Arterial stiffness increases with age and has been associated with hypertension, diabetes mellitus (DM), end-stage renal disease and atherosclerosis (Avolio et al 1983; Roman et al 1992; Girerd et al 1994; London et al 1990; Wada et al 1994; van Popele et al 2001). Arterial stiffness has recently been established as a predictor of cardiovascular events (Benetos et al 1997; Franklin et al 1999; Boutouyrie et al 2002; Mattace-Raso et al 2006). The extent of the increase in stiffness may depend on various factors, such as genetic variations.

Inflammatory markers have been found to be associated with arterial stiffness (Mattace-Raso et al 2004; Kullo et al 2005; Tomiyama et al 2005; Vlachopoulos et al 2005). Further study into variations in genes in the inflammatory pathway in relation to arterial stiffness may provide more information on potential pathophysiological mechanisms. Indeed, significant heritability estimates have been found for arterial stiffness (Sayed-Tabatabaei et al 2005).

Interleukin-6 (IL-6) is a pleiotropic cytokine with many different functions. It plays an important role in the acute-phase response and inflammatory cascade, such as up-regulation of acute-phase proteins as CRP (Castell et al 1990; Heinrich et al 1990; Nabata et al 1990; Yudkin et al 1999). An association between IL-6 levels and increased arterial stiffness has been described (Diamant et al 2005; Mahmud and Feely 2005; Vlachopoulos et al 2005). Also elevated CRP levels have been shown to be associated with increasing pulse pressure and increased arterial stiffness (Mattace-Raso et al 2004; Kullo et al 2005; Tomiyama et al 2005; Vlachopoulos et al 2005). Fishman and colleagues (1998) detected a functional polymorphism in the promoter region of the human IL-6 gene (G -> C, 174 bp upstream from the start site). This polymorphism appears to influence the IL-6 gene transcription and is associated with elevated CRP levels (Fishman et al 1998; Basso et al 2002; Jenny et al 2002; Vickers et al 2002; Sie et al 2006).

To the best of our knowledge, the IL-6 gene has not been studied in relation to arterial stiffness. We studied the association of the IL-6 –174 G/C promoter polymorphism, measures of arterial stiffness and levels of CRP and IL-6 within the Rotterdam Study.

The Rotterdam Study is an ongoing prospective cohort study including 7983 participants of 55 years and older. Its general aims are to investigate determinants of chronic diseases (Hofman et al 1991). During the first phase of this study (1990–1993), all inhabitants of a Rotterdam suburban area (Ommoord) aged 55 years and over were invited to participate in this study. The response rate was 78%. The third examination phase took place from 1997–1999, during which measures of arterial stiffness were performed. Approval of the Medical Ethics Committee of the Erasmus University Rotterdam was obtained for the Rotterdam Study. From all participants written informed consent was acquired. A more in depth description of the Rotterdam Study was published previously (Hofman et al 1991).

Carotid-femoral PWV (m/s) was measured using an automatic device (Complior, Colson) and was calculated as the ratio between the distance travelled by the pulse wave and the foot-to-foot time delay.

Common carotid artery distensibility was assessed by measuring the vessel wall motion of the right common carotid artery using a duplex scanner (ATL Ultramark IV, operating frequency 7.5 MHz) connected to a vessel wall movement detector system (Hoeks et al 1990; Kool et al 1994). The cross-sectional arterial wall distensibility coefficient (DC) (1/MPa) was calculated as a measure of arterial stiffness. A decreased distensibility coefficient implies increased carotid stiffness.

Pulse pressure (PP) (mm Hg) was defined as the difference between systolic and diastolic blood pressure, using the mean systolic and diastolic blood pressure of two measurements obtained by measuring blood pressure on the right arm using a random-zero sphygmomanometer.

Details on all measures of stiffness, have been described previously (van Popele et al 2001; Mattace-Raso et al 2006).

General characteristics are described in Table 1 (ln-transformed data are back transformed). Genotype and allele proportions were in Hardy Weinberg equilibrium (p = 0.18).

Table 1 Population characteristics by IL6 –174 G/C genotype

The C-allele of the –174 G/C polymorphism (frequency: 40%) was significantly associated with an increased PWV, with evidence for an allele-dose effect (Figure 1). After adjustment for age, gender, MAP, and heart rate, this trend was significant (model 1, p = 0.016), and remained significant after further adjustment for cardiovascular risk factors (model 2, p = 0.024), and atherosclerosis (model 3, p = 0.018). The association was lost after additional adjustment for levels of IL-6 and CRP (data not shown). Those with the CC-genotype had a (significantly) 0.35 m/s higher PWV than those with the wildtype GG genotype (model 1, p = 0.013; model 2, p = 0.018; model 3, p = 0.013) (Figure 1).

Figure 1 Association of IL-6 –174 G/C and measures of arterial stiffness.

In the analyses of the association of the IL-6 polymorphism and the carotid distensibility coefficient no significant trends (model 1, p = 0.701, model 2, p = 0.579, model 3, p = 0.597), or differences between the genotypes were found (Figure 1).

The C-allele of the –174 G/C polymorphism, however, was significantly associated with an increased pulse pressure, with evidence for an allele-dose effect (Figure 1). After adjustment for age and gender this trend was significant (model 1, p = 0.036), and remained significant after further adjustment for cardiovascular risk factors (model 2, p = 0.049) and atherosclerosis (model 3, p = 0.041). Those with the CC-genotype had a (significantly) 2.0 mm Hg higher pulse pressure than those with the wildtype GG genotype (model 1, p = 0.037; model 2, p = 0.048; model 3, p = 0.041) (Figure 1).

The association between the IL-6 –174 G/C polymorphism and PWV, the distensibility coefficient and pulse pressure was also analyzed in strata of age (<73 years and ≥73 years, with 73 years being the median age). These analyses yielded essentially the same findings as the age-combined analyses (data not shown).

After adjustment for age, gender, MAP, and heart rate, the level of CRP was significantly associated with PWV (regression coefficient [natural log transformed] 0.208 mg/L, 95% CI 0.119–0.298) (Table 2). This association remained significant after adjustment for other cardiovascular risk factors and measures of atherosclerosis (Table 2). An inverse relation of CRP levels and the distensibility coefficient was shown, but was lost after full adjustment (Table 2).

Table 2 Association of pulse wave velocity (PWV) and distensibility coefficient (DC) with levels of C-reactive protein

IL-6 levels, although only available for a small random sample (n = 433), were also analyzed. The analyses suggested a tendency towards a positive, although nonsignificant, association with PWV, also after adjustment (Table 3). A significant inverse association was found between IL-6 levels and the distensibility coefficient within model 1 (Table 3). After full adjustment, this association remained near significant (Table 3).

Table 3 Association of pulse wave velocity (PWV) and distensibility coefficient (DC) with levels of interleukin-6

All analyses stratified by gender yielded no results essentially different from the overall analyses.

We studied a well-known functional polymorphism in the promoter region of the Interleukin-6 gene, –174 G/C in relation to arterial stiffness. The C-allele of the polymorphism was significantly associated with increased PWV and PP. CRP levels were significantly associated with PWV. Findings on levels of IL-6 also suggested (although not significantly) a tendency toward an association with arterial stiffness.

The –174 G/C polymorphism is located in the promotor of the IL-6 gene and may influence gene-transcription. In some studies no effect of this polymorphism on IL-6 and CRP levels was found (Nauck et al 2002; Bennet et al 2003; Bennermo et al 2004; Lieb et al 2004). However, other studies describe it as influencing IL-6 and CRP levels (Fishman et al 1998; Brull et al 2001; Basso et al 2002; Jenny et al 2002; Vickers et al 2002; Bruunsgaard et al 2004). Indeed, in a previous study in the Rotterdam Study, the C-allele was associated with increased CRP levels, but not with IL-6 levels (Sie et al 2006). However, we consider an association with IL-6 levels biologically plausible, and contribute our (non-significant) finding to a very small number of IL-6 samples (Sie et al 2006).

Interleukin-6 is a pleiotropic cytokine with many different inflammatory functions, eg, up-regulation of CRP (Castell et al 1990; Heinrich et al 1990; Nabata et al 1990; Yudkin et al 1999). IL-6 levels have been associated with arterial stiffness. Diamant and colleagues (2005) described a positive association of IL-6 and CRP levels and increased arterial stiffness in a small Dutch population (n = 32) in both type 2 DM patients as well as in healthy controls. Mahmud and Feely (2005) described an association of IL-6 and CRP levels and PWV in a study of Irish hypertensives (n = 78). In a healthy Greek population (n = 100), Vlachopoulos and colleagues (2005) also showed IL-6 and CRP levels to be correlated with PWV. In a much larger study of 9867 healthy Americans, CRP levels were associated with pulse pressure, a manifestation of arterial stiffness (Abramson et al 2002). Within the Rotterdam Study, an association between CRP levels and PWV was shown in 866 subjects, which was confirmed in our larger sample of 2995 subjects (Mattace-Raso et al 2004). Further, Kullo and colleagues (2005) found a relation between CRP levels and PWV in an American population (n = 214).

As CRP and IL-6 levels are associated with arterial stiffness, our findings of a significant association of the polymorphism and increased PWV is biologically plausible. Our significant findings for PP are in concordance herewith. We found no association between the polymorphism and the distensibility coefficient. PWV and the distensibility coefficient both reflect other anatomical regions (the aorta and carotids, respectively), which may contribute to discordances in findings on stiffness. It is also possible, however, that a small effect may have gone undetected due to limitations in sample size.

To assess the influence of age on the relation between the IL-6 polymorphism and arterial stiffness, we also performed analyses stratified by age. However, apart from significance (significance was preserved for PWV in the ≥73 years group and for PP in the <73 years group), the analyses yielded the same findings in the same direction and patterns as in the “age-combined” analyses. Therefore, differences in significance may be more likely due to the reduction in group sizes, than due to strong age-dependent influence on the relation between the IL-6 gene polymorphism and arterial stiffness.

We also found a significant association of CRP levels and PWV. The number of currently available CRP levels within the Rotterdam Study was more than three times larger than the number available at the time of our previous analyses; the results of our present study are in concordance with these earlier findings (Mattace-Raso et al 2004). Our findings suggest IL-6 levels to be positively related to PWV, although this association was not significant. IL-6 has been described to be too unstable in time (plasma half-life of less than 2 hours) to be precisely measured (Riches et al 1992; Waage et al 1989). This might explain the lack of a significant association between PWV and IL-6 levels. In addition we have to remark, that the number of subjects with IL-6 measurements (random sample) was very small.

Our study is based on a large ongoing population-based study in a relatively homogeneous population, as 98% of the participants in our study are Caucasian and are all living in the same suburb of Rotterdam. We adjusted all analyses for established cardiovascular risk factors. The distensibility coefficient has a strong correlation with MAP; a higher MAP in the artery stretches the elastin and collagen fibres in the arterial wall, making the arteries less distensible. Therefore, the analyses were adjusted for MAP.

To interpret the findings correctly, several methodological aspects of the measures of arterial stiffness need to be discussed. First, pulse waves in the carotid artery and the femoral artery travel in opposite directions, while measurements of carotid-femoral PWV is based on the assumption that the pulse wave travels from the carotid artery to the femoral artery. In this way, measuring the distance between the carotid and the femoral artery led to an overestimation of the distance between the sites of the pulse waves, resulting in overestimation of the velocity of the pulse waves. However, variations in anatomy are limited and this error may be considered similar for all subjects examined, therefore we do not think it has seriously biased our results. Second, the distance between the carotid and the femoral artery may be overestimated in (especially adipose) subjects when this distance is measured by tape. To avoid this error we adjusted the analyses for BMI. Third, in computing the carotid distensibility coefficient, we used the brachial PP rather than the carotid PP. Information on comparisons between the carotid and the brachial PP indicates that the carotid PP is lower than the brachial PP but the differences are relatively small (Waddell et al 2001).

Some general remarks: data on measures of stiffness were not available for all subjects who visited the research center. However, missing information was primarily due to logistic reasons, which is likely to be random and thus will not have biased our results. Second, the cross-sectional design of our study limits the ability to infer a causal relationship between the IL-6 –174 G/C polymorphism, inflammation, and arterial stiffness. Any associations do not necessarily imply causality and should be interpreted with reservation. Further, we interpreted our results also in the context of literature and (known) pathophysiology. However, we are also aware that some findings were not significant and may be chance findings. Finally, because our study was performed in a population of predominantly elderly Caucasian subjects, the generalizability of our findings to younger individuals or other ethnicities remains uncertain.

In conclusion, the C-allele of the interleukin-6 –174 G/C promoter polymorphism is associated with increased PWV and PP. Our results consolidate the finding that CRP levels are associated with PWV in an elderly Dutch population.

This study was supported by NWO (Netherlands Organization for Scientific Research) under grant no. 904-61–196 and under the ASPASIA grant no. 015.000.090, by the Center for Medical Systems Biology and by the European Commission under grant QLK6-CT-2002-02629 (GENOMOS).