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Inflammation plays an important role in the pathogenesis of atherosclerosis, and low-grade chronic systemic inflammation is thought to be related to adverse cardiovascular outcomes.¹ In the past decade, epidemiological studies have repeatedly shown an association between coronary heart disease and periodontal disease. Observational studies have shown that periodontitis is associated with an increased risk of myocardial infarction and stroke, although a causal link has not yet been proven.² Periodontitis involves infection induced by a variety of gram-negative bacteria that invade superficial and deeper gingival tissues.³ Chronic periodontitis is characterized by multiple episodes of bacteraemia events that can allow species associated with periodontal disease to migrate to atheromatous plaques. This study was undertaken to ascertain the presence of DNA of periodontal bacteria (Tannerella forsythia, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis and Actinobacillus action-mycetemcomitans) in coronary atheromatous plaques and to compare them with periodontal bacteria from the subgingival plaque samples obtained from the same patients.

Subjects
From May 2006 to March 2007, a total of 51 patients scheduled for coronary artery bypass graft were enrolled in this study. The age of the patients ranged from 38 to 72 years. All the patients were hospitalized at the Department of Cardiovascular Surgery of the First Affiliated Hospital, Xinjiang Medical University.

Methods
The coronary atheromatous plaques were obtained surgically one week after the periodontal examination. The atheromatous samples were placed in a sterile tube with 10 ml of saline solution and immediately transferred to the Laboratory of Biochemical Research, Xinjiang Medical University. Odontological examinations were performed by a periodontist who determined a clinical diagnosis of periodontitis by Armitage's criteria.⁴ A sample of subgingival plaques was collected from the deepest pocket of each patient and then transferred to a sterile tube and sent to the laboratory. Both surgical specimens and samples of subgingival plaques were frozen at −70ºC. The samples of subgingival plaques and specimens of atheromatous plaques were examined with the polymerase chain reaction (PCR) technique using specific primers for periodontal bacteria.

Laboratory procedure
Approximately 100 mg of tissues was harvested from the atherosclerotic plaques and then homogenized and subjected to DNA extraction. DNA was extracted by the Chelex-100 method. The extracted DNA was measured by a spectrophotometer.

Primers design
The primers were designed in accordance with those used by Cairo et al,⁵ Ashimoto et al⁶ and Kimura et al (Table).⁷ These primers were synthesized by Sangon Bio-tek (Shanghai, China). All of the primer sequences were compared with the GenBank to ensure their specificity.

Positive controls
The strains A. actinomycetemcomitans ATCC 29523, T. forsythia ATCC43037, P. gingivalis ATCC 33277, F. nucleatum ATCC25586 and P. intermedia ATCC 25611 were cultured on Shaedler Anaerobe Agar incubated in a chamber for 7 days at 37ºC in an anaerobic atmosphere created with the AnaeroGen system. Colonies obtained from cultures were suspended in 500 µl of sterile water. DNA was extracted by the chelex-100 method and quantified by a spectrophotometer and used as the positive control.

PCR conditions
PCR amplification was carried out in a 37.5-µl reaction mixature containing 10 × buffer 3.75 µl, 25 mmol/L MgCl₂ 2.25 µl, 10 mmol/L of each dNTP 0.6 µl, Taq DNA polymerase 2U, 1.0 µl（10 pmol/µl）of each primer pair and 10–30 ng of DNA template. The PCR protocol consisted of a preliminary denaturation step (94ºC for 5 minutes), 33 cycles of annealing temperature for each pair of primers (Table) for 1 minute, extension (72ºC for 1 minute) and denaturation (94ºC for 30 seconds) and a final elongation (7 minutes at 72ºC).

Analysis of PCR products
The PCR products were analyzed by electrophoresis in 2% agarose gel (Sangon, China), stained with ethidium bromide (0.5 µg/ml) and photographed under ultraviolet light (Bio-Rad, Doc2000, USA). The presence of a DNA band at the expected size confirmed the presence of the periodontal pathogens (Table and Figure 1).

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Table. Primers used to identify the bacteria

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Figure 1. Identification of bacterial species from atheromatous plaques and subgingival plaques by polymerase chain reaction (PCR). Lanes 2,15: PUC19DNA/MspI(Hpall)-(molecular weight marker); lanes 1,14: negative control (H2O); lanes 3, 5, 8, 11: positive control (A. a ATCC 29523, T. f ATCC43037, P. g ATCC 33277, F. n ATCC 25586); lanes 4,6,9,12: clinical isolates from subgingival plaque (A. a, T. f, P. g, F. n); lanes 7,10,13: clinical isolates from atheromatous plaqued (T. f, P. g, F. n).

Analysis of DNA sequencing
DNA sequencing was performed to ascertain the sequence of periodontal bacteria DNA, and it was compared with type strains sequences from the GenBank by blast to ensure their sequence homology.

All subjects were affected by advanced chronic periodontitis. The sequences of periodontal bacteria DNA in atheromatous plaques could be ascertained by DNA sequencing such as DNA sequences of the amplified products of Tannerella forsythia and Porphyromonas gingivalis (Figures 2 and 3). They were compared with type strains sequences from the GenBank by blast to confirm their sequence homology.

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Figure 2. DNA sequencing of the amplified product of Tannerella forsythia.

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Figure 3. DNA sequencing of the amplified product of Porphyromonas gingivalis.

The DNA of at least one of the target bacteria was detected in each subgingival sample. The prevalence of T. forsythia, F. nucleatum, P. intermedia, P. gingivalis, and A. actinomycetemcomitans was 84%, 78%, 59%, 39% and 22%, respectively.

The DNA of periodontal bacteria was detected in the atheromatous plaques except A. actinomycetemcomitans. The prevalence of T. forsythia, F. nucleatum, P. intermedia and P. gingivalis was 31%, 12%, 18%, and 33%, respectively.

In 13 of 51 cases, T. forsythia was found in periodontal samples and atheromatous plaque samples. In 10 cases, P. gingivalis was present in periodontal samples and atheromatous plaque samples. P. intermedia and F. nucleatum were present in periodontal samples and atheromatous plaque samples in 5, 4 cases, respectively (Figure 1).

In recent years, an increasing number of epidemiological studies have indicated that periodontitis may increase the risk of cardiovascular events.⁸ This effect may be due to the direct effect of periodontal pathogens or their products on endothelial cells via transient bacteremia.⁹ In cell cultures, invasive P. gingivalis and fimbria stimulate endothelial cell activation, a necessary initial event in the development of atherogenesis.¹⁰

Higashi et al¹¹ reported that periodontitis is associated with endothelial dysfunction in subjects without cardiovascular risk factors, as illustrated by a decrease in NO bioavailability, and that systemic inflammation as a cause of endothelial dysfunction may lead to cardiovascular diseases. A case-control study showed that periodontitis is associated with endothelial dysfunction, and that 6 months after therapy, the benefits in oral health may be associated with improvement in endothelial function.¹² Experimental animal models (ApoE mice) demonstrated that P. gingivalis chronic inoculations increased lipid profiles, enhanced atheroma formation, and facilitated the calcification of aortal atherosclerotic plaques.¹³

In the present study, subgingival plaque samples and atheromatous plaque specimens were examined with the PCR technique using specific primers for periodontal bacteria. The DNA of at least one of the target bacteria was detected in each subgingival sample. The prevalence of T. forsythia, F. nucleatum, P. intermedia, P. gingivalis, and A. actinomycetemcomitans was 84%, 78%, 59%, 39% and 22%, respectively. The DNA of periodontal bacteria except A. actinomycetemcomitans was detected in the atheromatous plaques. The sequences of periodontal bacteria DNA in the atheromatous plaques could be ascertained by DNA sequencing and they were compared with type strains sequences from the GenBank by blast to confirm their sequence homology. The prevalence of T. forsythia, F. nucleatum, P. intermedia and P. gingivalis was 31%, 12%, 18%, and 33%, respectively. In 13 of 51 cases, T. forsythia was found in periodontal samples and atheromatous plaque samples. In 10 cases, P. gingivalis was present in periodontal samples and atheromatous plaque samples. P. intermedia and F. nucleatum were present in periodontal samples and atheromatous plaque samples in 5, 4 cases, respectively.

The presence of periodontal bacteria DNA in coronary atheromatous plaques and subgingival plaque samples of the same patients was confirmed by this study and thus a correlation was established between putative bacteria contributing to atheromatous plaques and species associated with periodontal disease. The data of this study were consistent with those reported by Haraszthy et al¹⁴ (PCR-amplified 16S rDNA and DNA species-specific probes, 30% positive for T. forsythia, 26% for P. gingivalis, 14% for P. intermedia) and Ishiara et al¹⁵ (PCR-amplified 16S rRNA, 21.6% positive for P. gingivalis, 5.9% for T. forsythia). Thus a correlation may be established between coronary heart disease and periodontal disease. The presence of periodontal bacteria in atheromatous plaques and periodontal samples of the same cases supports the potential role of this periodontopathogenic bacterial species in some steps of the atherogenesis process or as a contributor of a different mechanism that worsens this disease. The link found in this study supports the concept that there may be a true inflammatory link between atherogenesis and periodontitis.

1. Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis. Circulation 2002; 105: 1135-1143. [PubMed]

2. Scannapieco FA, Bush RB, Paju S. Associations between periodontal disease and risk for atherosclerosis, cardiovascular disease, and stroke. A systematic review. Ann Periodontol 2003; 8: 38-53. [PubMed]

3. Pihlstrom BL, Michalowicz BS, Johnson NW. Periodontal diseases. Lancet 2005; 366: 1809-1820. [PubMed]

4. Armitage GC, Wu YF, Wang HY, Sorrell J, Di Giovine FS, Duff GW. Low prevalence of a periodontitis associated interkeukin-1 composite genotype in individuals of Chinese heritage. J Periodontol 2000; 71: 164-171. [PubMed]

5. Cairo F, Gaeta C, Dorigo W, Oggioni MR, Pratesi C, Pini Prato GP, et al. Periodontal pathogens in atheromatous plaques. A controlled clinical and laboratory trial. J Periodont Res 2004; 39: 442-446.[PubMed]

6. Ashimoto A, Chen C, Bakker I, Slots J. Polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesions. Oral Microbiol Immunol 1996; 11: 266-273. [PubMed]

7. Kimura S, Ooshima T, Takiguchi M, Sasaki Y, Amano A, Morisaki I, et al. Periodontopathic bacterial infection in childhood. J Periodontol 2002; 73: 20-26. [PubMed]

8. Beck JD, Offenbacher S. The association between periodontal diseases and cardiovascular diseases: a state-of-the-science review. Ann Periodontol 2001; 6: 9-15.[PubMed]

9. Geerts SO, Nys M, De MP, Charpentier J, Albert A, Legrand V, et al. Systemic release of endotoxins induced by gentle mastication: association with periodontitis severity. J Periodontol 2002; 73: 73-78. [PubMed]

10. Takahashi Y, Davey M, Yumoto H, Gibson FC 3rd, Genco CA. Fimbria-dependent activation of pro-inflammatory molecules in Porphyromonas gingivalis infected human aortic endothelial cells. Cell Microbiol 2006; 8: 738-757. [PubMed]

11. Higashi Y, Goto C, Jitsuiki D, Umemura T, Nishioka K, Hidaka T, et al. Periodontal infection is associated with endothelial dysfunction in healthy subjects and hypertensive patients. Hypertension 2008; 51: 446-453. [PubMed]

12. Tonetti MS, D'Aiuto F, Nibali L, Donald A, Storry C, Parkar M, et al. Treatment of periodontitis and endothelial function. N Engl J Med 2007; 356: 911-920.[PubMed]

13. Li L, Messas E, Batista EL Jr, Levine RA, Amar S. Porphyromonas gingivalis infection accelerates the progression of atherosclerosis in a heterozygous apolipoprotein E-deficient murine model. Circulation 2002; 105: 861-867. [PubMed]

14. Haraszthy VI, Zambon JJ, Trevisan M, Zeid M, Genco RJ. Identification of periodontal pathogens in atheromatous plaques. J Periodontol 2000; 71: 1554-1560.[PubMed]

15. Ishihara K, Nabuchi A, Ito R, Miyachi K, Kuramitsu HK, Okuda K. Correlation between detection rates of periodontopathic bacterial DNA in coronary stenotic artery plaque and in dental plaque samples. J Clin Microbiol 2004; 42: 1313-1315. [PubMed]