Chronic Oral Infection with Porphyromonas gingivalis Accelerates Atheroma Formation by Shifting the Lipid Profile

BACKGROUND: Recent studies have suggested that periodontal disease increases the risk of atherothrombotic disease. Atherosclerosis has been characterized as a chronic inflammatory response to cholesterol deposition in the arteries. Although several studies have suggested that certain periodontopathic bacteria accelerate atherogenesis in apolipoprotein E-deficient mice, the mechanistic link between cholesterol accumulation and periodontal infection-induced inflammation is largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We orally infected C57BL/6 and C57BL/6.KOR-Apoe(shl) (B6.Apoeshl) mice with Porphyromonas gingivalis, which is a representative periodontopathic bacterium, and evaluated atherogenesis, gene expression in the aorta and liver and systemic inflammatory and lipid profiles in the blood. Furthermore, the effect of lipopolysaccharide (LPS) from P. gingivalis on cholesterol transport and the related gene expression was examined in peritoneal macrophages. Alveolar bone resorption and elevation of systemic inflammatory responses were induced in both strains. Despite early changes in the expression of key genes involved in cholesterol turnover, such as liver X receptor and ATP-binding cassette A1, serum lipid profiles did not change with short-term infection. Long-term infection was associated with a reduction in serum high-density lipoprotein (HDL) cholesterol but not with the development of atherosclerotic lesions in wild-type mice. In B6.Apoeshl mice, long-term infection resulted in the elevation of very low-density lipoprotein (VLDL), LDL and total cholesterols in addition to the reduction of HDL cholesterol. This shift in the lipid profile was concomitant with a significant increase in atherosclerotic lesions. Stimulation with P. gingivalis LPS induced the change of cholesterol transport via targeting the expression of LDL receptor-related genes and resulted in the disturbance of regulatory mechanisms of the cholesterol level in macrophages. CONCLUSIONS/SIGNIFICANCE: Periodontal infection itself does not cause atherosclerosis, but it accelerates it by inducing systemic inflammation and deteriorating lipid metabolism, particularly when underlying hyperlidemia or susceptibility to hyperlipidemia exists, and it may contribute to the development of coronary heart disease

Effect of <it>Porphyromonas gingivalis</it> infection on post-transcriptional regulation of the low-density lipoprotein receptor in mice

<p>Abstract</p> <p>Background</p> <p>Periodontal disease is suggested to increase the risk of atherothrombotic disease by inducing dyslipidemia. Recently, we demonstrated that proprotein convertase subtilisin/kexin type 9 (PCSK9), which is known to play a critical role in the regulation of circulating low-density lipoprotein (LDL) cholesterol levels, is elevated in periodontitis patients. However, the underlying mechanisms of elevation of PCSK9 in periodontitis patients are largely unknown. Here, we explored whether <it>Porphyromonas gingivalis,</it> a representative periodontopathic bacterium, -induced inflammatory response regulates serum PCSK9 and cholesterol levels using animal models.</p> <p>Methods</p> <p>We infected C57BL/6 mice intraperitoneally with <it>Porphyromonas gingivalis</it>, a representative strain of periodontopathic bacteria, and evaluated serum PCSK9 levels and the serum lipid profile. PCSK9 and LDL receptor (LDLR) gene and protein expression, as well as liver X receptors (<it>Lxrs</it>), inducible degrader of the LDLR (<it>Idol</it>), and sterol regulatory element binding transcription factor (<it>Srebf</it>)<it>2</it> gene expression, were examined in the liver.</p> <p>Results</p> <p><it>P. gingivalis</it> infection induced a significant elevation of serum PCSK9 levels and a concomitant elevation of total and LDL cholesterol compared with sham-infected mice. The LDL cholesterol levels were significantly correlated with PCSK9 levels. Expression of the <it>Pcsk9</it>, <it>Ldlr</it>, and <it>Srebf2</it> genes was upregulated in the livers of the <it>P. gingivalis</it>-infected mice compared with the sham-infected mice. Although <it>Pcsk9</it> gene expression is known to be positively regulated by sterol regulatory element binding protein (SREBP)2 (human homologue of Srebf2), whereas <it>Srebf2</it> is negatively regulated by cholesterol, the elevated expression of <it>Srebf2</it> found in the infected mice is thought to be mediated by <it>P. gingivalis</it> infection.</p> <p>Conclusions</p> <p><it>P. gingivalis</it> infection upregulates PCSK9 production via upregulation of <it>Srebf2</it>, independent of cholesterol levels. Further studies are required to elucidate how infection regulates <it>Srebf2</it> expression and subsequently influences lipid metabolism.</p

Comparison of relative gene expression levels in the aorta between the control group and the infected group or between the short-term and long-term infected groups (N = 5 in each group).

<p>The relative quantity of experimental mRNA was normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The box plots present medians and the 25th and 75th percentiles as boxes and the 10th and 90th percentiles as whiskers. (* <i>P</i><0.01, Mann-Whitney U-test)</p

Effects of oral infection with <i>P. gingivalis</i> on aortic atherosclerosis in wild-type and B6.Apoeshl mice.

<p>(A) Representative aortas from wild-type and B6.Apoeshl mice are depicted. (B) Aortic atherosclerosis expressed as a percentage of the total area (N = 8 in each group). Box plots present medians and the 25th and 75th percentiles as boxes and the 10th and 90th percentiles as whiskers. Significant differences were observed between the infected group and the sham-infected group (* <i>P</i><0.01, Mann-Whitney U-test). (C) Representative aortic sinus cross sections from wild-type and B6.Apoeshl mice. Original magnifications, 40×.</p

Serum lipid profile associated with infection period in wild-type and B6.Apoeshl mice.

<p>Values are indicated as mg/dL.</p><p>CM indicates chylomicron; VLDL, very low density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein.</p><p>N = 5 in each group.</p

Lipopolysaccharide from <i>P. gingivalis</i> and <i>E. coli</i> inhibit cholesterol efflux and enhance cholesterol uptake.

<p>(A) Peritoneal macrophages from wild-type and B6.Apoeshl mice were loaded with BODIPY-cholesterol and treated with vehicle, 1 µg/ml <i>P. gingivalis</i> LPS or 0.1 µg/ml <i>E. coli</i> LPS in the presence or absence of 1 µM GW3965. Data are presented as apoA1-dependent cholesterol efflux. (B) Cholesterol uptake was estimated as the total of cellular and effluxed cholesterol. The results are shown as the mean <b>±</b> S.D. of three independent experiments. There were significant differences found between the wild-type and B6.Apoeshl macrophages (unpaired t-test; § <i>P</i><0.05) and between control and LPS-treated macrophages in the absence (paired t-test; * <i>P</i><0.05) or presence of GW3965 (paired <i>t</i>-test; † <i>P</i><0.05).</p

Effects of oral infection with <i>P. gingivalis</i> on serum levels of interleukin (IL)-6 (A), serum amyloid A (SAA; B), and anti-<i>P. gingivalis</i> antibody (C; N = 5 in each group).

<p>All experiments were performed in triplicate wells for each condition and repeated at least twice. Representative data are shown. Box plots present medians and the 25th and 75th percentiles as boxes and the 10th and 90th percentiles as whiskers. Significant differences were observed between the infected group and the sham-infected group (* <i>P</i><0.05; ** <i>P</i><0.01, Mann-Whitney U-test).</p

Comparison of the relative gene expression levels in the liver between the control group and the infected groups or between the short-term and long-term infected groups (N = 5 in each group).

<p>The relative quantity of mRNA was normalized to the relative quantity of GAPDH mRNA. The box plots present medians and the 25th and 75th percentiles as boxes and the 10th and 90th percentiles as whiskers. (* <i>P</i><0.05; ** <i>P</i><0.01, Mann-Whitney U-test)</p

β2-Microglobulin and Neutrophil Gelatinase-Associated Lipocalin, Potential Novel Urine Biomarkers in Periodontitis: A Cross-Sectional Study in Japanese

Objectives. Several serum biomarkers have been reported to increase in periodontitis patients as possible mediators linking periodontal inflammation to systemic diseases. However, the relationship between periodontitis and urine biomarkers is still unclear. The aim of this cross-sectional study was to investigate potential urine biomarkers of periodontitis in a Japanese population. Materials and Methods. This study included 108 male subjects, and microbiological and clinical parameters were evaluated as a periodontitis marker. The correlation between nine urine biomarkers (typically used to diagnose kidney disease) and periodontal parameters was analyzed. Based on the findings, β2-microglobulin (β2-MG) and neutrophil gelatinase-associated lipocalin (NGAL) were selected for comparison and multivariate regression analysis, and the Kruskal–Wallis test followed by Bonferroni correction was used to identify differences in their concentrations between the three periodontitis groups (severe, moderate, and no/mild periodontitis). Results. β2-MG and NGAL exhibited a significant correlation with clinical parameters of periodontitis. The prevalence of clinical parameters such as bleeding on probing and number of sites with probing depth (PD) ≥ 6 mm were greater in the β2-MG high group (≥300 μg/g creatinine) than in the normal group (P=0.017 and 0.019, respectively). Multivariate regression analysis indicated that the number of sites with PD ≥ 6 mm was independently associated with urine β2-MG. Moreover, the number of sites with the clinical attachment level (CAL) ≥ 6 mm was greater in the NGAL high group (highest quartile) (P=0.041). Multivariate regression analysis showed that the number of sites with CAL ≥ 6 mm was associated independently with urine NGAL. Finally, β2-MG was significantly higher in the severe periodontitis subjects compared to the no/mild periodontitis subjects. Conclusion. The significant association between urine β2-MG or NGAL and periodontitis was revealed. These biomarkers can potentially be used to screen for or diagnose periodontitis. This trial is registered with the UMIN Clinical Trials Registry UMIN000013485