Presence of fatty liver and the relationship between alcohol consumption and markers of inflammation.

Background and Aims. Local and systemic inflammation represent a major feature of atherosclerotic cardiovascular disease (CVD) and are also linked to nonalcoholic fatty liver disease (NAFLD). Studies indicate that NAFLD might be a risk factor for CVD whereas low-to-moderate alcohol consumption is associated with lower cardiovascular morbidity and mortality compared to abstainers and heavy drinkers. We hypothesize that FLD interacts with the effect of alcohol intake on markers of inflammation, and thus potentially on cardiovascular risk. Methods and Results. We evaluated alcohol consumption, markers of inflammation and sonographic criteria of FLD in 515 subjects, representing a subsample of a cross-sectional population based study (Echinococcus multilocularis and Internal Diseases in Leutkirch (EMIL) Study). Presence of FLD was markedly reduced in subjects drinking 0-20 g alcohol/d (19%), compared to nondrinkers (35%) and heavy drinkers (34-44.9%). Serum concentrations of inflammatory markers were substantially higher in subjects with FLD. However, presence of FLD showed no effect on the association between alcohol consumption and inflammatory biomarkers. Conclusions. Based on data from a population-based sample, there is no evidence for a link between FLD, alcohol consumption, and inflammatory cardiovascular risk markers. However, larger prospective studies are needed to confirm this.

Antifibrotic role of HGF in sarcoidosis.

BACKGROUND: Pulmonary sarcoidosis has a variable course ranging from self-limiting disease to progressive fibrosis. Activation of fibroblasts, myofibroblast transformation, and matrix production may contribute to pulmonary damage in sarcoidosis. These processes are influenced by pulmonary cytokines which can be measured in bronchoalveolar lavage fluid (BALF). In order to clarify the incompletely understood fibrotic process in sarcoidosis, we classified activity of sarcoidosis according to WASOG criteria, measured TNF-α, IL-6, and HGF in BALF, and assessed the effect of HGF and BALF on proliferation and matrix production of human lung fibroblasts. RESULTS: BALF was obtained from 34 consecutive patients with sarcoidosis. BALF of active sarcoidosis contained elevated levels of TNF-α, HGF, and IL-6 and stimulated fibroblast proliferation. BALF of inactive sarcoidosis, but not of active sarcoidosis, stimulated the production of matrix proteins. HGF levels in inactive sarcoidosis were below those of control patients. HGF suppressed TGF-ß-induced matrix expression and transformation of fibroblasts into myofibroblasts. CONCLUSION: Prevention of TGF-ß-induced myofibroblast transformation may account for the inhibitory effect of HGF on matrix production. The strong fibrogenic effect of BALF of inactive sarcoidosis corresponds to the worse clinical course of inactive sarcoidosis compared with active disease and may be related to a lack of protective HGF.

Influence of Storage and Inter- and Intra-Assay Variability on the Measurement of Inflammatory Biomarkers in Population-Based Biobanking.

BACKGROUND: In the present study, we examined the effect of sample storage on the reproducibility of several inflammatory biomarkers, including high-sensitivity C-reactive protein (hsCRP), high-sensitivity interleukin-6 (hsIL6), and high-sensitivity tumor necrosis factor alpha (hsTNFα). In addition, we assessed inter- and intra-assay variability between collaborating biobanks. METHODS: In total, 240 fasting plasma samples were obtained from the LifeLines biobank. Samples had been stored for less than 2 or more than 4 years at -80°C. Measurements were performed at three different laboratories. hsCRP was measured by immunonephelometry and ELISA, hsIL6, and hsTNFα samples were measured with ELISAs from two different manufacturers. For confirmation, similar analyses were performed on samples obtained from a subpopulation of 80 obese individuals. Passing-Bablok regression analysis and Bland-Altman plots were used to compare the results. RESULTS: We observed good stability of samples stored at -80°C. hsCRP measured on the day of blood draw was similar to levels measured after more than 4 years of storage. There were small interlaboratory differences with the R&D ELISAs for hsIL6 and hsTNFα. We found a linear correlation between the Bender Medsystems ELISA and the R&D ELISA for hsIL6, with significantly higher levels measured with the R&D ELISA. Over 90% of hsTNFα samples measured with the IBL ELISA were below the detection limit of 0.13 ng/L, rendering this assay unsuitable for large-scale analysis. Similar results were found in the confirmation study. CONCLUSION: In summary, plasma hsCRP showed good stability in samples stored for either less than 2 years or more than 4 years at -80°C. Both the R&D and Bender Medsystems for hsIL6 measurement yielded similar results. The IBL hsTNFα assay is not suited for use in biobanking samples. Assays for the measurement of inflammatory biomarker assays should be rigorously tested before large sample sets are measured.

Lipoprotein-associated phospholipase A2 adds to risk prediction of incident coronary events by C-reactive protein in apparently healthy middle-aged men from the general population: results from the 14-year follow-up of a large cohort from southern Germany.

BACKGROUND: Chronic inflammation represents an essential feature of the atherosclerotic process. Lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme mainly produced by monocytes/macrophages, generates potent proinflammatory products. METHODS AND RESULTS: Plasma concentrations of Lp-PLA2 were determined by ELISA in 934 apparently healthy men aged 45 to 64 years sampled from the general population in 1984 and followed up until 1998. During this period, 97 men experienced a coronary event diagnosed according to the MONICA (MONItoring of trends and determinants in CArdiovascular disease) protocol. Baseline levels of Lp-PLA2 were higher in subjects who experienced an event than in event-free subjects (295+/-113 versus 263+/-79 ng/mL, P<0.01). Lp-PLA2 was positively correlated with total cholesterol (R=0.30, P<0.0001) and age (R=0.12, P=0.001), was only slightly correlated with HDL cholesterol (R=0.09, P=0.005) and C-reactive protein R=0.06, P=0.06), but was not correlated with body mass index or blood pressure. In a Cox model, a 1-SD increase in Lp-PLA2 was associated with risk of future coronary events (hazard ratio [HR] 1.37, 95% CI 1.16 to 1.62). After controlling for potential confounders, the HR was attenuated but remained statistically significant (HR 1.23, 95% CI 1.02 to 1.47). Further inclusion of C-reactive protein in the model did not appreciably affect its predictive ability (HR 1.21, 95% CI 1.01 to 1.45). CONCLUSIONS: Elevated levels of Lp-PLA2 appeared to be predictive of future coronary events in apparently healthy middle-aged men with moderately elevated total cholesterol, independent of CRP. This suggests that Lp-PLA2 and CRP may be additive in their ability to predict risk of coronary heart disease.

Association between Lp-PLA2 and coronary artery disease: focus on its relationship with lipoproteins and markers of inflammation and hemostasis.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) generates pro-inflammatory molecules from oxidized LDL. We examined the association between Lp-PLA2 plasma concentrations and risk of stable coronary artery disease (CAD) in a large case-control study and further assessed the relationship between Lp-PLA2 and various lipid, inflammatory and hemostatic parameters. Lp-PLA2 concentrations were measured in 312 patients with CAD and in 479 age- and gender-matched blood donors. Various sensitive inflammatory and hemostatic markers and a complete lipoprotein profile were obtained. Lp-PLA2 concentrations were significantly higher in cases than in controls (296.1 ng/mL versus 266.0 ng/mL, p<0.0001). In multivariable logistic regression, the age- and gender-adjusted OR for the presence of CAD was 1.61 (95% CI, 1.07-2.44) if the top quartile of the Lp-PLA2 distribution was compared to the bottom quartile. Adjustment for traditional cardiovascular risk factors and statin use resulted in an OR of 2.04 (95% CI, 1.19-3.48). After additional controlling for vWF, the OR was slightly attenuated but still remained statistically significant (OR 1.91; 95% CI, 1.12-3.28). Thus, elevated Lp-PLA2 concentrations were associated with the presence of stable CAD, independent of various biochemical markers. Our results support the hypothesis that Lp-PLA2 may be a novel, independent risk marker for CAD.

Essential role of calcium in vascular endothelial growth factor A-induced signaling: mechanism of the antiangiogenic effect of carboxyamidotriazole.

Vascular endothelial growth factor-A (VEGF-A) plays a major role in tumor angiogenesis and raises the concentration of intracellular free calcium ([Ca2+]i). Carboxyamidotriazole (CAI), an inhibitor of calcium influx and of angiogenesis, is under investigation as a tumoristatic agent. We studied the effect of CAI and the role of [Ca2+]i in VEGF-A signaling in human endothelial cells. VEGF-A induced a biphasic [Ca2+]i signal. VEGF-A increased the level of intracellular inositol 1,4,5-trisphosphate (IP3), which suggests that VEGF-A releases Ca2+ from IP3-sensitive stores and induces store-operated calcium influx. Reduction of either extracellular or intracellular free Ca2+ inhibited VEGF-A-induced proliferation. CAI inhibited IP3 formation, both phases of the calcium signal, nitric oxide (NO) release, and proliferation induced by VEGF-A. CAI prevented neither activation of VEGF receptor-2 (VEGFR-2) (KDR/Flk-1), phospholipase C-g, or mitogen-activated protein kinase (MAP kinase) nor translocation of nuclear factor of activated T cells (NFAT). We conclude that calcium signaling is necessary for VEGF-A-induced proliferation. MAP kinase activation occurs independently of [Ca2+]i but is not sufficient to induce proliferation in the absence of calcium signaling. Inhibition of the VEGF-A-induced [Ca2+]i signal and proliferation by CAI can be explained by inhibition of IP3 formation and may contribute to the antiangiogenic action of CAI. Calcium-dependent NO formation may represent a link between calcium signaling and proliferation.

Effect of drinking on adiponectin in healthy men and women: a randomized intervention study of water, ethanol, red wine, and beer with or without alcohol.

OBJECTIVE: Moderate alcohol consumption is associated with reduced incidence of type 2 diabetes and cardiovascular mortality and increases adiponectin concentrations, but effects might differ according to sex and beverage consumed. RESEARCH DESIGN AND METHODS: A total of 72 healthy individuals (22-56 years) were enrolled in this randomized controlled crossover trial. After washout, two interventions for 3 weeks followed: ethanol (concentration 12.5%), beer (5.6%), or red wine (12.5%) equivalent to 30 g ethanol/day for men and 20 g/day for women or the same de-alcoholized beverages or water. Adiponectin was measured by sandwich enzyme-linked immunosorbent assay. RESULTS: Among women, adiponectin significantly increased after consuming red wine (29.8%, P < 0.05) and increased among men after ethanol solution (17.4%, P < 0.05) and consuming beer (16.1%, P < 0.05). De-alcoholized beverages had no substantial effect on adiponectin concentrations. CONCLUSIONS: Moderate amounts of ethanol-containing beverages increased adiponectin concentrations, but sex-specific effects might depend on type of beverage consumed.

Variability of serial lipoprotein-associated phospholipase A2 measurements in post myocardial infarction patients: results from the AIRGENE Study Center Augsburg.

BACKGROUND: Of the numerous emerging biomarkers for coronary heart disease (CHD), lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), an enzyme involved in lipid metabolism and inflammatory pathways, seems to be a promising candidate. Implementation of Lp-PLA(2) measurement into clinical practice, however, requires data on the reliability of such measurements. METHODS: We measured Lp-PLA(2) concentrations by ELISA in blood samples drawn from 200 post-myocardial infarction patients (39-76 years) at 6 monthly intervals between May 2003 and February 2004, for a total of 1143 samples. We estimated analytical, within-individual, and between-individual variation, the critical difference, and the intraclass correlation coefficient of reliability (ICC) to assess the reliability of serial Lp-PLA(2) measurements. RESULTS: The mean (SD) plasma Lp-PLA(2) concentration for the study participants was 188.7 (41.8) microg/L, with no significant difference between men and women. The analytical CV for Lp-PLA(2) was 4.4%, the within-individual biological CV was 15%, and the between-individual CV was 22%. The ICC was 0.66. An important part of the total variation in plasma Lp-PLA(2) concentration was explained by the between-individual variation (as a percentage of the total variance, 66.1%), whereas the within-individual variance was 31.3%. The analytical variance was as low as 2.6%. CONCLUSIONS: Between-individual variation in Lp-PLA(2) concentration was substantially greater than within-individual variation. In general, our data demonstrate considerable stability and good reproducibility of serial Lp-PLA(2) measurements, results that compared favorably with those for the more commonly measured lipid markers.

Prevalence of non-alcoholic fatty liver and characteristics in overweight adolescents in the general population.

Overweight and obesity among children and adolescents are increasing. Fatty liver disease (FLD) is an emerging problem in this age group. We investigated prevalence of overweight and non-invasive FLD and associated clinical characteristics in a representative population-based sample of 378 children and adolescents aged 12-20 years who were randomly selected from the general population in Leutkirch, Southern Germany. Overweight was defined as having a body mass index above the 90th percentile for the respective age and sex. About 15% of female (29 out of 194) and 12% of male participants (22/182) were overweight. Among females, only one non-overweight individual showed signs of FLD but in more than one third of the overweight males (8/22) signs of FLD were present. Overweight subjects in general had an unfavourable lipid profile and abnormal concentrations of obesity-related hormones such as significantly lower concentrations of adiponectin and increased levels of inflammatory markers including C-reactive protein and fibrinogen. Overweight males with signs of FLD showed even more severe altered metabolic responses compared to those who were overweight without signs of liver injury. FLD was not explained by alcohol consumption or other chronic liver disease. In this sample of children and adolescents representative of the general population a high prevalence of non-alcoholic fatty liver disease (NAFLD) is found in overweight males. These individuals showed the most severe metabolic alterations compared to non-overweight and overweight individuals without NAFLD indicating even higher risk for future overweight and obesity-related diseases such as type 2 diabetes and cardiovascular disease.