Periodontal disease and inflammatory blood cytokines in patients with stable coronary artery disease

ABSTRACT Periodontal disease has been associated with elevations of blood cytokines involved in atherosclerosis in systemically healthy individuals, but little is known about this association in stable cardiovascular patients. The aim of this study was to assess the association between periodontal disease (exposure) and blood cytokine levels (outcomes) in a target population of patients with stable coronary artery disease (CAD). Material and Methods This cross-sectional study included 91 patients with stable CAD who had been under optimized cardiovascular care. Blood levels of IL-1β, IL-6, IL-8, IL-10, IFN-γ, and TNF-α were measured by Luminex technology. A full-mouth periodontal examination was conducted to record probing depth (PD) and clinical attachment (CA) loss. Multiple linear regression models, adjusting for gender, body mass index, oral hypoglycemic drugs, smoking, and occurre:nce of acute myocardial infarction were applied. Results CAD patients that experienced major events had higher concentrations of IFN-γ (median: 5.05 pg/mL vs. 3.01 pg/mL; p=0.01), IL-10 (median: 2.33 pg/mL vs. 1.01 pg/mL; p=0.03), and TNF-α (median: 9.17 pg/mL vs. 7.47 pg/mL; p=0.02). Higher numbers of teeth with at least 6 mm of CA loss (R2=0.07) and PD (R2=0.06) were significantly associated with higher IFN-γ log concentrations. Mean CA loss (R2=0.05) and PD (R2=0.06) were significantly related to IL-10 concentrations. Elevated concentrations of TNF-α were associated with higher mean CA loss (R2=0.07). Conclusion Periodontal disease is associated with increased systemic inflammation in stable cardiovascular patients. These findings provide additional evidence supporting the idea that periodontal disease can be a prognostic factor in cardiovascular patients.


INTRODUCTION
initiation and development of atherosclerosis 19 . Atherosclerotic lesions are comprised of various cytokines, which participate in the plaque formation cascade in vessel walls 12 (IL)-6, tumor necrosis factor (TNF)-a, and interferon blood from cardiovascular patients 22,29 , and the IL-10 concentration is increased in unstable compared with stable atheromas 18 . Moreover, IL-6 and TNF-a have direct effects on the production and release of C-reactive protein (CRP) 20 and, consequently, have been used as predictors of future cardiovascular events in cardiac patients 11 .
Periodontal diseases are caused by bacteria attached to teeth in organized biofilms and is characterized by bleeding, loss of tooth surrounding tissues, and tooth mobility, being the major cause of halitosis and tooth loss. Periodontal diseases may be expressed using various clinical parameters and factors and systemic conditions 26 . Probing depth and clinical attachment loss are the most frequent health/disease status, and they may be expressed as individual averages or by the number of teeth affected per individual.
One of the possible mechanism linking periodontal diseases to cardiovascular diseases (CVD) is the periodontal etiopathogenic process 14,22 . Studies have demonstrated that periodontitis, which is the destructive form of periodontal disease, increases systemic levels of some of the aforementioned CVD-related cytokines 3,15 . Most evidence supporting this claim has been provided by studies conducted in otherwise systemically healthy individuals, suggesting that periodontal disease may be a risk factor for CVD. On the other hand, there is little information about the systemic effects of periodontal disease on patients with stable CVD, with few studies evaluating the association between periodontal disease and a small number of patients 2,8,10,17,28 .
To the best of the authors' knowledge, no study has evaluated the association between periodontal parameters and blood levels of a broad range of cytokines, assessed by Multiplex technology, in cardiovascular patients. Such studies may indicate and destruction in the occurrence of future cardiovascular events on these patients. Therefore, the aim of this study was to assess the associations between periodontal disease and blood cytokine levels in patients with stable CAD who were under standard cardiovascular care.

MATERIAL AND METHODS
This cross-sectional observational study was conducted with stable CAD patients who had been receiving cardiovascular care for at least six months in a tertiary care cardiovascular clinic at a university hospital in Brazil. To be included in the study, all occurrence of at least one of the following events six months before entering the study: documented history of myocardial infarction (MI), stable angina, or ischemia in noninvasive tests; surgical or percutaneous myocardial revascularization and lesion size greater than 50% in at least one major coronary artery, as assessed by angiography; and presence of angina and positive results of noninvasive testing of ischemia. Additional inclusion criteria included the presence of at least four teeth, no periodontal treatment, and no use of antibiotics the study.
Overall, 239 consecutive patients were evaluated from April to December 2011. Among them, 78 (32.6%) individuals had less than four teeth, 30 (12.6%) refused participation, and 17 (7.1%) lived outside the city and were not available for clinical examination. Moreover, 16 (6.7%) did not attend the clinical examination appointment. Clinical examinations were performed in 98 (41.0%) individuals; however, seven did not undergo present investigation comprised 91 individuals.
The study protocol was approved by the institutional review boards of the University Hospital and the Federal University of Rio Grande do Sul. All participants read and signed an informed consent form before entering the study.

Cardiovascular care
All patients received cardiovascular tertiary care including medication and counseling. Regarding drug prescription, the protocol of the cardiovascular care in this clinic includes statins for the majority of patients. When appropriate, oral hypoglycemic drugs, insulin, acetylsalicylic acid, and antihypertensives were also prescribed. Counseling mainly includes daily exercise, smoking cessation, and dietary therapy. Most patients in this sample were using statins (92.3%), acetylsalicylic acid (90.1%), and antihypertensive drugs (93.4%). Only 13.1% and 9.9% were using insulin and antidepressives, respectively.

Data collection
We applied a structured questionnaire to collect data about age, gender, and smoking exposure. We obtained medical history, medication use, weight, and height from the hospital records of the patients.
Clinical periodontal parameters were assessed with a manual periodontal probe (Williams probe, Newmar, São Paulo, SP, Brazil) by two calibrated examiners (weighted kappa values ranging from 0.80 and 0.90) who were unaware of the cardiovascular Visible plaque (VP), gingival recession (GR), probing depth (PD), and bleeding on probing (BOP) were recorded at six sites per tooth in all present teeth, excluding third molars. Clinical attachment (CA) loss was obtained as the sum of GR and PD.

Blood samples
A trained nurse collected 20 mL of blood from the antecubital fossa of each individual. Fasting blood samples were obtained between 7:00 am and 12:00 pm to control for possible diurnal variations. Half of the blood (10 mL) was stored in EDTA-containing Eppendorf tubes and was immediately centrifuged for analysis. Plasma from the remaining 10 mL Samples were stored for two years on average.

Cardiovascular status
The cardiovascular status of patients was assessed by identifying known blood markers of risk for cardiovascular events. High-sensitivity CRP, glucose, glycated hemoglobin, triglycerides (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) were measured as previously described 8 . Low-density lipoprotein cholesterol (LDL-C) was calculated by the Friedwald formula.
Participants were further divided into those having medium-and long-term past history of major cardiovascular events using a combination of information about the occurrence of cardiovascular events and time elapsed since the event. Patients that have experienced acute myocardial infarction or myocardial revascularization (angioplasty or surgery) in the last nine years were included in the high status group, whereas if the events occurred before 10 years or more, the individuals were Systemic cytokines methodology in combination with the 3.1 Xponent software package (Luminex Corp., Austin, TX, USA ). The MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel-07 kit (HCYTOMAG-60k, EMD Millipore, Saint Charles, USA) was used following the manufacturer's instructions. Results were expressed as standard curve units in pg/mL. All samples were analyzed at the same time under standardized experimental conditions.
In summary, before starting the immunoassay, samples were completely thawed, mixed by vortexing, and centrifuged at 1000g for 10 minutes to remove particulates. All kit reagents were of wash buffer was added to each of the 96 wells in the plate, which was then sealed, mixed on a plate shaker for 10 minutes at room temperature, inverted, and tapped several times onto absorbent towels to decant and remove the residual wash buffer. The standard curve and control wells received Assay buffer alone was used as the background (0 per well. Each antibody-bead vial was sonicated for 30 each antibody-bead vial were added to the mixing to each well. The plate was sealed and incubated overnight (18 hours) at 4°C with agitation on a plate shaker. Well contents were gently removed, and the aliquot of detection antibodies was added to each well and incubated for 1 hour at room temperature with agitation on a plate shaker. Without further added to each well, followed by incubation for another 30 minutes with agitation on a plate shaker. Washing with wash buffer was repeated twice.
The beads were re-suspended on a plate shaker for 5 minutes, and the plate was run on Luminex. The calculating cytokine concentrations in the samples.

Statistical analysis
The outcomes of the present study were . Cardiovascular blood markers (CRP, TG, lipids, glucose, glycated hemoglobin) were expressed as means and standard deviations (SD) to describe the Numbers of teeth with PD and CA loss of 6 mm or as having severe periodontitis in the presence of two or more interproximal sites with CA loss of 6 mm or greater and at least one interproximal site with PD of 5 mm or greater in nonadjacent teeth 7 .
Multiple linear regression models were used to study the association between periodontal status and systemic levels of cytokines. In these models, mean CA loss, mean PD, and numbers of teeth with CA loss of at least 6 mm and PD of at least 6 mm. Cytokine concentrations were log-transformed periodontal status as the main independent variable and adjusting them for gender, body mass index (BMI), use of oral hypoglycemic drugs, lifetime smoking exposure (in packyears), and time elapsed since the occurrence of acute myocardial infarction.
(SE), and adjusted R 2 values were determined. It has been suggested that the levels of triglycerides may modify the production of cytokines 5 . Thus, an comparisons according to the TG control status, with 150 mg/dL as the cut-off.
Secondary analysis was performed using diagnostic tests to estimate the magnitude severe periodontitis that may predict higher levels of systemic cytokines. Cytokine levels were dichotomized by the median value. Sensitivity, values (PPV and NPV, respectively) were calculated for severe periodontitis and each cytokine.
Statistical analyses were performed with a statistical package (STATA SE for Macintosh version 13, StataCorp, College Station, USA). The individual was the unit of analysis. The alpha level was set at 5%.

RESULTS
The mean age of the sample was 62.9 (SD: 9.9) years and the average lifetime smoking exposure equaled 21.8 (SD: 31.9) packyears. Table 1  A total of 52 (57.1%) patients experienced acute myocardial infarction, and more than half of these patients had this major event during the nine years before the study (medium-term past history of events). CAD patients in the medium-term past history, compared with the long-term group, had and TNF-( Table 2). loss (R 2 =0.07) and PD of at least 6 mm (R 2 =0.06) concentrations. Mean CA loss (R 2 =0.05) and PD (R 2 associated with higher mean CA loss (R 2 =0.07). found for IL-6, IL-8, and TNF-. Diagnostic tests of severe periodontitis as a predictor of cytokine concentrations above the median are shown in Table 5

DISCUSSION
The present study demonstrated that periodontal periodontal condition, as measured by CA loss and PD, was associated with increased blood levels of . These cytokines were also related to major events in the studied sample. These notion that periodontal disease may be a prognostic factor in cardiovascular patients.

Table 5-
The role of IL-10 in the cascade of atherosclerosis and cardiovascular events remains controversial, with some studies indicating that high IL-10 concentrations may have a protective effect on MI 9,23 and other longitudinal observational studies 4,16 reporting that baseline elevated blood IL-10 levels increase the risk for MI. This controversy seems to have been clarified by a recently published meta-analysis 13 , which demonstrated that high ILnew cardiovascular events over time in patients with acute coronary syndrome. If IL-10 is truly related to new major cardiovascular events, then periodontal disease might have a detrimental effect on atherosclerosis by increasing the levels of IL-10, as demonstrated in this study. Nevertheless, the possible effect of other confounders not assessed in the present study and which may be related to the association found between periodontal status and IL-10 should not be discarded.
collagen production and smooth cell proliferation, thereby altering the plaque composition and stability 30 IL-6 production 27 . In the present study, clinical parameters of periodontal inflammation and disease may act in early stages of the cascade of atherogenic events in cardiovascular patients by . The IL-6 cytokine is directly involved in the hepatic production of CRP 20 . Periodontal disease was not associated with blood levels of IL-6 in the present sample of cardiovascular patients. However, very low levels of this cytokine were detected in our patients, probably due to the cardiovascular care received by using statins. Such low levels may have hindered possible associations between periodontal status and this cytokine. association between periodontal tissue breakdown and some cytokines in this study. The systemic have also suggested some modulatory effect of elevated TG levels on the production of some cytokines when stimulus came from P. gingivalis lipopolysaccharides 5 . Taken together, these data may indicate that the effect of periodontal disease levels of cardiovascular control. Nevertheless, this effect should be explored in the future for better of this patient population.
A diagnosis of severe periodontitis was correlated with above-median concentrations of cytokines by in an attempt to provide a better estimation of the clinical relevance of the associations found in the linear regression models that used log-transformed data (which cannot be directly interpreted). It was evidenced that performing a periodontal examination for establishing the presence of periodontitis may be recommended because sensitivity values were close to 80%, indicating that the presence of periodontitis will most often be related to abovemedian cytokine concentrations. On the other hand, when the diagnosis of periodontitis was already above-median concentrations of cytokines were low (PPV range: 51.5-55.9). When the diagnosis (66.7) compared with other cytokines. There is extensive evidence suggesting that periodontal disease may be a risk factor for CVD 26 . In contrast, evidence is still emerging to determine if periodontal disease may be a prognostic factor in patients already diagnosed with coronary disease. Risk factors and prognostic factors differ in that whereas the second relates to the course of an existing disease 1 present study in chronic CAD patients. Thus, the main focus here was to study periodontal disease in the presence of cardiovascular disease, potentially attributing a perspicuous factor in the disease process. It will be reasonable to assume that such marker (periodontal disease) could play a prognostic role in the history of CVD. Noteworthy, this study is by Luminex in CAD patients. Previous studies support a possible role of periodontal disease in periodontal parameters and IL-6 and TNF- 10,17,25 .
Findings of the present study should be analyzed in view of its possible limitations. The results of the because of the loss of statistical power. Another limitation may include the cross-sectional nature, which precludes causal inferences from being drawn from the associations. Luminex analyses were performed in uniplicate because of costs, and this may have lowered the precision of the readings. The storage time of the samples of two years may be a limitation; however, there is evidence that the during this period of time 6,31 .
This study applied well-established methodologies, measured by the Multiplex technology that allows fast, reliable, and accurate quantification of biomarkers in the blood. The analyses controlled for possible confounding effects by applying multivariable models. Moreover, periodontal examinations were conducted using a full-mouth protocol of six sites per tooth to avoid assessment bias 24 . A broad range of periodontal parameters was and to associate parameters with blood levels of cytokines.

CONCLUSIONS
Periodontal disease is associated with increased Greater CA loss and PD were associated with higher , even after controlling for important confounders.