Circulation levels of acute phase proteins pentraxin 3 and serum amyloid A in atherosclerosis have correlations with periodontal inflamed surface area

Abstract Objectives One of the plausible mechanisms in the relationship between periodontitis and coronary artery disease (CAD) is the systemic inflammatory burden comprised of circulating cytokines/mediators related to periodontitis. This study aims to test the hypothesis that periodontal inflamed surface area (PISA) is correlated with higher circulating levels of acute phase reactants (APR) and pro-inflammatory cytokines/mediators and lower anti-inflammatory cytokines/mediators in CAD patients. Material and Methods Patients aged from 30 to 75 years who underwent coronary angiography with CAD suspicion were included. Clinical periodontal parameters (probing depth - PD, clinical attachment loss, and bleeding on probing - BOP) were previously recorded and participants were divided into four groups after coronary angiography: Group 1: CAD (+) with periodontitis (n=20); Group 2: CAD (+) without periodontitis (n=20); Group 3: CAD (-) with periodontitis (n=21); Group 4: CAD (-) without periodontitis (n = 16). Serum interleukin (IL) −1, −6, −10, tumor necrosis factor (TNF)-α, serum amyloid A (SAA), pentraxin (PTX) 3, and high-sensitivity C-reactive protein (hs-CRP) levels were measured with ELISA. Results Groups 1 and 3 showed periodontal parameter values higher than Groups 2 and 4 (p<0.0125). None of the investigated serum parameters were statistically significantly different between the study groups (p>0.0125). In CAD (-) groups (Groups 3 and 4), PISA has shown positive correlations with PTX3 and SAA (p<0.05). Age was found to predict CAD significantly according to the results of the multivariate regression analysis (Odds Ratio: 1.17; 95% Confidence Interval: 1.08-1.27; p<0.001). Conclusions Although age was found to predict CAD significantly, the positive correlations between PISA and APR in CAD (-) groups deserve further attention, which might depend on the higher PISA values of periodontitis patients. In further studies conducted in a larger population, the stratification of age groups would provide us more accurate results.


Introduction
Periodontitis and atherosclerotic cardiovascular diseases, coronary artery diseases (CAD), comprise a major health problem with their high prevalence and mortality rates for the latter 6 . The most biologically plausible mechanism in the relationship between periodontitis and CAD was suggested to be the entry of oral bacteria into the circulation, resulting Thus, PTX3 and CRP were suggested to be associated with atherosclerotic lesions 20 . Serum amyloid A (SAA) was suggested to enhance the local effect and to be a more valuable biomolecular diagnosis for acute myocardial infarctions than the other APR 18 .
The increase of APR in circulation and in periodontal tissues of periodontitis patients was also reported by various studies 11,13,28 . This response was suggested to influence atherosclerosis within the endothelium 1 . The correlations between plasma levels of IL-6 and TNF-α and cardiovascular risk factors were also determined 17 .
The decreased levels of the anti-inflammatory cytokine IL-10 in acute coronary syndrome were found to be associated with the increased cardiovascular risk and clinical instability 3 .
There is a need for a reliable measurement system that evaluates the periodontal inflammation determine the groups to which the patients will belong.

Statistical analysis
The variables were investigated using visual (histograms, probability plots) and analytical methods (Kolmogorov-Smirnov/Shapiro-Wilks's test) to determine whether they are normally distributed. All variables were determined not normally distributed (p<0.05). The parameters are shown as the median (minimum-maximum values). The Kruskal-Wallis test was used to determine the between-group differences for the continuous parameters, and chi-square test was used for the categorical parameters. A p value less than 0.05 was considered to show significant results. To avoid Type-I errors, a Bonferroni correction was applied, and differences between the group pairs were investigated, using the Mann-Whitney U test, with a p value less than 0.0125, being considered significant. Spearman's Rank-order correlation test was used to define the correlations between variables. For multivariate analysis, the possible factors identified with univariate analysis were further entered into the logistic regression analysis to determine the independent predictors of CAD. Hosmer-Lemeshow goodness of fit statistics were used to assess model fit ( YTL, New Turkish Lira; †significant difference between CAD (+) P (+) and CAD (-) P (-); ‡ significant difference between CAD (-) P (+) and CAD (-)P (-); § The average national income is 25. 13  hip ratio (p>0.0125, Table 1). The CAD (+) groups (Group 1 and 2) are significantly older than the CAD (-) groups (Groups 3 and 4), (p<0.0125, Table 1).
The periodontal parameters, PISA and PESA values of the study groups are shown in Table 2.  Table 1).
The serum cytokine and APR levels are in Table 3.
None of the investigated parameters were statistically significantly different between the study groups (p>0.0125).
In Table 4, the statistically significant correlations between the investigated parameters are shown.
Correlations between the parameters were tested in groups with CAD (Groups 1 and 2, n=40), in groups without CAD (Groups 3 and 4, n=37), and in the whole group (N=77). In the CAD (+) groups (Groups 1 and 2), PD showed a significant negative correlation with serum IL-10 level (p<0.05). In CAD (-) groups (Groups 3 and 4), the PISA value showed significant correlations with PTX3 and SAA (p<0.05).
In Table 5

Discussion
In this cross-sectional study, the effect of the magnitude of the periodontal inflamed area on the serum APR and pro-/anti-inflammatory cytokines/ mediators in patients with/without CAD was evaluated.  reactions with elevated levels of CRP and SAA share various common risk factors involving smoking, high BMI, and older age 10 . In this study, the study groups were not statistically significantly different regarding blood lipid levels, BMI, and smoking status ( Table   1). The correlations mentioned were found in CAD (-) patients who had an age lower than the CAD (+) groups. Despite the younger age in the CAD (-) patients, SAA has shown a significant correlation with PISA values, suggesting that the periodontal status might have affected the serum SAA levels. Adjustment for age does not alter the statistical comparative results; there was no significant difference regarding the circulating cytokines and APR between the study groups (p>0.0125, data not shown). However, multivariate logistic regression analysis revealed that only age has the predictability to having CAD among the investigated parameters in this study (Table 5). Our regression model was good fitted when the factors such as smoking, education, income and BMI were included (p=0.150). In a study conducted with an older population, it was suggested that the change in CRP levels with age depends mainly on the socioeconomic profile (SEP) which is mediated by metabolic alterations and health-risk behavior rather than age only 9 . After adjustments for age, sex, acute infection and chronic inflammatory conditions, very high CRP was associated with lower social position, depressive symptoms, physical inactivity, smoking, and alcohol abstinence in a geriatric population 16  Angiotensin-converting enzyme (ACE) inhibitors were reported to have an anti-inflammatory effect, and the usage of the drugs was reported to cause a decrease in the production of IL-1, IL-6, TNF-α, IL-8, CRP, IL-12, interferon-γ, E-selectin, intracellular adhesion molecule-1, vascular cell adhesion molecule-1, monocyte chemoattractant molecule-1, and matrix metalloproteinase 9, along with the increased production of IL-10 21 . The positive correlation between IL-10 and PD in the entire study group (N=77) was surprising when the anti-inflammatory role of IL-10 in periodontal disease was considered. However, IL-10 seems to be a protective factor in atherosclerosis against the effects of pathogens 33 . Also, it is known that increased serum IL-10 levels are associated with a positive prognosis in acute coronary syndrome patients 8 . The increase in IL-10 in myocardial infarct patients was found to be correlated with systemic pro-inflammatory activity evaluated with thrombosis, plaque rupture and heart destruction related to IL-6 and TNF-α plasma concentrations 3 . Bing, et al. 8 (2015) found lower levels of IL-10 in healthy patients when compared to patients with CAD (+) and periodontitis and patients with only periodontitis without CAD. Therefore, the positive correlation between PD and IL-10 in the whole study group is an important finding. This may be the result of the effort with the increased synthesis (levels) of IL-10 to protect/prevent atherosclerosis in the whole study group in this study, which had patients with coronary artery symptoms and a potential diagnosis of CAD.
Besides the aforementioned strengths, the cross-sectional nature of our study complicates the comments about the effect of periodontitis in the development and progression of CAD, in other words, in terms of causality, which might be considered as a limitation. Another limitation was the small sample size, although we have determined the sample size for each group before the study.

Conclusion
When summarized, this study has revealed an important correlation within its limitations. The results of our study should be evaluated as preliminary.
However, when the strengths of this study were considered, it can be suggested that our study has driven some APR (PTX3 and SAA) forward in the relationship between CAD and periodontitis. This should be investigated in more detail in interventional studies to clarify the relationship between periodontitis and CAD, and to develop strategies in the primary and secondary prevention of CAD.