Do inflammation and procoagulation biomarkers contribute to the metabolic syndrome cluster?

Context The metabolic syndrome (MetS), in addition to its lipid, metabolic, and anthropomorphic characteristics, is associated with a prothrombotic and the proinflammatory state. However, the relationship of inflammatory biomarkers to MetS is not clear. Objective To study the association between a group of thrombotic and inflammatory biomarkers and the MetS. Methods Ten conventional MetS risk variables and ten biomarkers were analyzed. Correlations, factor analysis, hexagonal binning, and regression of each biomarker with the National Cholesterol Education Program (NCEP) MetS categories were performed in the Family Heart Study (n = 2,762). Results Subjects in the top 75% quartile for plasminogen activator inhibitor-1 (PAI1) had a 6.9 CI95 [4.2–11.2] greater odds (p < 0.0001) of being classified with the NCEP MetS. Significant associations of the corresponding top 75% quartile to MetS were identified for monocyte chemotactic protein 1 (MCP1, OR = 2.19), C-reactive protein (CRP, OR = 1.89), interleukin-6 (IL6, OR = 2.11), sICAM1 (OR = 1.61), and fibrinogen (OR = 1.86). PAI1 correlated significantly with all obesity and dyslipidemia variables. CRP had a high correlation with serum amyloid A (0.6) and IL6 (0.51), and a significant correlation with fibrinogen (0.46). Ten conventional quantitative risk factors were utilized to perform multivariate factor analysis. Individual inclusion, in this analysis of each biomarker, showed that, PAI1, CRP, IL6, and fibrinogen were the most important biomarkers that clustered with the MetS latent factors. Conclusion PAI1 is an important risk factor for MetS. It correlates significantly with most of the variables studied, clusters in two latent factors related to obesity and lipids, and demonstrates the greatest relative odds of the 10 biomarkers studied with respect to the MetS. Three other biomarkers, CRP, IL6, and fibrinogen associate also importantly with the MetS cluster. These 4 biomarkers can contribute in the MetS risk assessment.


Introduction
Metabolic syndrome (MetS) is a cluster of physiologic markers that includes obesity, insulin resistance, dyslipidemia, and hypertension. The National Cholesterol Education Program ATP III (NCEP) considers a proinflammatory and prothrombotic state as characteristic of metabolic syndrome (MetS) [1]. A group of biomarkers, products of proinflammatory and prothrombotic states, may play an important role in the cascade of the biochemical processes in the MetS development [2,3]. It has been suggested that imbalance in the energy associated with obesity, can drive all aspects of MetS, including the proinflammatory and prothrombotic states [3].
Inflammation and thrombosis are considered to influence the pathogenesis of coronary heart disease. Inflammation in general is associated with increased levels of leucocytes, fibrinogen, and C reactive protein (CRP) as well as other biomarkers. For example, Interleukin-6 (IL6) is considered an important inducer of the hepatic secretion of CRP. Plasma levels of IL6 and CRP have been reported to predict type 2 diabetes in humans [4]. It is assumed that the up-regulation of proinflammatory cytokines may trigger an impaired endothelial response and the production of other substances such as plasminogen activator inhibitor 1 (PAI1). The inflammation may serve as a stimulus to control procoagulation factors, downregulate procoagulants, and inhibit fibrinolysis [5][6][7].
Although several studies have used multivariate factor analysis as a tool for characterizing MetS clusters of risk variables [8][9][10][11], only a few have also included inflammatory markers [10,[12][13][14]. Sakkinen et al. [12] studied 322 nondiabetic elderly. The traits were 11 conventional MetS risk variables and 10 procoagulation, inflammation and fybrinolysis biomarkers. They found that PAI1 clustered with the "body mass" latent factor while CRP, fibrinogen, DDimer, and a few other biomarkers clustered as an "inflammation" latent factor. PAI1 was also associated with the "insulin/glucose" factor. They hypothesized that obesity is related to impaired fibrinolysis. Meigs [9] commented on the failure of inflammatory markers to contribute substantially to the "body mass" factor, a surprising finding considering the fact that CRP was reported to be associated with BMI. For example, Laaksonen et al. [15] showed that middle-aged non-diabetic men with CRP levels ≥ 3 mg/l at baseline (with low grade inflammation), were 3 times more likely to develop MetS, but the risk weakened when they adjusted the data for BMI. Similar findings for three ethnicities were described by Hanley et al. [14] (n = 1,087). They found PAI1 contributed to a "metabolic syndrome" factor, whereas fibrinogen and CRP contributed to an "inflammation" factor. Also, Yudkin et al. [10] (n = 393) reported that fibrinogen, CRP, and IL6 contributed together in a separate "inflam-mation" factor, but with a very low contribution in the "metabolic syndrome" factor. Recently, Tang et al. [13] utilized two biomarkers as part of the risk factors for MetS. Studying data in the Family Heart Study, they found factor 1 as the most representative of MetS where PAI1 was an important contributor.
We studied ten conventional MetS risk variables in the NHLBI Family Heart Study (Time 2, see Methods). Their combinations in a multivariate factor analysis generated factors of MetS. A subgroup of the conventional variables in our study represented the set of the variables utilized to qualitatively characterize MetS (NCEP definition, see Methods). Consequently, the 10 conventional variables served as a representative construct for MetS. Adding ten prothrombotic and proinflammatory biomarkers in the study provided an ample vision to assess the role of ten biomarkers in the MetS cluster.

Sampled population
The NHLBI Family Heart Study (FHS-Time 2) is a familybased study that investigates genetic and non-genetic causes of coronary calcification, atherosclerosis, and cardiovascular risk factors during a 2 nd visit of selected FHS families. The white cohort (n = 2,762, including 19 participants with race undefined or "other") was recruited through 2002-2003 at five centers. Details of this study are reported elsewhere [16,17]. The exact numbers of subjects analyzed varied, because some of the biomarker assays were available only for a smaller group of participants (see the corresponding tables and figures for the exact sample sizes).

Risk variables
Ten MetS conventional risk variables were studied: body mass index (BMI, kg/m 2 ), waist circumference (WAIST, cm), waist to hip ratio (WHR), percent body fat (PBF), fasting (≥ 12 hours) glucose (GLUC, mg/dl), high density lipoprotein cholesterol (HDLC, mg/dl), tryglicerides (TG, mg/dl), low density lipoprotein cholesterol (LDLC, mg/ dl), and systolic (SBP, mm Hg) and diastolic blood pressure (DBP, mm Hg). PBF was measured as the bioelectric impedance of the body and was calculated based on the Lukaski formula [18]. The venipuncture blood samples were drawn and collected at each field center. Blood biochemistries were quantified at the Central Biochemistry Laboratory at Fairview-University Medical Center in Minneapolis, MN, described in detail elsewhere [17].
Ten inflammatory, prothrombotic, or fibrinolysis biomarkers were tested for their contribution to the MetS, respectively CRP, fibrin fragment D-Dimer (DDIMER), fibrinogen, interleukin-2 soluble receptor alpha (IL2SR), IL6, monocyte chemotactic protein 1 (MCP1), matrix metalloproteinase (MMP3), PAI1, serum amyloid A (SAA), and soluble intercellular adhesion molecule 1 (sICAM1) ( Table 1). Eight of the biochemical assays on the inflammatory markers were performed at the Laboratory for Clinical Biochemistry Research, University of Vermont, Colchester, VT. PAI1 and fibrinogen assays were performed at the Central Biochemistry Laboratory at Fairview-University Medical Center in Minneapolis, MN. The following assays were carried out: CRP (mg/l) was measured using the BNII nephelometer from Dade Behring utilizing a particle enhanced immunonepholometric assay. Intra-assay coefficient of variation (IACV) ranged from 2.3 -4.4% and inter-assay coefficient of variation (IRCV) ranged from 2.1 -5.7%.
Fibrinogen (mg/dl) concentrations were quantified by the STAR automated coagulation analyzer (Diagnostica Stago), and the clotting method of Clauss [20]. In this method, the level of fibrinogen is directly correlated to the clotting time of a diluted plasma sample in the presence of excess thrombin. Participants' fibrinogen results were then validated based on control results. The IACV was 4% and the IRCV was 4%.
MMP3 (ng/ml) was measured by an ultra-sensitive, solidphase sandwich ELISA using a polyclonal antibody specific for both the pro-and active forms of MMP-3 (Quantikine Human MMP-3 (total) Immunoassay; R&D Systems, Minneapolis, MN). The IACV and IRCV ranged from 5.7-6.4% and 7.0-8.6%, respectively. PAI1 (ng/ml) assay is sensitive to free PAI1 (both latent and active), but not PAI1 in complex with t-PA. It is done as a two-site ELISA. The analytical CV for this assay was 3.47% [23][24][25].

Metabolic syndrome
A grouping of at least any 3 of the 5 following MetS categories beyond the NCEP defined thresholds in a subject, was considered as a clinical expression of the NCEP MetS: Abdominal obesity, given as large WAIST for men >102 cm and > 88 cm for women; high fasting GLUC ≥ 110 mg/ dl or medication for diabetes; high TG ≥ 150 mg/dl; low HDLC < 40 mg/dl in men or < 50 mg/dl in women; high blood pressure (SBP/DBP) ≥ 130/≥ 85 mm Hg or antihypertensive medication use [1].

Statistical analysis
Variables of interest were expressed as mean ± SD (Table  1). In addition, four statistical techniques were applied to investigate the contribution of 10 biomarkers onto the conventional MetS risk variables clusters and/or to the MetS categories.
First, the multivariate factor analysis was performed with the FACTANAL function, with the maximum likelihood estimate option and the "Varimax" rotation, in Splus version 6.2 of Insightful Corp., Seattle, WA. This method was performed first on 10 conventional MetS risk variables. After, one biomarker was added in the analysis, subject to the sample sizes available, to discover any cluster of each biomarker onto the identified latent factors. The acceptance of an extra factor in the model was made by applying two general rules, the p-value of the model had to be < 0.05, and the last factor had to have at least two contributing risk variables (with a contributing coefficient about 0.4 or above). We stopped beyond the 5-th factor because the additional factors represented only single variables. The contributing coefficients for the standardized variables correspond to correlation coefficients of the original risk variables to the latent factors. A contributing coefficient value in the tables greater/equal to 0.4 was highlighted in bold and is considered as having a substantial influence on the factor. In advance to factor analysis, distributions of all risk variables were checked for their fit to a normal distribution. Variables BMI, WHR, HDLC, TG, CRP, DDIMER, fibrinogen, IL2SR, IL6, MCP1, MMP3, PAI1, SAA, and SICAM1, were natural log transformed. GLUC was expressed as the inverse of a power transformation (1/GLUC 2 ). Risk variables went also through covariate adjustments, in a stepwise regression in SAS v. 9.1.3 for Linux OS, for age, age 2 , age 3 and recruitment center, within gender. The residuals were standardized to mean 0 and variance 1.
Second, Pearson correlations were produced on the above preprocessed data, by using the PROC CORR of SAS (Table 2).
Third, a binary association between the PRESENCE/ ABSENCE of a participant in the top 75% quartile, as well as in the 50% of the data for each biomarker and if CLAS-SIFIED/NOT with MetS was evaluated through logistic regression (PROC LOGISTIC of SAS).
In addition, a graphical technique named "hexagonal binning" was implemented [28]. For its methodology, results, and discussion see the Appendix.

Results
Participants between ages 30 and 93 had a mean BMI of 28.9 ± 5.68 (Table 1). PAI1 levels were relatively high (39.58 ± 44.36) with an interquartile range (IR) of 46 between the 25% quartile (q 25 ) and 75% quartile (q 75 ) (see Table 1 and Figure 1). The CRP mean level was 3.61 ± 5.65. The distribution is shown in Figure 2 which indi-cates an IR of CRP of 3.4 in a sample of 2,685 participants. The central 50% of the data were more spread around the mean for PAI1 than for CRP. Using the ratio of IR/SD we found that fibrinogen, sICAM1, PAI1, IL6, and MMP3 compared to BMI, had a similar spread of the central 50% of the data. In contrast, the central 50% of the data were less spread around the mean compared to BMI for SAA, CRP, DDIMER, IL2sr, and MCP1.
It is interesting to note that PAI1 correlated significantly with all obesity variables (with BMI, 0.53; PBF, 0.51; WAIST, 0.56), IL6 (0.49), dyslipidemia, even with SBP and DBP, reaching almost the lowest but still significant correlation with fibrinogen (  Figure 2).
The above correlation matrix reflected in the results of factor analysis. The factor analysis with "Varimax" rotation on the 10 conventional risk variables produced 4 latent factors. The first factor was a contribution of BMI, WAIST, PBF, and more modest contributions of WHR and GLUC. The second factor was mostly SBP and DBP contributions. The third factor was contributed by TG, HDLC, with less contribution of GLUC, LDLC, and PBF. The last factor represented a contribution of WHR and WAIST. The MetS cluster of latent factors explained about 60% of the total variance of the conventional risk variables.    -T2  BMI  PBF  WAIST  WHR  GLUC  LDLC  HDLC  TG  SBP  DBP  CRP  SICAM1  FIB  PAI1  IL2SR  IL6  MCP1  MMP3   25th percentile; and a black color annotates the 50% central data of PAI1 distribution. The locations of these points in Figure 4   For presentation clarity, 1 data point above 2500 pg/ml for MCP1, and 3 data points above 100 mg/l for SAA were removed from the graph.

Discussion
Inflammation, procoagulation and fibrinolysis biomarkers have been recently hypothesized to be associated with MetS [12] and the development of atherosclerosis [29]. In our study, PAI1 represented an important contributor to the obesity and lipids factors and associated with the NCEP MetS. Similar to our findings, PAI1 had a significant contribution in the "Metabolic syndrome" factor in the Hanly et al. [14] and in the Tang et al. [13], and in the "Body mass" and lesser in the "Insulin/glucose" factors in Sakkinen et al. studies [12]. PAI1 was strongly associated with MetS components such as BMI, TG, a homeostasis model assessment of insulin resistance, hs-CRP, and For presentation clarity 2 data points above 70 mg/l for CRP, and 1 data point above 6000 pg/ml for IL2SR were removed from the graph.  [30]. PAI1 prevents plasmin generation and is considered a primary inhibitor of fibrinolysis [31], and of extracellular matrix degradation [32]. Similar to CRP, PAI1 is lowered by weight loss and drugs that improve insulin sensitivity. PAI deficient mice were resistant to diet induced obesity, explained by increased energy expenditure [33]. PAI1 is induced by many proinflammatory and pro-oxidant factors. For example, TNFα (tumor necrosis factor alpha) is an inducer of the increased levels of PAI1 [34][35][36][37]. Transgenic mice with elevated levels of PAI1 in plasma developed venous occlusions [38]. Transgenic mice with overexpression of human PAI1 were found to develop macrovascular coronary thrombosis and subendocardial myocardial infarction [39]. Smith et al. [40] concluded that PAI1 together with fibrinogen, DDIMER were associated significantly with risk of CVD. Consequently, PAI1 has important functions and is a biomarker that can increase the prediction for MetS in addition to the conventional risk factors.
Mertens et al. [7] investigated the association of 5 biomarkers with the MetS as defined by the NCEP criteria in 520 overweight and obese subjects, concluded that PAI1 is a true component of MetS. However, questions remain open whether PAI1 genes contribute to the MetS development or represent a step in the cascade of the biochemical pathways of MetS. It has been proposed that as a result of obesity and hypoxia white adipose tissue overspills inflammation markers [41]. Skurk and Hauner [42] reviewed obesity and impaired fibrinolysis and reported that increased fat cell size and adipose tissue mass associate with higher offerings of PAI1 in circulation. Moreover, Liang et al. [33] studying cultured adipocytes from PAI1 (+/+) and PAI1(-/-) mice, found that PAI1 deficiency had a protective role against insulin resistance, and was also associated with up-and down-regulation of some other important genes.   [4], Ridker et al. [43], and Biasucci et al. [44] summarized that CRP predicts incident myocardial infraction, stroke, peripheral arterial disease, sudden cardiac death, and confers additional prognostic value at all levels of MetS. In contrast, Lloyd-Jones and Greenland [45] argued that there is no advantage in adding CRP to the standard risk model for coronary vascular disease (CVD). The CRP predictive values were also explored by Pankow et al. [46] who found that BMI, WHR and the prevalent diabetes together explained 30% and 22% of inter-individual variability in the CRP levels respectively in women and men. In our study CRP contributes a small fraction to the obesity factor (a loading of 0.35) and less to the lipids factors (a loading of 0.14). Quite the oppo-site, CRP clustered well in an independent latent factor with fibrinogen (a loading of 0.8, data not shown). The trends in our correlation findings for CRP matched with the ones found in other publications. For example, CRP had a high correlation 0.47 (Sakkinen et al. [12]) and 0.5 (Hanley et al.[14]) with fibrinogen, as two markers of the acute-phase response.
Another interesting biomarker with significant findings in our data was IL6. On severe exercise, IL6 is released in large quantities from the skeletal muscle [47]. IL6 has been suggested to be involved in the insulin sensitivity in mice [48,49], and recently it is suggested that IL6 might influence glucose tolerance [50,51]. Elevated levels of IL6 are found to be a predictor of CVD [52]. In our data, IL6 A three-dimensional presentation of relations among PAI1, HDLC, and TG, which accounts for only a small number of rela-tionships of PAI1 with the classical risk factors of MetS  sICAM1 is an important adhesion molecule and is implicated to mediate the firm adhesion of leukocytes into endothelium [53]. sICAM1 in our data correlated positively and significantly with PAI1 and CRP. Other studied biomarkers such as MCP1, SAA, and IL2SR had significant correlations with CRP. Of them, SAA is an acute-phase protein that exhibits a strong rise in blood in response to inflammation. In our data the SAA response to inflammation was depicted with its highest significant correlation (0.6) to CRP, but was not associated with MetS.
It has been hypothesized that a chronic, mild inflammatory state is the ground of MetS. Besides, Dancan and Schmidt [54] suggested that chronic activation of the innate immune system can be related to MetS. Recently, Forsythe et al. [55] showed that low carbohydrate diet decreased greatly TNF-α, IL6, IL8, MCP1, ICAM, and PAI1. Based on these and several other reported facts, we speculate that some of these biomarkers are not only biochemical steps in the cascades of inflammation, fibrinolysis and prothrombosis, but also markers of obesity and dyslipidemia. DDIMER and MMP3 in our data could not be used as direct predictors of the NCEP MetS, while PAI1, CRP, IL6, and fibrinogen can convey additional information on individuals classified with MetS. In particular PAI1, a key component in the biochemical cascade of fibrinolysis, may simultaneously influence fibrinolysis and coagulation which may contribute in the arterial remodeling and in the atherogenesis [29].
To our surprise, LDLC was not a good measure of the relationships with the biomarkers studied. Its correlation coefficients with biomarkers were practically insignificant. Therefore, we would suggest that future studies of MetS use HDLC and TG as more reliable measures for the lipids component.
Our study has also limitations. We studied only 10 biomarkers. Their sample size depended on the cost of the biochemical assays. We have previously reported that about 25% of the FHS Time 2 participants studied used anti-hyperlipidemics, and that these medications were a confounder when studying the MetS prevalence [56]. It is known that such treatments affect concentrations in plasma of a few biomarkers studied. However, at present no medication summaries for biomarkers were available and thus, unfeasible to account for those effects.
In conclusions, PAI1 significantly correlated with most of the MetS conventional variables. PAI1 clustered in obesity and lipids factors, and participants in the PAI1 top 75% percentile had higher odds, than other 9 biomarkers, to be classified with MetS. PAI1 is an important risk factor for MetS. Three other biomarkers, CRP, IL6, and fibrinogen associate importantly with MetS cluster. In contrast DDIMER, SSA, and MMP3 did not associate directly with MetS cluster.
biomarkers, but the biomarker relation trend with categories of MetS remained flat. This reinforced our previous conclusions that with the studied sample it is less probable that DDIMER and MMP3 contributed in the development or were associated with MetS. In the BMI graph one can see that the frequency of subjects with MetS increased as the BMI values augmented. From the Figure 1 it is clear more than half of the subjects with BMI above 31.9 (q 75 ) were classified with MetS. Although a small group of subjects were with BMI less than 25 (q 25 ), they also were classified with MetS. This shows that MetS is not only result of obesity in combination with other components of MetS, but it can also be a hypertension-dyslipidemic-hyperglycemic combination. For the PAI1 biomarker, (Figure 1), subjects with PAI1 greater than 54.5 (q 75 ) were predominantly classified with MetS. In conclusion, hexagonal binning was a spatial graphical attempt to better characterize and understand the contributions of each biomarker studied in relation to MetS NCEP categories.
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