Study design and participants:

A case-control study was proposed to evaluate oral and systemic variables and analytical parameters in patients with or without cardiovascular disease.

Cases - Study Group (SG): They were recruited from CVD patients who are cared for at the Vascular Risk Unit of the University Hospital of Bellvitge, with a maximum of 3 months from the cardiovascular event. Patients who had suffered an acute myocardial infarction, angina with demonstrated coronary ischemia, arteriopathy of the lower extremities with intermittent claudication or an atherothrombotic stroke were included.

Controls - Control Group (CG): They were recruited from patients who visited the Dental Hospital of the University of Barcelona (Fundación Josep Finestres. Faculty of Medicine and Health Sciences). They had no cardiovascular history and were matched for age and sex.

Each patient underwent a detailed medical history, a thorough oral examination, a blood test with unconventional cardiovascular risk factors, and an orthopantomography (OPG). The blood analysis was carried out at the blood extraction department of the Bellvitge Hospital and the OPG at the Dental Hospital of the University of Barcelona. Radiographic status was diagnosed using digital orthopantomographies taken by 2 proficient technicians with more than a decade of experience (Promax, Planmeca, class 1, type B, 80 KHz, Planmeca, Helsinki, Finland). All the teeth except the third molars were taken into account.

All participants provided written informed consent before entering the study, and the study was approved by the Ethics Committee of the Bellvitge University Hospital Research Institute (IDIBELL - Reference: PR187/15).

Individuals with missing follow-up data were excluded from the study. Additionally, individuals with missing information on any of the covariates included in the multivariable regression models were also excluded Figure 3.

Assessment of PD, DC and AP:

An expert examiner carried out the oral examination.

Oral hygiene was assessed using the Silness and Löe plaque index 20, the absence or presence of plaque on dental surfaces was assessed with an exploration probe. According to the numerical results, we divided oral hygiene as: i) good (0.0– 0.65), ii) regular (0.66–1.85) and iii) poor (1.86–3.0).

Periodontal health was assessed using the community periodontal index of treatment needs (CPITN) 21. Once the individual results were found, it was correlated with the PD. i) If CPITN <1, no PD; ii) If CPITN 1-1.9, mild PD; iii) If CPITN 2-2.9, moderate PD; iv) If CPITN> 2.9, severe PD was scored.

Caries was analyzed using the decayed, missing and/or filled teeth index (DMFT)22.

The AP was analyzed using the OPG, evaluating it according to Örstavik 23. Where we defined 1 as normal periapical structure, 2 as small changes in bone structure, 3 as changes in bone structure with some mineral loss, 4 as periodontitis with well-defined radiolucent area and 5 as severe periodontitis with exacerbated signs. Furthermore, they were finally grouped into AP <3 and AP ≥ 3, depending on the severity of the lesions observed radiographically.

Definition and assessment of other relevant covariates:

Weight, height, body mass index and abdominal perimeter were measured. History of hypertension, diabetes and hypercholesterolemia, use of tobacco and medication were assessed. Systolic and diastolic blood pressures were measured. Levels of high sensitivity CRP, Hcy, fasting blood glucose, triglycerides, total cholesterol, low density lipoprotein cholesterol (c-LDL) levels, high density lipoprotein cholesterol (c-HDL) levels, Apolipoprotein A (apo A1), and apo B were assessed in fasting blood samples at the central laboratory of the Bellvitge Hospital.

For the present analysis, diabetes was defined as any of the following: presence of a positive history of diabetes at baseline, the use of glucose-lowering medications at baseline, baseline glycosylated hemoglobin (HbA1c) levels ≥6%, and/or baseline fasting blood glucose levels ≥126 mg/dL. Hypertension was defined as any of the following: presence of a positive history of hypertension at baseline, the use of hypertension-lowering medication at baseline, and/or baseline systolic and diastolic blood pressures ≥ 140-90 mmHg. Finally, dyslipidaemia was defined as any of the following: presence of a positive history of dyslipidaemia at baseline, the use of cholesterol-lowering medication at baseline, and/or baseline fasting blood total cholesterol levels ≥200 mg / dL, or c-LDL levels> 130mg / dl or c-HDL levels <40mg / dl. Every smoker was defined as either being current smoker or former smoker.

Statistical Analyses:

A descriptive statistical analysis was carried out for all the variables. The number of valid cases, mean, standard deviation, and 25th and 75th percentiles (P25-P75) described continuous variables. On the other hand, the categorical variables were described by absolute and relative frequencies of each category over the total of valid values (N). In case of missing values, their number per group were described.

Comparisons of categorical variables were made using the Student's t-test for independent data or the χ2 test as applicable. In the case of intra-group comparisons to evaluate the evolution, the Student's t-test was used for paired data.

All analyses were performed on the data set using all available information with intention to treat criteria.

Study population:

Of the 104 cases and 52 controls participants, we excluded 1 duplicated individual and 6 who didn’t get the OPG done. This yielded a final study population of 99 cases and 50 controls included in the analyses Figure 3.

Baseline characteristics of the study participants:

The baseline demographic and clinical characteristics of the study participants are summarized in Table 1. Overall, median age was 50.47 years, 30.2% were women and 70.5% were overweight or obese. At baseline, 81.7% were current or ex-smokers, 16.2% had diabetes, 41.7% had hypertension and 53.7% had dyslipidemia. Regarding the drug treatments, 17.4% were treated with oral antidiabetic drugs, 45% were on antihypertensive drugs and 67.1% were on statin therapy (100% of the study group used statins).

Analytical parameters:

The most relevant analytical parameters of the study participants are summarized in Table 2. Most of the analytical parameters studied are significantly higher in the SG than in the CG. It should be noted that although the observed values of CRP are higher in the SG than in the CG, 3.09 mg/L and 2.15 mg/L respectively, these differences were not statistically significant. On the other hand, CG patients presented significantly higher c-LDL values compared to SG patients, 3.32 mmol/L vs. 2.06 mmol/L, respectively. It should also be emphasized that SG patients presented significantly higher plasma values of Hcy and Lp(a), when compared to the healthy patients group [(Hcy: 14.25 vs 10.92; p = 0.0032) (Lp(a): 124.53 vs 52.84; p <0.0001)].

Oral and dental pathology:

In Table 3, oral health data comparing both groups is presented. In this section, some patients were excluded because they did not have teeth needed for indexing or because they were edentulous. In the case of oral hygiene, 7 individuals from SG were discarded; to analyze the CPITN and its relationship with PD, 6 patients from SG and 1 from CG were excluded, and for assessment of the DMFT and AP, 6 SG patients were discarded. 77.2% of the study group presented poor or deficient oral hygiene, which is statistically significant when compared to the control group. Furthermore, the group with CVD presented higher number of cases and worse periodontal status (p <0.001), and a greater number of AP (1.02 vs. 0.32) than the group without CVD. On the other hand, we did not find statistically significant differences regarding the caries index (DMFT), between both groups (1.07 vs. 0.68, respectively).

Multivariable-adjusted associations between oral and dental pathology and CRP, Hcy, Lp(a):

In Table 4, we describe the multivariate regression models of each oral variable in relation to the analytical parameters. We did not find any oral variable that was significantly related to CRP, Hcy or Lp(a) concentrations.

CRP is a non-specific inflammation biomarker related to CVD risk 24. Several studies highlight a relationship between PD and CRP, concluding that patients with higher PD severity had higher plasma CRP levels 25, 26. However, in other studies27 such as in this research work, we did not find significant differences between the CRP concentrations between both groups, although a tendency was observed for the SG to have a higher CRP level than the CG. It should be noted that in a study on the relationship between dental pathology and CRP concentrations27, CRP was measured in crevicular fluid and not in blood serum as in the vast majority of studies. Likewise, we must consider that SG patients have greater protection against inflammation primarily due to pharmacological treatments, particularly statins, as well as due to the recommendations of secondary prevention specialists regarding exercise and diet, factors that have a clear influence on CRP concentrations 28.

Regarding cholesterol concentrations, we observed that SG patients present plasma concentrations of total Cholesterol, c-HDL, c-LDL, apo A1 and apo B lower than those of CG. These differences can be attributed to the fact that 100% of SG patients are treated with hypolipidemic drugs, while only 2% of CG patients were treated with these drugs. Similar differences between CVD patients and controls were observed in two similarly designed case-control studies 29, 30. It should be noted that statins very markedly decrease c-LDL and apo B, but they hardly modify c-HDL and apo A1. Therefore, although both excess of c-LDL and apo B, as well as deficiency of c-HDL and apo A1 are associated with atherosclerosis; it is expected to find lower values ​​of c-LDL and apo B in SG patients, all treated with statins, and lower concentrations of c-HDL and apo A1 are maintained. The latter related to their disease (also because CVD patients more frequently have obesity, diabetes and metabolic syndrome which are disorders related to a decrease in c-HDL and apoA1) and not corrected by the effect of statins.

Furthermore, we also found that patients who have suffered an atherothrombotic cardiovascular event have higher levels of Lp(a) and homocysteine ​​than CG. This data is in agreement with those observed in other studies on the relationship between Lp(a)15, 19 and homocysteine ​​with CVD14, 31. Performing multivariate regression models for all oral variables, we did not find that these variables are related to the concentrations of Lp(a) and homocysteine; that is, we observed that neither Lp(a) nor homocysteine ​​are found to increase with respect to any of the oral variables, or vice versa. As cited previously, homocysteine ​​is an amino acid derived from methionine that has a proinflammatory and prothrombotic effect and its concentrations are increased in patients with atherothrombotic diseases18 . It could be increased in those patients who had more oral inflammation, represented by a higher PD, a greater number of caries and/or a greater number of AP, but it is not a good marker of inflammation. As we have mentioned, in our study, patients with greater dental and/or periodontal pathology do not have higher plasma homocysteine ​​levels, therefore, in our study, it could not be considered a marker related to oral pathology. In previous studies, PD patients have higher levels of Hcy and after the periodontal treatment alone 32, 33 or in conjunction with folic acid therapy 27, their plasma levels decrease.

The function of Lp(a) is not well known. Containing a c-LDL molecule, it transports cholesterol in the plasma. A characteristic fact is its affinity for oxidized phospholipids that are highly proinflammatory and proatherogenic, since they attract inflammatory cells to the vessel walls and stimulate the proliferation of smooth muscle cells 34, 35. Lp(a) is an acute phase reactant in addition to an atherothrombotic risk factor, and is increased in inflammatory diseases. For this reason, it seems that the treatment of PD not just with curettage but also with a subgingival dose of doxycycline could contribute to a decrease in systemic inflammation, which would be associated with a decrease in CRP and Lp(a) concentrations 36.

Patients with demonstrated CVD from a minimum of 3 months have been included in this study. These patients are treated with statins and other drugs with anti-inflammatory effects that cause a decrease in the concentrations of CRP, Hcy and Lp (a).

Patients who have suffered an atherothrombotic accident present more dental and periodontal pathology (DC, PD or AP) than those without a history of CVD.

CVD patients have higher levels of C-reactive protein, Homocysteine ​​and Lp(a).

The concentrations of CRP, homocysteine ​​and Lp(a) are not related to the degree of dento-periodontal pathology (such as PD, caries or AP).

Table 1

Table 3

	Overall(N = 142)	Study group(N = 92)	Control group(N = 50)	P Value
Oral hygiene (%)				<0.001*
Good	57 (40.1)	21 (22.8)	36 (72.0)
Mild	54 (38.0)	41 (44.6)	13 (26.0)
Deficient	31 (21.8)	30 (32.6)	1 (2.0)
	N = 142	N= 93	N =49
CPITN - PD (%)				<0.001*
No PD	58 (40.8)	22 (23.7)	36 (73.5)
Mild PD	27 (19.0)	22 (23.7)	5 (10.2)
Moderate PD	26 (18.3)	22 (23.7)	4 (8.2)
Severe PD	31 (21.8)	27 (28.9)	4 (8.2)
	N=143	N=93	N=50
DMFT	0.93 (2.09)	1.07 (2.16)	0.68 (1.94)	0.287
AP	0.78 (1.14)	1.02 (1.29)	0.32 (0.55)	<0.001*
	N = 149	N= 99	N =50
AP max (%)				0.046*
AP max < 3	95 (63.7)	57 (57.6)	38 (76)
AP max ≥ 3	54 (36.3)	42 (42.5)	12 (24)

Data presented as mean (SD) or N (%).

Figure 2

Figure 3

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