, we have identified the nuclear lncRNA Neat1 as part of a conserved oxygen-sensitive feedback mechanism by regulation of miRNA processing and a potential target in cardioprotection.
Assuntos
Citoproteção/genética , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Processamento Pós-Transcricional do RNA/genética , RNA Longo não Codificante/metabolismo , Animais , Hipóxia Celular/genética , Modelos Animais de Doenças , Regulação para Baixo/genética , Feminino , Técnicas de Silenciamento de Genes , Precondicionamento Isquêmico , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RatosRESUMO
Atrial natriuretic peptide (ANP) represents an attractive therapeutic target in hypertension and heart failure. The biologically active form of ANP is produced by the cardiac serine protease corin, and modulation of its activity might therefore represent a novel approach for ANP augmentation. MicroRNAs (miRNAs) are pervasive regulators of gene expression, but their potential role in regulating corin activity has not been elucidated. Our aim was to systematically identify and characterize miRNA regulators of corin activity in human cardiomyocytes. An assay for measuring serine protease activity in human induced pluripotent stem cell (iPS)-derived cardiomyocytes was used to perform a comprehensive screening of miRNA family inhibitors (n = 42). miRNA 1-3p (miR-1-3p) was identified as a potent inhibitor of corin activity. The interaction between miR-1-3p and a specific target site in the CORIN 3' untranslated region (3' UTR) was confirmed through argonaute 2 (AGO2)-RNA immunoprecipitation and reporter assays. Inhibition of miR-1-3p resulted in upregulation of CORIN gene and protein expression, as well as a concomitant increase in extracellular ANP. Additionally, miR-1-3p was found to interact with and inhibit the expression of several transcriptional activators of ANP gene expression. In conclusion, we have identified a novel regulator of corin activity and ANP biogenesis in human cardiomyocytes that might be of potential future therapeutic utility.
Assuntos
Fator Natriurético Atrial/genética , MicroRNAs/genética , Serina Endopeptidases/genética , Regiões 3' não Traduzidas/genética , Fator Natriurético Atrial/biossíntese , Linhagem Celular , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Coração/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , MicroRNAs/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Serina Endopeptidases/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The cardiac hormone atrial natriuretic peptide (ANP) is a central regulator of blood volume and a therapeutic target in hypertension and heart failure. Enhanced ANP activity in such conditions through inhibition of the degradative enzyme neprilysin has shown clinical efficacy but is complicated by consequences of simultaneous accumulation of a heterogeneous array of other hormones. Targets for specific ANP enhancement have not been available. Here, we describe a cis-acting antisense transcript (NPPA-AS1), which negatively regulates ANP expression in human cardiomyocytes. We show that NPPA-AS1 regulates ANP expression via facilitating NPPA repressor RE1-silencing transcription factor (REST) binding to its promoter, rather than forming an RNA duplex with ANP mRNA. Expression of ANP mRNA and NPPA-AS1 was increased and correlated in isolated strained human cardiomyocytes and in hearts from patients with advanced heart failure. Further, inhibition of NPPA-AS1 in vitro and in vivo resulted in increased myocardial expression of ANP, increased circulating ANP, increased renal cGMP, and lower blood pressure. The effects of NPPA-AS1 inhibition on NPPA expression in human cardiomyocytes were further marked under cell-strain conditions. Collectively, these results implicate the antisense transcript NPPA-AS1 as part of a physiologic self-regulatory ANP circuit and a viable target for specific ANP augmentation.
Assuntos
Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Cromatina , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Insuficiência Cardíaca , Humanos , Masculino , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Neprilisina , RNA Mensageiro/metabolismo , Fatores de TranscriçãoRESUMO
Dysregulation of receptor tyrosine kinase-induced pathways is a critical step driving the oncogenic potential of brain cancer. In this study, we investigated the role of two members of the Sprouty (Spry) family in brain cancer-derived cell lines. Using immunoblot analyses we found essential differences in the pattern of endogenous Spry3 and Spry4 expression. While Spry4 expression was mitogen-dependent and repressed in a number of cells from higher malignant brain cancers, Spry3 levels neither fluctuated in response to serum withdrawal nor were repressed in glioblastoma (GBM)-derived cell lines. In accordance to the well-known inhibitory role of Spry proteins in fibroblast growth factor (FGF)-mediated signaling, both Spry proteins were able to interfere with FGF-induced activation of the MAPK pathway although to a different extent. In response to serum solely, Spry4 exerts its role as a negative regulator of MAPK activation. Ectopic expression of Spry4 inhibited proliferation and migration of GBM-originated cells, positioning it as a tumor suppressor in brain cancer. In contrast, elevated Spry3 levels accelerated both proliferation and migration of these cell lines, while repression of Spry3 levels using shRNA caused a significant diminished growth and migration velocity rate of a GBM-derived cell line. This argues for a tumor-promoting function of Spry3 in GBMs. Based on these data we conclude that Spry3 and Spry4 fulfill different if not opposing roles within the cancerogenesis of brain malignancies.
Assuntos
Neoplasias Encefálicas/metabolismo , Carcinogênese/metabolismo , Glioblastoma/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Humanos , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
Heart failure (HF) is a major public health problem characterized by inability of the heart to maintain sufficient output of blood. The systematic characterization of circulating proteins across different stages of HF may provide pathophysiological insights and identify therapeutic targets. Here we report application of aptamer-based proteomics to identify proteins associated with prospective HF incidence in a population-based cohort, implicating modulation of immunological, complement, coagulation, natriuretic and matrix remodeling pathways up to two decades prior to overt disease onset. We observe further divergence of these proteins from the general population in advanced HF, and regression after heart transplantation. By leveraging coronary sinus samples and transcriptomic tools, we describe likely cardiac and specific cellular origins for several of the proteins, including Nt-proBNP, thrombospondin-2, interleukin-18 receptor, gelsolin, and activated C5. Our findings provide a broad perspective on both cardiac and systemic factors associated with HF development.
Assuntos
Insuficiência Cardíaca/sangue , Transplante de Coração , Proteoma/análise , Adulto , Idoso , Aptâmeros de Peptídeos/metabolismo , Biomarcadores/sangue , Biomarcadores/metabolismo , Seio Coronário/metabolismo , Seio Coronário/patologia , Feminino , Perfilação da Expressão Gênica , Insuficiência Cardíaca/epidemiologia , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/cirurgia , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Proteoma/metabolismo , Proteômica/métodos , Fatores de RiscoRESUMO
AIMS: Exosome-mediated microRNA transfer is a recently discovered mode of cell-to-cell communication, in which microRNAs act as paracrine molecules, exerting their regulatory effects in recipient cells. T cells and endothelial cells are two main players in the mechanism of acute cellular cardiac rejection. The aim of this study was to investigate the role of exosomal microRNAs in the crosstalk between T cells and endothelial cells and its implications for the molecular mechanisms that drive acute cellular rejection in heart transplantation. METHODS AND RESULTS: Exosomes isolated from serum samples of heart transplant patients with and without acute cardiac allograft rejection were profiled and showed enrichment of miR-142-3p, miR-92a-3p, miR-339-3p and miR-21-5p. Treatment of endothelial cells with the respected serum exosomes resulted the increased of miR-142-3p level in endothelial cells. Using T cells isolated from healthy donors and activated with either anti-CD3/CD28 antibody or IL-2/PHA, we could show that miR-142-3p is released from activated cells, is contained in exosomes and can be transferred to human vascular endothelial cells in vitro. Transcriptome analysis of endothelial cells treated with activated T cell supernatant with or without exosomes was used to identify mRNA targets of transferred miR-142-3-p. Overexpression of miR-142-3p in endothelial cells resulted in a significant down-regulation of RAB11FIP2, and interaction of miR-142-3p with its predicted target site was confirmed with a reporter assay. Moreover, treatment of endothelial cells with serum exosomes from heart transplant patients with acute cellular rejection resulted in down-regulation of RAB11FIP2 expression and increase in vascular endothelial permeability. CONCLUSION: We have identified a novel mechanism whereby miR-142-3p, a microRNA enriched in exosomes during acute cellular rejection, is transferred to endothelial cells and compromises endothelial barrier function via down-regulation of RAB11FIP2. This study sheds new light on the interaction between host immune system and cardiac allograft endothelium during acute cellular rejection.
Assuntos
Permeabilidade Capilar , Proteínas de Transporte/metabolismo , Exossomos/metabolismo , Rejeição de Enxerto/sangue , Transplante de Coração/efeitos adversos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/sangue , Linfócitos T/metabolismo , Regiões 3' não Traduzidas , Doença Aguda , Adulto , Idoso , Aloenxertos , Sítios de Ligação , Proteínas de Transporte/genética , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Regulação para Baixo , Exossomos/imunologia , Feminino , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Humanos , Ativação Linfocitária , Masculino , Proteínas de Membrana/genética , MicroRNAs/genética , Pessoa de Meia-Idade , Comunicação Parácrina , Transdução de Sinais , Linfócitos T/imunologia , Transfecção , Regulação para Cima , Proteínas rab de Ligação ao GTPRESUMO
BACKGROUND: Ischemic preconditioning (IPC) protects the heart from prolonged ischemic insult and reperfusion injury through a poorly understood mechanism. Post-translational modifications of histone residues can confer rapid and drastic switches in gene expression in response to various stimuli, including ischemia. The aim of this study was to investigate the effect of histone methylation in the response to cardiac ischemic preconditioning. METHODS AND RESULTS: We used cardiac biopsies from mice subjected to IPC to quantify global levels of 3 of the most well-studied histone methylation marks (H3K9me2, H3K27me3, and H3K4me3) with Western blot and found that H3K9me2 levels were significantly increased in the area at risk compared to remote myocardium. In order to assess which genes were affected by the increase in H3K9me2 levels, we performed ChIP-Seq and transcriptome profiling using microarray. Two hundred thirty-seven genes were both transcriptionally repressed and enriched in H3K9me2 in the area at risk of IPC mice. Of these, Mtor (Mechanistic target of rapamycin) was chosen for mechanistic studies. Knockdown of the major H3K9 methyltransferase G9a resulted in a significant decrease in H3K9me2 levels across Mtor, increased Mtor expression, as well as decreased autophagic activity in response to rapamycin and serum starvation. CONCLUSIONS: IPC confers an increase of H3K9me2 levels throughout the Mtor gene-a master regulator of cellular metabolism and a key player in the cardioprotective effect of IPC-leading to transcriptional repression via the methyltransferase G9a. The results of this study indicate that G9a has an important role in regulating cardiac autophagy and the cardioprotective effect of IPC.
Assuntos
Autofagia/genética , Repressão Epigenética , Código das Histonas/genética , Histona-Lisina N-Metiltransferase/metabolismo , Precondicionamento Isquêmico Miocárdico , Serina-Treonina Quinases TOR/genética , Animais , Western Blotting , Imunoprecipitação da Cromatina , Epigênese Genética , Metilação , Camundongos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Androgen-deprivation therapy (ADT) for prostate cancer has been associated with increased risk for development of cardiovascular events and recent pooled analyses of randomized intervention trials suggest that this primarily is the case for patients with pre-existing cardiovascular disease treated with gonadotropin-releasing hormone receptor (GnRH-R) agonists. In the present study we investigated the effects of the GnRH-R agonist leuprolide and the GnRH-R antagonist degarelix on established atherosclerotic plaques in ApoE(-/-) mice. A shear stress modifier was used to produce both advanced and more stable plaques in the carotid artery. After 4 weeks of ADT, increased areas of necrosis was observed in stable plaques from leuprolide-treated mice (median and IQR plaque necrotic area in control, degarelix and leuprolide-treated mice were 0.6% (IQR 0-3.1), 0.2% (IQR 0-4.4) and 11.0% (IQR 1.0-19.8), respectively). There was also evidence of increased inflammation as assessed by macrophage immunohistochemistry in the plaques from leuprolide-treated mice, but we found no evidence of such changes in plaques from control mice or mice treated with degarelix. Necrosis destabilizes plaques and increases the risk for rupture and development of acute cardiovascular events. Destabilization of pre-existing atherosclerotic plaques could explain the increased cardiovascular risk in prostate cancer patients treated with GnRH-R agonists.