Prognostic Value of Circulating MicroRNA-210 Levels in Patients with Moderate to Severe Aortic Stenosis

<div><p>Background</p><p>Circulating micro-RNAs have been proposed as a novel class of cardiovascular (CV) biomarkers, but whether they meet analytical requirements and provide additional information to establish risk indices have not been established. miR-210 levels are increased in subjects with low VO₂ max, which is a recognized risk factor in patients with aortic stenosis (AS), and we hypothesized that circulating miR-210 levels may be increased in patients with AS and associated with a poor prognosis.</p><p>Methods</p><p>We measured circulating miR-210 levels by real-time PCR in 57 patients with moderate to severe AS and in 10 age- and gender-matched healthy controls. The merit of miR-210 as a biomarker was assessed according to established criteria, including by comparing miR-210 levels with NT-proBNP and miR-22 levels, which is another miRNA biomarker candidate.</p><p>Results</p><p>All patients and control subjects had miR-210 levels within the range of detection (Cq<35) and the analytical variability was low. Circulating miR-210 levels were 2.0±0.2 [mean±SEM] fold increased in AS patients compared to controls (p = 0.002), whereas miR-22 levels were not differently expressed in the AS patients (0.12±0.06 fold increase, p = 0.45). The increase in miR-210 levels in AS patients was comparable to the increment in NT-proBNP levels: [AUC] 0.82 (95% CI 0.70–0.90) vs. 0.85 (0.75–0.93), respectively, p = 0.71. During a median follow-up of 1287 days, 15 patients (26%) died. There was a significant association between higher circulating levels of miR-210 and increased mortality during follow-up: hazard ratio [supra- vs. inframedian levels] 3.3 (95% CI 1.1–10.5), p = 0.039. Adjusting for other risk indices in multivariate analysis did not attenuate the prognostic merit of circulating miR-210 levels.</p><p>Conclusion</p><p>Circulating miR-210 levels are increased in patients with AS and provide independent prognostic information to established risk indices. Analytical characteristics were also excellent supporting the potential of micro-RNAs as novel CV biomarkers.</p></div

Echocardiographical data in the patients with aortic stenosis and control subjects.

<p>LV, left ventricular; and LVEF, left ventricular ejection fraction.</p><p><b>*</b>n = 53 for E/e’ and n = 52 for E/A ratio.</p

Assosciation between miR-210 levels and mortality during follow-up with miR-210 levels divided by the median (p = 0.029 by the log-rank test).

<p>Assosciation between miR-210 levels and mortality during follow-up with miR-210 levels divided by the median (p = 0.029 by the log-rank test).</p

Extraction from serum and RT-qPCR of the spike-in control cel-miR-39 were performed with minimal variation between samples (CV 2.4% for AS patients [#1–36 and #38–58] and 2.2% for the control subjects [#59–74]), which indicates that miRNAs seem to meet the analytical requirements proposed for novel biomarkers.

<p>Data are presented as the absolute Cq values.</p

Correlations in AS patients and control subjects (Ctr) between miR-210 levels and clinical and echocardiographical parameters of myocardial structure and function.

<p>BMI, body mass index; NT-proBNP, N-terminal pro-B-type natriuretic peptide; and miR, micro-RNA; LV, left ventricular; and LVEF, left ventricular ejection fraction.</p><p><b>*</b>n = 53 for E/e’ and n = 52 for E/A ratio.</p

Predictors for mortality during follow-up by univariate and multivariate Cox proportional hazard regression analysis.

<p>BMI, body mass index; NYHA class, New York Heart Association functional class; COPD, chronic obstructive pulmonary disease; ACEI, angiotensin-converting enzyme inhibitor; AII-blocker, angiotensin II blocker; LV, left ventricular; LVEF, left ventricular ejection fraction NT-proBNP, N-terminal pro-B-type natriuretic peptide; and miR, micro-RNA.</p><p>NT-proBNP levels were transformed by the natural logarithm prior to regression analysis due to a right-skewed distribution.</p

Baseline data stratified according to treatment allocation.

<p>NYHA: New York Heart Association; TIA: transient ischemic attack; ACEI: angiotensin converting enzyme inhibitor; ARB: angiotensin receptor blocker.</p

Correlation between left ventricular ejection fraction as measured by echocardiography and as measured by magnetic resonance imaging.

<p>Data from baseline and follow-up are pooled. The Pearson correlation coefficient between results obtained by magnetic resonance imaging (MRI) and results obtained by echocardiography was 0.82 (p < 0.001).</p

Results from cardiac imaging exams, New York Heart Association (NYHA) classification and quality of life measurements.

<p>*This row represents left ventricular ejection fraction (LVEF) by magnetic resonance imaging (MRI) or, if the latter result was not available at both baseline and follow-up, LVEF by echocardiography (E/D, N = 18). ^† p < 0.05 for difference between baseline and follow-up. CI: confidence interval; EDV: end diastolic left ventricular internal volume; ESV: end systolic left ventricular internal volume; NT-proBNP: N-terminal pro-B-type natriuretic peptide; LDL: low density lipoprotein; MLHFQ: Minnesota Living with Heart Failure Questionnaire; VAS: visual analogue scale.</p

Changes in markers of inflammation and extracellular matrix turnover.

<p>Changes in C-reactive protein (CRP), soluble tumor necrosis factor receptor type 1 (sTNF-R1), and procollagen type I and III N-terminal pro-peptides (PINP and PIIINP) stratified by treatment allocation. p-values for between-group differences in changes were computed by independent group t-tests. While PINP increased more in patients treated with rosuvastatin as compared to patients treated with placebo (p  =  0.03), treatment did not affect any of the other markers of inflammation or matrix remodeling. Boxes: 25–75 percentiles; whiskers: 5–95 percentiles.</p