Deregulation of microRNA-181c in cerebrospinal fluid of patients with clinically isolated syndrome is associated with early conversion to relapsing-remitting multiple sclerosis.

BACKGROUND: MiRNA-181c, miRNA-633 and miRNA-922 have been reported to be deregulated in multiple sclerosis. OBJECTIVES: To investigate the association between miRNA-181c, miRNA-633 and miRNA-922 and conversion from clinically isolated syndrome (CIS) to relapsing-remitting multiple sclerosis (RRMS); and to compare microRNAs in cerebrospinal fluid (CSF) and serum with regard to dysfunction of the blood-CSF barrier. METHODS: CSF and serum miRNA-181c, miRNA-633 and miRNA-922 were retrospectively determined by quantitative real-time polymerase chain reaction in CIS patients with (CIS-RRMS) and without (CIS-CIS) conversion to RRMS within 1 year. RESULTS: Thirty of 58 CIS patients developed RRMS. Cerebrospinal fluid miRNA-922, serum miRNA-922 and cerebrospinal fluid miRNA-181c were significantly higher in CIS-RRMS compared to CIS-CIS (P=0.027, P=0.048, P=0.029, respectively). High levels of cerebrospinal fluid miRNA-181c were independently associated with conversion from CIS to RRMS in multivariate Cox regression analysis (hazard ratio 2.99, 95% confidence interval 1.41-6.34, P=0.005). A combination of high cerebrospinal fluid miRNA-181c, younger age and more than nine lesions on magnetic resonance imaging showed the highest specificity (96%) and positive predictive value (94%) for conversion from CIS to RRMS. MiRNA-181c was higher in serum than in cerebrospinal fluid (P <0.001), while miRNA-633 and miRNA-922 were no different in cerebrospinal fluid and serum. Cerebrospinal fluid/serum albumin quotients did not correlate with microRNAs in cerebrospinal fluid (all P>0.711). CONCLUSIONS: Cerebrospinal fluid miRNA-181c might serve as a biomarker for early conversion to RRMS. Moreover, our data suggest an intrathecal origin of microRNAs detected in the cerebrospinal fluid.

Circulating miR-let7a levels predict future diagnosis of chronic thromboembolic pulmonary hypertension.

Distinct patterns of circulating microRNAs (miRNAs) were found to be involved in misguided thrombus resolution. Thus, we aimed to investigate dysregulated miRNA signatures during the acute phase of pulmonary embolism (PE) and test their diagnostic and predictive value for future diagnosis of chronic thromboembolic pulmonary hypertension (CTEPH). Microarray screening and subsequent validation in a large patient cohort (n = 177) identified three dysregulated miRNAs as potential biomarkers: circulating miR-29a and miR-720 were significantly upregulated and miR-let7a was significantly downregulated in plasma of patients with PE. In a second validation study equal expression patterns for miR-29a and miR-let7a regarding an acute event of recurrent venous thromboembolism (VTE) or deaths were found. MiR-let7a concentrations significantly correlated with echocardiographic and laboratory parameters indicating right ventricular (RV) dysfunction. Additionally, circulating miR-let7a levels were associated with diagnosis of CTEPH during follow-up. Regarding CTEPH diagnosis, ROC analysis illustrated an AUC of 0.767 (95% CI 0.54-0.99) for miR-let7a. Using logistic regression analysis, a calculated patient-cohort optimized miR-let7a cut-off value derived from ROC analysis of ≥ 11.92 was associated with a 12.8-fold increased risk for CTEPH. Therefore, miR-let7a might serve as a novel biomarker to identify patients with haemodynamic impairment and as a novel predictor for patients at risk for CTEPH.

Circulating microRNAs in Fabry Disease.

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.

Differential effects of organic nitrates on endothelial progenitor cells are determined by oxidative stress.

OBJECTIVE: Reduced levels and impaired function of endothelial progenitor cells (EPCs) foster development and progression of atherosclerotic lesions. Endothelial nitric oxide synthase (eNOS)-derived NO regulates EPC mobilization and function. Organic nitrates release NO, and therefore may favorably affect EPC biology. METHODS AND RESULTS: We compared the effects of 2 different nitrates on circulating EPC numbers and function. Treatment of rats with pentaerythritol-trinitrate (PETriN) or isosorbide dinitrate (ISDN) increased circulating EPC levels. EPC from ISDN- but not PETriN-treated animals displayed impaired migratory capacity and increased reactive oxygen species formation in EPCs. In vitro treatment with ISDN reduced migration and incorporation of human EPCs into vascular structures on matrigel, whereas PETriN improved EPC function. ISDN, but not PETriN, increased NADPH oxidase-mediated oxidative stress in cultured human EPCs. Addition of polyethylene-glycolated superoxide dismutase or diphenyliodonium normalized both ISDN-induced superoxide anion production and impaired migratory capacity of EPCs. CONCLUSIONS: Long-acting nitrates increase levels of circulating EPCs, but differ in their effects on EPC function dependent on the induction of intracellular oxidative stress. Organic nitrates that improve EPC function may confer long-term cardiovascular protection based on their beneficial effects on EPC biology.

Circulating microRNA-132 levels improve risk prediction for heart failure hospitalization in patients with chronic heart failure.

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.

Preclinical Development of a MicroRNA-Based Therapy for Elderly Patients With Myocardial Infarction.

BACKGROUND: Aging populations show higher incidences of myocardial infarction (MI) and heart failure (HF). Cardiac remodeling post-MI leads to progressive impaired cardiac function caused by a disarray of several processes including derailed autophagy. Microribonucleic acids (miRNAs) are known to be key players in cardiovascular disease but their involvement in cardiac autophagy and aging is not well understood. OBJECTIVES: This study sought to identify new miRNA candidates that regulate cardiac autophagy and aging. METHODS: We exploited a high-throughput, fluorescence-activated cell sorting-based green fluorescent protein-LC3 detection method to measure the autophagic flux in cardiomyocytes after transfection of a precursor miRNA library consisting of 380 miRNAs. This was followed by a series of molecular and in vivo studies. RESULTS: Together with additional expression screenings, we identified miR-22 as an abundant and strong inhibitor of the cardiac autophagy process. Cardiac miR-22 expression levels increased during aging of mice as well as in aging neonatal cardiomyocytes in vitro by a P53-dependent mechanism. Inhibition of miR-22 in aging cardiomyocytes in vitro activated autophagy and inhibited cellular hypertrophy. Pharmacological inhibition of miR-22 post-MI in older mice activated cardiac autophagy, prevented post-infarction remodeling, and improved cardiac function compared with control subjects. Interestingly, similar effects were less pronounced in younger mice with significantly lower cardiac miR-22 expression levels. In addition, circulating levels of miR-22 in 154 patients with systolic HF were highly associated with early mortality. CONCLUSIONS: We concluded that miR-22 is an important regulator of cardiac autophagy and a potential therapeutic target, especially in the older myocardium. Finally, circulating miR-22 provides prognostic information for HF patients, highlighting miR-22 as a promising therapeutic and biomarker candidate for cardiovascular disorders.

miR-21 promotes fibrosis in an acute cardiac allograft transplantation model.

AIMS: Cardiac transplantation is the only curative therapy for end-stage heart failure. Fibrosis is one of the major causes for impaired function of cardiac allografts. MicroRNAs, a class of small non-coding RNAs, play a critical role in the development of cardiovascular disease, but the role of microRNAs in cardiac allograft failure is not well understood. METHODS AND RESULTS: To uncover a role of microRNAs during cardiac graft fibrosis, we generated global microRNA profiles in allogeneic (BALB/c in C57BL/6N) and isogeneic (C57BL/6N in C57BL/6N) murine hearts after transplantation. miR-21 together with cardiac fibrosis was increased in cardiac allografts compared with isografts. Likewise, patients with cardiac rejection after heart transplantation showed increased cardiac miR-21 levels. miR-21 was induced upon treatment with IL-6 in a monocyte cell line. Overexpression of miR-21 in this monocyte cell line activated a fibrotic gene programme and promoted monocyte-to-fibrocyte transition together with activation of chemokine (C-C) motif ligand 2 (monocyte chemoattractant protein 1) via the phosphatase and tensin homologue/activator protein 1 regulatory axis. In vivo, both genetic and pharmacological inhibition of miR-21 successfully reduced fibrosis and fibrocyte accumulation in cardiac allografts. CONCLUSION: Thus, inhibition of miR-21 is a novel strategy to target fibrosis development in cardiac allografts.

MicroRNAs in Serum and Bile of Patients with Primary Sclerosing Cholangitis and/or Cholangiocarcinoma.

BACKGROUND AND AIM: Patients with primary sclerosing cholangitis (PSC) are at high risk for the development of cholangiocarcinoma (CC). Analysis of micro ribonucleic acid (MiRNA) patterns is an evolving research field in biliary pathophysiology with potential value in diagnosis and therapy. Our aim was to evaluate miRNA patterns in serum and bile of patients with PSC and/or CC. METHODS: Serum and bile from consecutive patients with PSC (n = 40 (serum), n = 52 (bile)), CC (n = 31 (serum), n = 19 (bile)) and patients with CC complicating PSC (PSC/CC) (n = 12 (bile)) were analyzed in a cross-sectional study between 2009 and 2012. As additional control serum samples from healthy individuals were analyzed (n = 12). The miRNA levels in serum and bile were determined with global miRNA profiling and subsequent miRNA-specific polymerase chain reaction-mediated validation. RESULTS: Serum analysis revealed significant differences for miR-1281 (p = 0.001), miR-126 (p = 0.001), miR-26a (p = 0.001), miR-30b (p = 0.001) and miR-122 (p = 0.034) between patients with PSC and patients with CC. All validated miRNAs were significantly lower in healthy individuals. MiR-412 (p = 0.001), miR-640 (p = 0.001), miR-1537 (p = 0.003) and miR-3189 (p = 0.001) were significantly different between patients with PSC and PSC/CC in bile. CONCLUSIONS: Patients with PSC and/or CC have distinct miRNA profiles in serum and bile. Furthermore, miRNA concentrations are different in bile of patients with CC on top of PSC indicating the potential diagnostic value of these miRNAs.

MicroRNA signatures differentiate preserved from reduced ejection fraction heart failure.

AIMS: Differentiation of heart failure with reduced (HFrEF) or preserved (HFpEF) ejection fraction independent of echocardiography is challenging in the community. Diagnostic strategies based on monitoring circulating microRNA (miRNA) levels may prove to be of clinical value in the near future. The aim of this study was to identify a novel miRNA signature that could be a useful HF diagnostic tool and provide valuable clinical information on whether a patient has HFrEF or HFpEF. METHODS AND RESULTS: MiRNA biomarker discovery was carried out on three patient cohorts, no heart failure (no-HF), HFrEF, and HFpEF, using Taqman miRNA arrays. The top five miRNA candidates were selected based on differential expression in HFpEF and HFrEF (miR-30c, -146a, -221, -328, and -375), and their expression levels were also different between HF and no-HF. These selected miRNAs were further verified and validated in an independent cohort consisting of 225 patients. The discriminative value of BNP as a HF diagnostic could be improved by use in combination with any of the miRNA candidates alone or in a panel. Combinations of two or more miRNA candidates with BNP had the ability to improve significantly predictive models to distinguish HFpEF from HFrEF compared with using BNP alone (area under the receiver operating characteristic curve >0.82). CONCLUSION: This study has shown for the first time that various miRNA combinations are useful biomarkers for HF, and also in the differentiation of HFpEF from HFrEF. The utility of these biomarker combinations can be altered by inclusion of natriuretic peptide. MiRNA biomarkers may support diagnostic strategies in subpopulations of patients with HF.

Blood-based microRNA signatures differentiate various forms of cardiac hypertrophy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is caused by mutations in different structural genes and induces pathological hypertrophy with sudden cardiac death as a possible consequence. HCM can be separated into hypertrophic non-obstructive and obstructive cardiomyopathy (HNCM/HOCM) with different clinical treatment approaches. We here distinguished between HNCM, HOCM, cardiac amyloidosis and aortic stenosis by using microRNA profiling and investigated potential interactions between circulating miRNA levels and the most common mutations in MYH7and MYBPC3 genes. METHODS: Our study included 4 different groups: 23 patients with HNCM, 28 patients with HOCM, 47 patients with aortic stenosis and 22 healthy controls. Based on previous findings, 8 different cardiovascular known microRNAs (miR-1, miR-21, miR-29a, miR-29b, miR-29c, miR-133a, miR-155 and miR-499) were studied in serum of all patients and compared with clinically available patient data. RESULTS: We found miR-29a levels to be increased in patients with HOCM and correlating markers of cardiac hypertrophy. This was not the case in HNCM patients. In contrast, we identified miR-29c to be upregulated in aortic stenosis but not the other patient groups. ROC curve analysis of miR-29a/c distinguished between HOCM patients and aortic stenosis patients. MiR-29a and miR-155 levels discriminated HNCM patients from patients with senile cardiac amyloidosis. MiR-29a increased mainly in HOCM patients with a mutation in MYH7, whereas miR-155 was decreased in hypertrophic cardiomyopathy patients with a mutation in MYBPC3. CONCLUSION: We demonstrated that miR-29a and miR-29c show a specific signature to distinguish between aortic stenosis, hypertrophic non-obstructive and obstructive cardiomyopathies and thus could be developed into clinically useful biomarkers.