Karin Willeit

Association of MicroRNAs and YRNAs With Platelet Function

RATIONALE Platelets shed microRNAs (miRNAs). Plasma miRNAs change on platelet inhibition. It is unclear whether plasma miRNA levels correlate with platelet function. OBJECTIVE To link small RNAs to platelet reactivity. METHODS AND RESULTS Next-generation sequencing of small RNAs in plasma revealed 2 peaks at 22 to 23 and 32 to 33 nucleotides corresponding to miRNAs and YRNAs, respectively. Among YRNAs, predominantly, fragments of RNY4 and RNY5 were detected. Plasma miRNAs and YRNAs were measured in 125 patients with a history of acute coronary syndrome who had undergone detailed assessment of platelet function 30 days after the acute event. Using quantitative real-time polymerase chain reactions, 92 miRNAs were assessed in patients with acute coronary syndrome on different antiplatelet therapies. Key platelet-related miRNAs and YRNAs were correlated with platelet function tests. MiR-223 (rp=0.28; n=121; P=0.002), miR-126 (rp=0.22; n=121; P=0.016), and other abundant platelet miRNAs and YRNAs showed significant positive correlations with the vasodilator-stimulated phosphoprotein phosphorylation assay. YRNAs, miR-126, and miR-223 were also among the small RNAs showing the greatest dependency on platelets and strongly correlated with plasma levels of P-selectin, platelet factor 4, and platelet basic protein in the population-based Bruneck study (n=669). A single-nucleotide polymorphism that facilitates processing of pri-miR-126 to mature miR-126 accounted for a rise in circulating platelet activation markers. Inhibition of miR-126 in mice reduced platelet aggregation. MiR-126 directly and indirectly affects ADAM9 and P2Y12 receptor expression. CONCLUSIONS Levels of platelet-related plasma miRNAs and YRNAs correlate with platelet function tests in patients with acute coronary syndrome and platelet activation markers in the general population. Alterations in miR-126 affect platelet reactivity.

Extracellular matrix proteomics identifies molecular signature of symptomatic carotid plaques

BACKGROUND. The identification of patients with high-risk atherosclerotic plaques prior to the manifestation of clinical events remains challenging. Recent findings question histology- and imaging-based definitions of the “vulnerable plaque,” necessitating an improved approach for predicting onset of symptoms. METHODS. We performed a proteomics comparison of the vascular extracellular matrix and associated molecules in human carotid endarterectomy specimens from 6 symptomatic versus 6 asymptomatic patients to identify a protein signature for high-risk atherosclerotic plaques. Proteomics data were integrated with gene expression profiling of 121 carotid endarterectomies and an analysis of protein secretion by lipid-loaded human vascular smooth muscle cells. Finally, epidemiological validation of candidate biomarkers was performed in two community-based studies. RESULTS. Proteomics and at least one of the other two approaches identified a molecular signature of plaques from symptomatic patients that comprised matrix metalloproteinase 9, chitinase 3-like-1, S100 calcium binding protein A8 (S100A8), S100A9, cathepsin B, fibronectin, and galectin-3-binding protein. Biomarker candidates measured in 685 subjects in the Bruneck study were associated with progression to advanced atherosclerosis and incidence of cardiovascular disease over a 10-year follow-up period. A 4-biomarker signature (matrix metalloproteinase 9, S100A8/S100A9, cathepsin D, and galectin-3-binding protein) improved risk prediction and was successfully replicated in an independent cohort, the SAPHIR study. CONCLUSION. The identified 4-biomarker signature may improve risk prediction and diagnostics for the management of cardiovascular disease. Further, our study highlights the strength of tissue-based proteomics for biomarker discovery. FUNDING. UK: British Heart Foundation (BHF); King’s BHF Center; and the National Institute for Health Research Biomedical Research Center based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London in partnership with King’s College Hospital. Austria: Federal Ministry for Transport, Innovation and Technology (BMVIT); Federal Ministry of Science, Research and Economy (BMWFW); Wirtschaftsagentur Wien; and Standortagentur Tirol.

Carotid Atherosclerosis and Incident Atrial Fibrillation

Objective—Atrial fibrillation (AF) and atherosclerotic vascular disease are closely entangled disorders and often coexist. Whether atherosclerosis predisposes to the development of AF has not been fully elucidated. Approach and Results—This study was performed within the framework of the Bruneck Study, a population-based survey with near-complete participation (932 of 1000), long-term follow-up (1990–2010), and thorough assessment of AF. The carotid arteries served as a window to systemic atherosclerosis and were scanned every 5 years. Pooled logistic regression and multistate proportional hazards models were used to identify risk predictors of incident AF and effects of AF on mortality. During follow-up, 118 new cases of AF were detected (incidence per 1000 person-years of 8.1; 95% confidence interval, 6.8–9.6). Individuals with atherosclerosis were more likely to develop AF than individuals without (odds ratio, 1.8; 95% confidence interval, 1.1–3.1; P=0.021). This finding applied to women and men and to both baseline and incident atherosclerosis during follow-up. Subjects with atherosclerosis and AF were significantly more likely to die than those with either condition alone (P=0.0034), and mortality in this group was ≈4-fold compared with individuals free of atherosclerosis and AF (hazard ratio, 4.2; 95% confidence interval, 2.6–6.8; P<0.0001). Conclusions—We found that subjects with carotid atherosclerosis are at high risk of developing AF.

Thrombolysis and clinical outcome in patients with stroke after implementation of the Tyrol Stroke Pathway: a retrospective observational study

BACKGROUND Intravenous thrombolysis for ischaemic stroke remains underused worldwide. We aimed to assess whether our statewide comprehensive stroke management programme would improve thrombolysis use and clinical outcome in patients. METHODS In 2008-09, we designed the Tyrol Stroke Pathway, which provided information campaigns for the public and standardised the entire treatment pathway from stroke onset to outpatient rehabilitation. It was commenced in Tyrol, Austria, as a long-term routine-care programme and aimed to include all patients with stroke in the survey area. We focused on thrombolysis use and outcome in the first full 4 years of implementation (2010-13). FINDINGS We enrolled 4947 (99%) of 4992 patients with ischaemic stroke who were admitted to hospitals in Tyrol; 675 (14%) of the enrollees were treated with alteplase. Thrombolysis administration in Tyrol increased after programme implementation, from 160 of 1238 patients (12·9%, 95% CI 11·1-14·9) in 2010 to 213 of 1266 patients (16·8%, 14·8-19·0) in 2013 (ptrend 2010-13<0·0001). Differences in use of thrombolysis in the nine counties of Tyrol in 2010 (range, 2·2-22·6%) were reduced by 2013 (12·1-22·5%). Median statewide door-to-needle time decreased from 49 min (IQR 35-60) in 2010 to 44 min (29-60) in 2013; symptomatic post-thrombolysis intracerebral haemorrhages occurred in 28 of 675 patients (4·1%, 95% CI 2·8-5·9) during 2010-13. In four Austrian states without similar stroke programmes, thrombolysis administration remained stable or declined between 2010 and 2013 (mean reduction 14·4%, 95% CI 10·9-17·9). Although the 3-month mortality was not affected by our programme (137 [13%] of 1060 patients in 2010 vs 143 [13%] of 1069 patients in 2013), 3-month functional outcome significantly improved (modified Rankin Scale score 0-1 in 375 [40%] of 944 patients in 2010 vs 493 [53%] of 939 in 2013; score 0-2 in 531 [56%] patients in 2010 and 615 [65%] in 2013; ptrend 2010-13<0·0001). INTERPRETATION During the period of implementation of our comprehensive stroke management programme, thrombolysis administration increased and clinical outcome significantly improved, although mortality did not change. We hope that these results will guide health authorities and stroke physicians elsewhere when implementing similar programmes for patients with stroke. FUNDING Reformpool of the Tyrolean Health Care Fund.

Heme Oxygenase-1 Gene Promoter Microsatellite Polymorphism Is Associated With Progressive Atherosclerosis and Incident Cardiovascular Disease

Objective—The enzyme heme oxygenase-1 (HO-1) exerts cytoprotective effects in response to various cellular stressors. A variable number tandem repeat polymorphism in the HO-1 gene promoter region has previously been linked to cardiovascular disease. We examined this association prospectively in the general population. Approach and Results—Incidence of stroke, myocardial infarction, or vascular death was registered between 1995 and 2010 in 812 participants of the Bruneck Study aged 45 to 84 years (49.4% males). Carotid atherosclerosis progression was quantified by high-resolution ultrasound. HO-1 variable number tandem repeat length was determined by polymerase chain reaction. Subjects with ≥32 tandem repeats on both HO-1 alleles compared with the rest of the population (recessive trait) featured substantially increased cardiovascular disease risk (hazard ratio [95% confidence interval], 5.45 [2.39, 12.42]; P<0.0001), enhanced atherosclerosis progression (median difference in atherosclerosis score [interquartile range], 2.1 [0.8, 5.6] versus 0.0 [0.0, 2.2] mm; P=0.0012), and a trend toward higher levels of oxidized phospholipids on apolipoprotein B-100 (median oxidized phospholipids/apolipoprotein B level [interquartile range], 11364 [4160, 18330] versus 4844 [3174, 12284] relative light units; P=0.0554). Increased cardiovascular disease risk in those homozygous for ≥32 repeats was also detected in a pooled analysis of 7848 participants of the Bruneck, SAPHIR, and KORA prospective studies (hazard ratio [95% confidence interval], 3.26 [1.50, 7.33]; P=0.0043). Conclusions—This study found a strong association between the HO-1 variable number tandem repeat polymorphism and cardiovascular disease risk confined to subjects with a high number of repeats on both HO-1 alleles and provides evidence for accelerated atherogenesis and decreased antioxidant defense in this vascular high-risk group.

Plasma Concentrations of Afamin Are Associated With Prevalent and Incident Type 2 Diabetes: A Pooled Analysis in More Than 20,000 Individuals

OBJECTIVE The human vitamin E–binding glycoprotein afamin is primarily expressed in the liver and has been associated with prevalent and incident metabolic syndrome. These data were in line with observations in transgenic mice. We thus investigated whether afamin concentrations are associated with prediabetes, type 2 diabetes, and insulin resistance (IR). RESEARCH DESIGN AND METHODS Individual-level baseline (n = 20,136) and follow-up data (n = 14,017) of eight prospective cohort studies were investigated. Study-level data were combined using random-effects meta-analyses. Main outcomes were prevalent and incident type 2 diabetes, prediabetes, and IR. Discrimination and reclassification of participants was analyzed for incident type 2 diabetes. RESULTS Mean afamin concentrations between studies ranged from 61 to 73 mg/L. The eight studies included 1,398 prevalent and 585 incident cases of type 2 diabetes. Each increase of afamin by 10 mg/L was associated with prevalent type 2 diabetes (odds ratio [OR] 1.19 [95% CI 1.12–1.26], P = 5.96 × 10−8). Afamin was positively associated with IR assessed by HOMA-IR (β 0.110 [95% CI 0.089–0.132], P = 1.37 × 10−23). Most importantly, afamin measured at baseline was an independent predictor for 585 incident cases of type 2 diabetes (OR 1.30 [95% CI 1.23–1.38], P = 3.53 × 10−19) and showed a significant and valuable gain in risk classification accuracy when added to this extended adjustment model. CONCLUSIONS This pooled analysis in >20,000 individuals showed that afamin is strongly associated with IR, prevalence, and incidence of type 2 diabetes independent of major metabolic risk factors or parameters. Afamin might be a promising novel marker for the identification of individuals at high risk for the development of type 2 diabetes.

Do Women With Atrial Fibrillation Experience More Severe Strokes?: Results From the Austrian Stroke Unit Registry

Background and Purpose— Ischemic strokes associated with atrial fibrillation (AF) are more severe than those of other cause. We aim to study potential sex effects in this context. Methods— In this cross-sectional study, 74 425 adults with acute ischemic stroke from the Austrian Stroke Unit Registry were included between March 2003 and January 2016. In 63 563 patients, data on the National Institutes of Health Stroke Scale on admission to the stroke unit, presence of AF, vascular risk factors, and comorbidities were complete. Analysis was done by a multivariate regression model. Results— Stroke severity in general increased with age. AF-related strokes were more severe than strokes of other causes. Sex-related differences in stroke severity were only seen in stroke patients with AF. Median (Q25,75) National Institutes of Health Stroke Scale score points were 9 (4,17) in women and 6 (3,13) in men (P<0.001). The interaction between AF and sex on stroke severity was independent of age, previous functional status, vascular risk factors, and vascular comorbidities and remained significant in various subgroups. Conclusions— Women with AF do not only have an increased risk of stroke when compared with men but also experience more severe strokes.

Association Between Vascular Cell Adhesion Molecule 1 and Atrial Fibrillation

Importance Accumulating evidence links inflammation and atrial fibrillation (AF). Objective To assess whether markers of systemic and atrial inflammation are associated with incident AF in the general population. Design, Setting, and Participants The Bruneck Study is a prospective, population-based cohort study with a 20-year follow-up (n = 909). The population included a random sample of the general community aged 40 to 79 years. Levels of 13 inflammation markers were measured at baseline in 1990. Findings were replicated in a case-control sample nested within the prospective Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR) study (n = 1770). Data analysis was performed from February to May 2016. Exposures Levels of 13 inflammation markers. Main Outcomes and Measures Incident AF over a 20-year follow-up period in the Bruneck Study. Results Of the 909 participants included in the Bruneck Study, mean [SD] age was 58.8 (11.4) years and 448 (49.3%) were women. Among the 880 participants free of prevalent AF (n = 29) at baseline, 117 developed AF during the 20-year follow-up period (incidence rate, 8.2; 95% CI, 6.8-9.6 per 1000 person-years). The levels of soluble vascular cell adhesion molecule 1 (VCAM-1) and osteoprotegerin were significantly associated with incident AF (hazard ratio [HR], 1.49; 95% CI, 1.26-1.78; and 1.46; 95% CI, 1.25-1.69, respectively; P < .001 with Bonferroni correction for both), but osteoprotegerin lost significance after age and sex adjustment (HR, 1.05; 95% CI, 0.87-1.27; P > .99 with Bonferroni correction). Matrix metalloproteinase 9, metalloproteinase inhibitor 1, monocyte chemoattractant protein-1, P-selectin, fibrinogen, receptor activator of nuclear factor-&kgr;B ligand, high-sensitivity C-reactive protein, adiponectin, leptin, soluble intercellular adhesion molecule 1, and E-selectin all fell short of significance (after Bonferroni correction in unadjusted and age- and sex-adjusted analyses). The HR for a 1-SD higher soluble VCAM-1 level was 1.34 (95% CI, 1.11-1.62; Bonferroni-corrected P = .03) in a multivariable model. The association was of a dose-response type, at least as strong as that obtained for N-terminal pro-B-type natriuretic peptide (multivariable HR for a 1-SD higher N-terminal pro-B-type natriuretic peptide level, 1.15; 95% CI, 1.04-1.26), internally consistent in various subgroups, and successfully replicated in the SAPHIR Study (age- and sex-adjusted, and multivariable odds ratios for a 1-SD higher soluble VCAM-1 level, 1.91; 95% CI, 1.24-2.96, P = .003; and 2.59; 95% CI, 1.45-4.60; P = .001). Conclusions and Relevance Levels of soluble VCAM-1, but not other inflammation markers, are significantly associated with new-onset AF in the general community. Future studies should address whether soluble VCAM-1 is capable of improving AF risk classification beyond the information provided by standard risk scores.

Familial hypercholesterolaemia in patients with ischemic stroke or transient ischemic attack

Identification of patients with familial hypercholesterolaemia (FH) is a prerequisite for the appropriate management of their excess cardiovascular risk. It is currently unknown how many patients with acute ischaemic stroke or transient ischaemic attack (TIA) are affected by FH and whether systematic screening for FH is warranted in these patients.

Circulating Wnt inhibitory factor 1 levels are associated with development of cardiovascular disease

BACKGROUND AND AIMS Wnt signaling is involved in atherosclerotic plaque formation directly and indirectly by modulating cardiovascular risk factors. We investigated whether circulating concentrations of Wnt inhibitors are associated with cardiovascular events in subjects with intermediate cardiovascular risk. METHODS 904 non-diabetic subjects participating in the SAPHIR study were assessed. In the SAPHIR study, middle-aged women without overt atherosclerotic disease at study entry were followed up for 10 years. 88 patients of our study cohort developed cardiovascular disease at follow-up (CVD group). Subjects of the CVD group were 1:2 case-control matched for age, sex, BMI and smoking behavior with subjects without overt cardiovascular disease after a 10 year-follow-up (control group). 18 patients of the CVD group and 19 subjects of the control group were retrospectively excluded due to fulfilling exclusion criteria. Baseline circulating sclerostin, dickkopf (DKK)-1, secreted frizzled-related protein (SFRP)-1 and Wnt inhibitory factor (WIF)-1 levels were assessed by ELISA. RESULTS Baseline systemic SFRP-1 and WIF-1 levels were significantly higher in patients with cardiovascular events (n = 70) when compared to healthy controls (n = 157) while DKK-1 and sclerostin levels were similar in both groups. Logistic regression analysis revealed WIF-1 as a significant predictor of future cardiovascular events. CONCLUSIONS Our data suggest that increased SFRP-1 and WIF-1 levels precede the development of symptomatic atherosclerotic disease. Assessment of systemic WIF-1 levels, which turned out to be independently associated with CVD, might help to early identify patients at intermediate cardiovascular risk.