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1.
Sci Rep ; 14(1): 16459, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-39013942

RESUMO

Beneficial effects of sodium glucose co-transporter 2 inhibitors (SGLT2is) in cardiovascular diseases have been extensively reported leading to the inclusion of these drugs in the treatment guidelines for heart failure. However, molecular actions especially on non-myocyte cells remain uncertain. We observed dose-dependent inhibitory effects of two SGLT2is, dapagliflozin (DAPA) and empagliflozin (EMPA), on inflammatory signaling in human umbilical vein endothelial cells. Proteomic analyses and subsequent enrichment analyses discovered profound effects of these SGLT2is on proteins involved in mitochondrial respiration and actin cytoskeleton. Validation in functional oxygen consumption measurements as well as tube formation and migration assays revealed strong impacts of DAPA. Considering that most influenced parameters played central roles in endothelial to mesenchymal transition (EndMT), we performed in vitro EndMT assays and identified substantial reduction of mesenchymal and fibrosis marker expression as well as changes in cellular morphology upon treatment with SGLT2is. In line, human cardiac fibroblasts exposed to DAPA showed less proliferation, reduced ATP production, and decelerated migration capacity while less extensive impacts were observed upon EMPA. Mechanistically, sodium proton exchanger 1 (NHE1) as well as sodium-myoinositol cotransporter (SMIT) and sodium-multivitamin cotransporter (SMVT) could be identified as relevant targets of SGLT2is in non-myocyte cardiovascular cells as validated by individual siRNA-knockdown experiments. In summary, we found comprehensive beneficial effects of SGLT2is on human endothelial cells and cardiac fibroblasts. The results of this study therefore support a distinct effect of selected SGLT2i on non-myocyte cardiovascular cells and grant further insights into potential molecular mode of action of these drugs.


Assuntos
Compostos Benzidrílicos , Fibroblastos , Glucosídeos , Células Endoteliais da Veia Umbilical Humana , Inibidores do Transportador 2 de Sódio-Glicose , Humanos , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Compostos Benzidrílicos/farmacologia , Glucosídeos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Trocador 1 de Sódio-Hidrogênio/metabolismo , Trocador 1 de Sódio-Hidrogênio/antagonistas & inibidores , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos
2.
Front Psychiatry ; 13: 1020530, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36506422

RESUMO

Objective: Major depressive disorder (MDD) constitutes a main risk factor for suicide. Suicide risk in psychiatric patients is primarily determined by often unreliable, self-reported information. We assessed serum levels of three microRNAs (miRNAs), previously demonstrated to be dysregulated in post-mortem brain samples of suicide victims, as potential peripheral biomarkers for suicidality. Methods: All study participants were diagnosed with MDD according to Diagnostic and Statistical Manual of Mental Disorders, 5th edition criteria. Suicidality, defined as acute suicide risk or suicide attempt within one week prior to study entry, was assessed by clinical interview. Relative serum levels of miR-30a, miR-30e, and miR-200a, normalized to U6, were measured by quantitative real-time PCR in MDD inpatients with (MDD/SI, N = 19) and without (MDD, N = 31) acute suicide risk. Median age and gender distribution were comparable in both groups. Results: Levels of miR-30a, miR-30e, and miR-200a were significantly elevated in MDD/SI compared to MDD. Subgroup analysis of the MDD/SI group showed that levels of miR-30e and miR-200a were significantly higher and miR-30a was increased by trend in patients admitted following a suicide attempt (N = 7) compared to patients with acute suicide risk but without recent suicide attempt (N = 12). Additionally, use of two databases for in silico transcription factor-miRNA interaction prediction indicated early growth response protein (EGR) 1 as potential transcriptional regulator for all three miRNAs. Conclusion: This study demonstrates suicide risk in MDD patients to be associated with increased levels of miR-30a, miR-30e, and miR-200a. Thus, these miRNAs might constitute potential biomarkers to predict suicidal behavior in MDD patients.

3.
Front Cardiovasc Med ; 9: 877450, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35783871

RESUMO

Background: Constant supply of oxygen is crucial for multicellular tissue homeostasis and energy metabolism in cardiac tissue. As a first response to acute hypoxia, endothelial cells (ECs) promote recruitment and adherence of immune cells to the dysbalanced EC barrier by releasing inflammatory mediators and growth factors, whereas chronic hypoxia leads to the activation of a transcription factor (TF) battery, that potently induces expression of growth factors and cytokines including platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). We report a hypoxia-minded, targeted bioinformatics approach aiming to identify and validate TFs that regulate angiogenic signaling. Results: A comprehensive RNA-Seq dataset derived from human ECs subjected to normoxic or hypoxic conditions was selected to identify significantly regulated genes based on (i) fold change (normoxia vs. hypoxia) and (ii) relative abundancy. Transcriptional regulation of this gene set was confirmed via qPCR in validation experiments where HUVECs were subjected to hypoxic conditions for 24 h. Screening the promoter and upstream regulatory elements of these genes identified two TFs, KLF5 and SP1, both with a potential binding site within these regions of selected target genes. In vitro, siRNA experiments confirmed SP1- and KLF5-mediated regulation of identified hypoxia-sensitive endothelial genes. Next to angiogenic signaling, we also validated the impact of TFs on inflammatory signaling, both key events in hypoxic sensing. Both TFs impacted on inflammatory signaling since endogenous repression led to increased NF-κB signaling. Additionally, SP1 silencing eventuated decreased angiogenic properties in terms of proliferation and tube formation. Conclusion: By detailed in silico analysis of promoter region and upstream regulatory elements for a list of hypoxia-sensitive genes, our bioinformatics approach identified putative binding sites for TFs of SP or KLF family in vitro. This strategy helped to identify TFs functionally involved in human angiogenic signaling and therefore serves as a base for identifying novel RNA-based drug entities in a therapeutic setting of vascularization.

4.
Sci Rep ; 11(1): 12456, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-34127686

RESUMO

The family of RNA-binding proteins (RBP) functions as a crucial regulator of multiple biological processes and diseases. However, RBP function in the clinical setting of idiopathic pulmonary fibrosis (IPF) is still unknown. We developed a practical in silico screening approach for the characterization of RBPs using multi-sources data information and comparative molecular network bioinformatics followed by wet-lab validation studies. Data mining of bulk RNA-Sequencing data of tissues of patients with IPF identified Quaking (QKI) as a significant downregulated RBP. Cell-type specific expression was confirmed by single-cell RNA-Sequencing analysis of IPF patient data. We systematically analyzed the molecular interaction network around QKI and its functional interplay with microRNAs (miRs) in human lung fibroblasts and discovered a novel regulatory miR-506-QKI axis contributing to the pathogenesis of IPF. The in silico results were validated by in-house experiments applying model systems of miR and lung biology. This study supports an understanding of the intrinsic molecular mechanisms of IPF regulated by the miR-506-QKI axis. Initially applied to human lung disease, the herein presented integrative in silico data mining approach can be adapted to other disease entities, underlining its practical relevance in RBP research.


Assuntos
Fibrose Pulmonar Idiopática/genética , Pulmão/patologia , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/genética , Estudos de Casos e Controles , Células Cultivadas , Biologia Computacional , Conjuntos de Dados como Assunto , Fibroblastos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Fibrose Pulmonar Idiopática/patologia , Pulmão/citologia , Cultura Primária de Células , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/genética , Proteínas de Ligação a RNA/metabolismo , Análise de Célula Única
5.
Sci Rep ; 11(1): 1755, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33469076

RESUMO

Hypertrophic cardiomyopathy (HCM) is one of the most common hereditary heart diseases and can be classified into an obstructive (HOCM) and non-obstructive (HNCM) form. Major characteristics for HCM are the hypertrophy of cardiomyocytes and development of cardiac fibrosis. Patients with HCM have a higher risk for sudden cardiac death compared to a healthy population. In the present study, we investigated the abundancy of selected proteins as potential biomarkers in patients with HCM. We included 60 patients with HCM and 28 healthy controls and quantitatively measured the rate of a set of 92 proteins already known to be associated with cardiometabolic processes via protein screening using the proximity extension assay technology in a subgroup of these patients (20 HCM and 10 healthy controls). After validation of four hits in the whole cohort of patients consisting of 88 individuals (60 HCM patients, 28 healthy controls) we found only one candidate, c-KIT, which was regulated significantly different between HCM patients and healthy controls and thus was chosen for further analyses. c-KIT is a tyrosine-protein kinase acting as receptor for the stem cell factor and activating several pathways essential for cell proliferation and survival, hematopoiesis, gametogenesis and melanogenesis. Serum protein levels of c-KIT were significantly lower in patients with HCM than in healthy controls, even after adjusting for confounding factors age and sex. In addition, c-KIT levels in human cardiac tissue of patients with HOCM were significant higher compared to controls indicating high levels of c-KIT in fibrotic myocardium. Furthermore, c-KIT concentration in serum significantly correlated with left ventricular end-diastolic diameter in HOCM, but not HCM patients. The present data suggest c-KIT as a novel biomarker differentiating between patients with HCM and healthy population and might provide further functional insights into fibrosis-related processes of HOCM.


Assuntos
Cardiomiopatia Hipertrófica/sangue , Cardiomiopatia Hipertrófica/diagnóstico , Proteínas Proto-Oncogênicas c-kit/sangue , Adulto , Idoso , Biomarcadores/sangue , Ecocardiografia , Feminino , Fibrose/patologia , Ventrículos do Coração/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/patologia
6.
J Mol Cell Cardiol ; 150: 91-100, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33127387

RESUMO

BACKGROUND: Myocardial fibrosis is a hallmark of the failing heart, contributing to the most common causes of deaths worldwide. Several microRNAs (miRNAs, miRs) controlling cardiac fibrosis were identified in recent years; however, a more global approach to identify miRNAs involved in fibrosis is missing. METHODS AND RESULTS: Functional miRNA mimic library screens were applied in human cardiac fibroblasts (HCFs) to identify annotated miRNAs inducing proliferation. In parallel, miRNA deep sequencing was performed after subjecting HCFs to proliferating and resting stimuli, additionally enabling discovery of novel miRNAs. In-depth in vitro analysis confirmed the pro-fibrotic nature of selected, highly conserved miRNAs miR-20a-5p and miR-132-3p. To determine downstream cellular pathways and their role in the fibrotic response, targets of the annotated miRNA candidates were modulated by synthetic siRNA. We here provide evidence that repression of autophagy and detoxification of reactive oxygen species by miR-20a-5p and miR-132-3p explain some of their pro-fibrotic nature on a mechanistic level. CONCLUSION: We here identified both miR-20a-5p and miR-132-3p as crucial regulators of fibrotic pathways in an in vitro model of human cardiac fibroblast biology.


Assuntos
Fibroblastos/metabolismo , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/genética , Miocárdio/citologia , Análise de Sequência de RNA , Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Sequência de Bases , Fibrose , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Regulação da Expressão Gênica , Humanos , Inativação Metabólica/genética , MicroRNAs/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Superóxido Dismutase/metabolismo
7.
Circulation ; 141(9): 751-767, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-31948273

RESUMO

BACKGROUND: Myocardial fibrosis is a hallmark of cardiac remodeling and functionally involved in heart failure development, a leading cause of deaths worldwide. Clinically, no therapeutic strategy is available that specifically attenuates maladaptive responses of cardiac fibroblasts, the effector cells of fibrosis in the heart. Therefore, our aim was to develop novel antifibrotic therapeutics based on naturally derived substance library screens for the treatment of cardiac fibrosis. METHODS: Antifibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts, subsequent validation, and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of human cardiac fibroblasts, modulation of apoptosis, and extracellular matrix expression. In vitro findings were confirmed in vivo with an angiotensin II-mediated murine model of cardiac fibrosis in both preventive and therapeutic settings, as well as in the Dahl salt-sensitive rat model. To investigate the mechanism underlying the antifibrotic potential of the lead compounds, treatment-dependent changes in the noncoding RNAome in primary human cardiac fibroblasts were analyzed by RNA deep sequencing. RESULTS: High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds. CONCLUSIONS: We identified the molecules bufalin and lycorine as drug candidates for therapeutic applications in cardiac fibrosis and diastolic dysfunction.


Assuntos
Alcaloides de Amaryllidaceae/farmacologia , Bufanolídeos/farmacologia , Cardiomiopatias/prevenção & controle , Fármacos Cardiovasculares/farmacologia , Fibroblastos/efeitos dos fármacos , Fenantridinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , Cardiomiopatias/fisiopatologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Diástole , Modelos Animais de Doenças , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Ensaios de Triagem em Larga Escala , Humanos , Hipertensão/complicações , Hipertensão/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Ratos Endogâmicos Dahl , Selenoproteína P/genética , Selenoproteína P/metabolismo , Função Ventricular Esquerda/efeitos dos fármacos
8.
Sci Rep ; 9(1): 15277, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31649303

RESUMO

Fabry disease is an X-linked deficiency of the lysosomal hydrolase alpha-galactosidase A (alpha-Gal). This results in an accumulation of globotriaosylceramide (GL-3/Gb3) in a variety of cells with subsequent functional impairment. The continuous progress of FD often leads to decreased quality of life and premature death caused by multi-organic complications. The overall aim of our study was to determine the amount of circulating miRNAs in Fabry patients and to test whether ERT would alter the level of individual circulating miRNAs. We used miRNA sequencing by the HTG EdgeSeq System to identify the circulating miRNA pool from Fabry patients with and without enzyme replacement therapy (n = 6). In total, 296 miRNAs in serum of patients were identified. Among them 9 miRNAs were further evaluated in extra serum samples (n = 31) using real-time qPCR and 6 of them showed significant differential expression. The resulting miRNA pattern may help to better understand mechanisms involved in the beneficial effects of ERT and these new miRNA markers could help to estimate the efficacy of ERT or to identify Fabry patients with specific need for ERT.


Assuntos
MicroRNA Circulante/sangue , Terapia de Reposição de Enzimas/métodos , Doença de Fabry/sangue , alfa-Galactosidase/uso terapêutico , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Adulto Jovem
9.
Neurol Neuroimmunol Neuroinflamm ; 6(6): e623, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31575652

RESUMO

OBJECTIVE: To validate a previously discovered microRNA (miRNA) panel in the CSF of patients with MS, we now tested the diagnostic value of CSF-derived candidate miRNAs in a case-control study in a larger MS cohort. METHODS: The levels of miR-181c, miR-633, and miR-922 were analyzed in the CSF of 218 patients with MS and 211 patients with other neurologic diseases (OND) by real-time quantitative reverse transcriptase PCR. RESULTS: CSF levels of both miR-181c (p < 0.001 and miR-633 p < 0.001) were higher in patients with MS patients compared with patients with OND. Both miR-181c (area under the curve [AUC] 0.75, 95% CI 0.70-0.80, p < 0.001) and miR-633 (AUC 0.75, 95% CI 0.68-0.83, p < 0.001) differentiated MS from OND. Combining both miRNAs yielded a sensitivity of 62% and specificity of 89% to differentiate MS from OND. miR-922 was not confirmed to be differentially expressed in this validation cohort. CONCLUSIONS: The results of this so far largest study on CSF-based miRNAs confirm the diagnostic value of miR-181c and miR-633 for MS. The present study may help to extend the diagnostic tools for patients with suspected MS and may add further knowledge to the pathology of the disease. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that CSF-derived miR-181c and miR-633 distinguish patients with MS from patients with OND.


Assuntos
MicroRNAs/líquido cefalorraquidiano , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/diagnóstico , Adulto , Idoso , Biomarcadores/líquido cefalorraquidiano , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
10.
Mol Ther Nucleic Acids ; 18: 363-374, 2019 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-31634682

RESUMO

Long non-coding RNAs (lncRNAs) have potential as novel therapeutic targets in cardiovascular diseases, but detailed information about the intercellular lncRNA shuttling mechanisms in the heart is lacking. Here, we report an important novel crosstalk between cardiomyocytes and fibroblasts mediated by the transfer of lncRNA-enriched extracellular vesicles (EVs) in the context of cardiac ischemia. lncRNA profiling identified two hypoxia-sensitive lncRNAs: ENSMUST00000122745 was predominantly found in small EVs, whereas lncRNA Neat1 was enriched in large EVs in vitro and in vivo. Vesicles were taken up by fibroblasts, triggering expression of profibrotic genes. In addition, lncRNA Neat1 was transcriptionally regulated by P53 under basal conditions and by HIF2A during hypoxia. The function of Neat1 was further elucidated in vitro and in vivo. Silencing of Neat1 in vitro revealed that Neat1 was indispensable for fibroblast and cardiomyocyte survival and affected fibroblast functions (reduced migration capacity, stalled cell cycle, and decreased expression of fibrotic genes). Of translational importance, genetic loss of Neat1 in vivo resulted in an impaired heart function after myocardial infarction highlighting its translational relevance.

11.
Cardiovasc Res ; 115(12): 1804-1810, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30843048

RESUMO

AIMS: Delayed re-endothelialization after balloon angioplasty in patients with coronary or peripheral artery disease impairs vascular healing and leads to neointimal proliferation. In the present study, we examined the effect of RNA-binding motif protein 38 (Rbm38) during re-endothelialization in a murine model of experimental vascular injury. METHODS AND RESULTS: Left common carotid arteries of C57BL/6 mice were electrically denudated and endothelial regeneration was evaluated. Profiling of RNA-binding proteins revealed dysregulated expression of Rbm38 in the denudated and regenerated areas. We next tested the importance of Rbm38 in human umbilical vein endothelial cells (HUVECS) and analysed its effects on cellular proliferation, migration and apoptosis. Rbm38 silencing in vitro demonstrated important beneficial functional effects on migratory capacity and proliferation of endothelial cells. In vivo, local silencing of Rbm38 also improved re-endothelialization of denuded carotid arteries. Luciferase reporter assay identified miR-98 and let-7f to regulate Rbm38 and the positive proliferative properties of Rbm38 silencing in vitro and in vivo were mimicked by therapeutic overexpression of these miRNAs. CONCLUSION: The present data identified Rbm38 as an important factor of the regulation of various endothelial cell functions. Local inhibition of Rbm38 as well as overexpression of the upstream regulators miR-98 and let-7f improved endothelial regeneration in vivo and thus may be a novel therapeutic entry point to avoid endothelial damage after balloon angioplasty.


Assuntos
Lesões das Artérias Carótidas/terapia , Proliferação de Células , Células Endoteliais/metabolismo , Proteínas de Ligação a RNA/metabolismo , Reepitelização , Regiões 3' não Traduzidas , Animais , Apoptose , Sítios de Ligação , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Movimento Celular , Modelos Animais de Doenças , Células Endoteliais/patologia , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Fatores de Tempo
12.
J Cardiovasc Transl Res ; 12(5): 447-458, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30840186

RESUMO

Calcification of aortic valves leads to aortic stenosis mainly in elderly individuals, but the underlying molecular mechanisms are still not understood. Here, we studied microRNA (miR, miRNA) expression and function in healthy and stenotic human aortic valves. We identified miR-21, miR-24, and miR-143 to be highly upregulated in stenotic aortic valves. Using luciferase reporter systems, we found direct binding of miR-143 to the 3'UTR region of the matrix gla protein (MGP), which in turn is a key factor to sustain homeostasis in aortic valves. In subsequent experiments, we demonstrated a therapeutic potential of miRNA regulation during calcification in cardiac valvular interstitial cells. Collectively, our data provide evidence that deregulated miR expression contributes to the development of stenotic valve disease and thus form novel therapeutic opportunities of this severe cardiovascular disease.


Assuntos
Estenose da Valva Aórtica/metabolismo , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Calcinose/metabolismo , MicroRNAs/metabolismo , Regiões 3' não Traduzidas , Idoso , Idoso de 80 Anos ou mais , Animais , Estenose da Valva Aórtica/genética , Estenose da Valva Aórtica/patologia , Sítios de Ligação , Calcinose/genética , Calcinose/patologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Masculino , MicroRNAs/genética , Osteogênese , Transdução de Sinais , Sus scrofa , Regulação para Cima , Proteína de Matriz Gla
13.
Sci Rep ; 9(1): 20350, 2019 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-31889077

RESUMO

Hypertrophic cardiomyopathy (HCM) is one of the most common hereditary heart diseases and is associated with a high risk of sudden cardiac death. HCM is characterized by pronounced hypertrophy of cardiomyocytes, fiber disarray and development of fibrosis and can be divided into a non-obstructive (HNCM) and obstructive form (HOCM) therefore requiring personalized therapeutic therapies. In the present study, we investigated the expression patterns of several circulating circular RNAs (circRNAs) as potential biomarkers in patients with HCM. We included 64 patients with HCM and 53 healthy controls to the study and quantitatively measured the expression of a set of circRNAs already known to be associated with cardiac diseases (circDNAJC6) and/or being highly abundant in blood (circTMEM56 and circMBOAT2). Abundancy of circRNAs was then correlated to relevant clinical parameters. Serum expression levels of circRNAs DNAJC6, TMEM56 and MBOAT2 were downregulated in patients with HCM. The inverse association between circRNA levels and HCM remained unchanged even after adjusting for confounding factors. All circRNAs, evaluated separately or in combination, showed a robust discrimination capacity when comparing control subjects with HCM, HNCM or HOCM patients (AUC from 0.722 to 0.949). Two circRNAs, circTMEM56 and circDNAJC6, significantly negatively correlated with echocardiographic parameters for HOCM. Collectively, circulating circRNAs DNAJC6, TMEM56 and MBOAT2 can distinguish between healthy and HCM patients. In addition, circTMEM56 and circDNAJC6 could serve as indicators of disease severity in patients with HOCM. Thus, circRNAs emerge as novel biomarkers for HCM facilitating the clinical decision making in a personalized manner.


Assuntos
Cardiomiopatia Hipertrófica/diagnóstico , Cardiomiopatia Hipertrófica/genética , Ácidos Nucleicos Livres , RNA Circular/sangue , Biomarcadores , Cardiomiopatia Hipertrófica/sangue , Estudos de Casos e Controles , Ecocardiografia , Feminino , Testes de Função Cardíaca , Humanos , Masculino , Razão de Chances , Curva ROC
14.
Sci Rep ; 8(1): 17584, 2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30498227

RESUMO

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

15.
PLoS One ; 13(10): e0204235, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30332417

RESUMO

INTRODUCTION: Amyloidosis is caused by dysregulation of protein folding resulting in systemic or organ specific amyloid aggregation. When affecting the heart, amyloidosis can cause severe heart failure, which is associated with a high morbidity and mortality. Different subtypes of cardiac amyloidosis exist e.g. transthyretin cardiac amyloidosis and senile cardiac amyloidosis. Today, diagnostics is primarily based on cardiac biopsies and no clinically used circulating blood-based biomarkers existing. Therefore, our aim was to identify circulating microRNAs in patients with different forms of amyloidosis. METHODS: Blood was collected from healthy subjects (n = 10), patients with reduced ejection fraction (EF < 35%; n = 10), patients affected by transthyretin cardiac amyloidosis (n = 13) as well as senile cardiac amyloidosis (n = 11). After performing TaqMan array profiling, promising candidates, in particular miR-99a-5p, miR-122-5p, miR-27a-3p, miR-221-3p, miR-1180-3p, miR-155-5p, miR-339-3p, miR-574-3p, miR-342-3p and miR-329-3p were validated via quantitative real time PCR. RESULTS: The validation experiments revealed a significant upregulation of miR-339-3p in patients affected with senile cardiac amyloidosis compared to controls. This corresponded to the array profiling results. In contrast, there was no deregulation in the other patient groups. CONCLUSION: MiR-339-3p was increased in blood of patients with senile cardiac amyloidosis. Therefore, miR-339-3p is a potential candidate as biomarker for senile cardiac amyloidosis in future studies. Larger patient cohorts should be investigated.


Assuntos
Amiloidose/genética , Cardiopatias/genética , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Regulação para Cima , Idoso , Idoso de 80 Anos ou mais , Neuropatias Amiloides Familiares/sangue , Neuropatias Amiloides Familiares/genética , Amiloidose/sangue , Estudos de Casos e Controles , MicroRNA Circulante/genética , Feminino , Perfilação da Expressão Gênica , Marcadores Genéticos , Cardiopatias/sangue , Humanos , Masculino , Pessoa de Meia-Idade
16.
Eur J Heart Fail ; 20(1): 78-85, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29027324

RESUMO

AIMS: Non-coding microRNAs (miRNAs) are critically involved in cardiovascular pathophysiology. Since they are measurable in most body fluids, they have been proposed as circulating biomarkers. We examined the prognostic value of a specific candidate miRNA in a large cohort of patients with chronic heart failure (HF) enrolled in a multicentre clinical trial. METHODS AND RESULTS: Plasma levels of miR-132 were measured using miRNA-specific PCR-based technologies at randomization in 953 patients with chronic, symptomatic HF from the GISSI-Heart Failure trial. The association with fatal (all-cause and cardiovascular death) and non-fatal events (time to first admission to hospital for cardiovascular reasons or worsening of HF) and the incremental risk prediction were estimated in adjusted models. Higher circulating miR-132 levels were independently associated with younger age, better renal filtration, ischaemic aetiology of HF, more severe HF symptoms, higher diastolic blood pressure, higher cholesterol, and male sex. After extensive adjustment for demographic, clinical, and echocardiographic risk factors and baseline NT-proBNP concentrations, miR-132 remained associated only with HF hospitalizations (hazard ratio 0.79, 95% confidence interval 0.66-0.95, P = 0.01) and improved its risk prediction with the continuous net reclassification index (cNRI 0.205, P = 0.001). CONCLUSION: In well characterized patients with chronic HF, circulating miR-132 levels rise with the severity of HF. Lower circulating miR-132 levels improved risk prediction for HF readmission beyond traditional risk factors, but not for mortality. MiR-132 may be helpful to intensify strategies aimed at reducing re-hospitalization, which has a substantial health and economic burden in HF.


Assuntos
Ácidos Graxos Ômega-3/administração & dosagem , Insuficiência Cardíaca/sangue , Hospitalização/tendências , MicroRNAs/sangue , Medição de Risco/métodos , Rosuvastatina Cálcica/administração & dosagem , Volume Sistólico/fisiologia , Administração Oral , Idoso , Biomarcadores/sangue , MicroRNA Circulante/sangue , Relação Dose-Resposta a Droga , Método Duplo-Cego , Ecocardiografia , Feminino , Seguimentos , Alemanha/epidemiologia , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/epidemiologia , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Itália/epidemiologia , Masculino , MicroRNAs/genética , Morbidade/tendências , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Risco , Volume Sistólico/efeitos dos fármacos , Taxa de Sobrevida/tendências
17.
Sci Rep ; 7(1): 14747, 2017 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-29116107

RESUMO

Although several risk factors such as infarct size have been identified, the progression of heart failure (HF) remains difficult to predict in clinical practice. Using an experimental rat model of post-myocardial infarction (MI), we previously identified 45 proteins differentially modulated during HF by proteomic analysis. This study sought to identify microRNAs (miRNAs) able to regulate these proteins and to test their relevance as biomarkers for HF. In silico bioinformatical analysis selected 13 miRNAs related to the 45 proteins previously identified. These miRNAs were analyzed in the rat and in cohorts of patients phenotyped for left ventricular remodeling (LVR). We identified that 3 miRNAs, miR-21-5p, miR-23a-3p and miR-222-3p, and their target Mn superoxide dismutase (SOD2) were significantly increased in LV and plasma of HF-rats. We found by luciferase activity a direct interaction of miR-222-3p with 3'UTR of SOD2. Transfection of human cardiomyocytes with miR-222-3p mimic or inhibitor induced respectively a decrease and an increase of SOD2 expression. Circulating levels of the 3 miRNAs and their target SOD2 were associated with high LVR post-MI in REVE-2 patients. We demonstrated for the first time the potential of microRNAs regulating SOD2 as new circulating biomarkers of HF.


Assuntos
Insuficiência Cardíaca/sangue , MicroRNAs/sangue , Superóxido Dismutase/sangue , Regiões 3' não Traduzidas , Animais , Biomarcadores/sangue , Modelos Animais de Doenças , Ventrículos do Coração/enzimologia , Masculino , Prognóstico , Proteômica/métodos , Processamento Pós-Transcricional do RNA , Ratos , Ratos Wistar , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Remodelação Ventricular
18.
PLoS One ; 12(8): e0183828, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28854245

RESUMO

Alloreactive CD8+ T-cells mediate the curative graft-versus-leukaemia effect, the anti-viral immunity and graft-versus-host-disease (GvHD) after allogeneic stem cell transplantation (SCT). Thus, immune reconstitution with CD8+ T-cells is critical for the outcome of patients after allogeneic SCT. Certain miRNAs such as miR-146a or miR-155 play an important role in the regulation of post-transplant immunity in mice. While some miRNAs e.g. miR-423 or miR-155 are regulated in plasma or full blood during acute GvHD also in man, the relevance and expression profile of miRNAs in T-cells after allogeneic SCT is unknown. miR-625-3p has recently been described to be overexpressed in colorectal malignancies where it promotes migration, invasion and apoptosis resistance. Since similar regulative functions in cancer and T-cells have been described for an increasing number of miRNAs, we assumed a role for the cancer-related miR-625-3p also in T-cells. Here, we studied miR-625-3p expression selectively in CD8+ T-cells both in vitro and during immune reconstitution after allogeneic SCT in man. T-cell receptor stimulation lead to miR-625-3p upregulation in human CD8+ T-cells in vitro. Maintenance of elevated miR-625-3p expression levels was dependent on ongoing T-cell proliferation and was abrogated by withdrawal of interleukin 2 or the mTOR inhibitor rapamycin. Finally, miR-625-3p expression was analyzed in human CD8+ T-cells purified from 137 peripheral blood samples longitudinally collected from 74 patients after allogeneic SCT. miR-625-3p expression was upregulated on day 25 and on day 45, i.e. during the early phase of CD8+ T-cell reconstitution after allogeneic SCT and subsequently declined with completion of CD8+ T-cell reconstitution until day 150. In conclusion, this study has shown for the first time that miR-625-3p is regulated in CD8+ T-cells during proliferation in vitro and during early immune reconstitution after allogeneic SCT in vivo. These results warrant further studies to identify the targets and function of miR-625-3p in CD8+ T-cells and to analyze its predictive value for an effective immune reconstitution.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , MicroRNAs/genética , Transplante de Células-Tronco/métodos , Regulação para Cima , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células/genética , Feminino , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Ativação Transcricional , Transplante Homólogo
19.
Stem Cell Res ; 20: 70-75, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28314201

RESUMO

MicroRNAs (miRNAs) repeatedly have been demonstrated to play important roles in the generation of induced pluripotent stem cells (iPSCs). To further elucidate the molecular mechanisms underlying transcription factor-mediated reprogramming we have established a model, which allows for the efficient screening of whole libraries of miRNAs modulating the generation of iPSCs from murine embryonic fibroblasts. Applying this model, we identified 14 miRNAs effectively inhibiting iPSC generation, including miR-132 and miR-212. Intriguingly, repression of these miRNAs during iPSC generation also resulted in significantly increased reprogramming efficacy. MiRNA target evaluation by qRT-PCR, Western blot, and luciferase assays revealed two crucial epigenetic regulators, the histone acetyl transferase p300 as well as the H3K4 demethylase Jarid1a (KDM5a) to be directly targeted by both miRNAs. Moreover, we demonstrated that siRNA-mediated knockdown of either p300 or Jarid1a recapitulated the miRNA effects and led to a significant decrease in reprogramming efficiency. Thus, conducting a full library miRNA screen we here describe a miRNA family, which markedly reduces generation of iPSC and upon inhibition in turn enhances reprogramming. These miRNAs, at least in part, exert their functions through repression of the epigenetic modulators p300 and Jarid1a, highlighting these two molecules as an endogenous epigenetic roadblock during iPSC generation.


Assuntos
Reprogramação Celular , Epigenômica/métodos , Fibroblastos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , MicroRNAs/metabolismo , Fatores de Transcrição/metabolismo , Regiões 3' não Traduzidas , Animais , Antagomirs/metabolismo , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Fibroblastos/metabolismo , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Camundongos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição de p300-CBP/antagonistas & inibidores , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismo
20.
Transplantation ; 101(1): e26-e33, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27653298

RESUMO

BACKGROUND: Cardiac allograft vasculopathy (CAV) remains the leading cause of morbidity and mortality after orthotopic heart transplantation (OHT). Because of its clinically silent progression and lack of symptoms, detection is often difficult and invasive coronary angiography is performed routinely. To date, there are no established noninvasive biomarkers available for prediction of CAV in transplanted patients.MicroRNAs (miRNAs) are highly conserved, small noncoding RNA molecules that negatively regulate gene expression. As they are detectable in peripheral blood, recent studies have suggested miRNAs as biomarkers for various cardiovascular diseases. Thus, we hypothesized that circulating miRNAs may serve as noninvasive biomarkers for CAV. METHODS: To determine the regulation of circulating miRNAs, we performed miRNA profiling studies in plasma samples of OHT patients with confirmed high-degree CAV and a matched control group consisting of patients without any signs of CAV at least 5 years after OHT. Candidate miRNAs were verified by quantitative reverse transcriptase polymerase chain reaction. RESULTS: Microarray analysis revealed 5 candidate miRNAs (miR-34a, miR-98, miR-155, miR-204, miR-628-5p) that were differentially regulated in plasma samples of patients with CAV and therefore were selected for verification by quantitative reverse transcriptase polymerase chain reaction. In CAV patients, plasma levels of miR-628-5p and miR-155 were significantly increased (P = 0.001 and P = 0.028, respectively). A miR628-5p value above 1.336 was able to predict CAV with a sensitivity of 72% and a specificity of 83%. CONCLUSIONS: For the first time, the present study identifies the circulating miRNA miR-628-5p as a novel potential biomarker of CAV in patients after OHT.


Assuntos
Doença da Artéria Coronariana/genética , Transplante de Coração/efeitos adversos , MicroRNAs/genética , Adulto , Idoso , Aloenxertos , Área Sob a Curva , Estudos de Casos e Controles , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/diagnóstico , Feminino , Perfilação da Expressão Gênica/métodos , Marcadores Genéticos , Humanos , Masculino , MicroRNAs/sangue , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Projetos Piloto , Valor Preditivo dos Testes , Curva ROC , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Resultado do Tratamento , Regulação para Cima , Adulto Jovem
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