1Department of Epidemiology, The German Centre for Research on Ageing, University of Heidelberg, Germany
2Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Austria
3Department of Internal Medicine IICardiology, University of Ulm Medical Center, Robert-Koch Str. 8, 89081 Ulm, Germany
Received 22 December 2004; revised 20 April 2005; accepted 28 April 2005; online publish-ahead-of-print 2 June 2005.
* Corresponding author. Tel: +49 731 500 24465; fax: +49 731 500 33872. E-mail address: wolfgang.koenig{at}medizin.uni-ulm.de
See page 1579 for the editorial comment on this article (doi:10.1093/eurheartj/ehi374)
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Abstract |
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Methods and results Three hundred and twelve patients aged 4068 with angiographically confirmed stable CHD and 476 age- and gender-matched controls were included in this casecontrol study. Adiponectin serum concentrations (adiponectin, R&D Systems, Wiesbaden, Germany), markers of inflammation and haemostasis, and an extensive lipid profile were determined. Adiponectin serum concentrations were lower in CHD patients when compared with age- and gender-matched controls, both in men (median 4.95 vs. 5.58 µmol/L, P=0.004) and in women (median 9.64 vs. 11.60 µmol/L, P=0.018). Adiponectin was strongly correlated with lipoproteins and apolipoproteins, in particular HDL-cholesterol (HDL-C), and to a lesser degree with markers of inflammation such as C-reactive protein, IL-6, or markers of coagulation or fibrinolysis. When compared with subjects with adiponectin serum concentrations in the lower quintile, the OR for CHD was 0.52 (95% CI 0.280.95) in the upper one after adjustment for covariates (P<0.007 for trend). After additional adjustment for HDL-C the association was strongly reduced, reflecting the close association between adiponectin and HDL-C.
Conclusion Adiponectin serum concentrations may have an important role in the development of CHD. The protective effect of high serum concentration may partly be mediated by effects on the metabolism of lipoproteins, especially on the metabolism of HDL.
Key Words: Adiponectin Coronary heart disease Inflammation Lipids Casecontrol study
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Introduction |
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Increasing evidence supports the notion that the adipocyte may have an active endocrine function; it produces several cytokines [among them interleukin-6 (Il-6) and tumour necrosis factor- (TNF-
)] and adiponectin, a 30 kDa adipocyte complement-related protein.5 Adiponectin levels in serum are mainly determined by the size and amount of adipocytes. Highest serum concentrations are found in subjects with only few body fat. Adiponectin has insulin sensitizing and anti-atherogenic effects4,5 and lower serum levels have been reported in patients with coronary heart disease.6,7
A variety of inflammatory and other biochemical markers potentially related to atherogenesis have been identified,8,9 some of which may also originate from the adipose tissue. Currently, we are beginning to understand the potential pathophysiological role of adiponectin in atherosclerosis. A reciprocal association with several pro-inflammatory cytokines such as C-reactive protein,10 an important determinant of vascular disease, has been reported. Furthermore, hypoadiponectinaemia has been closely linked to endothelial dysfunction11 and may be involved in lipid meatobolism.12 Whether adiponectin is an independent risk factor in this signalling network or mainly associated with other inflammatory proteins such as Il-6 or C-reactive protein or other cardiovascular risk factors is yet unclear. The further elucidation of the pathogenetic role of adiponectin may allow to identify subjects at increased risk for coronary heart disease (CHD), and subsequently lead to more focused preventive measures and eventually to a specific treatment (e.g. application of recombinant adiponectin).
We investigated the association of adiponectin serum concentrations with a variety of established sociodemographic and laboratory risk markers for CHD and determined the correlation with various acute phase proteins, inflammation-associated cytokines, intracellular adhesion molecule-1 (ICAM-1), and various lipoproteins. Furthermore, we analysed data of a casecontrol study in patients with stable CHD in order to investigate the association of serum adiponectin concentrations with the risk of CHD, after careful adjustment for other established risk factors under special consideration of various pathogenic pathways.
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Methods |
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The control group consisted of subjects who were occasional blood donors at the local Red Cross Centre serving the University Hospitals of Ulm. None of the controls had a history of definite or suspected CHD, and they did not report infections or surgery within the previous 4 weeks. Response was 479 of 570 in controls (84%) of whom 477 could be included in this analysis (from two controls, no adiponectin measurements were available).
Frequency matching for age and gender was performed and a casecontrol ratio of 1:1.5 was intended. All subjects underwent standardized interviews conducted by trained interviewers. The primary objective of the study was to assess the effect of various infectious agents on CHD-risk13 and to investigate the role of other emerging risk factors. All subjects gave written informed consent and the study was approved by the Ethics Committee of the University of Ulm.
Laboratory methods
Venous blood was drawn in the morning under standardized conditions and a complete blood cell count was done (Coulter STKS chamber, Coulter Co., Krefeld, Germany). Within 30 min, the remaining blood was centrifuged at 3000g for 10 min, immediately aliquoted and frozen at 70°C until analysis. In cases, blood drawing was done before the angiographic procedure.
Adiponectin serum concentrations were determined by a commercially available ELISA (adiponectin, R&D Systems, Wiesbaden, Germany). In addition, the following markers of inflammation and haemostasis were determined by ELISA: IL-6 and TNF- (Quantikine, R&D Systems, Wiesbaden, Germany), ICAM-1 (Diaclone, Besancon, France), plasminogen-activator-inhibitor-1 (PAI-1) activity (Immuno, Heidelberg, Germany), and von Willebrand factor (vWF) (Haemochrom, Essen, Germany). In addition, C-reactive protein determinations were done by an immunoradiometric assay (range 0.0510 mg/L).14 Fibrinogen was measured by immunonephelometry (Dade Behring, Marburg, Germany). Serum amyloid A (SAA) was also determined by immunonephelometry (Dade Behring, Marburg, Germany) and finally, measurement of plasma viscosity (PV) was done in a HarknessCoulter viscometer (Coulter Electronics, Luton, UK). Plasma levels of lipoprotein-associated phospholipase A2 (Lp-PLA2) were determined with a commercial Lp-PLA2-ELISA kit (PLACTM Test) was supplied by diaDexus Inc. (South San Francisco, CA, USA). Inter-assay coefficients of variation were 10.9% for adiponectin, 7.0% for IL-6, 17.9% for TNF-
, 14.2% for ICAM-1, 12.0% for C-reactive protein, 7.4% for SAA, 5.0% for fibrinogen, 11.0% for PAI-1, 15.8% for vWF, 2.0% for PV, and 9.6% for Lp-PLA2. HDL-cholesterol (HDL-C) concentrations were determined by routine enzymatic methods.
Statistical methods
Sociodemographic and medical characteristics of CHD cases and control subjects are presented in a descriptive fashion. Mean concentrations of adiponectin were calculated in cases and controls and compared using the Wilcoxon rank sum test. In addition, the distribution of adiponectin (in quintiles of controls) was compared among cases and controls and quantified by a MantelHaenszel 2 test after adjustment for age and gender.
Mean concentrations of adiponectin were calculated for age, gender, and body mass index (BMI) after adjustment for casecontrol status; also for various levels of sociodemographic and other established CHD risk factors after adjustment for age, gender, BMI, and casecontrol status by a general linear regression method. Partial spearman correlation coefficients were calculated for adiponectin serum concentrations and lipoproteins, apolipoproteins, acute phase proteins, and other suggested laboratory CHD risk markers after adjustment for age, gender, BMI, and casecontrol status. A two sided P-value of 0.05 or less was considered to be statistically significant, if not indicated otherwise.
Furthermore, we used unconditional logistic regression to assess the association of adiponectin serum concentrations (in quintiles) with CHD, while simultaneously controlling for age and gender and additionally controlling for BMI, duration of school education, cigarette smoking (pack-years), alcohol consumption, history of hypertension, and history of diabetes mellitus (which are all established risk factors for CHD). The linearity assumption was assessed for continuous variables by a goodness-of-fit test (significance level =0.1). To further elucidate the pathogenic link between adiponectin and CHD risk, we also included the basic model several laboratory markers, which represent defined changes in the pathogenic processes of atherogenesis and showed an association with adiponectin (all variables considered with P<0.1 in bivariate analysis). These were triglyceride-values, HDL-C values, apo-lipoproteins (ApoA1, ApoA2, ApoB, ApoC2, ApoC3, and ApoE), markers of inflammation (C-reactive protein, IL-6 and TNF-
, and leukocyte count), and PAI-1 and D-dimers. All statistical procedures were carried out with the SAS® statistical software package (release 8.2, Cary, NC USA: SAS Institute Inc., 1999).
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Results |
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Table 4 shows the correlation between adiponectin serum concentrations and lipid variables, markers of coagulation, fibrinolysis and inflammation, and Lp-PLA2. There were statistically significant positive correlations of adiponectin concentrations seen with HDL, ApoA1, and ApoA2. Furthermore, negative correlations were seen with PAI-1, C-reactive protein, TNF-, and leukocyte count. There were no statistically significant correlations with fibrinogen, D-dimers, vWF, ICAM-1, IL-6, PV, albumin, and Lp-PLA2.
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Discussion |
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Our data are in line with other reports describing an associaton between low levels of adiponectin serum concentration and risk of CHD. Kumada et al.6 estimated a two-fold risk for ischaemic heart disease associated with low adiponectin levels. Furthermore, a reduced risk of subsequent acute myocardial infarction associated with higher levels of adiponectin in serum at baseline was described in a nested casecontrol study,7 notably, this association was also reduced after adjustment for covariates, but persisted after additional adjustment for lipids, glycemic status, and C-reactive protein.
Various physiological mechanisms of adiponectin have been identified in vitro which could play a role in atherogenesis.6 Systemically measurable markers of low-grade inflammation are important predictors of CHD risk4 and are increased in patients with stable CHD. As the adipose tissue itself at least, in part, generates these markers (TNF-, IL-6 or C-reactive protein as well as of PAI-1), a direct link between adiponectin and these pro-inflammatory cytokines seems conceivable.4 In addition, TNF-
and IL-6 are also negatively related to insulin sensitivity,15,16 whereas adiponectin has a positive effect on it. In addition, TNF-
has also been suggested as a strong inhibitor of adiponectin promoter activity17 and may explain partly the inverse association between the amount of accumulated visceral fat, increased TNF-
secretion, and decreased adiponectin levels.
Although the physiological role of adiponectin has not yet been fully elucidated, it may well be involved in the regulation of many of the inflammatory processes or in the lipid metabolisms, which are contributing to atherosclerosis. However, we found only moderate correlations between adiponectin serum concentrations and these inflammatory factors, of which correlations with TNF- and C-reactive protein were the strongest.
Among the many risk factors involved in atherogenesis disorders of lipoprotein metabolism are considered to play the most important role.18 In particular, high LDL and low HDL concentrations are main determinants of CHD risk. We indeed found the strongest correlations of adiponectin serum concentrations with HDL levels. The correlation to ApoA1, which is essential to HDL-formation was of similar strength. Similar findings were also reported by others.19 Most notably, plasma levels of adiponectin and its relationship to serum HDL and triglyceride concentrations were independent from BMI12 and this supports the suggestion that adiponectin levels play a role in the regulation of lipid metabolism. Furthermore, in our study, the association between adiponectin and CHD was abrogated by including HDL in the model adjusted for conventional risk factors. The most important anti-atherogenic function of HDL considered is its participation in the reverse cholesterol transport, which delivers excess cholesterol from systemic vasculature to the liver for disposal as bile salt.20 In addition, HDL also has antioxidant and anti-inflammatory properties.21
The biochemical mechanisms linking adiponectin and HDL metabolism have not been clarified so far. Adiponectin also seems to have a key role in the metabolic syndrome,22 an accumulation of multiple risk factors. It may therefore represent the link between obesity (or even more important visceral fat accumulation), insulin resistance, and diabetes. Low concentrations of adiponectin may lead to insulin resistance.23 Insulin resistance, in turn, may lower the concentration of HDL through different mechanisms, directly and indirectly. First, insulin may directly stimulate the transcriptional activity of ApoA1, the major apolipoprotein of HDL.24 Secondly, insulin may decrease the production of VLDL25 and enhances the expression of lipoprotein lipase.26 Insulin resistance thus may raise the concentration of triglyceride-rich lipoproteins in the circulation, which may alter the formation and remodelling of HDL particles. Altogether, this raises the possibility that low adiponectin concentrations may cause low HDL-C and that the pro-atherogenic effects of low adiponectin may be mediated by its effects on HDL metabolism. As we found a closer association of adiponectin with HDL than with inflammatory markers, the lipoprotein effects of adiponectin may be more important than the anti-inflammatory links with TNF-, IL-6, or C-reactive protein,27 or the suggested effects on ICAM-1,28 an adhesion molecule that regulates the attachment and transmigration of leukocytes across the vascular endothelium.
The present study has several limitations which should be addressed. CHD was defined invasively by coronary angiography in cases, but for ethical reasons, no coronary angiogram could be obtained in controls. Although we excluded controls with a history or characteristic symptoms of CHD, the presence of asymptomatic CHD cannot be definitely ruled out; however, the prevalence of asymptomatic CHD cases seems to be low in a middle-aged population.29 Furthermore, the choice of blood donors as controls can be considered as suboptimal, as they might be healthier than the target population the cases were drawn from. We tried to minimize this potential bias by carrying out multivariable adjustments for a variety of covariates. As always in casecontrol studies in which exposure and outcome are collected at one point in time, it is difficult to assess the time-sequence of the described associations and therefore, it is highly desirable to replicate our results in prospective studies. The limited sample size may also be a reason why the relationship of adiponectin with markers of inflammation did not reach statistical significance.
Despite these limitations, the current study provides evidence that adiponectin serum concentrations may have an important role in the development of CHD, and it raises the possibility that the protective effect of high serum concentrations may partly be mediated by effects on the lipid metabolism, especially on the HDL levels.
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References |
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