Siegfried Weger

Plasma MicroRNA Profiling Reveals Loss of Endothelial MiR-126 and Other MicroRNAs in Type 2 Diabetes

Rationale: MicroRNAs (miRNAs) have been implicated in the epigenetic regulation of key metabolic, inflammatory, and antiangiogenic pathways in type 2 diabetes (DM) and may contribute to common disease complications. Objective: In this study, we explore plasma miRNA profiles in patients with DM. Methods and Results: Total RNA was extracted from plasma samples of the prospective population-based Bruneck study. A total of 13 candidate miRNAs identified by microarray screening and miRNA network inference were quantified by quantitative PCR in all diabetic patients of the Bruneck study and age- and sex-matched controls (1995 evaluation, n=80 each). Quantitative PCR assessment revealed lower plasma levels of miR-20b, miR-21, miR-24, miR-15a, miR-126, miR-191, miR-197, miR-223, miR-320, and miR-486 in prevalent DM, but a modest increase of miR-28-3p. Findings emerged as robust in multivariable analysis and were independent of the standardization procedure applied. For endothelial miR-126, results were confirmed in the entire Bruneck cohort (n=822) in univariate (odds ratio [95% confidence interval], 0.38 [0.26 to 0.55]; P=2.72×10−7) and multivariate analyses (0.57 [0.37 to 0.86]; P=0.0082). Importantly, reduced miR-15a, miR-29b, miR-126, miR-223, and elevated miR-28-3p levels antedated the manifestation of disease. Most differences in miRNA levels were replicated in plasma obtained from hyperglycemic Lepob mice. High glucose concentrations reduced the miR-126 content of endothelial apoptotic bodies. Similarly in patients with DM, the reduction of miR-126 was confined to circulating vesicles in plasma. Conclusions: We reveal a plasma miRNA signature for DM that includes loss of endothelial miR-126. These findings might explain the impaired peripheral angiogenic signaling in patients with DM.

Telomere Length and Risk of Incident Cancer and Cancer Mortality

CONTEXT Telomeres are essential to preserve the integrity of the genome. Critically short telomeres lead to replicative cell senescence and chromosomal instability and may thereby increase cancer risk. OBJECTIVE To determine the association between baseline telomere length and incident cancer and cancer mortality. DESIGN, SETTING, AND PARTICIPANTS Leukocyte telomere length was measured by quantitative polymerase chain reaction in 787 participants free of cancer at baseline in 1995 from the prospective, population-based Bruneck Study in Italy. MAIN OUTCOME MEASURES Incident cancer and cancer mortality over a follow-up period of 10 years (1995-2005 with a follow-up rate of 100%). RESULTS A total of 92 of 787 participants (11.7%) developed cancer (incidence rate, 13.3 per 1000 person-years). Short telomere length at baseline was associated with incident cancer independently of standard cancer risk factors (multivariable hazard ratio [HR] per 1-SD decrease in log(e)-transformed telomere length, 1.60; 95% confidence interval [CI], 1.30-1.98; P < .001). Compared with participants in the longest telomere length group, the multivariable HR for incident cancer was 2.15 (95% CI, 1.12-4.14) in the middle length group and 3.11 (95% CI, 1.65-5.84) in the shortest length group (P < .001). Incidence rates were 5.1 (95% CI, 2.9-8.7) per 1000 person-years in the longest telomere length group, 14.2 (95% CI, 10.0-20.1) per 1000 person-years in the middle length group, and 22.5 (95% CI, 16.9-29.9) per 1000 person-years in the shortest length group. The association equally applied to men and women and emerged as robust under a variety of circumstances. Furthermore, short telomere length was associated with cancer mortality (multivariable HR per 1-SD decrease in log(e)-transformed telomere length, 2.13; 95% CI, 1.58-2.86; P < .001) and individual cancer subtypes with a high fatality rate. CONCLUSION In this study population, there was a statistically significant inverse relationship between telomere length and both cancer incidence and mortality.

Prospective Study on Circulating MicroRNAs and Risk of Myocardial Infarction

OBJECTIVES This study sought to explore the association between baseline levels of microRNAs (miRNAs) (1995) and incident myocardial infarction (1995 to 2005) in the Bruneck cohort and determine their cellular origin. BACKGROUND Circulating miRNAs are emerging as potential biomarkers. We previously identified an miRNA signature for type 2 diabetes in the general population. METHODS A total of 19 candidate miRNAs were quantified by real-time polymerase chain reactions in 820 participants. RESULTS In multivariable Cox regression analysis, 3 miRNAs were consistently and significantly related to incident myocardial infarction: miR-126 showed a positive association (multivariable hazard ratio: 2.69 [95% confidence interval: 1.45 to 5.01], p = 0.002), whereas miR-223 and miR-197 were inversely associated with disease risk (multivariable hazard ratio: 0.47 [95% confidence interval: 0.29 to 0.75], p = 0.002, and 0.56 [95% confidence interval: 0.32 to 0.96], p = 0.036). To determine their cellular origin, healthy volunteers underwent limb ischemia-reperfusion generated by thigh cuff inflation, and plasma miRNA changes were analyzed at baseline, 10 min, 1 h, 5 h, 2 days, and 7 days. Computational analysis using the temporal clustering by affinity propagation algorithm identified 6 distinct miRNA clusters. One cluster included all miRNAs associated with the risk of future myocardial infarction. It was characterized by early (1 h) and sustained activation (7 days) post-ischemia-reperfusion injury and consisted of miRNAs predominantly expressed in platelets. CONCLUSIONS In subjects with subsequent myocardial infarction, differential co-expression patterns of circulating miRNAs occur around endothelium-enriched miR-126, with platelets being a major contributor to this miRNA signature.

Cellular Aging Reflected by Leukocyte Telomere Length Predicts Advanced Atherosclerosis and Cardiovascular Disease Risk

Objective—To determine the association between leukocyte telomere length (TL) and atherosclerosis and its clinical sequelae stroke and myocardial infarction. Methods and Results—Within the scope of the prospective population-based Bruneck Study, leukocyte TL was measured by quantitative polymerase chain reaction in 800 women and men aged 45 to 84 years (in 1995). The manifestation of cardiovascular disease (CVD) (1995–2005) and the progression of atherosclerosis (1995–2000) were carefully assessed. The TL was shorter in men than in women (age-adjusted mean [95% CI], 1.41 [1.33 to 1.49] versus 1.55 [1.47 to 1.62]; P=0.02) and inversely correlated to age (r=−0.22, P<0.001) and family history of CVD (P=0.03). Participants with CVD events during follow-up (n=88) had significantly shorter telomeres (age- and sex-adjusted mean [95% CI], 1.25 [1.08 to 1.42] versus 1.51 [1.45 to 1.57]; P<0.001). In multivariable Cox models, baseline TL emerged as a significant and independent risk predictor for the composite CVD end point and its individual components (myocardial infarction and stroke); however, this was not the case for de novo stable angina and intermittent claudication. Subjects in the top and bottom TL tertile group differed in their CVD risk by a factor of 2.72 (95% CI, 1.41 to 5.28), which is the risk ratio attributable to a 13.9-year difference in chronological age. Remarkably, in our atherosclerosis progression model, TL was strongly associated with advanced, but not early, atherogenesis. All findings were consistent in women and men. Conclusion—Our findings indicate a differential role of telomere shortening in the various stages of atherosclerosis, with preferential involvement in advanced vessel pathology and acute vascular syndromes.

Endothelial Progenitor Cells, Cardiovascular Risk Factors, Cytokine Levels and Atherosclerosis – Results from a Large Population-Based Study

Background EPC number and functionality are assumed to reflect the endogenous vascular repair capacity with the EPC pool declining in higher ages and being exhausted by unfavorable life-style and risk factors. This intriguing and clinically highly relevant concept, however, has so far been derived from small case-control studies and patient series. Methodology and Principle Findings In the population-based Bruneck Study EPC number and EPC-colony forming units (EPC-CFU) were assessed as part of the fourth follow-up evaluation (2005) in 571 and 542 subjects, respectively. EPC number declined with age (p = 0.013), was significantly lower in women (p = 0.006) and higher in subjects on statin, hormone replacement or ACE inhibitor/angiotensin-receptor blockers, and correlated positively with moderate alcohol consumption. Unexpectedly, a positive relation between EPC number and several vascular risk factors emerged. In a step forward multivariate linear regression analysis EPC number was independently related with SDF1α, MMP-9, triglycerides, alcohol consumption, and Hba1c. EPC-CFU in turn was related to SDF1α and diastolic blood pressure. Moreover, EPC number showed a significant positive association with the Framingham risk score (P = 0.001). Finally, there was an inverse association between EPC number and common carotid artery intima-media thickness (p = 0.02) and the carotid artery atherosclerosis score (p = 0.059). Conclusions Our population-based data confirm the decline of EPC number with advancing age and lend first epidemiological support to a role of SDF-1α and MMP9 in EPC differentiation, mobilization and homing, but are conflict with the view that EPC number is unfavorably affected by cardiovascular risk factors. EPC number increases with the cardiovascular risk estimated by the Framingham risk score (FRS), which in the absence of similar changes for EPC-CFU. Finally, we demonstrate a significant inverse association between EPC number and extent of carotid atherosclerosis even though this association was only of moderate strength and not entirely consistent in other vascular territories.

Leucocyte Telomere Length and Risk of Type 2 Diabetes Mellitus: New Prospective Cohort Study and Literature-Based Meta-Analysis

Background Short telomeres have been linked to various age-related diseases. We aimed to assess the association of telomere length with incident type 2 diabetes mellitus (T2DM) in prospective cohort studies. Methods Leucocyte relative telomere length (RTL) was measured using quantitative polymerase chain reaction in 684 participants of the prospective population-based Bruneck Study (1995 baseline), with repeat RTL measurements performed in 2005 (n = 558) and 2010 (n = 479). Hazard ratios for T2DM were calculated across quartiles of baseline RTL using Cox regression models adjusted for age, sex, body-mass index, smoking, socio-economic status, physical activity, alcohol consumption, high-density lipoprotein cholesterol, log high-sensitivity C-reactive protein, and waist-hip ratio. Separate analyses corrected hazard ratios for within-person variability using multivariate regression calibration of repeated measurements. To contextualise findings, we systematically sought PubMed, Web of Science and EMBASE for relevant articles and pooled results using random-effects meta-analysis. Results Over 15 years of follow-up, 44 out of 606 participants free of diabetes at baseline developed incident T2DM. The adjusted hazard ratio for T2DM comparing the bottom vs. the top quartile of baseline RTL (i.e. shortest vs. longest) was 2.00 (95% confidence interval: 0.90 to 4.49; P = 0.091), and 2.31 comparing the bottom quartile vs. the remainder (1.21 to 4.41; P = 0.011). The corresponding hazard ratios corrected for within-person RTL variability were 3.22 (1.27 to 8.14; P = 0.014) and 2.86 (1.45 to 5.65; P = 0.003). In a random-effects meta-analysis of three prospective cohort studies involving 6,991 participants and 2,011 incident T2DM events, the pooled relative risk was 1.31 (1.07 to 1.60; P = 0.010; I 2 = 69%). Conclusions/Interpretation Low RTL is independently associated with the risk of incident T2DM. To avoid regression dilution biases in observed associations of RTL with disease risk, future studies should implement methods correcting for within-person variability in RTL. The causal role of short telomeres in T2DM development remains to be determined.

Vascular Cell Adhesion Molecule 1 as a Predictor of Severe Osteoarthritis of the Hip and Knee Joints

OBJECTIVE Osteoarthritis (OA) is a leading cause of pain and physical disability in middle-aged and older individuals. We undertook this study to determine predictors of the development of severe OA, apart from age and overweight. METHODS Joint replacement surgery due to severe hip or knee OA was recorded over a 15-year period in the prospective Bruneck cohort study. Demographic characteristics and lifestyle and biochemical variables, including the level of soluble vascular cell adhesion molecule 1 (VCAM-1), were assessed at the 1990 baseline visit and tested as predictors of joint replacement surgery. RESULTS Between 1990 and 2005, hip or knee joint replacement due to OA was performed in 60 subjects. VCAM-1 level emerged as a highly significant predictor of the risk of joint replacement surgery. Intervention rates were 1.9, 4.2, and 10.1 per 1,000 person-years in the first, second, and third tertiles, of the VCAM-1 level, respectively. In multivariable logistic regression analysis, the adjusted relative risk of joint replacement surgery in the highest versus the lowest tertile group of VCAM-1 level was 3.9 (95% confidence interval 1.7-8.7) (P<0.001). Findings were robust in various sensitivity analyses and were consistent in subgroups. Addition of the VCAM-1 level to a risk model already including age, sex, and body mass index resulted in significant gains in model discrimination (C statistic) and calibration and in more accurate risk classification of individual participants. CONCLUSION The level of soluble VCAM-1 emerged as a strong and independent predictor of the risk of hip and knee joint replacement due to severe OA. If our findings can be reproduced in other epidemiologic cohorts, they will assist in routine risk classification and will contribute to a better understanding of the etiology of OA.

Common CCR5-del32 Frameshift Mutation Associated With Serum Levels of Inflammatory Markers and Cardiovascular Disease Risk in the Bruneck Population

Background and Purpose— Atherosclerosis is a progressive inflammatory disease and can develop in large arteries such as carotid and femoral arteries or medium-sized muscular arteries of the heart. Previous predominantly experimental studies suggested an important role of chemokines in the development of atherosclerosis. The main aim of this study was to examine potential effect of the CCR5-del32 mutation on systemic inflammation, intima-media thickness in carotid and femoral arteries, and on the indices of cardiovascular disease. Methods— In the present study, we have examined the association of a common functional 32-bp frameshift deletion mutation in a chemokine receptor (CCR5) in relation to inflammation and atherosclerosis. CCR5 is a G protein-coupled receptor involved in inflammatory response and regulation of leukocytes activation and migration. Genetic screening of this mutation was carried out on a well-known and previously described cohort of Bruneck (n=826) using polymerase chain reaction. Results— Screening was successful in 810 subjects of whom 7 were homozygous, 102 were heterozygous, and 701 were normal. The mutation was associated with significantly lower levels of C-reactive protein in a dose-dependent manner. Moreover, CCR5-del32 was associated with a significantly lower carotid intima-media thickness in the common carotid artery (del32/del32, 837±8 &mgr;m; wt/del32, 909±21 &mgr;m; wt/wt, 958±8 &mgr;m; P=0.007 after multivariable adjustment). Furthermore, incident cardiovascular disease (1995 to 2005) was markedly reduced in del32 homozygotes and heterozygotes subjects compared with wild-type homozygotes (del32/del32=0%, wt/del32=7.8%, wt/wt=14.8%, P=0.020). Findings equally applied to coronary artery and cerebrovascular disease. Conclusions— The chemokine receptor CCR5-del32 frameshift mutation is associated with low levels of C-reactive protein, decreased intima-media thickness, and cardiovascular disease risk. These findings are consistent with the hypothesis that the chemokine receptor CCR5 is involved in the mediation of low-grade systemic inflammation and may play a role in human atherosclerosis and cardiovascular disease.

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.

Haptoglobin 2-2 genotype is not associated with cardiovascular risk in subjects with elevated glycohemoglobin-results from the Bruneck Study.

Background Haptoglobin (Hp) is an abundant plasma protein with antioxidant properties. The Hp 2‐2 genotype has previously been linked to coronary heart disease risk in individuals with elevated glycosylated hemoglobin (HbA1c). We investigated the association of Hp and HbA1c with cardiovascular disease (CVD) in the longitudinal, population‐based Bruneck Study. Methods and Results Hp genotype was determined by polymerase chain reaction according to standard procedures and HbA1c concentration by a Diabetes Control and Complications Trial‐aligned assay. HbA1c was measured in 1995, 2000, and 2005. Occurrence of the combined CVD endpoint of myocardial infarction or stroke was recorded between 1995 and 2010. Outcome analyses employed the Cox proportional hazards model with HbA1c category as time‐varying covariate. At baseline in 1995, 806 subjects (male sex, 49.3%; age, mean±standard deviation, 62.70±11.08 years) were included. During follow‐up, 123 subjects experienced at least 1 CVD event (48 suffered myocardial infarction, 68 stroke, and 7 both). Among subjects with HbA1c≥6.5% (≥48 mmol/mol), those with the Hp 2‐2 genotype did not show an elevated risk of incident CVD compared with those with other genotypes (age‐ and sex‐adjusted hazard ratio [95% CI], 0.47 [0.19, 1.13], P=0.092) and a null association was also observed in subjects with HbA1c<6.5% (1.10 [0.75, 1.62], P=0.629) (P for interaction=0.082). Conclusions Subjects with the Hp 2‐2 genotype and elevated HbA1c compared with subjects with other Hp genotypes and elevated HbA1c did not show increased CVD risk.