Martin M Dokter

Reproducibility of telomere length assessment: an international collaborative study.

Background: Telomere length is a putative biomarker of ageing, morbidity and mortality. Its application is hampered by lack of widely applicable reference ranges and uncertainty regarding the present limits of measurement reproducibility within and between laboratories. Methods: We instigated an international collaborative study of telomere length assessment: 10 different laboratories, employing 3 different techniques [Southern blotting, single telomere length analysis (STELA) and real-time quantitative PCR (qPCR)] performed two rounds of fully blinded measurements on 10 human DNA samples per round to enable unbiased assessment of intra- and inter-batch variation between laboratories and techniques. Results: Absolute results from different laboratories differed widely and could thus not be compared directly, but rankings of relative telomere lengths were highly correlated (correlation coefficients of 0.63–0.99). Intra-technique correlations were similar for Southern blotting and qPCR and were stronger than inter-technique ones. However, inter-laboratory coefficients of variation (CVs) averaged about 10% for Southern blotting and STELA and more than 20% for qPCR. This difference was compensated for by a higher dynamic range for the qPCR method as shown by equal variance after z-scoring. Technical variation per laboratory, measured as median of intra- and inter-batch CVs, ranged from 1.4% to 9.5%, with differences between laboratories only marginally significant (P = 0.06). Gel-based and PCR-based techniques were not different in accuracy. Conclusions: Intra- and inter-laboratory technical variation severely limits the usefulness of data pooling and excludes sharing of reference ranges between laboratories. We propose to establish a common set of physical telomere length standards to improve comparability of telomere length estimates between laboratories.

Short-term vitamin D3 supplementation lowers plasma renin activity in patients with stable chronic heart failure: An open-label, blinded end point, randomized prospective trial (VitD-CHF trial)

BACKGROUND Many chronic heart failure (CHF) patients have low vitamin D (VitD) and high plasma renin activity (PRA), which are both associated with poor prognosis. Vitamin D may inhibit renin transcription and lower PRA. We investigated whether vitamin D3 (VitD3) supplementation lowers PRA in CHF patients. METHODS AND RESULTS We conducted a single-center, open-label, blinded end point trial in 101 stable CHF patients with reduced left ventricular ejection fraction. Patients were randomized to 6 weeks of 2,000 IU oral VitD3 daily or control. At baseline, mean age was 64 ± 10 years, 93% male, left ventricular ejection fraction 35% ± 8%, and 56% had VitD deficiency. The geometric mean (95% CI) of 25-hydroxyvitamin D3 increased from 48 nmol/L (43-54) at baseline to 80 nmol/L (75-87) after 6 weeks in the VitD3 treatment group and decreased from 47 nmol/L (42-53) to 44 nmol/L (39-49) in the control group (P < .001). The primary outcome PRA decreased from 6.5 ng/mL per hour (3.8-11.2) to 5.2 ng/mL per hour (2.9-9.5) in the VitD3 treatment group and increased from 4.9 ng/mL per hour (2.9-8.5) to 7.3 ng/mL per hour (4.5-11.8) in the control group (P = .002). This was paralleled by a larger decrease in plasma renin concentration in the VitD3 treatment group compared to control (P = .020). No significant changes were observed in secondary outcome parameters, including N-terminal pro-B-type natriuretic peptide natriuretic peptide and fibrosis markers. CONCLUSIONS Most CHF patients had VitD deficiency and high PRA levels. Six weeks of supplementation with 2,000 IU VitD3 increased 25-hydroxyvitamin D3 levels and decreased PRA and plasma renin concentration.

Renal handling of galectin-3 in the general population, chronic heart failure, and hemodialysis.

Background Galectin‐3 is a biomarker for prognostication and risk stratification of patients with heart failure (HF). It has been suggested that renal function strongly relates to galectin‐3 levels. We aimed to describe galectin‐3 renal handling in HF. Methods and Results In Sprague–Dawley rats, we infused galectin‐3 and studied distribution and renal clearance. Furthermore, galectin‐3 was measured in urine and plasma of healthy controls, HF patients and hemodialysis patients. To mimic the human situation, we measured galectin‐3 before and after the artificial kidney. Infusion in rats resulted in a clear increase in plasma and urine galectin‐3. Plasma galectin‐3 in HF patients (n=101; mean age 64 years; 93% male) was significantly higher compared to control subjects (n=20; mean age 58 years; 75% male) (16.6 ng/mL versus 9.7 ng/mL, P<0.001), while urinary galectin‐3 in HF patients was comparable (28.1 ng/mL versus 35.1 ng/mL, P=0.830). The calculated galectin‐3 excretion rate was lower in HF patient (2.3 mL/min [1.5 to 3.4] versus 3.9 mL/min [2.3 to 6.4] in control subjects; P=0.005). This corresponded with a significantly lower fractional excretion of galectin‐3 in HF patients (2.4% [1.7 to 3.7] versus 3.0% [1.9 to 5.5]; P=0.018). These differences, however, were no longer significant after correction for age, gender, diabetes, and smoking. HF patients who received diuretics (49%) showed significantly higher aldosterone and galectin‐3 levels. Hemodialysis patients (n=105; mean age 63 years; 65% male), without urinary galectin‐3 excretion, had strongly increased median plasma galectin‐3 levels (70.6 ng/mL). Conclusions In this small cross‐sectional study, we report that urine levels of galectin‐3 are not increased in HF patients, despite substantially increased plasma galectin‐3 levels. The impaired renal handling of galectin‐3 in patients with HF may explain the described relation between renal function and galectin‐3 and may account for the elevated plasma galectin‐3 in HF.

Galectin‐3 and left ventricular reverse remodelling after surgical mitral valve repair

Mitral valve repair in patients with functional mitral regurgitation (FMR) has been associated with beneficial left ventricular (LV) reverse remodelling. Recently, galectin‐3 emerged as a marker of myocardial inflammation and fibrosis which may influence LV remodelling after surgery. The aim of the current study was to evaluate the association between pre‐operative galectin‐3 levels and LV reverse remodelling in heart failure patients with significant FMR who underwent mitral valve repair.

Corrigendum to "Reproducibility of Telomere Length Assessment - An International Collaborative Study" [International Journal of Epidemiology 2014, doi: 10.1093/ije/dyu191]

Background: Telomere length is a putative biomarker of ageing, morbidity and mortality. Its application is hampered by lack of widely applicable reference ranges and uncertainty regarding the present limits of measurement reproducibility within and between laboratories. Methods: We instigated an international collaborative study of telomere length assessment: 10 different laboratories, employing 3 different techniques [Southern blotting, single telomere length analysis (STELA) and real-time quantitative PCR (qPCR)] performed two rounds of fully blinded measurements on 10 human DNA samples per round to enable unbiased assessment of intraand inter-batch variation between laboratories and techniques. Results: Absolute results from different laboratories differed widely and could thus not be compared directly, but rankings of relative telomere lengths were highly correlated (correlation coefficients of 0.63–0.99). Intra-technique correlations were similar for Southern blotting and qPCR and were stronger than inter-technique ones. However, VC The Author 2014. Published by Oxford University Press on behalf of the International Epidemiological Association 1673 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. International Journal of Epidemiology, 2015, 1673–1683 doi: 10.1093/ije/dyu191 Advance Access Publication Date: 19 September 2014

Galectin-3 and sST2 in prediction of left ventricular ejection fraction after myocardial infarction.

BACKGROUND Fibrosis is a pivotal event in infarct repair and progressive remodeling after myocardial infarction (MI). Biomarkers may be used to monitor fibrosis, and therefore we evaluated the predictive value of galectin-3 and sST2 for cardiac remodeling after MI. METHODS Plasma galectin-3 and sST2 were measured in patients admitted with primary percutaneous coronary intervention (PCI) for acute MI, at baseline and at 4months. Left ventricular ejection fraction (LVEF) and infarct size were measured after 4months with cardiac MRI (CMR). RESULTS In total, 247 patients had blood samples and CMR data available (mean age 57.7±11.6years; 79.8% male). Increased baseline galectin-3 (≥17.8ng/mL) identified patients with lower LVEF (50.3% (±9.1) vs. non-elevated galectin-3 55.0% (±8.0); P<0.001), and larger infarct size (13.8g. (±12.9) vs. 8.6g. (±8.7); P=0.002) after 4months. Elevated sST2 (≥35.0ng/mL) did not predict decreased LVEF or larger infarct size. Furthermore we showed that at baseline, galectin-3 was an independent predictor for LVEF (β=-0.18; P=0.005) and infarct size (β=0.18; P=0.004). We repeated the analyses using median values of galectin-3 (13.4ng/mL) and sST2 (30.3ng/mL) as a cut point, and this validated our results. CONCLUSION The fibrosis biomarker galectin-3, but not sST2, taken immediately after MI, predicts LVEF and infarct size after 4months. We hypothesize that galectin-3 may play a role in the pathophysiology of cardiac remodeling after acute MI.

The liver X receptor agonist AZ876 protects against pathological cardiac hypertrophy and fibrosis without lipogenic side effects

Liver X receptors (LXRs) transcriptionally regulate inflammation, metabolism, and immunity. Synthetic LXR agonists have been evaluated for their efficacy in the cardiovascular system; however, they elicit prolipogenic side effects which substantially limit their therapeutic use. AZ876 is a novel high‐affinity LXR agonist. Herein, we aimed to determine the cardioprotective potential of LXR activation with AZ876.

Sodium restriction on top of renin-angiotensin-aldosterone system blockade increases circulating levels of N-acetyl-seryl-aspartyl-lysyl-proline in chronic kidney disease patients.

Objective: Sodium restriction potentiates the efficacy of the rennin–angiotensin–aldosterone system (RAAS)-blockade and improves long-term cardiovascular and renal protection, even independent of the better blood pressure control. The mechanisms underlying the potentiation of cardiorenal protection by sodium restriction are incompletely understood. RAAS-blockade with angiotensin-converting enzyme (ACE) inhibitors increases circulating levels of the anti-inflammatory and antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), which is assumed to contribute to its therapeutic effects. We hypothesized that sodium restriction on top of RAAS-blockade further increases AcSDKP, as a possible explanation for the enhanced effects of RAAS-blockade during sodium restriction. Methods: To test this hypothesis, we performed a secondary analysis of a randomized clinical trial investigating 46 nondiabetic chronic kidney disease (CKD) patients (age 50 ± 13 years, 80% men) with overt proteinuria and mild to moderate renal insufficiency. Patients were subjected, in a crossover design, to four double-blind 6-week study periods with either regular sodium diet (194 ± 49 mmol Na+/day) or low sodium diet (102 ± 52 mmol Na+/day) on top of either lisinopril (40 mg/day; single RAAS-blockade) or lisinopril plus valsartan (320 mg/day; dual RAAS-blockade). Results: Sodium restriction significantly increased circulating levels of AcSDKP during single and dual RAAS-blockade (P = 0.032 and 0.042, respectively). Linear mixed-model analysis confirmed that AcSDKP levels were increased in response to sodium restriction, irrespective of sex, age, creatinine clearance, blood pressure, BMI, single or dual RAAS-blockade, treatment sequence and other dietary factors, that is calcium and protein (P = 0.020). Conclusion: In patients with nondiabetic CKD, we demonstrated that sodium restriction, on top of single and dual RAAS-blockade, increases circulating levels of the anti-inflammatory and antifibrotic peptide AcSDKP. The rise in AcSDKP may contribute to the increased protection of RAAS-blockade during sodium restriction.