Association of Periodontal Status and Concentration with Severity of Coronary Artery Disease in Angiography Patients

Introduction

Periodontitis is a chronic inflammatory disease characterized by accumulation of dental plaque and progressive destruction of alveolar bone, leading to tooth mobility and eventual tooth loss, which in turn affects quality of life [1]. Periodontitis is the sixth most prevalent human disease [2]. Data from the National Health and Nutrition Examination Survey (NHANES) 2009–2014 indicate that nearly 50% of adults have periodontitis, with 7.8% exhibiting severe forms [3]. Globally, severe periodontitis affects approximately 11% of the population, impacting around 743 million individuals [4,5]. Periodontitis has increasingly been recognized as a potential factor in the development and progression of various systemic conditions [6], including cardiovascular diseases [7], type II diabetes [8], chronic obstructive pulmonary disease, [9] and cognitive impairment [10].

Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality globally, especially coronary artery disease (CAD), which is a significant public health problem [11,12]. Following strong epidemiological evidence, consensus was established between periodontology and cardiology researchers investigating potential pathways linking periodontal and CVD [13,14]. Periodontal pathogens are presumed to directly enter the bloodstream or indirectly affect cardiovascular health by elevating inflammatory mediators [15]. Initiated through inflammatory and immune reactions, endothelial dysfunction is the initial and most critical step in atherosclerosis [16]. Cytokines and bacteria from periodontitis can instigate or accelerate endothelial injury, leading to atheroma lesions [17].

Porphyromonas gingivalis (Pg), a gram-negative anaerobic bacterium and the primary pathogen causing periodontitis [18], is thought to play a significant role in the etiology of periodontal disease [19] and is frequently detected in atherosclerotic plaques [20]. Despite this, few clinical studies have directly examined the impact of Pg in CAD patients.

Therefore, the aim of this research was to examine the concentration of Pg DNA in oral and blood specimens, and to analyze the associations among Pg concentration, periodontal status, and the severity of CAD, with the goal of optimizing early risk assessment and guiding clinical management of CAD patients.

Material and Methods

STUDY POPULATION:

This cross-sectional study, conducted from April 2023 to September 2023 at the Department of Cardiology at the Second Hospital of Tianjin Medical University, included consecutive patients referred for diagnostic coronary angiography. All participants provided written informed consent to take part in the research. The research protocol and the informed consent process were approved by the Ethics Review Committee for Human Studies at the Second Hospital of Tianjin Medical University, Tianjin, China (approval ID: KY2021K143). The study protocol was in accordance with the ethics principles outlined in the 1975 Declaration of Helsinki.

The inclusion criteria for the case group were a degree of coronary artery stenosis exceeding 50% and diagnosis with CAD. The control group was comprised of individuals admitted to the hospital during the same period due to chest discomfort, who underwent coronary angiography and showed no obstructions, thereby excluding CAD. The diagnostic criteria for CAD were in accordance with the current CAD guidelines [21]. The exclusion criteria were: 1) 8 or fewer remaining teeth in the oral cavity, 2) NYHA Class III or Killip Class II heart failure signs, 3) severe liver disease, 4) renal failure with hemodialysis (serum creatinine >176.8 μmol/L or 2.0 mg/dL), 5) rheumatic and immune system disorders, 6) malignant tumors, 7) patients deemed to have poor compliance, and 8) severe cognitive dysfunction.

CLINICAL STATUS:

Comprehensive medical records were gathered for all participants. All participants received coronary angiography, which was conducted by a Philips DSA system (Integris BH, 5000; Philips, Netherlands). The degree of coronary artery stenosis was assessed independently by 2 operators. The severity of CAD was evaluated using the Gensini scoring system [22,23].

PERIODONTAL STATUS:

The periodontal evaluation of each participant included was conducted within the first 2 days after admission and prior to the coronary angiography. The assessment was performed using a dental mirror, dental tweezers, and a Williams periodontal probe at the Department of Cardiology with the patient positioned in bed and a portable lighting source. Each participant received a comprehensive periodontal assessment conducted by a qualified dentist. The examination included all teeth, except for the third molars. The number of missing teeth and teeth with mobility was documented. Tooth mobility was reported as a percentage of the total number of teeth. The presence or absence of supragingival plaque was documented on 4 surfaces of each tooth (mesio-buccal, disto-buccal, mesio-lingual, and disto-lingual). The plaque index was recorded as the percentage of tooth surfaces covered by plaque. This record serves as an indicator of the infectious burden related to periodontal tissues [24].

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Oral specimens were collected before coronary angiography using paper points that were inserted to periodontal pockets of the first molars in the 4 quadrants and left for 15 seconds. If the first molar was absent, specimens were collected from the adjacent teeth instead. Four paper points of each patient were moved to a single tube and stored at −80°C. Venous blood was drawn and centrifuged for 10 minutes at 2500 rpm to isolate the plasma. Blood samples were stored at −80°C until all specimens were collected.

To each tube of oral samples, we added 50 μL of Nucleic Acid Releaser (YB91202; Shanghai Yu Bo Biotech Co., Ltd., China) thoroughly vortexed to ensure complete mixing at room temperature. After standing for 10 minutes, the paper points were removed and samples were resuspended in preparation for qPCR. According to the kit instructions (DP339; Tiangen Biotech Co., Ltd., Beijing, China), circulating cell-free DNA (cfDNA) was purified from blood specimens for qPCR.

The quantification of Pg DNA was measured by qPCR and targeted primer probes designed by commercial laboratories (YB7C42900Y, Shanghai Yu Bo Biotech Co., Ltd., China). The PCR reaction consisted of the following components: 10 μL of 2×Probe qPCR MagicMix, 3 μL of probe-primer mixture, and 7 μL of DNA sample. The formulated mixture was transferred to the PCR strip tubes. Amplification was conducted using the Bio-Rad CFX96™ real-time PCR system, with an initial denaturation step at 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 60 seconds. The amplification outcomes were evaluated using CFX Maestro™ software.

STATISTICAL ANALYSIS:

Continuous variables are presented as means±standard deviations. If the data met the criteria for normal distribution and equal variance, the intergroup differences were assessed by a t test; otherwise, the Wilcoxon rank sum test was used. Categorical variables are presented as counts (percentages), and intergroup differences were compared using Pearson’s chi-square test or Fisher’s exact test, depending on the expected frequencies. LASSO regression was employed to identify the most relevant predictors by applying a penalty term to the model, thereby shrinking the coefficients of less important variables towards zero. Logistic regression analysis was employed to assess the associations among periodontal status, Pg concentration, and CAD. All statistical analyses were conducted as 2-tailed tests, with P<0.05 regarded as statistically significant. SPSS (version 25.0) and R (version 3.6.1) were used for statistical analysis.

Results

COMPARISON OF BASIC CLINICAL DATA:

A total of 131 patients participated in the research. The CAD group consisted of 78 CAD patients, with an average age of 64.04±11.52 years, who had coronary artery stenosis exceeding 50% as verified by coronary angiography. The control group consisted of 53 non-CAD patients, with an average age of 56.3±11.00 years, who were confirmed to have no coronary artery stenosis via coronary angiography. Statistically significant differences (P<0.05, Table 1) were observed between the CAD and control groups in terms of the proportion of male patients (42 [54%] vs 12 [23%]), the proportion of female patients (36 [46%] vs 41 [77%]), medical history of hypertension (58 [74%] vs 17 [32%]), and diabetes prevalence (25 [32%] vs 7 [13%]). Similarly, the cardiac ultrasound results and laboratory indexes demonstrated significant differences between the CAD and control groups (Table 1).

Periodontal examinations were conducted on 131 patients, with the results presented in Table 1. Compared to the control group, the CAD group displayed a higher average number of missing teeth (CAD: 4.513±5.08 vs control: 2.83±4.85), teeth with mobility (CAD: 4.013±2.63 vs control: 1.245±1.86), and teeth with plaque (CAD: 11.22±3.78 vs control: 6.58±2.07). Additionally, the CAD group had a significantly higher percentage of teeth with mobility (CAD: 19.31±15.24% vs control: 6.838±13.77%) and plaque (CAD: 50.78±20.44% vs control: 28.23±13.36%). These findings demonstrated statistically significant differences compared to the control group.

IDENTIFICATION OF CHARACTERISTIC FACTORS:

LASSO regression analysis was performed on the remaining independent variables, with the occurrence of CAD serving as the dependent variable (Figure 1). LASSO regression helps in compressing variable coefficients to prevent overfitting and address severe multicollinearity issues [25]. The analysis, with a lambda value of 0.1074 (minimum mean square error), resulted in the reduction of 18 independent variables to 3, which included: sex, age, and medical history of hypertension.

ASSOCIATION BETWEEN PERIODONTAL STATUS AND THE OCCURRENCE OF CAD:

The association between periodontal status and the occurrence of CAD was evaluated using univariate and multivariate logistic regression analyses. The occurrence of CAD (Yes: 1; No: 0) served as the dependent variable, and periodontal status was the primary independent variable of interest, adjusted for other covariates. The results indicated that all of the periodontal status indicators except for the number of missing teeth were independent risk factors for CAD. This association was consistent across multiple models (Figure 2). These findings suggest that all of the periodontal status indicators, except for the number of missing teeth, were significantly associated with an increased risk of CAD after adjusting for potential confounders.

ASSOCIATION BETWEEN PERIODONTAL STATUS AND SEVERITY OF CAD:

The severity of CAD was categorized into mild (Gensini score <21) and moderate-to-severe (Gensini score ≥21) groups. The number of missing teeth, the percentage of teeth with mobility, and the percentage of plaque were significantly associated with the severity of CAD in univariate logistic regression analysis. These associations remained significant after adjusting for potential confounding factors (Figure 3).

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The association between Pg concentration (measured in cfDNA and oral cavity) and the occurrence of CAD was evaluated by univariate and multivariate logistic regression analysis. The results, presented in Figure 4, show that Pg infection, measured in blood samples, was significantly associated with an increased risk of CAD incidence, with odds ratio (OR)=1.058, 95% confidence interval (CI) 1.013–1.104, in Model 1, P<0.05; and Model 2: OR=1.086, 95% CI 1.018–1.157, P<0.05.

Discussion

This study is the first application of qPCR to detect the concentration of Pg DNA in plasma and oral specimens, and to analyze the relationships among periodontal health status, Pg concentration, and CAD. The primary findings of this study were: (1) the severity of periodontal status increased the risk of CAD and was associated with the severity of CAD; (2) a high concentration of free Pg DNA in plasma was a significate risk factor for CAD, with clinical significance in assessing the risk of CAD.

This study investigated the correlation between the periodontal status of patients with chronic periodontitis (CP) and the occurrence and severity of CAD. This status represented the cumulative destructive outcomes of long-standing chronic periodontitis. The degree of periodontal destruction in CAD patients was significantly greater compared to individuals with normal coronary arteries. This observation has also been validated in similar clinical studies [7]. The relationship between PD and CAD has been extensively studied in cohort and case-control studies [26]. A recent systematic review that synthesized all available evidence from observational studies [15] concluded that individuals with serious PD had a higher risk of CAD compared to patients without PD. Compared to healthy individuals, patients with PD had a significantly higher (25%) risk of CAD [27]. Furthermore, findings from several interventional studies demonstrated that specific preventive oral hygiene measures, such as regular tooth brushing and comprehensive oral health care, including effective self-care oral hygiene practices [28], dental care [29], routine oral examinations [30], and periodontal therapy [31,32], can reduce the incidence of CAD.

Chronic periodontitis (CP), a chronic inflammatory disease caused by periodontal pathogens, involves microorganisms and their products in dental plaque. In particular, during the advanced stages of CP, the microorganisms and their products are more likely to enter the bloodstream through activities such as chewing and various dental procedures (eg, scaling, surgical treatments). Once in the bloodstream, they can reach distant organs, leading to bacteremia and systemic inflammation [33], mainly via the impact of periodontal pathogens on the development of subacute endocarditis, CAD, atherosclerosis, and ischemic infarction [34,35]. The primary mechanism by which periodontal pathogens influenced the development of CAD is the direct invasion of endothelial cells [15,20]. Pg can inhibit the proliferation of vascular endothelial cells, inducing apoptosis, and thereby disrupting the endothelial barrier [36]. cfDNA refers to DNA fragments present in the bloodstream without a nucleus. These DNA fragments typically originate from the apoptosis or necrosis of normal cells, as well as from infected cells. In this study, the detection of Pg cfDNA concentrations in plasma indicated that Pg had invaded the circulatory system. Several studies also confirmed the presence of numerous periodontal pathogens in human atherosclerotic plaques [37,38]. Among these, Pg was the most frequently detected bacterium in atherosclerotic plaques of patients [20]. The review provided by the European Federation of Periodontology (EFP) and the American Academy of Periodontology (AAP) demonstrated that periodontal pathogens can induce and facilitate atherosclerosis [39]. In animal disease models in mice [40], rabbits [41], and pigs [42], periodontal bacteria, especially Pg, were also shown to promote atherosclerosis. In mice, recurrent intravenous administration of Pg can exacerbate the development of atherosclerosis, and the obstruction lesions were 2 times larger than in the control group [43].

In conclusion, Pg exerted an independent impact on the development of CAD by directly entering the circulatory system. Currently, there was insufficient evidence to demonstrate that periodontal therapy can reduce Pg concentrations to further prevent the progression of CAD [13,44]. However, as clinicians, we should emphasize the periodontal health of cardiovascular disease patients, providing oral hygiene instruction (OHI) and implementing appropriate preventive and therapeutic measures. In practice, it is challenging for clinicians to promptly assess and obtain detailed periodontal health information, such as the number of teeth with mobility, the number of teeth with plaque, and the probing bleeding index, in patients admitted for CAD. Nonetheless, determining the levels of related pathogens in patient blood samples might provide valuable insights into the incidence and severity of CAD, guiding further clinical interventions.

This study had several limitations. It was a single-center study with a relatively limited sample size. As with any observational study, it was not possible to definitively establish a causal relationship between Pg and CAD. Future research should consider collecting larger samples from multiple centers to enhance the generalizability and representativeness of the findings. Moreover, in this study, the concentration of Pg in oral cavity was not independently associated with the occurrence of CAD, which was different from previous findings [45,46]. Despite our efforts to ensure the consistency and accuracy of oral sampling, the sampling process in bedridden hospitalized patients may have influenced the reliability of the results. Future studies should utilize more precise sampling methods and tools to reduce measurement errors. Finally, further investigation and validation were needed to elucidate the mediating role of Pg in the association between PD and the risk of CAD.

Conclusions

In conclusion, this study found that the severity of periodontal status increased the risk of CAD and was correlated with the severity of CAD; high Pg DNA concentration in plasma was a significate risk factor of CAD, with a clinical significance in assessing the possibility of CAD occurrence. In addition, the results further strengthen the epidemiological association between periodontal disease (PD) and CAD. To confirm these findings and to determine whether treating PD and eliminating periodontal pathogens can inhibit the incidence or recurrence of CAD, multicenter, prospective, and randomized clinical trials are needed.