This is a very interesting and relevant study that demonstrates the ability of epigallocatechin-3-gallate (EGCG), apolyphenol extract from green tea, to protect against Porphyromonas gingivalis-induced atherosclerosis. The experimental approach is straightforward and the results provide evidence for the mechanism accounting for the protective effect
Green tea epigallocatechin-3-gallate attenuates Porphyromonas gingivalis-induced atherosclerosis
Article first published online: 15 NOV 2012
© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved
Pathogens and Disease
Volume 67, Issue 1, pages 76–83, February 2013
How to Cite
Cai, Y., Kurita-Ochiai, T., Hashizume, T. and Yamamoto, M. (2013), Green tea epigallocatechin-3-gallate attenuates Porphyromonas gingivalis-induced atherosclerosis. Pathogens and Disease, 67: 76–83. doi: 10.1111/2049-632X.12001
- Issue published online: 12 FEB 2013
- Article first published online: 15 NOV 2012
- Accepted manuscript online: 19 OCT 2012 06:20AM EST
- Manuscript Accepted: 27 AUG 2012
- Manuscript Revised: 24 AUG 2012
- Manuscript Received: 26 JUL 2012
- Grants-in-Aid for Scientific Research. Grant Number: 22390398
- Porphyromonas gingivalis ;
- apolipoprotein E knockout mice;
The purpose of this study was to determine whether epigallocatechin-3-gallate (EGCG) ameliorates Porphyromonas gingivalis-induced atherosclerosis. EGCG is a polyphenol extract from green tea with health benefits and P. gingivalis is shown here to accelerate atheroma formation in a murine model. Apolipoprotein E knockout mice were administered EGCG or vehicle in drinking water; they were then fed high-fat diets and injected with P. gingivalis three times a week for 3 weeks. Mice were then killed at 15 weeks. Atherosclerotic plaques in the proximal aorta were determined by Oil Red O staining. Atherosclerosis risk factors in serum, liver or aorta were analysed using cytokine antibody arrays, enzyme-linked immunosorbent assay and real-time PCR. Atherosclerotic lesion areas of the aortic sinus caused by P. gingivalis infection decreased in EGCG-treated groups, wherein EGCG reduced the production of C-reactive protein, monocyte chemoattractant protein-1, and oxidized low-density lipoprotein (LDL), and slightly lowered LDL/very LDL cholesterol in P. gingivalis-challenged mice serum. Furthermore, the increase in CCL2, MMP-9, ICAM-1, HSP60, CD44, LOX-1, NOX-4, p22phox and iNOS gene expression levels in the aorta of P. gingivalis-challenged mice were reduced in EGCG-treated mice. However, HO-1 mRNA levels were elevated by EGCG treatment, suggesting that EGCG, as a natural substance, inhibits P. gingivalis-induced atherosclerosis through anti-inflammatory and antioxidative effects.
Atherosclerosis is a multifactor cardiovascular disease, and is a leading cause of mortality and morbidity in Western countries. Atherosclerosis is related to physiological and behavioral risk factors such as age, gender, hypertension, hypercholesterolemia, obesity, diabetes, smoking and a sedentary lifestyle (Stocker & Keaney, 2004). Studies have indicated that chronic inflammatory responses and oxidative stress conditions are associated with the pathogenesis of atherosclerosis (Galkina & Ley, 2009). Therefore, chronic inflammatory and immune responses activated by infectious agents may damage the vessel wall and may be a trigger for the onset of atherosclerosis (Rosenfeld & Campbell, 2011; Tufano et al., 2012). Periodontitis increases the risk of atherosclerosis, and previous studies have suggested that chronic infection with a periodontal pathogen, such as Porphyromonas gingivalis, is associated with an increased risk of atherosclerosis (Hayashi et al., 2010). Indeed, P. gingivalis was detected more frequently than other periodontal bacteria in atherosclerotic plaques (Pucar et al., 2007).
Porphyromonas gingivalis promotes platelet aggregation, increases systemic inflammatory markers, invades endothelium and vascular smooth muscle cells, and alters endothelial function (Hayashi et al., 2010). Furthermore, P. gingivalis accelerates the progression of atherosclerosis in homo- and heterozygous apolipoprotein E (ApoE)-deficient mice, rabbits and pigs (Hayashi et al., 2010). Previous studies have indicated that early activation of inflammatory mediators in response to P. gingivalis challenge may be associated with P. gingivalis-accelerated atherosclerosis (Koizumi et al., 2008; Fukasawa et al., 2012). Therefore, oral inflammatory diseases and systemic inflammation caused by P. gingivalis infection may contribute to atherosclerosis.
Green tea is a popular drink worldwide, and consumption has been suggested to prevent the development of a variety of diseases, including diabetes, hypertension, cancer and cardiovascular diseases (Wolfram, 2007). The effects of green tea are attributed to its abundant and biologically active catechin, epigallocatechin-3-gallate (EGCG), which has antioxidative (Feng et al., 2001), anti-inflammatory (Tipoe et al., 2007), antitumorigenic (Mukhtar & Ahmad, 2000) and antiangiogenic (Cao & Cao, 1999) effects. Inflammation plays an important role in the initiation and/or development of atherosclerosis. Oxidative stress, or oxidation of low-density lipoprotein (ox-LDL), is used as a marker of inflammation (Stocker & Keaney, 2004). Therefore, this study was performed to determine whether the continuous intake of EGCG alleviates the development of atherosclerotic lesions induced by P. gingivalis in ApoE-deficient mice, and to assess its effects on gene expression and protein synthesis in the aorta and serum, focusing on possible anti-inflammatory and antioxidant activities.
Materials and methods
Porphyromonas gingivalis strain 381 was cultured on anaerobic blood agar plates (Becton Dickinson) in a model 1024 anaerobic system (Forma Scientific) with 10% H2, 80% N2 and 10% CO2 for 3–5 days. Cultures were then inoculated into brain heart infusion broth (Difco), supplemented with 5 μg hemin mL−1 and 0.4 μg menadione mL−1, and grown for 2 days, reaching an optical density at 660 nm (OD660) of 0.8, corresponding to 109 CFU mL−1. The cultured cells were then centrifuged at 8000 g for 20 min at 4 °C and diluted with phosphate-buffered saline (PBS) for intravenous (i.v.) infection.
Mice and treatments
Six-week-old male ApoE knockout (ApoE-KO) mice, obtained from Jackson Laboratories, were divided randomly into three groups (n = 5 for each group). The institutional Animal Care and Use Committee of Nihon University approved all animal protocols. The mice were supplemented for 7 weeks via their drinking water, with (1) and (2) a placebo (distilled water) or (3) 0.02% solution of EGCG. EGCG was administered at a dose of 0.02% in drinking water, which is comparable with common human dietary intake. This dose corresponds to an equivalent intake in humans of 100 mg day−1, a value within the range of intake measured in several populations (11–121 mg day−1) (Erdman et al., 2007). Green tea-derived EGCG (95% purity as determined by HPLC) was purchased from Sigma-Aldrich (Cat. no. E4143) and dissolved in distilled water. After 2 weeks of housing, mice were given a high-fat diet (HFD) containing 16.5% fat, 22.6% protein and 45.1% carbohydrate. The mice were inoculated intravenously three times per week for 3 weeks with (1) PBS (100 μL per mouse) or (2) and (3) P. gingivalis (108 CFU 100 μL−1 per mouse). Food and drink was provided to mice ad libitum. All mice were monitored daily until death. At the age of 15 weeks, the mice from each group were killed, and tissue and blood samples were collected.
Quantification of the atherosclerotic lesion area
Blood was collected into heparinized syringes from the orbital veins of mice anesthetized with Isozol (Nichi Iko). The heart and aortic tree were then perfused through the left ventricle with ice-cold 0.9% PBS for 10 min. The heart was then carefully dissected and removed. The upper half of the heart containing the aortic origin was separated and embedded in Tissue-Tek OCT compound (Fisher Scientific) in cryomolds, and cryostat sections were prepared (Paigen et al., 1987). Using a modified version of the method of Paigen et al. (1987), cryosections of the aortic arch for atherosclerotic plaque accumulation by Oil Red O staining were examined. The lesion area was then quantified under a microscope (BX51; Olympus), interfaced with a CCD camera and an image analysis system (Lumina Vision; Mitani Co.). Briefly, cross-sectional areas from three images were summed to obtain the total lesion area per slide, and the percentage of the aortic lumen occupied by lesions per section was calculated. Slides were analysed in a blinded manner, and the total lesion area, as well as the percentage of the aortic lumen occupied by lesions, was averaged over 15 sections per animal, expressed as the mean lesion area and the percentage of the lumen of the proximal aorta occupied by lesions per section, per animal.
Serum antibody array
Serum samples from blood collected at death (15 weeks) were analysed using a cytokine antibody array (RayBio Mouse Atherosclerosis Ab array I; Ray Biotech) consisting of 22 different atherosclerosis-related cytokine antibodies, as per the manufacturer's recommendation. Signal intensities from the bound cytokines were measured using Light Capture II equipped with CS analyser (ATTO). Biotin-conjugated IgG served as a positive control at six spots, where it was used to identify the membrane orientation and to normalize the results from different membranes that were being compared. For each spot, the net optical density level was determined by subtracting the background optical level from the total raw optical density and the level of each cytokine was represented as a percentage of the positive control.
Serum ELISA assay
Each serum sample from blood collected after euthanasia (15 weeks) was screened by high-sensitivity enzyme-linked immunosorbent assay (ELISA) for C-reactive protein (CRP) (Kamiya), monocyte chemoattractant protein 1 (MCP-1) (Thermo Scientific), interleukin-8 (IL-8) homolog and MIP-2 (R&D Systems), and oxidized in low-density lipoprotein (ox-LDL) (Cusabio Biotech, Newark, DE). Serum LDL/very low-density lipoprotein (VLDL) cholesterol was measured using a quantitative kit (BioVision).
Quantitative real-time PCR
Total RNA was purified from liver and aorta tissues using an RNeasy Plus Mini Kit or RNeasy Fibrous Tissue Kit (Qiagen) and reverse-transcribed with Oligo (dT) primers using Primescript RT Reagent Kit reverse transcriptase (Takara Bio) to generate cDNA. Quantitative real-time PCR (qPCR) analysis was performed using the Thermal Cycler Dice real-time PCR system (Takara Bio) in accordance with the manufacturer's protocol. Briefly, the reactions contained 12.5 μL of 2× SYBR Green (Takara Bio), each primer at 100 nM and 30 ng of reverse-transcribed RNA. The PCR conditions were as follows: 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. Dissociation curve analysis was then performed to confirm specificity. Each gene was tested in triplicate. Primer details are given in Table 1. Target RNA levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA.
Results are expressed as the mean ± SD. Data were analysed using the two-tailed Student's test or one-way anova, followed by Tukey–Kramer multiple tests. P < 0.05 was considered to indicate statistical significance.
Histomorphometric analysis of the aortic sinus
A cryosection of the aortic sinus was examined for atherosclerotic plaque accumulation by Oil Red O staining. Histomorphological analysis revealed an increase in atherosclerotic plaque accumulation in P. gingivalis-challenged mice compared with sham-treated mice, with the percentage of the total lumen of the proximal aorta occupied by lesions showing the same pattern (Fig. 1a and b, 1267 ± 281 vs. 750 ± 281 mm2, P < 0.05; Fig. 1a and c, 9.13 ± 0.06 vs. 5.50 ± 0.03%, P < 0.05). In contrast, treatment with EGCG reduced atherosclerotic plaque accumulation in the P. gingivalis-infected group (Fig. 1a and b, 1267 ± 281 vs. 602 ± 245 mm2, P < 0.05; Fig. 1a and c, 9.13 ± 0.06 vs. 4.35 ± 0.05%, P < 0.05).
Serum cytokine and atherosclerotic risk factor level
To analyse the levels of systemic inflammation in serum from P. gingivalis-infected mice, semiquantitative membrane-based antibody arrays were utilized to detect atherosclerotic mediators. Porphyromonas gingivalis challenge increased serum levels of the proinflammatory cytokines IL-1α, IL-1β, IL-6, MCP-1, and tumor necrosis factor α (TNFα), as well as other inflammatory mediators, basic fibroblast growth factor (bFGF), CD40, macrophage colony-stimulating factor (M-CSF), macrophage inflammatory protein 3α (MIP-3α) and P-selectin, as compared with sham-treated mice (Fig. 2). The increase in inflammatory cytokine and mediator levels caused by infection with P. gingivalis was reduced in EGCG-treated mice. Individual cytokine ELISA was performed to characterize the magnitude of other atherosclerotic risk factors. Serum samples obtained from P. gingivalis-challenged mice expressed increased levels of other inflammatory mediators, including CRP, IL-8, ox-LDL and LDL/VLDL cholesterol, as well as MCP-1, compared with sham-treated mice, although the increased IL-8 level was not statistically significant (Fig. 3; CRP, 4.1 ± 0.4 vs. 7.0 ± 0.3 ng mL−1, P < 0.05; IL-8, 58.3 ± 7.2 vs. 67.1 ± 17.0 pg mL−1, P > 0.05; ox-LDL, 4.0 ± 0.01 vs. 6.7 ± 0.1 μmol mL−1, P < 0.05; LDL/VLDL, 2.48 ± 0.02 vs. 3.2 ± 0.7 μg μL−1, P < 0.05; MCP-1, 330.0 ± 67.2 vs. 493.4 ± 95.1 pg mL−1, P < 0.05). In contrast, EGCG treatment decreased the inflammatory mediators, although the decreased LDL/VLDL level was not statistically significant (Fig. 3; CRP, 7.0 ± 0.4 vs. 4.0 ± 0.04 ng mL−1, P < 0.05; IL-8, 67.1 ± 17.0 vs. 43.7 ± 9.4 pg mL−1, P < 0.05; ox-LDL, 6.7 ± 0.1 vs. 4.5 ± 0.2 μmol mL−1, P < 0.05; LDL/VLDL, 3.2 ± 0.7 vs. 2.2 ± 0.5 μg μL−1, P = 0.058; MCP-1, 493.4 ± 95.1 vs. 339.8 ± 94.2 pg mL−1, P < 0.05).
Real-time PCR detection of inflammatory and oxidative stress mediators
To examine the involvement of various inflammatory and oxidative stress mediators in atherosclerosis activated by P. gingivalis challenge, the level of gene expression in the liver and aorta in each group of mice was detected by real-time PCR. In the aorta (Fig. 4), P. gingivalis induced an increase in mRNA expression of inflammatory mediators, such as CCL2 (P < 0.05), matrix metalloproteinase-9 (MMP-9) (P < 0.05), intercellular adhesion molecule-1 (ICAM-1) (P < 0.05), heat shock protein 60 (HSP60) (P < 0.05) and CD44 (P < 0.01) compared with the sham-treated group (Fig. 4a). In contrast, EGCG treatment significantly decreased the expression of CCL2 (P < 0.05), MMP-9 (P < 0.01), ICAM-1 (P < 0.05), HSP60 (P < 0.01) and CD44 (P < 0.01).
Previous studies have shown that ox-LDL increases lectin-like oxidized LDL receptor-1 (LOX-1) expression (Paigen et al., 1987). Endothelial NADPH oxidase is a major source of reactive oxygen species (ROS) in vascular endothelial cells, and atherogenic levels of LDL have been shown to induce a marked increase in NADPH oxidase-generated ROS (Rueckschloss et al., 2001). Therefore, the expression of oxidative stress mediators was investigated. Porphyromonas gingivalis significantly increased mRNA expression of LOX-1 (P < 0.01), NOX-4 (P < 0.05), p22 phox (P < 0.05) and inducible nitric oxide synthase (iNOS) (P < 0.05) compared with the sham-treated group (Fig. 4b). NOX-1 and NOX-2 mRNA levels showed a slight increase (P > 0.05; Fig. 4b). EGCG treatment significantly decreased the expression of LOX-1 (P < 0.01), NOX-1 (P < 0.05), NOX-2 (P < 0.05), NOX-4 (P < 0.05), p22 phox (P < 0.01) and iNOS (P < 0.01). However, expression of heme oxygenase-1 (HO-1), an antioxidant endoplasmic reticulum stress protein, was significantly increased by EGCG treatment (P < 0.05). In the liver, EGCG treatment reduced CRP and HMG CoA (3-hydroxy-3-methylglutaryl coenzyme A) expressions activated with P. gingivalis, although no significant differences were observed between P. gingivalis-challenged and PBS-inoculated groups (data not shown).
In this study, we found that infection of atherosclerosis-prone ApoE-KO mice with P. gingivalis accelerated atherosclerosis, which was associated with an increased plaque lipid content and elevated serum proinflammatory cytokines, including IL-1α, IL-1β, IL-6, MCP-1 and TNFα, as well as other inflammatory mediators, such as βFGF, CD40, M-CSF, MIP-3α, P-selectin, CRP, ox-LDL and LDL/VLDL cholesterol levels, compared with sham-treated mice. Furthermore, P. gingivalis challenge increased the expression of inflammatory- and oxidative stress-related mediators, such as CCL2, MMP-9, ICAM-1, HSP60, CD44, LOX-1, NOX-4, p22 phox and iNOS compared with the sham-treated group in the aorta. In contrast, consumption of EGCG decreased atherosclerotic lesions, proinflammatory cytokines, and inflammatory- and oxidative stress-related mediators in the serum and aorta induced by P. gingivalis.
Porphyromonas gingivalis induced a local inflammatory response resulting in oral bone destruction, characteristic of chronic periodontitis (Cutler et al., 1995). Chronic inflammation associated with P. gingivalis infection is initiated at the vascular endothelium (Moughal et al., 1992). Periodontal pathogens are translocated and released from the sulcus into the bloodstream, and such transient bacteremia may cause a secondary infection in distant tissues or organs, including arteries. The pathogenicity of P. gingivalis is attributed to various virulence factors, such as capsule, fimbriae, hemagglutinins, lipopolysaccharide (LPS) and potent proteolytic enzymes, such as gingipains (Holt et al., 1999). Porphyromonas gingivalis invasion is mediated through upregulation of adhesion molecules, such as ICAM-1, VCAM-1, and P- and E-selectins, only in the presence of fimbriae. The activation of adhesion molecules is required to bind leukocytes to endothelium, which initiates transmigration and atherogenesis (Khlgatian et al., 2002). Porphyromonas gingivalis LPS triggers inflammatory pathways through cytokine production, such as TNFα, IL-1 and PGE2. In contrast, gingipains mediate atherosclerosis by selective proteolysis of apolipoprotein B-100 and oxidative modification of LDL (Hashimoto et al., 2006; Bengtsson et al., 2008). Previous studies have suggested that oxidative damage to vascular cells and oxidation of LDL contributes to atherosclerosis (Stocker & Keaney, 2004). Therefore, P. gingivalis and its virulence factors play a role in inflammation and oxidative stress, resulting in atherosclerosis.
In contrast, EGCG treatment reduced inflammation, oxidative stress and atherosclerotic lesion development caused by P. gingivalis infection. Dietary polyphenols are bioactive molecules that can inhibit the development of atherosclerosis. Previous studies have shown that polyphenol-rich beverages, such as red wine (Waddington et al., 2004), dealcoholized red wine (Stocker & O'Halloran, 2004) and tea (Miura et al., 2001), can inhibit atherosclerosis in ApoE-KO mice.
Inflammation is a key process in atherosclerosis (Libby, 2002; Stocker & Keaney, 2004). Inflammatory mediators include cell adhesion molecules, cytokines, chemokines and growth factors that direct the recruitment of inflammatory cells, including monocyte/macrophages, neutrophils and T-lymphocytes, which modulate cellular signaling, cell growth, differentiation and a variety of other cellular processes. Our results indicated that EGCG inhibited the production and expression of proinflammatory cytokines and inflammatory mediators secreted or induced by P. gingivalis. As EGCG inhibits lesion formation by P. gingivalis, the anti-inflammatory property probably contributes to the antiatherogenic effect of EGCG. Hypercholesterolemia is a risk factor for atherosclerosis, and EGCG may protect against the disease by exerting hypocholesterolemic effects, although EGCG had no significant effect in this study on LDL/VLDL cholesterol concentrations after P. gingivalis infection.
Lipid peroxidative damage may be a critical step in the pathogenesis of atherosclerosis (Stocker & Keaney, 2004). oxLDL is important in the development of atherosclerosis, and the primary endothelial receptor of oxLDL, LOX-1, is responsible for the binding, internalization and degradation of oxLDL in endothelial cells. We observed increased oxLDL production in serum and LOX-1 expression in the aorta in P. gingivalis-challenged ApoE-KO mice, compared with sham-infected controls. Therefore, following the invasion of vascular endothelial cells, these cells recruit monocytes, which in the presence of elevated circulating lipids such as oxLDL form atheromas, a process that may be associated with P. gingivalis infection. The antioxidant activity of EGCG has led to the proposal that EGCG protection against atherosclerosis may involve its antioxidant properties (Hishikawa et al., 2005). EGCG inhibits atherosclerosis in ApoE-KO mice by reducing LDL susceptibility to oxidation (Hayek et al., 1997; Miura et al., 2001). Oxidative stress was also effectively attenuated by EGCG, as demonstrated by reduction of oxLDL concentration in serum and of LOX-1 and NADPH oxidase expression in the aorta.
While EGCG treatment reduced iNOS expression induced by P. gingivalis challenge, it increased HO-1 expression. NO inhibits the inflammatory proliferative reactions in vascular wall cells and exerts antithrombogenic and endothelial cell protective properties. However, high amounts of NO produced by iNOS, a reactive intermediate of NO containing a superoxide anion, are involved in proinflammatory reactions and tissue damage (Yamagishi & Matsui, 2011). iNOS is known to play a role in producing NO during inflammation, and thus contributes to the initiation and development of inflammatory cardiovascular disease, such as atherosclerosis. iNOS-KO mice decreased diet-induced atherosclerosis (Liu & Huang, 2008). In contrast, HO-1 has a protective role in inflammatory atherosclerotic disease (Stocker & Perrella, 2006), and inducers of HO-1 reduce lesion size in ApoE-KO mice (Wu et al., 2006), LDL receptor-deficient mice (Ishikawa et al., 2001a, b) and rabbits (Ishikawa et al., 2001a, b). Therefore, reduction in iNOS-mediated oxidative stress and inflammation, and increases in the levels of anti-oxidative enzyme by EGCG, may protect against lesion formation induced by P. gingivalis in ApoE-KO mice.
In conclusion, our results indicate that inflammation and oxidative modification play a role in atherosclerosis promotion in ApoE-KO mice after P. gingivalis infection, which can be prevented by treating with EGCG. Furthermore, our results indicated that the early activation of inflammatory and oxidative mediators in response to an infectious challenge may be associated with pathogen-accelerated atherosclerosis, suggesting that prior catechin consumption may be useful for the prevention of pathogen-accelerated atherosclerosis.
This study was supported by Grants-in-Aid for Scientific Research (22390398) from the Japan Society for the Promotion of Science, and by the ‘Strategic research Base Development’ Program (Japan[MEXT], 2010-2014[S1001024]) for Private Universities of the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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