Discussion
This study investigated the influence of severe periodontitis on the plasma inflammatory profile of individuals who had a first MI. To the best of our knowledge, this is the first comprehensive evaluation of circulating inflammatory markers aiming to find inflammatory markers in plasma that could potentially contribute to a mechanistic explanation of the association between periodontitis and MI. We found that patients who had an MI displayed altered expression of CCL19, TNFRSF9 and LAP TGF-β1. The presence of deep periodontal pockets in these patients was associated with increased expression of a set of proteins mainly related to regulation of intracellular signal transduction and chemotaxis.
Patients who had an MI presented increased expression of CCL19 and TNFRSF9 and decreased expression of latent TGF-β1. Interestingly, TNFRSF9 correlated significantly with the amount of radiographic bone loss. A previous study found that serum levels of CCL19 on admission acute coronary syndrome with associated with the development of heart failure.14 Similarly, elevated serum levels of soluble TNFRSF9 (sCD137) in patients with acute coronary syndrome was associated with increased short-term risk of major adverse cardiovascular events.15 Lower frequency of LAP+ regulatory T cells along with lower serum TGF-β1 have been shown in patients with acute coronary syndrome.16 The altered expression of these proteins in stable MI patients might partially account for the increased risk of another cardiovascular event in comparison with non-MI participants.17 Moreover, when we consider only MI patients, those with high residual cardiovascular risk (hs-CRP ≥2 mg/L) showed increased expression of IL-6, HGF, OSM and CSF-1 in comparison to those with low residual risk. This is not surprising as the relationship between CRP and IL-6 is well known and both have been shown to be predictors of adverse cardiovascular outcome after acute coronary syndrome.18 19
MI participants were selected based the periodontal status, where 50 of them were diagnosed with severe periodontitis and 50 did not have severe periodontitis. This led to a very low number of participants with severe periodontitis in the control (non-MI) group. Thus, we decided to restrict our analysis to patients with MI with and without periodontitis. We explored periodontitis based on two parameters, radiographic bone loss and the presence of deep pockets. When proteins in plasma from MI patients with and without severe periodontitis were compared, nine protein were significantly higher in plasma from patients with periodontitis. However, after adjustment for age and smoking, two factors that have a strong influence on the severity of periodontitis, only one protein, CCL23, remained associated with periodontitis.
Furthermore, when assessed according to the presence of at least two periodontal pockets ≥6 mm, periodontitis was associated with increased expression of five proteins, which could be related to the elevated WCC in these patients. These five proteins, IL-18R1, FGF-21, HGF, OSM, and CCL20, differed significantly also after adjustment for age and smoking. It is not surprising that the difference, after adjustment, between patients with and without deep periodontal pockets is bigger as compared with the difference between patients with and without bone loss. While radiographic bone loss is an expression of the severity of periodontitis over time, deep pockets can be seen as an indicator of ongoing periodontal inflammation, which could have a stronger influence on the plasma proteins. Worth noting is that none of the five proteins that differed between patients with and without periodontitis differed between patients with and without MI. However, all of them correlated significantly with hs-CRP, while two of them, HGF and OSM, were increased in patients with MI with high residual risk. Most of these proteins associated with either bone loss or deep pockets have previously been related to periodontitis, such as increased concentrations of HGF and OSM in serum from periodontitis patients20 21 and upregulation of IL-18R1, CCL20 and CCL23 in monocytes stimulated by Porphyromonas gingivalis lipopolysaccharide.22 23 More importantly, most of them have also been associated with increased risk of CVD,24–27 which suggests they might be part of the pathophysiologic mechanisms explaining the increased risk of MI in periodontitis.
We also assessed the associations of important health covariates, such as age, sex, smoking, BMI, systolic BP and HbA1c, with the inflammatory proteins. We found that 24 out of 71 proteins (33.8%) were associated to at least one covariate. Age had the biggest impact on the protein profile, showing an effect on the expression of 15 proteins, which are mainly related to chemokine signalling. Sex, on the other hand, was associated with five proteins, all higher in males than in females. Age has been previously shown to influence the plasma levels of a broad range of proteins some of which were also found in the current study such as IL-8, HGF, Flt3L, OPG, CXCL9 and CXCL10.28 These associations might help to explain the age-associated emergence and the sex discrepancies of chronic inflammatory diseases, including CVD. Smoking had a considerable impact on the protein profile, with seven proteins associated to it. Two of the proteins identified in this study, HGF and MMP-10, have also been associated with smoking in a previous study.29 Interestingly, systolic BP had no significant effect on any protein. On the contrary, a previous study reported a large influence of systolic BP on biomarkers levels, many of which were also affected by age.28 Most of the MI participants in the current study had been treated with renin–angiotensin inhibitors and β-blockers lowering the BP, which could have influenced its association with the proteins.
We also found that diabetes was related to increased expression of nine proteins, IL-18R1, CCL11, CDCP1, OPG, HGF, TGF-α, CCL20, IL-6 and FGF-21. This is in line with a previous study reporting a major shift in the serum proteome in type 2 diabetes, partially reflecting inflammatory processes and extracellular matrix alterations.30 Similarly, plasma expression of HGF, IL-6, FGF-21, CDCP1 and IL-18R1 have been identified among the top 30 proteins associated with newly diagnosed type 2 diabetes.31 It is interesting to note that four of the proteins increased in diabetes were also increased in patients with MI deep periodontal pockets. We have previously reported in the PAROKRANK cohort that undetected diabetes was more frequent among participants with severe periodontitis.12 This was reflected in the sample randomly selected for the current study, where most of diabetes participants had undiagnosed disease at the time of sampling. Moreover, we believe diabetes did not play a major role in driving the differences between MI patients with and without deep periodontal pockets, as its prevalence was similar between the groups. Rather, the similarities in the altered proteins might indicate common pathways in which periodontitis and diabetes increase the risk of CVD.
This study provides limited evidence to a possible mechanistic explanation for the association between MI and periodontitis. There could be several explanations for this, such as the assessment of circulating proteins during the stable rather than acute phase of MI. Almost all patients with MI was treated with drugs having an anti-inflammatory effect such as aspirin and statins, which can be seen in the relatively low hs-CRP levels and its similar levels between patients with and without severe periodontitis. A meta-analysis has shown that patients with periodontitis have increased levels of CRP.32 However, we have not seen this increase in this study, which could be an effect of the medication. Another limitation of the current study could be the lack of genetic analysis as some biomarkers have a significant genetic component.28 Furthermore, as this was a cross-sectional study, we cannot establish any causal claim between the altered proteins, periodontitis and MI. A longitudinal study to investigate these relationships is warranted. Nevertheless, the well-characterised cohort and the unbiased assessment of a large panel of biomarkers are strengths of the study.
Altogether, we conclude that patients with MI with periodontitis have increased systemic inflammatory activity and reveal a set of proteins that might be part of the biological mechanisms linking periodontitis to CVD.