Molecular inflammation and oxidative stress are shared mechanisms involved in both myocardial infarction and periodontitis

Molecular inflammation and oxidative stress are shared mechanisms involved in both myocardial infarction and periodontitis. Carmen M. Díaz1, Beatriz Bullon1, Rafael J. Ruiz-Salmerón2, Patricia Fernández-Riejos3, Ana Fernández-Palacín4, Maurizio Battino5,6,7, Mario D. Cordero8, José L Quiles8,9, Alfonso Varela-López8, Pedro Bullón1 1Department of Oral Medicine and Periodontology, University of Seville, Sevilla, Spain. 2Heart Center, Virgen Macarena University Hospital, Sevilla, Spain. 3Department of Clinical Biochemistry, Virgen Macarena University Hospital, Sevilla, Spain. 4Department of Statistics, School of Medicine, University of Seville, Sevilla, Spain. 5Dipartimento di Scienze Cliniche Specialistiche ed Odontostomatologiche—Sez. Biochimica, Università Politecnica delle Marche, Ancona, Italy. 6Nutrition and Food Science Group, Department of Analytical and Food Chemistry, CITACA, CACTI, University of Vigo, Vigo. Spain. 7International Research Center for Food Nutrition and Safety, Jiangsu University, Zhenjiang, China 8Institute of Nutrition and Food Technology "José Mataix Verdú", Department of Physiology, Biomedical Research Center, University of Granada, Granada, Spain. 9College of Food Science and Technology, Northwest University, Xi’an, Shaanxi, China. Corresponding author: Pedro Bullón. Department or Oral Medicine and Periodontology, University of Seville, Sevilla, Spain. c/ Avicena s/n, 41009 Sevilla, Spain. Email address: [email protected] Keywords: autophagy, interleukin-1beta, lipid peroxidation. Abstract Background and Objective. Our aims were to improve the understanding of the pathogenic relationship between cardiovascular diseases and periodontitis and to generate new perspectives in the prevention and treatment of acute myocardial infarction (AMI) and periodontitis. The present study evaluates possible differences in inflammation, oxidative stress and autophagy markers among subject suffering AMI, periodontitis or both, to explore possible common pathogenic mechanisms. Material and Methods. A total of 260 subjects were enrolled in the study, 106 subjects that survived to a first AMI (AMI group) and 154 subjects had no cardiac events in their clinical record (control group). A questionnaire was used to assess age, height, weight, blood pressure and heart rate. The clinical probing depth, clinical attachment loss, number of remaining teeth and average number of sites with bleeding on probing were assessed. Lipid peroxidation and protein levels of phosphorylated AMP-activated protein kinase (p-AMPK) and microtubule-associated proteins 1A/1B-light chain 3-II (LC3-II) were determined in isolated peripheral blood mononuclear cells by thiobarbituric acid reactive substances (TBARS) assay and western-blot, respectively. Plasma levels of interleukin- 1β were determined using a commercial ELISA kit. All the obtained variables were compared between subjects suffering an AMI with or without periodontitis and control subject periodontal healthy or with periodontitis. Results. A higher proportion of subjects suffering AMI+periodontitis than only AMI (without periodontitis) was found. Higher levels of TBARS were found in subjects with periodontitis than in subjects without periodontitis in both AMI and control subjects. Positive correlations between IL-1β levels and TBARS and between IL-1β levels and LC3-II were found only in control subjects. Conclusions. Results from the present study are consistent with the suggestion of periodontitis as a potential risk factor for AMI. Periodontitis association with circulating lipid peroxides in both AMI and control subjects were found. The absence of differences in IL-1β levels between AMI subjects (only AMI vs. AMI+periodontitis) suggests that oxidative stress could be the main pathogenic link between AMI and periodontitis. 1. INTRODUCTION According to the World Health Organization (WHO), 17.9 million people die each year from cardiovascular diseases (CVDs), an estimated 31% of all deaths worldwide.1 This implies that CVDs are the first cause of death globally with heart attacks and strokes producing 85% of all CVD deaths.1 Besides associated mortality, the remained high incidence of atherosclerotic CVDs also entails an important direct and indirect economic burden over the world.2 At public health level, the most cost-effective way to reduce the prevalence of a disease is via preventive measures.3 In this sense, most CVDs can be prevented by addressing behavioral risk factors such as tobacco use, unhealthy diet and obesity.1 Therefore, people with CVDs, but also people at high cardiovascular risk, need early detection and management. Classic risk factors explaining the occurrence of CVDs include hypertension, diabetes and hyperlipidemia.1 However, in some cases of CVD, the underlying reasons remain unknown. In addition, there are some inconsistencies regarding the importance of risk factors for the different CVDs,4 but the reasons are mostly unknown yet. Strategies to improve the prevention of CVDs require a better understanding of the pathogenic mechanisms and the impact of nontraditional risk factors. Increasing evidence suggests periodontitis as a potential risk factor for increased morbidity or mortality of several systemic conditions, including coronary atherosclerotic complications such as angina pectoris and acute myocardial infarction (AMI).5 Periodontitis, the advanced form of periodontal disease,6 is considered an inflammatory disease characterized by an abnormal host response to oral bacteria affecting approximately 10-15% of the population. Thus, periodontitis is the sixth most prevalent human disease globally.7 The inflammatory cascade is particularly important in the atherosclerotic process and in adverse cardiovascular events such as AMI and sudden cardiac death.8 On the other hand, different inflammatory factors released during periodontal inflammation7 can reach blood and promote systemic and arterial inflammation complicating the early atherosclerotic processes and advancing the atherosclerotic plaque development and the risk for plaque instability9 which can lead to atherothrombotic cardiovascular events such as myocardial infarction.10 Indirectly, proinflammatory cytokines and chemokines also could cause oxidative stress by recruiting polymorphonuclear leukocytes7, that produce reactive oxygen species (ROS),11,12 and increasing mitochondrial production of ROS.13 Actually, overwhelming evidence indicates that oxidative stress plays a central role in the periodontal tissue and alveolar bone destruction.11,1415-16 Likewise, abnormal peripheral blood mononuclear cells (PBMCs) performance observed in periodontitis subjects also may promote oxidative stress17 since an elevated production of ROS or inflammatory mediators at periodontal tissues could contribute to systemic oxidative stress resulting in oxidative damage at other areas. Periodontitis can be not only a risk factor for CVDs, but also a condition modifying other primary risk factors associated with CVDs. In this sense, a state of systemic inflammation as consequence of the periodontal inflammation would cause the stiffness of large arteries and increases the pulse wave velocity contributing to the pathogenesis of hypertension.18-22 Likewise; serum pro-inflammatory cytokines may have a vital role in the association between periodontitis and dyslipidemia23 favouring the underlying sub-intimal lipid accumulation. Furthermore, it could also be possible that periodontal disease and CVDs share the same risk factors and pathogenic mechanisms. It is proposed that the aggravation of hyperlipidemic state is also linked with periodontal inflammation by the up-regulation of serum and gingival crevicular fluid proinflammatory cytokines..23 Lastly, oxidative stress could be also a factor that facilitates both, diseases onset and/or progression by evoking immune responses resulting in a higher expression of pro-inflammatory cytokines24 or driving NLRP3 inflammasome activation2. Furthermore, oxidized-LDLs are also able to induce secretion of IL-1β by macrophages via ROS-dependent NLRP3 inflammasome activation26 linking dyslipidemia, oxidative stress and inflammatory diseases. Finally, autophagy that has shown to be crucial for the maintenance of intracellular homeostasis and development in most cells including cells of cardiovascular origin (cardiomyocytes, endothelial cells, and arterial smooth muscle cells) and periodontal cells has been related to oxidative stress and inflammation.27-28 Indeed, autophagic or mitophagic defects have been associated with an increased propensity of laboratory animals to spontaneously develop specific cardiovascular disorders, including multiple forms of cardiomyopathy.29 Likewise, an elevated autophagosome production has been evidenced by image, protein levels and gene expression analyses in periodontal ligament stem cells from periodontitis patients30 Achieving a better understanding of the pathogenic relationship between CVDs and periodontitis is a primary goal in the research for the prevention and treatment of atherosclerotic CVDs. Our aim was to improve the understanding of the pathogenic relationship between CVDs and periodontitis exploring possible differences in inflammation, oxidative stress, and autophagy markers. This aim would allow us to generate new perspectives in the prevention and treatment of AMI and periodontitis. The present study compares between AMI subjects (only AMI or AMI+periodontitis) and control subjects (healthy or only periodontitis). The AMI subjects are subjects who have survived to a first AMI, whereas the control subjects have no cardiac events in their clinical record. Clinical parameters and molecular markers of pathogenic mechanisms involved in AMI and/or periodontitis were evaluated. 2. MATERIAL AND METHODS 2.1. Patients Male subjects over thirty five years of age, who have not experienced a prior cardiac event and who had no significant coronary disease attending the Dental School of the University of Sevilla (Control) without clinical records of previous cardiac events or diabetes diagnosis and male subjects attending and receiving treatment at the cardiovascular rehabilitation service of the University Hospital “Virgen Macarena”, Sevilla, Spain, who had experienced and survived to a previous AMI were asked to participate in the study. Informed consent was obtained from each subject and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a prior approval by the Human Research Committee of Sevilla University. The sample size was determined for the comparison of a numerical variable between four groups defined by the combination of two dichotomous variables: cardiovascular (yes / no) and periodontitis (yes / no). Data regarding the variances of the means (0.01, 0.27 and 0.07) and the common variance of the groups (0.4) were obtained from a similar published study. Setting the values of α at 5% and powers at 90% (for factor A) and 99% (for factor B and for interaction) we need between 43 patients in each of the 4 groups. 2.2. Clinical examination 2.2.1. General data Demographic characteristics (age, height and weight), medical record (blood pressure, heart rate, waist circumference and fasting blood glucose (FBG) were recorded and body mass index (BMI) was calculated from standardized measurements of weight and height (weight in kg divided by height in square meters). 2.2.2. Oral examination The following periodontal parameters were recorded: clinical probing depth (PD), clinical attachment loss (CAL), number of remaining teeth, percentage of sites with bleeding on probing (BOP). All periodontal measures were assessed by the same examiner, whose assessments of the first ten subjects were calibrated for reproducibility before the study; an intra-subject correlation coefficient of 92% was found for PD. 2.2.4 Periodontitis definitions Periodontitis was defined as the presence of ≥2 interproximal sites with CAL ≥6 mm in different teeth and ≥1 interproximal site with PD ≥5 mm.32 The severity and extent of periodontal disease were also expressed as a percentage of sites with attachment loss of 4 mm or more, and subjects were categorized as follows: 0% = no periodontitis, >0%– 33% = mild periodontitis, >33%–67% = moderate periodontitis, and >67% = severe periodontitis.33 Therefore, the Control and AMI groups were subcategorized according to these definitions. 2.3. Blood sample collection Venous blood samples (5 ml) were collected into glass tubes containing K3-EDTA (Venoject, Terumo, Leuven, Belgium) using the venipuncture procedure. The samples were collected after a 12-h fasting period. All samples were immediately centrifuged at 1000 x g for 15 min at 4°C, and plasma was divided into aliquots of 250 µl for immediate analysis or stored at -80°C. PBMCs were isolated and concentrated by isopycnic centrifugation using Histopaque-1119 and Histopaque-1077 (Sigma Chemical Co., St. Louis, MO, USA). 2.4. Lipid peroxidation Lipid peroxidation in PBMCs was estimated with a thiobarbituric acid reactive substances (TBARS) assay. This assay is performed measuring the levels of malondialdehyde (MDA). The levels of TBARS were determined using a method based on a reaction with thiobarbituric acid (TBA). The reaction was performed at 90°C for 15 min at pH 2-3. The sample was incorporated into two volumes of cold 10% (w/v) trichloroacetic acid to precipitate proteins. This precipitate was pelleted by centrifugation anA total of 260 subjects were enrolled in the study, 106 subjects had no previous AMI history (control group) and 154 subjects had suffered AMI previously (AMI group).d an aliquot of the supernatant was reacted with an equal volume of 0.67% (w/v) TBA in a boiling water bath for 10 min. After cooling, the absorbance was read at 532 nm. The results were expressed as nmol MDA per mg of protein. 2.5. Serum interleukin-1β levels and inappropriate nutrition, could damage periodontal tissues.53,56 Because the present study and most previous studies are cross-sectional, it is still not clear whether the increase of oxidative stress is the cause or the consequence of periodontitis. In the present study, a positive correlation between lipid peroxidation and IL-1β levels was found in control subjects. This correlation can be explained because polymorphonuclear cells, whose activity and number is increased by high levels of proinflammatory cytokines, produce ROS via the respiratory burst mechanism as part of the defense response to infection. Moreover, these cytokines also interact with mitochondria to increase ROS production.44 Furthermore; oxidative stress is one of the factors that could explain the pathophysiological mechanism of inflammatory conditions occurring in CVD and periodontitis. ROS can evoke immune responses through redox-sensitive gene transcription factors such as NF-κB resulting in the expression of pro-inflammatory cytokines. In addition, oxidative metabolism dictates the inflammatory status of macrophages, effects that may be upstream of inflammasome activity and IL-1β release.57,58 Abnormal PBMC performance observed in periodontitis subjects may promote oxidative stress and alter cytokine homeostasis.17 Therefore, an elevated production of ROS at periodontal tissues could result in oxidative damage and IL-1β release. However, the relationship between TBARS and IL-1β was no statistically significant in AMI subjects. This result could be a consequence of IL-1β production during the AMI48 and, thus, it would not reflect the levels prior to the cardiac event. Alterations in autophagy or autophagy-dependent mechanisms have also been suggested in the pathogenesis of inflammatory disorders. An elevated autophagosome production has been evidenced by image, protein levels and gene expression analyses in periodontal ligament stem cells from periodontitis patients.59 Here, LC3-II levels in PBMC were used to evaluate this process. LC3-II is a conjugated form of LC3 protein used as membrane marker for autophagosome and autophagolysosomes.60 This marker in the present study would suggest that autophagy initiation is higher in only periodontitis subjects than in AMI+periodontitis subjects. Decreased levels of both LC3 gene expression and LC3-II protein have been reported in the peripheral leucocytes of patients with coronary artery disease. This fact indicates that autophagosome formation is decreased, supporting a positive relationship between this process and atherosclerosis at coronary artery level.61 Importantly, in another case-control study, beclin-1 and Atg7 gene expression suggested that autophagy initiation was lower in subjects with acute coronary syndrome than in those with stable angina pectoris and in control groups, emphasizing the importance of autophagy for atherosclerotic plaque stability.44 In the present study, LC3-II levels were lower in subjects that have suffered AMI than in those without cardiac event history, regardless periodontitis diagnosis. This suggests that maintaining a higher rate of autophagy initiation could contribute to decrease AMI risk. On the other hand, it seems that circulating levels of this marker can be altered by periodontal condition. Some studies have suggested that IL-1β induces autophagy with beneficial effects against oxidative stress in other cell types.27,29 The involvement of IL-1β in autophagy in PBMC would be supported by the correlation found in the present study between IL-1β and LC3-II levels in groups without AMI history (control subjects). Importantly, no significant correlation was found between the same markers when an AMI had occurred. Autophagy also can increase in response to the accumulation of oxidatively damaged cell structures. Moreover, some redox-sensitive pathways also result in autophagy.24 In turn, this process can protect cells against oxidative damage, regulating ROS generation and scavenging. Autophagy has also been correlated with mitochondrial production of ROS in PBMC obtained from periodontitis patients in a previous study. Concomitantly, a significantly positive correlation between mitochondrial ROS and autophagy makers was also observed.62 The values of LC3-II levels and TBARS obtained in our study show a correlation in the same sense, but only in AMI subjects. Therefore, ROS levels or oxidative damage would be the main stimulus triggering autophagy in this group. With regard to AMPK, the information available from the literature is rather scarce in relation to periodontitis. In the present study, PBMC levels of the more active form of AMPK (p-AMPK) are lower in subjects with periodontitis in the absence of AMI than in healthy subjects. Regarding cardiovascular system, this marker has been reported to be decreased in patients with acute coronary syndrome compared to control patients.44 In our study, differences were found between AMI subjects (only AMI vs. AMI+periodontitis). Interestingly, in the AMI group, AMPK levels are inversely correlated with TBARS, which would be in consistency with the role of AMPK in the mechanisms of activation of the antioxidant defense. However, no correlation between AMPK and IL-1β was noted, despite of some suggested roles for AMPK-dependent pathways in the NLRP3 inflammasome regulation.63 Several limitations of the study need to be addressed. One of the major strengths of the present study was the well-defined control and test groups. Many studies are prone to misclassify periodontal status by using measures to assess periodontitis known to underestimate the true extent and severity of periodontitis, including indices based on non-probing assessment and questionnaires, as well as assessments of partial-mouth probing.64 In the present study full-mouth recordings were used to define periodontitis and its severity. The latter makes possible to analyze if periodontitis prevalence is similar for different degrees of the disease. However, the association magnitude and significance may be influenced by the small sample size when the severity is high. Furthermore, the CVD-affected group consisted of a well-defined group of patients with coronary heart disease, as all test subjects had suffered an AMI. However, control subjects were not recruited at the same hospital as the AMI subjects. Unfortunately, as consequence of only including subjects that have survived to a first AMI, sample size was more reduced respect to subjects suffering AMIs in general population. In addition, this study only included subjects who were referred to a periodontal clinic in the control group leading to overrepresentation of periodontal disease compared with the general population. Other important limitation is the absence of female subjects. 5. CONCLUSIONS Results from the present study are consistent with the suggestion that periodontitis is a risk factor for AMI. Periodontitis association with peroxidative lipid damage in both control and AMI subjects suggests that oxidative stress could be a common pathogenic factor between both diseases. In addition, a positive correlation between lipid peroxidation and IL-1β levels was found in control subjects which could be a consequence of ROS production by polymorphonuclear cell stimulated by proinflammatory cytokines. However, no differences in IL-1β levels were found between AMI subjects. Still, current IL-1β levels could be different from levels prior to the cardiac event since such cytokine is also produced during AMI. Because our study and most of previous studies are cross-sectional, it is not clear whether the increase in oxidative stress is the cause or the consequence of periodontitis. ACKNOWLEDGMENTS This study was supported by Grupo de Investigacion Junta de Andalucia CTS113, Spain. Authors acknowledge Nutraceutical Translations for English language editing of this manuscript. 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