Friedrich Oberhollenzer

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.

Body Iron Stores and the Risk of Carotid Atherosclerosis Prospective Results From the Bruneck Study

BACKGROUND Fe2+ released from tissue iron stores may accelerate lipid peroxidation by virtue of its pro-oxidant properties and thus promote early atherogenesis. METHODS AND RESULTS The present prospective survey addresses the potential association between serum ferritin concentrations and the 5-year progression of carotid atherosclerosis as assessed by ultrasonographic follow-up evaluations. The study population comprises a random sample of 826 men and women 40 to 79 years old. Serum ferritin was one of the strongest risk predictors of overall progression of atherosclerosis. The main part of this association appeared to act through modification of the atherogenic potential of LDL cholesterol (OR [95% CI] for a 1-SD unit increase in ferritin at LDL levels of 2.5, 3.6, and 4.9 mmol/L: 1.55 [1.30 to 1.85], 1.77 [1.40 to 2.24], and 2.05 [1.50 to 2.80]; P=.0012 for effect modification). Changes in iron stores during the follow-up period modified atherosclerosis risk, in that a lowering was beneficial and further iron accumulation exerted unfavorable effects. All these findings applied equally to incident atherosclerosis and the extension of preexisting atherosclerotic lesions. The significance of prominent iron stores in the development of carotid stenosis was clearly less pronounced. Finally, ferritin and LDL cholesterol showed a synergistic association with incident cardiovascular disease and death (n=59). CONCLUSIONS The present study provided strong epidemiological evidence for a role of iron stores in early atherogenesis and suggests promotion of lipid peroxidation as the main underlying pathomechanism. This hypothesis could in part explain the sex difference in atherosclerotic vascular disease.

Serum Soluble Heat Shock Protein 60 Is Elevated in Subjects With Atherosclerosis in a General Population

BACKGROUND Work from our laboratory has proven that increased titers of anti-heat shock protein 60 (HSP60) antibodies are associated with atherosclerosis and that HSP60-reactive T-cells are present in atherosclerotic lesions. Recent studies from others demonstrated that HSP60 directly activates endothelial cells and macrophages. METHODS AND RESULTS To explore the possibility that HSP60 exists in the circulation, where it could exert its functions, we performed a population-based study with 826 subjects aged 40 to 79 years. The following items were measured in all participants: serum soluble HSP60 (sHSP60); anti-Escherichia coli lipopolysaccharide; anti-HSP65, anti-Chlamydia and anti-Helicobacter pylori antibodies; and a variety of acute phase reactants (C-reactive protein, alpha(1)-antitrypsin, and ceruloplasmin) and markers of systemic inflammation. Carotid atherosclerosis was assessed twice (1990 and 1995), and 15 other risk factors were evaluated. Our data show that levels of sHSP60 were significantly elevated in subjects with prevalent/incident carotid atherosclerosis and that these levels were correlated with common carotid artery intima/media thickness. Multiple logistic regression analysis documented these associations as independent of age, sex, and other risk factors. Interestingly, sHSP60 was also correlated with anti-lipopolysaccharide, anti-Chlamydia and anti-HSP60 antibodies, various markers of inflammation, and the presence of chronic infections. The risk of atherosclerosis associated with high sHSP60 levels was amplified when subjects had clinical and/or laboratory evidence of chronic infections. CONCLUSIONS Our data provide the first evidence of a strong correlation between sHSP60 and atherosclerosis, suggesting that sHSP60 may play important roles in activating vascular cells and the immune system during the development of atherosclerosis.

Association of endotoxemia with carotid atherosclerosis and cardiovascular disease: prospective results from the Bruneck Study.

OBJECTIVES Focus of the current study was on the significance of bacterial endotoxin, which shows a variety of pro-atherogenic properties and may occur at high concentration in the circulation of infected subjects. BACKGROUND The possibility of an infectious risk factor in atherogenesis and cardiovascular disease has stimulated research interest, but the nature of such process remains obscure. METHODS We measured plasma endotoxin levels (LAL assay) in a random population of 516 men and women 50 to 79 years old at the 1990 baseline evaluation (Bruneck Study). End points of this prospective survey were incident (early) atherosclerosis in the carotid arteries as assessed with high-resolution Duplex ultrasound (five-year follow-up rate, 98%) and incident cardiovascular disease (follow-up rate, 100%). RESULTS Median endotoxin concentration amounted to 14.3 pg/ml (range, 6.0 to 209.2 pg/ml). Subjects with levels beyond 50 pg/ml (90th percentile) faced a threefold risk of incident atherosclerosis (odds ratio [95% confidence interval] 2.9 [1.4-6.3]; p < 0.01). The risk associated with high endotoxin was most pronounced in subjects with chronic infections and in current and ex-smokers. Notably, smokers with low endotoxin levels and nonsmokers did not differ in their atherosclerosis risk, whereas smokers with high levels almost invariably developed new lesions. All findings emerged as independent of vascular risk factors. Similar results were obtained for incident cardiovascular disease. CONCLUSIONS The current study yields first epidemiologic evidence that endotoxemia constitutes a strong risk factor of early atherogenesis in subjects with chronic or recurrent bacterial infections and a link in the association between cigarette smoking and atherosclerotic disease.

Chronic Infections and the Risk of Carotid Atherosclerosis

Background—Chronic infections have been implicated in the pathogenesis of atherosclerosis, yet from an epidemiological perspective, this concept remains controversial. Methods and Results—The Bruneck Study is a prospective population-based survey on the pathogenesis of atherosclerosis. In 826 men and women 40 to 79 years old (1990 baseline), 5-year changes in carotid atherosclerosis were thoroughly assessed by high-resolution duplex scanning. The presence of chronic respiratory, urinary tract, dental, and other infections was ascertained by standard diagnostic criteria. Chronic infections amplified the risk of atherosclerosis development in the carotid arteries. The association was most pronounced in subjects free of carotid atherosclerosis at baseline (age-/sex-adjusted odds ratio [95% CI] for any chronic infection versus none, 4.08 [2.42 to 6.85]; P<0.0001) and applied to all types of chronic (bacterial) infections. It remained independently significant after adjustment for classic vascular risk attribu...

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.

Insulin sensitivity and regular alcohol consumption: large, prospective, cross sectional population study (Bruneck study)

Abstract Objectives: To assess the relation between regular alcohol consumption and insulin sensitivity, and to estimate the importance of insulin in the association of alcohol with multiple vascular risk factors and cardiovascular disease. Design: Prospective and cross sectional study of a large randomly selected population sample. Setting: Part of the Bruneck study 1990–5 (Bolzano province, Italy). Subjects: 820 healthy non-diabetic women and men aged 40–79 years. Main outcome measure: Concentrations of fasting and post-glucose insulin, cholesterol, apolipoproteins, triglycerides, Lp(a) lipoprotein, glucose, fibrinogen, and antithrombin III; blood pressure; insulin resistance estimated by the homeostasis model assessment. Results: Fasting insulin concentrations in those who did not drink alcohol and subjects reporting low (1–50 g/day), moderate (51–99 g/day), and heavy (>/=100 g/day) alcohol intake were 12.4, 10.0, 8.7, and 7.1 mU/l (P<0.001). Likewise, post-glucose insulin concentrations and estimates for insulin resistance assessed by the homeostasis model assessment decreased significantly with increasing amounts of regular alcohol consumption. These trends were independent of sex, body mass index, physical activity, cigarette smoking, medication, and diet (P<0.001). Regular alcohol intake predicted multiple changes in vascular risk factors over a five year period including increased concentrations of high density lipoprotein cholesterol and apolipoprotein A I; higher blood pressure; and decreased concentration of antithrombin III. These associations were in part attributable to the decrease in insulin concentrations observed among alcohol consumers. Conclusions: Low to moderate amounts of alcohol, when taken on a regular basis, improve insulin sensitivity. Insulin is a potential intermediate component in the association between alcohol consumption and vascular risk factors (metabolic syndrome). Key messages Regular alcohol consumption predicted multiple changes of vascular risk factors over a five year period This alcohol associated metabolic syndrome is in part attributable to the decline in insulin concentrations

Role of Lipoprotein(a) and Apolipoprotein(a) Phenotype in Atherogenesis Prospective Results From the Bruneck Study

BACKGROUND Experimental studies have suggested both atherogenic and thrombogenic properties of lipoprotein(a) [Lp(a)], depending on Lp(a) plasma concentrations and varying antifibrinolytic capacity of apolipoprotein(a) [apo(a)] isoforms. Epidemiological studies may contribute to assessment of the relevance of these findings in the general population. METHODS AND RESULTS This study prospectively investigated the association between Lp(a) plasma concentrations, apo(a) phenotypes, and the 5-year progression of carotid atherosclerosis assessed by high-resolution duplex ultrasound in a random sample population of 826 individuals. We differentiated early atherogenesis (incident nonstenotic atherosclerosis) from advanced (stenotic) stages in atherosclerosis that originate mainly from atherothrombotic mechanisms. Lp(a) plasma concentrations predicted the risk of early atherogenesis in a dose-dependent fashion, with this association being confined to subjects with LDL cholesterol levels above the population median (3.3 mmol/L). Apo(a) phenotypes were distributed similarly in subjects with and without early carotid atherosclerosis. In contrast, apo(a) phenotypes of low molecular weight emerged as one of the strongest risk predictors of advanced stenotic atherosclerosis, especially when associated with high Lp(a) plasma concentrations (odds ratio, 6.4; 95% CI, 2.8 to 14. 9). CONCLUSIONS Lp(a) is one of the few risk factors capable of promoting both early and advanced stages of atherogenesis. Lp(a) plasma concentrations predicted the risk of early atherogenesis synergistically with high LDL cholesterol. Low-molecular-weight apo(a) phenotypes with a putatively high antifibrinolytic capacity in turn emerged as one of the leading risk conditions of advanced stenotic stages of atherosclerosis.

Insulin resistance as estimated by homeostasis model assessment predicts incident symptomatic cardiovascular disease in caucasian subjects from the general population : The bruneck study

OBJECTIVE—The purpose of this study was to evaluate whether insulin resistance is associated to cardiovascular disease (CVD) and to understand whether this association can be explained by traditional and novel CVD risk factors associated with this metabolic disorder. RESEARCH DESIGN AND METHODS—We examined a sample representative of the population of Bruneck, Italy (n = 919; aged 40–79 years). Insulin-resistant subjects were those with a score in the top quartile of the homeostasis model assessment (HOMA) for insulin resistance (HOMA-IR). Risk factors correlated with insulin resistance included BMI, A1C, HDL cholesterol, triglycerides, blood pressure, high-sensitivity C-reactive protein (hsCRP), fibrinogen, oxidized LDL, vascular cell adhesion molecule-1 (VCAM-1), and adiponectin. Subjects without CVD at baseline were followed up for 15 years for incident CVD, a composite end point including fatal and nonfatal myocardial infarction and stroke, transient ischemic attack, and any revascularization procedure. RESULTS—During follow-up, 118 subjects experienced a first symptomatic CVD event. Levels of HOMA-IR were higher at baseline among subjects who developed CVD (2.8) compared with those remaining free of CVD (2.5) (P < 0.05). Levels of HOMA-IR also were significantly correlated (P < 0.05) with most CVD risk factors we evaluated. In Cox proportional hazard models, insulin-resistant subjects had an age-, sex-, and smoking-adjusted 2.1-fold increased risk (95% CI 1.3–3.1) of incident symptomatic CVD relative to non–insulin-resistant subjects. After sequential adjustment for physical activity and classic risk factors (A1C, LDL cholesterol, and hypertension) as well as BMI, HDL cholesterol, triglycerides, and novel risk factors, including fibrinogen, oxidized LDL, hsCRP, VCAM-1, and adiponectin, the association between HOMA-IR and incident CVD remained significant and virtually unchanged (hazard ratio 2.2 [95% CI 1.4–3.6], P < 0.001). CONCLUSIONS—HOMA-estimated insulin resistance is associated with subsequent symptomatic CVD in the general population independently of all classic and several nontraditional risk factors. These data suggest that insulin resistance may be an important target to reduce CVD risk.

Association of Serum-Soluble Heat Shock Protein 60 With Carotid Atherosclerosis. Clinical Significance Determined in a Follow-Up Study

Background and Purpose— Previous work has shown that soluble heat shock protein 60 (HSP60; sHSP60), present in circulating blood, is associated with carotid atherosclerosis. In the current evaluation, we tested the hypothesis that sHSP60 levels are associated with the progression of carotid arteriosclerosis, prospectively. Methods— The association of sHSP60 with early atherogenesis (5-year development and progression of nonstenotic carotid plaques) was investigated as part of the population-based prospective Bruneck Study. The current study focused on the follow-up period between 1995 and 2000 and, thus, included 684 subjects. Results— sHSP60 levels measured in 1995 and 2000 were highly correlated (r=0.40; P<0.001), indicating consistency over a 5-year period. Circulating HSP60 levels were significantly correlated with antilipopolysaccharide and anti-HSP60 antibodies. It was also elevated in subjects with chronic infection (top quintile group of HSP60, among subjects with and without chronic infection: 23.8% versus 17.0%; P=0.003 after adjustment for age and sex). HSP60 levels were significantly associated with early atherogenesis, both in the entire population (multivariate odds ratio, for a comparison between quintile group V versus I+II: 2.0 [1.2 to 3.5] and the subgroup free of atherosclerosis at the 1995 baseline: 3.8 [1.6 to 8.9]). The risk of early atherogenesis was additionally amplified when high-sHSP60 and chronic infection were present together. Conclusions— Our study provides the first prospective data confirming an association between high levels of sHSP60 and early carotid atherosclerosis. This possibly indicates an involvement of sHSP60 in activating proinflammatory processes associated with early vessel pathology.