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1.
Sci Rep ; 11(1): 19752, 2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34611227

RESUMO

Although metabolic syndrome (MetS) is linked to an elevated risk of cardiovascular disease (CVD), the cardiac-specific risk mechanism is unknown. Obesity, hypertension, and diabetes (all MetS components) are the most common form of CVD and represent risk factors for worse COVID-19 outcomes compared to their non MetS peers. Here, we use obese Yorkshire pigs as a highly relevant animal model of human MetS, where pigs develop the hallmarks of human MetS and reproducibly mimics the myocardial pathophysiology in patients. Myocardium-specific mass spectroscopy-derived metabolomics, proteomics, and transcriptomics enabled the identity and quality of proteins and metabolites to be investigated in the myocardium to greater depth. Myocardium-specific deregulation of pro-inflammatory markers, propensity for arterial thrombosis, and platelet aggregation was revealed by computational analysis of differentially enriched pathways between MetS and control animals. While key components of the complement pathway and the immune response to viruses are under expressed, key N6-methyladenosin RNA methylation enzymes are largely overexpressed in MetS. Blood tests do not capture the entirety of metabolic changes that the myocardium undergoes, making this analysis of greater value than blood component analysis alone. Our findings create data associations to further characterize the MetS myocardium and disease vulnerability, emphasize the need for a multimodal therapeutic approach, and suggests a mechanism for observed worse outcomes in MetS patients with COVID-19 comorbidity.


Assuntos
COVID-19/patologia , Suscetibilidade a Doenças , Síndrome Metabólica/patologia , Animais , Fatores de Coagulação Sanguínea/genética , Fatores de Coagulação Sanguínea/metabolismo , COVID-19/complicações , COVID-19/virologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dieta Hiperlipídica/veterinária , Modelos Animais de Doenças , Humanos , Imunidade Inata/genética , Síndrome Metabólica/complicações , Síndrome Metabólica/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Miocárdio/metabolismo , Estresse Oxidativo/genética , Agregação Plaquetária , Receptores Purinérgicos P2Y1/genética , Receptores Purinérgicos P2Y1/metabolismo , Sistema Renina-Angiotensina , Fatores de Risco , SARS-CoV-2/isolamento & purificação , Suínos , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
2.
Life Sci Alliance ; 4(12)2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34663679

RESUMO

Extracellular vesicles (EVs) mediate intercellular signaling by transferring their cargo to recipient cells, but the functional consequences of signaling are not fully appreciated. RBC-derived EVs are abundant in circulation and have been implicated in regulating immune responses. Here, we use a transgenic mouse model for fluorescence-based mapping of RBC-EV recipient cells to assess the role of this intercellular signaling mechanism in heart disease. Using fluorescent-based mapping, we detected an increase in RBC-EV-targeted cardiomyocytes in a murine model of ischemic heart failure. Single cell nuclear RNA sequencing of the heart revealed a complex landscape of cardiac cells targeted by RBC-EVs, with enrichment of genes implicated in cell proliferation and stress signaling pathways compared with non-targeted cells. Correspondingly, cardiomyocytes targeted by RBC-EVs more frequently express cellular markers of DNA synthesis, suggesting the functional significance of EV-mediated signaling. In conclusion, our mouse model for mapping of EV-recipient cells reveals a complex cellular network of RBC-EV-mediated intercellular communication in ischemic heart failure and suggests a functional role for this mode of intercellular signaling.

4.
Artigo em Inglês | MEDLINE | ID: mdl-33782736

RESUMO

Genitourinary complications following orthopaedic intervention are uncommon but well-described occurrences and exist on a spectrum of severity. These complications vary depending on the anatomic location and surgical approach, with surgery of the spine, hip, and pelvis of particular concern. Injuries to the urinary tract may present acutely with urinary retention or hematuria. However, they often have a delayed presentation with severe complications such as urinary fistula and recurrent infection. Delayed presentations may place the onus of timely and proper diagnosis on the orthopaedic provider, who may serve as the patient's primary source of long-term follow-up. Detailed knowledge of anatomy and at-risk structures is key to both preventing and identifying injury. Although iatrogenic injury is not always avoidable, early identification can help to facilitate timely evaluation and management to prevent long-term complications such as bladder dysfunction, obstructive renal injury, sexual dysfunction, and chronic pain.  Keywords: urologic injury, bladder injury, genitourinary injury, hip arthroplasty, pelvic ring injuries, spine surgery.

5.
Circ Res ; 128(1): e1-e23, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33092465

RESUMO

RATIONALE: Previous translational studies implicate plasma extracellular microRNA-30d (miR-30d) as a biomarker in left ventricular remodeling and clinical outcome in heart failure (HF) patients, although precise mechanisms remain obscure. OBJECTIVE: To investigate the mechanism of miR-30d-mediated cardioprotection in HF. METHODS AND RESULTS: In rat and mouse models of ischemic HF, we show that miR-30d gain of function (genetic, lentivirus, or agomiR-mediated) improves cardiac function, decreases myocardial fibrosis, and attenuates cardiomyocyte (CM) apoptosis. Genetic or locked nucleic acid-based knock-down of miR-30d expression potentiates pathological left ventricular remodeling, with increased dysfunction, fibrosis, and cardiomyocyte death. RNA sequencing of in vitro miR-30d gain and loss of function, together with bioinformatic prediction and experimental validation in cardiac myocytes and fibroblasts, were used to identify and validate direct targets of miR-30d. miR-30d expression is selectively enriched in cardiomyocytes, induced by hypoxic stress and is acutely protective, targeting MAP4K4 (mitogen-associate protein kinase 4) to ameliorate apoptosis. Moreover, miR-30d is secreted primarily in extracellular vesicles by cardiomyocytes and inhibits fibroblast proliferation and activation by directly targeting integrin α5 in the acute phase via paracrine signaling to cardiac fibroblasts. In the chronic phase of ischemic remodeling, lower expression of miR-30d in the heart and plasma extracellular vesicles is associated with adverse remodeling in rodent models and human subjects and is linked to whole-blood expression of genes implicated in fibrosis and inflammation, consistent with observations in model systems. CONCLUSIONS: These findings provide the mechanistic underpinning for the cardioprotective association of miR-30d in human HF. More broadly, our findings support an emerging paradigm involving intercellular communication of extracellular vesicle-contained miRNAs (microRNAs) to transregulate distinct signaling pathways across cell types. Functionally validated RNA biomarkers and their signaling networks may warrant further investigation as novel therapeutic targets in HF.


Assuntos
MicroRNAs/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Comunicação Parácrina , Função Ventricular Esquerda , Remodelação Ventricular , Animais , Apoptose , Células Cultivadas , Modelos Animais de Doenças , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Regulação da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , /metabolismo , Ratos Sprague-Dawley , Ratos Transgênicos , Transdução de Sinais
6.
Circ Res ; 127(5): 631-646, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32418505

RESUMO

RATIONALE: Cardiac CITED4 (CBP/p300-interacting transactivators with E [glutamic acid]/D [aspartic acid]-rich-carboxylterminal domain4) is induced by exercise and is sufficient to cause physiological hypertrophy and mitigate adverse ventricular remodeling after ischemic injury. However, the role of endogenous CITED4 in response to physiological or pathological stress is unknown. OBJECTIVE: To investigate the role of CITED4 in murine models of exercise and pressure overload. METHODS AND RESULTS: We generated cardiomyocyte-specific CITED4 knockout mice (C4KO) and subjected them to an intensive swim exercise protocol as well as transverse aortic constriction (TAC). Echocardiography, Western blotting, qPCR, immunohistochemistry, immunofluorescence, and transcriptional profiling for mRNA and miRNA (microRNA) expression were performed. Cellular crosstalk was investigated in vitro. CITED4 deletion in cardiomyocytes did not affect baseline cardiac size or function in young adult mice. C4KO mice developed modest cardiac dysfunction and dilation in response to exercise. After TAC, C4KOs developed severe heart failure with left ventricular dilation, impaired cardiomyocyte growth accompanied by reduced mTOR (mammalian target of rapamycin) activity and maladaptive cardiac remodeling with increased apoptosis, autophagy, and impaired mitochondrial signaling. Interstitial fibrosis was markedly increased in C4KO hearts after TAC. RNAseq revealed induction of a profibrotic miRNA network. miR30d was decreased in C4KO hearts after TAC and mediated crosstalk between cardiomyocytes and fibroblasts to modulate fibrosis. miR30d inhibition was sufficient to increase cardiac dysfunction and fibrosis after TAC. CONCLUSIONS: CITED4 protects against pathological cardiac remodeling by regulating mTOR activity and a network of miRNAs mediating cardiomyocyte to fibroblast crosstalk. Our findings highlight the importance of CITED4 in response to both physiological and pathological stimuli.


Assuntos
Cardiomegalia Induzida por Exercícios , Hipertrofia Ventricular Esquerda/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/metabolismo , Função Ventricular Esquerda , Remodelação Ventricular , Animais , Comunicação Celular , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Regulação da Expressão Gênica , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Miócitos Cardíacos/patologia , Ratos , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Transcriptoma
7.
Surgery ; 167(2): 493-498, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31493902

RESUMO

BACKGROUND: Cardiac surgery and cardiopulmonary bypass are associated with alterations in blood pressure in the perioperative period, which, if uncontrolled, can result in end organ damage or dysfunction. Microvessels, significant contributors to blood pressure, both in the myocardium and peripheral skeletal muscle, have diminished responsiveness to major mediators of vascular tone, including thromboxane and serotonin after cardiopulmonary bypass. Responsiveness of these vessels to ß-adrenergic stimulation, a major mediator of vascular tone, has not yet been studied. In this report, we investigated the role of ß-adrenergic receptors in vascular tone regulation in human skeletal muscle microvessels before and after ß-adrenergic stimulation. METHODS: Skeletal muscle microvessels were isolated from patients undergoing cardiac surgery before and after cardiopulmonary bypass. Vessels were exposed in an ex vivo model to the ß-adrenergic agonist isoproterenol, or the direct adenylyl cyclase activator, forskolin, and the selective ß-receptor antagonist ICI18.551 hydrochloride plus isoproterenol. Immunofluorescence of ß receptors and Western blotting were also performed. RESULTS: Microvessels showed diminished responsiveness to isoproterenol (10-6 to 10-4M) after cardiopulmonary bypass (n = 8/group, P = .01). Pretreatment with the selective ß-2 blocker ICI18.551 (10-6M) prevented isoproterenol-induced microvascular relaxation (P = .001). Forskolin-induced relaxation response was also significantly diminished after cardiopulmonary bypass (n = 4/group, P < .05 versus before cardiopulmonary bypass). No significant changes in the total protein expression of ß-1, ß-2, and ß-3 receptors were detected by western blotting or immunofluorescence. CONCLUSION: Microvessels isolated from human skeletal muscle show diminished responsiveness to isoproterenol and its downstream activator forskolin after cardiopulmonary bypass, suggesting there is an alteration in ß-adrenergic receptor responsive in adenylate cyclase. The relaxation response to isoproterenol was via activation ß-2 receptors without changes in ß-adrenergic receptor abundance.


Assuntos
Arteríolas/metabolismo , Ponte Cardiopulmonar/efeitos adversos , Receptores Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta , Antagonistas Adrenérgicos beta , Idoso , Idoso de 80 Anos ou mais , Arteríolas/efeitos dos fármacos , Colforsina , Feminino , Humanos , Técnicas In Vitro , Isoproterenol , Masculino , Músculo Esquelético/irrigação sanguínea
8.
PLoS One ; 14(12): e0225857, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31790488

RESUMO

Although the high-fat-diet-induced metabolic syndrome (MetS) is a precursor of human cardiac pathology, the myocardial metabolic state in MetS is far from clear. The discrepancies in metabolite handling between human and small animal models and the difficulties inherent in obtaining human tissue complicate the identification of the myocardium-specific metabolic response in patients. Here we use the large animal model of swine that develops the hallmark criteria of human MetS. Our comparative metabolomics together with transcriptomics and computational nonnegative matrix factorization (NMF) interpretation of the data exposes significant decline in metabolites related to the fatty acid oxidation, glycolysis, and pentose phosphate pathway. Behind the reversal lies decreased expression of enzymes that operate in the pathways. We showed that diminished glycogen deposition is a metabolic signature of MetS in the pig myocardium. The depletion of glycogen arises from disbalance in expression of genes that break down and synthesize glycogen. We show robust acetoacetate accumulation and activated expression of key enzymes in ketone body formation, catabolism and transporters, suggesting a shift in fuel utilization in MetS. A contrasting enrichment in O-GlcNAcylated proteins uncovers hexosamine pathway and O-GlcNAcase (OGA) expression involvement in the myocardial response to MetS. Although the hexosamine biosynthetic pathway (HBP) activity and the availability of the UDP-GlcNAc substrate in the MetS myocardium is low, the level of O-GlcNacylated proteins is high as the O-GlcNacase is significantly diminished. Our data support the perception of transcriptionally driven myocardial alterations in expression of standard fatty acids, glucose metabolism, glycogen, and ketone body related enzymes and subsequent paucity of their metabolite products in MetS. This aberrant energy metabolism in the MetS myocardium provide insight into the pathogenesis of CVD in MetS.


Assuntos
Redes e Vias Metabólicas , Síndrome Metabólica/metabolismo , Miocárdio/metabolismo , Animais , Colesterol na Dieta/efeitos adversos , Dieta , Glicosilação , Masculino , Metaboloma , Metabolômica , N-Acetilglucosaminiltransferases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Risco , Suínos , Aprendizado de Máquina não Supervisionado , beta-N-Acetil-Hexosaminidases/metabolismo
9.
J Physician Assist Educ ; 30(3): 155-158, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31385904

RESUMO

The Physician Assistant Clinical Knowledge Rating and Assessment Tool (PACKRAT) was developed to be an objective, comprehensive self-assessment tool for students. When the PACKRAT exam was initially developed, its goal was to help students determine what level of knowledge they currently possessed and what they had to learn prior to graduation. The purpose of this study was to review PACKRAT test-taker scores over the past 5 years and analyze the variations in test administration. Deidentified PACKRAT scores, exam length in minutes, and proctored or unproctored status, along with time extensions were analyzed from 2013 to 2018. Descriptive statistics and frequency counts were used to summarize the data. An independent samples t-test was used to determine if there was a difference in test-taker scores between proctored and unproctored exams. The 83,271 student test-taker exam data were analyzed, and time ranged from 180 to 360 minutes with a mean of 226.5 minutes. When comparing test-taker scores between proctored and unproctored exams, the mean scores were 145.02 and 144.77, respectively, with no significant difference. The Physician Assistant Clinical Knowledge Rating and Assessment Tool can be confidently used and compared to national scores whether a program administers the exam proctored or without a proctor. The average time taken for exam completion is close to the recommended 225 minutes that is suggested by Physician Assistant Education Association PACKRAT exam developers.


Assuntos
Avaliação Educacional/métodos , Assistentes Médicos/educação , Avaliação Educacional/normas , Humanos , Assistentes Médicos/normas , Assistentes Médicos/estatística & dados numéricos , Estudos Retrospectivos , Estudantes de Ciências da Saúde
10.
J Psychiatr Res ; 108: 7-23, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30419488

RESUMO

BACKGROUND: Depression independently predicts poor outcomes in heart failure (HF) patients, including increased mortality, morbidity and 30-day re-hospitalization. In this network meta-analysis, we compared different interventions designed to treat depression in HF. MATERIALS AND METHODS: Electronic searches were conducted using Ovid MEDLINE, EMBASE, CINAHL, Web of Science, and PsycINFO up to November 2016. Included randomized clinical trials (RCTs) compared interventions (Exercise therapy (ET), cognitive behavioral therapy (CBT) or antidepressant (AD) medications) for depression in heart failure patients. The primary outcome was change in depressive symptoms based on validated measures of depression. Network meta-analysis based on random effects model estimating standardized mean difference (SMD) with 95% confidence interval (CI), compared the effects of the 3 classes of interventions with respect to usual care or placebo control conditions. RESULTS: A total of 21 RCTs (including 4563 HF patients) reporting the effects of treating depression in HF patients were included in the analysis. In comparison to placebo or usual standard of care, ET (SMD -0.38; 95% CI -0.54 to -0.22) and CBT (SMD -0.29; 95% CI -0.58 to -0.01) were associated with reduction in depressive symptoms whereas AD (SMD -0.16; 95% CI -0.44 to 0.11) was less effective. CONCLUSIONS: This meta-analysis is suggestive of therapeutic benefit of ET and CBT in comparison to usual standard of care in treating depression in HF patients. However, comparison among the three interventions was not conclusive. Future randomized clinical trials are warranted to compare the therapeutic effects of ET, CBT and AD in such patients.


Assuntos
Depressão/complicações , Depressão/terapia , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/psicologia , Insuficiência Cardíaca/terapia , Humanos , Metanálise em Rede
11.
JAMA Cardiol ; 3(9): 871-876, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30090932

RESUMO

Importance: Mortality is high among patients heart failure (HF) who are receiving treatment, and therefore identifying new pathways rooted in preclinical cardiac remodeling phenotypes may afford novel biomarkers and therapeutic avenues. Circulating extracellular RNAs (ex-RNAs) are an emerging class of biomarkers with target-organ epigenetic effects relevant to myocardial biology, although large human investigations remain limited. Objective: To measure the association of highly expressed circulating ex-RNAs with left ventricular remodeling and incident HF in a community-based cohort. Design, Setting, and Participants: This is a prospective observational cohort study of individuals who were included in the eighth examination of the Framingham Offspring Cohort (2005-2008). Collected data include measurements of the left ventricle via electrocardiography, determination of circulating ex-RNAs in plasma, and incidence of heart failure. Data analysis was completed from December 2016 to June 2018. Exposures: A total of 398 circulating ex-RNA molecules in plasma were measured by reverse transcription polymerase chain reaction; disease ontology analysis was also performed. Main Outcomes and Measures: Echocardiographic indices of left ventricular (LV) remodeling and incident heart failure. Results: A total of 2763 participants of the Framingham Heart Study with measured ex-RNAs (mean [SD] age, 66.3 [9.0] years; 1499 [54.3%] female) were included in this study. Of this sample, 2429 to 2432 individuals had echocardiographic measures recorded (depending on the measurement). A total of 2681 individuals had HF status determined, of whom 116 (4.3%) experienced HF (median [interquartile range] follow-up, 7.7 [6.6-8.6] years). We identified 12 ex-RNAs associated with LV mass and at least 1 other echocardiographic phenotype (LV end-diastolic volume or left atrial dimension). Of these 12 ex-RNAs, 3 micro RNAs (miR-17, miR-20a, and miR-106b) were associated with a 15% reduction in long-term incident HF per 2-fold increase in circulating level during the follow-up period, after adjustments for age, sex, established HF risk factors, and prevalent or interim myocardial infarction. These 3 RNAs shared sequence homology and targeted a shared group of messenger RNAs that specified pathways relevant to HF (eg, transforming growth factor-ß signaling, growth/cell cycle, and apoptosis), and shared a disease association with hypertension in disease ontology analysis. Conclusions and Relevance: This study identifies a group of circulating, noncoding RNAs associated with echocardiographic phenotypes, long-term incident HF, and pathways relevant to myocardial remodeling in a large community-based sample. Further investigations into the functional biology of these ex-RNAs are warranted for surveillance for HF prevention.


Assuntos
Insuficiência Cardíaca/epidemiologia , Ventrículos do Coração/diagnóstico por imagem , MicroRNAs/sangue , Infarto do Miocárdio/epidemiologia , Remodelação Ventricular , Idoso , Ecocardiografia , Feminino , Insuficiência Cardíaca/genética , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/genética , Estudos Prospectivos , Análise de Sobrevida
12.
J Physician Assist Educ ; 29(3): 158-161, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30028357

RESUMO

The PAEA End of Rotation™ exams were developed to assess medical knowledge of the 7 core supervised clinical practice experiences, including Emergency Medicine, Family Medicine, Internal Medicine, General Surgery, Pediatrics, Women's Health, and Psychiatry and Behavioral Health. The examinations were created by experienced PA educators and national exam experts with continual review and content updates. This paper summarizes changes since the inception of the exam program, including test construction and development, reliability, validity, and scale scoring. They are built using content blueprints and topic lists that were developed by experienced PA educators and national examination experts. All examination items are peer reviewed by PA educators and statistically validated for accuracy and consistency by psychometricians who specialize in examination development. This article will review the changes since the inception of the exam program including test construction and development, reliability, validity and scale scoring. Also addressed is an update on the PAEA End of Curriculum™ examination.


Assuntos
Estágio Clínico/organização & administração , Avaliação Educacional/métodos , Assistentes Médicos/educação , Estágio Clínico/normas , Competência Clínica , Avaliação Educacional/normas , Humanos , Exame Físico/métodos , Exame Físico/normas , Reprodutibilidade dos Testes , Estudos Retrospectivos
13.
BMC Genomics ; 19(1): 331, 2018 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-29728066

RESUMO

BACKGROUND: Evolving interest in comprehensively profiling the full range of small RNAs present in small tissue biopsies and in circulating biofluids, and how the profile differs with disease, has launched small RNA sequencing (RNASeq) into more frequent use. However, known biases associated with small RNASeq, compounded by low RNA inputs, have been both a significant concern and a hurdle to widespread adoption. As RNASeq is becoming a viable choice for the discovery of small RNAs in low input samples and more labs are employing it, there should be benchmark datasets to test and evaluate the performance of new sequencing protocols and operators. In a recent publication from the National Institute of Standards and Technology, Pine et al., 2018, the investigators used a commercially available set of three tissues and tested performance across labs and platforms. RESULTS: In this paper, we further tested the performance of low RNA input in three commonly used and commercially available RNASeq library preparation kits; NEB Next, NEXTFlex, and TruSeq small RNA library preparation. We evaluated the performance of the kits at two different sites, using three different tissues (brain, liver, and placenta) with high (1 µg) and low RNA (10 ng) input from tissue samples, or 5.0, 3.0, 2.0, 1.0, 0.5, and 0.2 ml starting volumes of plasma. As there has been a lack of robust validation platforms for differentially expressed miRNAs, we also compared low input RNASeq data with their expression profiles on three different platforms (Abcam Fireplex, HTG EdgeSeq, and Qiagen miRNome). CONCLUSIONS: The concordance of RNASeq results on these three platforms was dependent on the RNA expression level; the higher the expression, the better the reproducibility. The results provide an extensive analysis of small RNASeq kit performance using low RNA input, and replication of these data on three downstream technologies.


Assuntos
Biblioteca Gênica , RNA/metabolismo , Encéfalo/metabolismo , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Fígado/metabolismo , MicroRNAs/análise , MicroRNAs/química , Placenta/metabolismo , Gravidez , Análise de Componente Principal , RNA/química , Kit de Reagentes para Diagnóstico , Análise de Sequência de RNA
14.
Methods Mol Biol ; 1740: 1-15, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29388131

RESUMO

Extracellular RNA (exRNA) has recently expanded as a highly important area of study in biomarker discovery and cancer therapeutics. exRNA consists of diverse RNA subpopulations that are normally protected from degradation by incorporation into membranous vesicles or by lipid/protein association. They are found circulating in biofluids, and have proven highly promising for minimally invasive diagnostic and prognostic purposes, particularly in oncology. Recent work has made progress in our understanding of exRNAs-from their biogenesis, compartmentalization, and vesicle packaging to their various applications as biomarkers and therapeutics, as well as the new challenges that arise in isolation and purification for accurate and reproducible analysis. Here we review the most recent advancements in exRNA research.


Assuntos
Espaço Extracelular/metabolismo , RNA/metabolismo , Animais , Vesículas Extracelulares/metabolismo , Humanos , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo
15.
Circ Heart Fail ; 11(2): e004278, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29438982

RESUMO

BACKGROUND: Plasma extracellular RNAs have recently garnered interest as biomarkers in heart failure (HF). Most studies in HF focus on single extracellular RNAs related to phenotypes and outcomes, and few describe their functional roles. We hypothesized that clusters of plasma microRNAs (miRNAs) associated with left ventricular (LV) remodeling in human HF would identify novel subsets of genes involved in HF in animal models. METHODS AND RESULTS: We prospectively measured circulating miRNAs in 64 patients with systolic HF (mean age, 64.8 years; 91% men; median LV ejection fraction, 26%) with serial echocardiography (10 months apart) during medical therapy. We defined LV reverse remodeling as a 15% reduction in LV end-systolic volume index. Using principal components analysis, we identified a component associated with LV reverse remodeling (odds ratio=3.99; P=0.01) that provided risk discrimination for LV reverse remodeling superior to a clinical model (C statistic, 0.58 for a clinical model versus 0.71 for RNA-based model). Using network bioinformatics, we uncovered genes not previously widely described in HF regulated simultaneously by >2 miRNAs. We observed increased myocardial expression of these miRNAs during HF development in animals, with downregulation of target gene expression, suggesting coordinate miRNA-mRNA regulation. Target mRNAs were involved in autophagy, metabolism, and inflammation. CONCLUSIONS: Plasma miRNAs associated with LV reverse remodeling in humans are dysregulated in animal HF and target clusters of genes involved in mechanisms implicated in HF. A translational approach integrating human HF, bioinformatics, and model systems may uncover novel pathways involved in HF. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT00351390.


Assuntos
Insuficiência Cardíaca Sistólica/sangue , Insuficiência Cardíaca/sangue , MicroRNAs/sangue , Disfunção Ventricular Esquerda/sangue , Remodelação Ventricular/fisiologia , Idoso , Idoso de 80 Anos ou mais , Terapia de Ressincronização Cardíaca/métodos , Progressão da Doença , Feminino , Insuficiência Cardíaca/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Volume Sistólico/fisiologia , Disfunção Ventricular Esquerda/fisiopatologia
16.
Am J Physiol Heart Circ Physiol ; 313(6): H1162-H1167, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-28916639

RESUMO

Exercise improves cardiometabolic and vascular function, although the mechanisms remain unclear. Our objective was to demonstrate the diversity of circulating extracellular RNA (ex-RNA) release during acute exercise in humans and its relevance to exercise-mediated benefits on vascular inflammation. We performed plasma small RNA sequencing in 26 individuals undergoing symptom-limited maximal treadmill exercise, with replication of our top candidate miRNA in a separate cohort of 59 individuals undergoing bicycle ergometry. We found changes in miRNAs and other ex-RNAs with exercise (e.g., Y RNAs and tRNAs) implicated in cardiovascular disease. In two independent cohorts of acute maximal exercise, we identified miR-181b-5p as a key ex-RNA increased in plasma after exercise, with validation in a separate cohort. In a mouse model of acute exercise, we found significant increases in miR-181b-5p expression in skeletal muscle after acute exercise in young (but not older) mice. Previous work revealed a strong role for miR-181b-5p in vascular inflammation in obesity, insulin resistance, sepsis, and cardiovascular disease. We conclude that circulating ex-RNAs were altered in plasma after acute exercise target pathways involved in inflammation, including miR-181b-5p. Further investigation into the role of known (e.g., miRNA) and novel (e.g., Y RNAs) RNAs is warranted to uncover new mechanisms of vascular inflammation on exercise-mediated benefits on health.NEW & NOTEWORTHY How exercise provides benefits to cardiometabolic health remains unclear. We performed RNA sequencing in plasma during exercise to identify the landscape of small noncoding circulating transcriptional changes. Our results suggest a link between inflammation and exercise, providing rich data on circulating noncoding RNAs for future studies by the scientific community.


Assuntos
MicroRNA Circulante/sangue , Exercício Físico , Inflamação/sangue , Síndrome Metabólica/sangue , RNA de Transferência/sangue , Adulto , Idoso , Animais , Ciclismo , MicroRNA Circulante/genética , Teste de Esforço/métodos , Feminino , Marcadores Genéticos , Nível de Saúde , Humanos , Inflamação/diagnóstico , Inflamação/genética , Masculino , Síndrome Metabólica/diagnóstico , Síndrome Metabólica/genética , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/sangue , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo , RNA de Transferência/genética , Fatores de Tempo
17.
Obesity (Silver Spring) ; 25(10): 1734-1744, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28834285

RESUMO

OBJECTIVE: Extracellular microRNAs (miRNAs) represent functional biomarkers for obesity and related disorders; this study investigated plasma miRNAs in insulin resistance phenotypes in obesity. METHODS: One hundred seventy-five miRNAs were analyzed in females with obesity (insulin sensitivity, n = 11; insulin resistance, n = 19; type 2 diabetes, n = 15) and without obesity (n = 12). Correlations between miRNA level and clinical parameters and levels of 15 miRNAs in a murine obesity model were investigated. RESULTS: One hundred six miRNAs were significantly (adjusted P ≤ 0.05) different between controls and at least one obesity phenotype, including miRNAs with the following attributes: previously reported roles in obesity and altered circulating levels (e.g., miR-122, miR-192); known roles in obesity but no reported changes in circulating levels (e.g., miR-378a); and no current reported role in, or association with, obesity (e.g., miR-28-5p, miR-374b, miR-32). The miRNAs in the latter group were found to be associated with extracellular vesicles. Forty-eight miRNAs showed significant correlations with clinical parameters; stepwise regression retained let-7b, miR-144-5p, miR-34a, and miR-532-5p in a model predictive of insulin resistance (R2 = 0.57, P = 7.5 × 10-8 ). Both miR-378a and miR-122 were perturbed in metabolically relevant tissues in a murine model of obesity. CONCLUSIONS: This study expands on the role of extracellular miRNAs in insulin-resistant phenotypes of obesity and identifies candidate miRNAs not previously associated with obesity.


Assuntos
Diabetes Mellitus Tipo 2/genética , Resistência à Insulina/genética , MicroRNAs/genética , Obesidade/genética , Adulto , Diabetes Mellitus Tipo 2/sangue , Feminino , Humanos , Pessoa de Meia-Idade , Obesidade/sangue
18.
Nat Commun ; 8: 15201, 2017 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-28541289

RESUMO

A number of microRNAs (miRNAs, miRs) have been shown to play a role in skeletal muscle atrophy, but their role is not completely understood. Here we show that miR-29b promotes skeletal muscle atrophy in response to different atrophic stimuli in cells and in mouse models. miR-29b promotes atrophy of myotubes differentiated from C2C12 or primary myoblasts, and conversely, its inhibition attenuates atrophy induced by dexamethasone (Dex), TNF-α and H2O2 treatment. Targeting of IGF-1 and PI3K(p85α) by miR-29b is required for induction of muscle atrophy. In vivo, miR-29b overexpression is sufficient to promote muscle atrophy while inhibition of miR-29b attenuates atrophy induced by denervation and immobilization. These data suggest that miR-29b contributes to multiple types of muscle atrophy via targeting of IGF-1 and PI3K(p85α), and that suppression of miR-29b may represent a therapeutic approach for muscle atrophy induced by different stimuli.


Assuntos
MicroRNAs/metabolismo , Atrofia Muscular/classificação , Atrofia Muscular/genética , Animais , Sequência de Bases , Linhagem Celular , Regulação da Expressão Gênica , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Atrofia Muscular/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Ratos Sprague-Dawley , Regulação para Cima/genética , Fator de Transcrição YY1/metabolismo
20.
Skelet Muscle ; 6(1): 41, 2016 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-27906108

RESUMO

BACKGROUND: Duchenne muscle dystrophy (DMD) is one of the most common lethal genetic diseases of children worldwide and is 100% fatal. Steroids, the only therapy currently available, are marred by poor efficacy and a high side-effect profile. New therapeutic approaches are urgently needed. METHODS: Here, we leverage PGC-1α, a powerful transcriptional coactivator known to protect against dystrophy in the mdx murine model of DMD, to search for novel mechanisms of protection against dystrophy. RESULTS: We identify heme oxygenase-1 (HO-1) as a potential novel target for the treatment of DMD. Expression of HO-1 is blunted in the muscles from the mdx murine model of DMD, and further reduction of HO-1 by genetic haploinsufficiency worsens muscle damage in mdx mice. Conversely, induction of HO-1 pharmacologically protects against muscle damage. Mechanistically, HO-1 degrades heme into biliverdin, releasing in the process ferrous iron and carbon monoxide (CO). We show that exposure to a safe low dose of CO protects against muscle damage in mdx mice, as does pharmacological treatment with CO-releasing molecules. CONCLUSIONS: These data identify HO-1 and CO as novel therapeutic agents for the treatment of DMD. Safety profiles and clinical testing of inhaled CO already exist, underscoring the translational potential of these observations.


Assuntos
Monóxido de Carbono/farmacologia , Heme Oxigenase-1/metabolismo , Distrofia Muscular de Duchenne/tratamento farmacológico , Adolescente , Adulto , Animais , Monóxido de Carbono/administração & dosagem , Monóxido de Carbono/uso terapêutico , Células Cultivadas , Criança , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo
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