Periodontitis and cardiovascular disease

Pirkko Pussinen, Ph.D. (Biochemist)
Senior scientist, Docent, Institute of Dentistry

The mechanisms behind the association between periodontitis and increased risk for cardiovascular diseases (CVD) are largely unknown. Therefore, our project aims to study this relationship by clinical and epidemiological studies as well as basic research. Our approaches are as follows:

1) Development of markers of periodontitis to be used in clinical and epidemiological studies.
i) Serology: In 2002 we have developed and validated a serological assay, which has a sensitivity and specificity of 71% and 90% for finding periodontitis by measuring the antibodies against major periodontal pathogens. The assay has been successfully used in several population-based epidemiological samples, which have been collected by the researchers of the National Public Health Institute in order to study risk factors for CVD. According to the results, high antibody levels to periodontal pathogens are associated with subclinical, prevalent, and future atherosclerosis.
ii) Lipopolysaccharide (LPS): We have in several studies successfully used LPS as a marker of systemic exposure to periodontal pathogens. We have shown that serum LPS concentration correlates positively with clinical periodontal status, antibody level against periodontal pathogens, as well as several systemic inflammation markers, which are considered risk factors for CVD. According to our results, high LPS concentration is an independent risk factor for incident CVD events (HR 1.8). In combination with high concentrations of inflammation markers, CRP, IL-6, or TNF-α, or low HDL cholesterol its predictive value for CVD increases still (HRs 2.2-3.9).
iii) Periodontal pathogens: We have recently set up a novel single copy gene -based quantitative real-time PCR (qPCR) assay to detect in oral and tissue samples five most important periodontal pathogens, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, Prevotella intermedia, and Tannerella forsythensis. All quantified pathogens alone were able to significantly distinguish between the subjects with and without periodontitis and provided ROC curve areas larger than 0.5. Furthermore, the total pathogen burden associated with periodontitis with an area of 0.821 (95% CI 0.76-0.89, p<0.001). The conventional PCR results concerning the presence of the pathogens in the saliva samples were compared to our qPCR results and we found the novel method to be superior in sensitivity.

2) Animal models. We have developed a mouse model to study the role of periodontal pathogen, Actinobacillus actinomycetemcomitans, in atherogenesis. The main results were as hypothesized that this periodontal pathogen induced i) systemic inflammation, which was shown by increases in the serum CRP and lipopolysaccharide-binding protein concentrations; ii) matrix remodelling, which was shown by increased expression of matrix metalloproteinase 9 in the aortic lesions and increased gelatinase activity in serum; iii) proatherogenic changes in lipoproteins including elevated triglyceride and cholesterol concentrations, induction of smaller-sized VLDL, LDL, and HDL particles; and iv) increases of LPS concentration in both VLDL and LDL classes. The early atherogenic lesion area and areas enriched by macrophages in the aortas were greater in the mice receiving pathogen compared to those receiving vehicle.  At the moment we are analysing samples from a mouse experiment, where the mice have been infected with two different pathogens causing chronic infections and CVD.

3) Basic research. Periodontitis is associated with endotoxemia, leakage of LPS deriving from periodontal pathogens into circulation. LPS is one of the potent stimulators of systemic inflammation and intima wall macrophage-derived foam cell formation, and therefore it is considered a proatherogenic compound. According to our earlier results, the structural variation in the LPS of A. actinomycetemcomitans determines the proatherogenic properties of the species. The proatherogenic properties of LPS are serotype-specific and associate with the periopathologic potential of the clonotype. We are investigating these phenomena using A. actinomycetemcomitans strains, whose LPS portion is mutated.

4) Clinical studies. The results from our clinical studies suggest that LPS at least partially deriving from periodontal pathogens promotes atherogenesis by interfering macrophage and mast cell cholesterol homeostasis. The infected area in periodontitis is associated via circulating LPS with macrophage activation, a hallmark of atherosclerosis. Periodontitis increases the risk for atherosclerosis also by decreasing concentrations and anti-atherogenic potency of HDL. In another clinical trial, our results suggested that long-term clarithromycin therapy is beneficial in prevention of recurrent cardiovascular events in non-periodontitis but not in periodontitis patients. According to our results, fewer CVD end points in clarithromycin group were seen in periodontal bacterium-negative, seronegative, edentulous, and non-periodontitis patients compared to those with bacterial, immunological, or radiological signs of periodontitis.

5) Genetic studies. We have found in both transplantation and unstable acute coronary syndrome (ACS) patients, that MHC haplotype (A3-B35-DRB1) alone, and with certain other polymorphisms, predispose to coronary artery disease, and some other haplotypes protect from it. Furthermore, these susceptibility haplotypes are linked to increased risk for having periodontitis, occurence of periodontal pathogens and seropositivity to them. We will now continue and broaden these studies by participating in the Corogene-study. The aim of the study is to identify gene loci and variations disclosing the association between periodontitis and coronary artery disease in patients with verified pathological changes in angiography. In the Corogene-study, 5000 patients were recruited, and in our substudy, we investigated 500 patients for their detailed dental status and systemic markers for periodontitis. A genetically interesting subsample of the total cohort will be genotyped for an informative set of haplotype tag SNPs derived from the HapMap European -derived sample. The genotyping chip to be used contains over 317,000 tag SNPs in close proximity of a gene, in evolutionary conserved regions, or changing amino acid, and a high density of SNPs across the MHC region. The genome-wide results and the candidate gene and -loci will be analysed by the Finnish Genome Center and the Sanger Institute.

The study will produce novel data on the causal role of periodontal pathogens in atherogenesis. Up to date, the mechanism behind the association of CVD and periodontitis or periodontal pathogens has not been clarified. Since severe periodontitis is relatively common affecting approximately 20% of middle-aged and elderly populations worldwide, its relevance to public health is extensive. Our preliminary results suggest that local infection by periodontal pathogens induces systemic inflammation resulting in abnormalities in cholesterol metabolism, and increases in antibodies and inflammation markers, which directly or indirectly increase the risk for CVD. According to the results, the systemic spread of LPS, i.e. endotoxemia, may be one of the most important atherogenic factors in periodontitis, since the major periodontal pathogens are Gram-negative.

Research group

Susanna Paju, DDS, PhD
Laura Lakio, DDS, PhD
Anita Tuomainen, MSc
Kati Hyvärinen, MSc
Elisa Kallio, BD
Airi Sinkko, lab technician

Selected publications

1. Pussinen PJ, Vilkuna-Rautiainen T, Alfthan G, Mattila K, Asikainen S. Multiserotype enzyme-linked immunosorbent assay as a diagnostic aid for periodontitis in large-scale studies. J Clin Microbiol 2002;40:512-518
2. Pussinen PJ, Jousilahti P, Alfthan G, Palosuo T, Asikainen S, Salomaa V. Antibodies to periodontal pathogens are associated with coronary heart disease. Arterioscler Thromb Vasc Biol 2003;23:1250-1254
3. Pussinen PJ, Jauhiainen M, Vilkuna-Rautiainen T, Sundvall J, Mattila K, Vesanen M, Palosuo T, Alfthan G, Asikainen S. Periodontitis decreases the antiatherogenic potency of high density lipoproteins. J Lipid Res 2004;45:139-147
4. Pussinen PJ, Vilkuna-Rautiainen T, Alfthan G, Palosuo T, Jauhiainen M, Sundvall J, Vesanen M, Mattila K, Asikainen S. Severe periodontitis enhances macrophage activation via increased serum lipopolysaccharide. Arterioscler Thromb Vasc Biol 2004;24:2174-2180
5. Pussinen PJ, Alfthan G, Rissanen H, Reunanen A, Asikainen S, Knekt P. Antibodies to periodontal pathogens and stroke risk. Stroke 2004;35:2020-2023
6. Pussinen PJ, Nyyssönen K, Alfthan G, Salonen R, Laukkanen JA, Salonen JT. Serum antibody levels to Actinobacillus actinomycetemcomitans predict the risk for coronary heart disease. Arterioscler Thromb Vasc Biol 2005;25:833-888
7. Paju S, Pussinen PJ, Sinisalo J, Mattila K, Doğan B, Ahlberg J, Valtonen V, Nieminen MS, Asikainen, S. Clarithromycin reduces recurrent cardiovascular events in subjects without but not with periodontitis. Atherosclerosis 2006;188:412-419
8. Lakio L, Lehto M, Tuomainen AM, Jauhiainen M, Malle E, Asikainen S, Pussinen PJ. Proatherogenic properties of lipopolysaccharide from periodontal pathogen, Actinobacillus actinomycetemcomitans. J Endotoxin Res 2006;12:57-648
9. Pussinen PJ, Tuomisto K, Jousilahti P, Sundvall J, Salomaa V. Endotoxemia, immune response to periodontal pathogens, and systemic inflammation associate with incident cardiovascular disease events. Arterioscler Thromb Vasc Biol 2007;27:1433-1439
10. Tuomainen AM, Jauhiainen M, Kovanen P, Metso J, Paju S, Pussinen PJ. Administration of live Actinobacillus actinomycetemcomitans induces matrix metalloproteinase-9 expression and proatherogenic lipoprotein profiles in apolipoprotein E –deficient mouse. Microbial Pathogen 2008;44:111-117
11. Palikhe A, Lokki M-L, Pussinen PJ, Paju S, Ahlberg J, Asikainen S, Seppänen M, Valtonen V, Nieminen MS, Sinisalo J. Lymphotoxin alpha LTA+496C allele is a risk factor for periodontitis in patients with coronary artery disease. Tissue Antigens 2008;71:530-737
12. Kallio KAE, Buhlin K, Jauhiainen M, Keva R, Tuomainen AM, Klinge B, Gustafsson A, Pussinen PJ. Lipopolysaccharide associates with proatherogenic lipoproteins in periodontitis patients. Innate Immunity 2008;14:247-253
13. Paju S, Pussinen PJ, Suominen-Taipale L, Hyvönen M, Knuuttila M. Könönen E. Detection of multiple pathogenic species in saliva associates with periodontal infection in adults. J Clin Microbiol 2009;47:235-238
14. Hyvärinen K, Laitinen S, Paju S, Hakala A, Suominen-Taipale L, Skurnik M, Könönen E, Pussinen PJ. Detection and quantification of five major periodontal pathogens by single copy gene -based real-time PCR. Innate Immunity, 2009