Genes, health-behavior and the psychosocial environment interact to determine whether or not, and, if at all, how rapidly silent atherosclerosis will progress to the clinical manifestation of CAD [34
]. The development of coronary artery sclerosis is a life-long process that probably has its onset in childhood [36
]. Particularly, as suggested by post mortem studies, inflammation-related endothelial damage plays an important role in atherosclerosis onset and progression early in life [37
]. The understanding of atherosclerosis as an inflammatory disease [1
] has kindled much interest in a number of genetic polymorphisms coding for inflammatory molecules potentially related to CAD [38
While plasma levels of the proinflammatory cytokine TNF-α are regulated by several polymorphisms of the TNF-α gene [39
], it is the TNF-α G-308A gene polymorphism which has been most intensely scrutinized as one candidate polymorphism underlying CAD [8
]. Plasma TNF-α levels predicted second myocardial infarction [40
], and have been associated with common carotid intima-media thickness [41
]. TNF-α also stimulates the liver to produce CRP [8
], which, itself, has been shown to predict coronary risk in numerous population based studies [7
Interestingly, blood cells from individuals who carry the A allele of the TNF-α G-308A gene polymorphism express more TNF-α in vitro
upon stimulation with lipopolysaccharide than cells from individuals being homozygous for the G allele [18
]. Despite this association, several studies did not find a significant association between the TNF-α G-308A gene polymorphism and incident CAD [19
]. There are, however, no studies examining whether established cardiovascular risk factors might interact with the TNF-α G-308A gene polymorphism in determining plasma levels of TNF-α and eventually CRP downstream in the inflammatory cascade. We thus investigated the effect of an interaction between smoking severity and the G-308A polymorphism of the TNF-α gene on plasma levels of these two proinflammatory markers. Our specific hypothesis was that there was a cumulative increase of TNF-α and CRP related to the TNF-α G-308A polymorphism in subjects who regularly smoke as compared to non-smokers.
In spite of two recent studies, which found an interaction between smoking and polymorphisms of molecules participating in the inflammatory response [14
], the results from the present study fail to support our hypothesis. More precisely, we found that the interaction between smoking status and the TNF-α G-308A polymorphism did not significantly affect plasma levels of TNF-α and CRP in both unadjusted and adjusted analyses. Also, there was no main effect for the polymorphism investigated in terms of plasma levels of TNF-α and CRP. On the other hand, although not an aim of our study, we confirmed previous findings of increased plasma CRP in regular smokers as compared to non-smokers [11
], while, rather unexpectedly, plasma levels of TNF-α were not different between smokers and non-smokers.
It must be noted that our findings are preliminary, and, they do not allow us to reject the overall hypothesis of a smoking-gene interaction modifying inflammatory processes contributing to atherosclerosis initiation and progression [14
]. For instance, because the number of homozygous carriers of the A allele in our study population was low reflecting low frequency of the AA genotype in the general population, we were unable to analyze whether there might be a "dose-response" relationship between the A allele dosage and CRP levels. Larger sample sizes are clearly needed to detect a potential difference in regulation of proinflammatory markers in plasma across the GG, AG, and AA polymorphism and with respect to their interaction with different cardiovascular risk factors. This reasoning becomes even more obvious with respect to the higher absolute difference in the mean estimates of plasma CRP levels between the GG and the GA/AA genotypes in smokers as compared to non-smokers (Figure). A highly powered study might raise the odds of this absolute difference to become statistically significant. Moreover, the biological plausibility of our hypothesis was straightforward given the important role of smoking, inflammation and their link in CAD [9
]. However, we do not know in how far interactions between smoking, the TNF-α G-308A polymorphism, and other polymorphisms of molecules involved in the inflammatory pathways not investigated in our study [14
] might affect plasma TNF-α and CRP levels in an unexpected way.
The lack of a difference in plasma TNF-α levels between subjects with the A allele as compared to those homozygous for the G allele stands in contrast to previous in vitro
]. However, aside from a power issue, our measured values of TNF-α levels only slightly exceeded the assay's sensitivity limit, incurring a larger chance of measurement error. We may speculate that a relation between gene variant, smoking, and circulating TNF-α levels might have been uncovered among patients with atherothrombotic disorders or other inflammatory conditions. Moreover, we measured systemic TNF-α; circulating TNF-α may not necessarily reflect TNF-α secretion at sites of confined subendothelial atherosclerotic lesions, where regulatory polymorphisms are most likely to affect reactions of immune cells. Finally, interactions also involving IL-6 polymorphisms [15
] may play a role in the association between smoking, elevated CRP, and increased CAD risk. Future studies thus may want to investigate whether IL-6 polymorphisms might be associated with plasma CRP levels and whether they interact with TNF-α and CRP in smokers.