ABSTRACT
Marfan syndrome is an autosomal dominant disease characterized by aneurysm and dilatation of the aortic root, tall stature, and ectopia lentis. These manifestations reflect excessive signaling of transforming growth factor beta (TGF-β). Moreover, cases are frequently associated with severe periodontitis, which is a chronic inflammation of the gingiva, periodontal ligament, and alveolar bone. Recently, angiotensin II receptor blockers (ARBs) were discovered to be an effective drug class that can prevent aortic aneurysm and dilation in Marfan syndrome by inhibiting TGF-β signaling. To investigate the effect of ARB on the progression of periodontitis, the application of a potent ARB, telmisartan, was examined in a mouse model of Marfan syndrome (MgΔ). Six-week-old male heterozygous MgΔ and wild-type mice were challenged with Porphyromonas gingivalis, which causes chronic periodontitis, with and without telmisartan application. After infection, alveolar bone resorption was measured by micro-computed tomography (μCT), and inflammatory cytokine levels were examined. Infection of Porphyromonas gingivalis induced alveolar bone resorption in both MgΔ and wild-type mice. The amount of resorption was significantly larger in the former than the latter. Immunoarray and enzyme-linked immunosorbent assay (ELISA) analyses demonstrated that interleukin-17 (IL-17) and tumor necrosis factor alpha (TNF-α) levels were significantly higher in infected MgΔ mice than infected wild-type mice. Telmisartan treatment significantly suppressed the alveolar bone resorption of infected MgΔ mice. Telmisartan also significantly decreased levels of TGF-β, IL-17, and TNF-α in infected MgΔ mice to levels seen in infected wild-type mice. This study suggests that ARB can prevent the severe periodontitis frequently seen in Marfan syndrome.
INTRODUCTION
Periodontitis is an inflammatory condition affecting the gingiva, periodontal ligament (PDL), and alveolar bone (1). Epidemiological studies indicate that approximately 15% of the adult population has an advanced form of periodontitis, causing multiple negative impacts on the quality of life (2, 3). Consequences of advanced periodontitis include negative esthetics and functional problems in occlusion, chewing, and speaking, and it can eventually result in tooth loss (4, 5). Porphyromonas gingivalis is an anaerobic, asaccharolytic Gram-negative bacterium that is strongly associated with chronic periodontitis showing alveolar bone loss (6).
Marfan syndrome is a systemic connective tissue disorder that affects about 1 in 5,000 individuals (7). This syndrome is caused in an autosomally dominant manner, and the responsible gene is FBN1, which encodes the extracellular matrix protein fibrillin-1 (7). FBN1 mutations lead to defects in multiple organs, including skeletal, cardiovascular, and ocular systems (8). The most serious problems occur in the cardiovascular system, such as mitral valve prolapse, mitral regurgitation, aneurysm, and dilation of the aortic root, which cause a short life expectancy in affected patients (9). Fibrillin-1 controls the bioavailability of endogenous transforming growth factor beta (TGF-β) by targeting the respective complexes to the extracellular cell matrix (10). Animal (11) and human (12) studies indicate that TGF-β signaling drives aneurysm progression in the aorta (13). In addition to these conditions, patients with Marfan syndrome are susceptible to advanced periodontitis (14, 15). Also, approximately 66% of patients with Marfan syndrome exhibited serious gingivitis compared with 11% of healthy volunteers (16). Since most Marfan syndrome patients have cardiovascular problems, the surgical replacement of aortic and mitral valve and aortic roots is often required (9). Because of this surgical replacement, it is essential to prevent dental focal infection, such as infectious endocarditis caused by the periodontitis and caries.
Recently, angiotensin II receptor blockers (ARBs) have been reported to inhibit the progression of aortic root dilation by suppressing TGF-β signaling (12, 17). Application of one ARB, losartan, has provided a remarkable benefit to Marfan syndrome patients by preventing cardiovascular conditions. From a dental point of view, it is essential to determine the effect of ARBs on the progression of periodontitis. Among ARBs, telmisartan has the longest half-life of any ARB and has 3,000 times higher binding affinity to AT1 than to AT2 (18–20). In this study, we took advantage of the drug action of telmisartan and examined its effect on the experimental periodontitis model. The results suggest that the ARBs can prevent the severe periodontitis frequently seen in Marfan syndrome.
MATERIALS AND METHODS
Animals.Six-week-old male MgΔ mice (21) were used in this study. The study was carried out in the specific pathogen-free unit of the animal facility in Tokyo Medical and Dental University. Mice were maintained in a 12-h light/dark cycle and received distilled water and food ad libitum. Homozygous MgΔ mice could survive for only up to 2 weeks, as previously reported (21). Therefore, only heterozygous MgΔ (MgΔ+) mice and wild-type (WT) mice were used as shown in this study. The heterozygous MgΔ mice were morphologically and histologically indistinguishable from the WT mice, as previously reported (21). Both groups of mice were identified by Southern analysis of BamHI-digested DNA, as previously reported (21). WT (n = 40) and heterozygous MgΔ (n = 40) mice were divided into groups of 8 mice as follows: mice (i) with or (ii) without Porphyromonas gingivalis infection were sacrificed 2 weeks after the infection; mice (iii) with or (iv) without bacterial infection were sacrificed 8 weeks after the infection; and (v) mice with bacterial infection and treated with telmisartan were sacrificed 8 weeks after the infection. Experimental procedures were approved by the Experimental Animal Committee of Tokyo Medical and Dental University (no. 100073).
Porphyromonas gingivalis infection.Porphyromonas gingivalis ATCC 33277 strains were grown on blood agar plates in an anaerobic chamber with 85% N2, 5% H2, and 10% CO2. After incubation at 37°C for 2 to 3 days, the bacterial cells were inoculated into Wilkins media (Oxoid) for 2 days under the same conditions, as previously reported (22). The bacteria were washed three times with sterile phosphate-buffered saline (PBS) before use. Infection was carried out as described by Baker et al. (23) and Lalla et al. (24). In brief, all animals were treated with antibiotics for 10 days (sulfamethoxazole-trimethoprim at 0.08% and 0.016%, respectively, in drinking water ad libitum). Three days following the withdrawal of antibiotics (day 14), mice were infected with the Porphyromonas gingivalis strain (109 CFU in 0.2 ml of PBS and 2% carboxymethylcellulose) or treated with vehicle only. The infection was performed by gavage on the maxillary gingiva bilaterally from the buccal side three times, once every other day. Telmisartan (5.0 mg/kg of body weight/day) was prepared in distilled water, and this solution was supplied as a drinking water for mice, as previously described (25, 26). Telmisartan was applied immediately after the infection, and the solution was changed every day. At 14 days (2 weeks) and 56 days (8 weeks) after the last infection, mice were anesthetized with 50 mg/ml sodium pentobarbital and exsanguinated by the heart puncture technique for blood collection. After the blood collection, maxillae were dissected. The same sample were used both for micro-computed tomography (μCT) and immunostaining.
Number of Porphyromonas gingivalis bacteria in infected mice.At 14 days after the Porphyromonas gingivalis infection, subgingival plaque samples of the left maxillary first molars were collected from wild-type and MgΔ mice (n = 5 for each genotype), as previously reported (27). The homogenized bacterial suspensions were prepared from plaque samples. A 100-μl volume of bacterial suspensions was streaked onto blood agar plated in an anaerobic chamber with 85% N2, 5% H2, and 10% CO2 for a week, and the CFU of cultivable Porphyromonas gingivalis was examined. The identification of Porphyromonas gingivalis was performed on the basis of colony morphology and by use of an API 20A system (bioMérieux, Inc.) (27).
Cytokine antibody array.A RayBio Mouse cytokine antibody array kit (RayBiotech) was used to detect active cytokine levels in mouse serum according to the manufacturer's instructions.
ELISA.Mice serum was extracted, and a TGF-β1 enzyme-linked immunosorbent assay (ELISA) kit (Promega), a mouse TNF-α ELISA kit (Invitrogen), and a mouse IL-17 ELISA kit (BenderMed) were used according to the instructions of the manufacturers to measure serum cytokine levels.
Immunostaining.After μCT images were taken, dissected maxillae were decalcified in Morse solution (10% sodium citrate, 20% formic acid; Wako) for 1 week and dehydrated and embedded in paraffin (Wako). Serial sagittal sections (5 μm thick) of the maxillary molars with surrounding tissue were prepared for the immunostaining. Immunostaining of cathepsin K was performed by using the rabbit polyclonal antibody against rat Cathepsin K (Abcam) and the rabbit ABC staining system (Santa Cruz Biotechnology), according to the instructions of the manufacturers.
To compare the osteoclast surface/bone surfaces (Oc.S/BS) in WT and MgΔ mice, cross sections (5 μm thick) of the mesial sides of the mesial roots were prepared from the maxillary first molars of each mouse. Eight mice of each genotype from each group were used. Two sections were prepared at identical levels from each side of the root and immunostained using the cathepsin K antibody, known as a marker of osteoclasts (28). Stained cells were counted as osteoclasts in a square (0.8 mm by 0.8 mm) adjacent to the tooth root in every section. The average value of Oc.S/BS from two sections was represented for each mouse. ImageJ software (ver. 1.4.3; National Institutes of Health) was used for measuring Oc.S and BS.
μCT analysis.Before decalcification, hemimaxillae were collected and prepared for bone loss measurements by μCT. Eight mice were used for each group. μCT images were taken using a benchtop microfocus X-ray CT system (InspeXio SMX-90CT; Shimadzu, Japan). Three-dimensional images were analyzed using ImageJ software.
To evaluate Oc.S/BS in WT and MgΔ mice, all specimens were placed in the same three-dimensional position. This was done reproducibly using ImageJ software. Both the right and left sides were measured in each mouse, and the average value was representative of one mouse. The total areas between the alveolar bone crest and the cement-enamel junction were measured and compared between groups.
Statistical analysis.The Student t test was used to compare the differences in CFU values of plaque samples from Porphyromonas gingivalis-infected wild-type and MgΔ mice. Analysis of variance (ANOVA) and the Tukey test were used to compare the differences between groups for the alveolar bone loss, levels of IL-17, TNF-α, and active TGF-β, and Oc.S/BS values. Statistical analysis was performed using SPSS version 13 (IBM). P values of less than 0.05 were considered significant.
RESULTS
Porphyromonas gingivalis infection increased alveolar bone resorption in WT and MgΔ mice.In order to examine alveolar bone resorption by infection in WT and MgΔ mice, periodontal tissues were challenged with Porphyromonas gingivalis. μCT images showed that apparent alveolar bone resorption was seen in both types of mice following infection (Fig. 1). In comparison to WT mice, increased resorption and a larger amount of tooth root protruding from the alveolar socket were observed in MgΔ mice (Fig. 1B and D). Quantitative analysis demonstrated that Porphyromonas gingivalis infection significantly increased bone resorption in uninfected WT and MgΔ mice by 23% and 45%, respectively (Fig. 1E). Compared to those two groups of infected mice, there was a significant higher value in infected MgΔ than infected WT mice. However, there was no significant difference between two uninfected groups of mice.
(A to D) μCT images of alveolar bone resorption in wild-type (WT) (A and B) and heterozygous MgΔ (MgΔ/+) (C and D) mice with and without Porphyromonas gingivalis infection. Data for infected mice are shown in panels B and D; data for uninfected mice are shown in panels A and C. (E) Alveolar bone loss was calculated and is represented as described in Materials and Methods. Each value is represented per 104 μm2. Data represent the mean values determined for eight mice, and error bars indicate standard errors of the means. Data from WT and MgΔ/+ mice are represented by open and stippled columns, respectively. a, not significantly different from uninfected WT mice; b, significantly different (P < 0.01) from uninfected WT mice; c, significantly different (P < 0.01) from uninfected MgΔ/+ mice; d, significantly different (P < 0.05) from infected WT mice.
Inflammatory cytokine levels in WT and heterozygous MgΔ mice infected with Porphyromonas gingivalis.In order to examine the inflammatory status after Porphyromonas gingivalis infection, cytokine antibody immunoarray analysis was performed on serum from WT and MgΔ mice 8 weeks after the infection. A total of 32 cytokines were examined, and the 12 cytokines were detected (Table 1). Serum levels of cutaneous T-cell-attracting chemokine (CTAK), eotaxin, granulocyte colony-stimulating factor (G-CSF), interleukin-2 (IL-2), IL-3, IL-4, IL-12, and IL-13 were higher in uninfected MgΔ mice than in uninfected WT mice. Serum levels of IL-2, IL-3, IL-4, IL-12, IL-13, and soluble tumor necrosis factor type I receptor (sTNFR1) were higher in infected WT mice than in uninfected WT mice. Serum levels of CTAK, eotaxin, IL-2, IL-3, IL-12, IL-13, IL-17, macrophage inflammatory protein-2 (MIP-2), tumor necrosis factor alpha (TNF-α), and sTNFR1 were higher in infected MgΔ mice than in uninfected MgΔ mice. Levels of CTAK, eotaxin, G-CSF, IL-2, IL-3, IL-13, IL-17, MIP-2, and TNF-α were all higher in infected MgΔ mice than in infected WT mice. Considering these differences, the levels of IL-17 and TNF α were specifically higher in infected MgΔ mice than in the other three groups of mice.
Cytokine immunoarray of wild-type and heterozygous MgΔ mice with and without Porphyromonas gingivalis infectiona
Next, serum levels of IL-17 (Fig. 2A and B) and TNF-α (Fig. 2C) were measured in uninfected and infected WT and MgΔ mice. IL-17 levels were significantly higher in infected MgΔ mice than in uninfected MgΔ and infected WT mice at 2 weeks after infection (Fig. 2A). At 8 weeks after infection, uninfected MgΔ and infected WT mice showed significant higher IL-17 levels than uninfected WT mice (Fig. 2B). Serum IL-17 levels were significantly higher in infected MgΔ mice than in uninfected MgΔ and infected WT mice. TNF-α levels could not be detected at 2 weeks after infection (data not shown). At 8 weeks after infection, TNF-α levels of uninfected MgΔ mice or infected WT mice were not significantly different from that of uninfected WT mice (Fig. 2C). However, TNF-α levels in infected MgΔ mice were significantly higher than in uninfected MgΔ and infected WT mice.
Interleukin-17 (IL-17) (A and B) and tumor necrosis factor alpha (TNF-α) (C) in the serum of heterozygous MgΔ (MgΔ/+) and wild-type (WT) mice after the infection with Porphyromonas gingivalis. Values from 2 weeks (A) and 8 weeks (B and C) after the infection are shown. Values of MgΔ/+ (black columns) and WT (open columns) mice with (denoted as Infection) and without Porphyromonas gingivalis infection are shown. Data are represented in picograms per milliliter. Data represent the mean values determined for eight mice, and error bars indicate the standard errors of the means. a, not significantly different from IL-17 levels of uninfected WT mice; b, not significantly different from IL-17 levels of uninfected WT mice; c, significantly different (P < 0.01) from IL-17 levels of uninfected MgΔ/+ mice; d, significantly different (P < 0.01) from IL-17 levels of infected WT mice; e, significantly different (P < 0.05) from IL-17 levels of uninfected WT mice; f, significantly different (P < 0.05) from IL-17 levels of uninfected WT mice; g, significantly different (P < 0.01) from IL-17 levels of uninfected MgΔ/+ mice; h, significantly different (P < 0.01) from IL-17 levels of infected WT mice; i, not significantly different from TNF-α levels of uninfected WT mice; j, not significantly different from TNF-α levels of uninfected WT mice; k, significantly different (P < 0.05) from TNF-α levels of uninfected MgΔ/+ mice; l, significantly different (P < 0.01) from TNF-α levels of infected WT mice.
At 14 days after the infection, the numbers of cultivable Porphyromonas gingivalis bacteria from wild-type and MgΔ mice were counted; the results were 3.64 ± 0.02 (log CFU) and 3.49 ± 0.02 (log CFU) in wild-type and MgΔ mice, respectively. There was no significant difference in values between two genotypes.
ARB reduced alveolar bone resorption and IL-17 and TNF-α levels.To examine the effect of ARB on alveolar bone resorption, telmisartan was applied to infected WT and MgΔ mice (Fig. 3). Increased alveolar bone resorption due to the infection was significantly reduced in MgΔ mice but not in WT mice treated with telmisartan (Fig. 3). Next, serum levels of IL-17 and TNF-α were measured in infected WT and MgΔ mice with and without telmisartan application (Fig. 4). Telmisartan application reduced serum levels of IL-17 (Fig. 4A) and TNF-α (Fig. 4B) in infected MgΔ mice to the levels observed in infected WT mice without telmisartan application. Telmisartan (10 μM) had no effect on the viability of the culture of Porphyromonas gingivalis (data not shown).
Alveolar bone resorption of Porphyromonas gingivalis-infected heterozygous MgΔ (MgΔ/+) and wild-type (WT) mice with (denoted as Telmisartan) and without the application of telmisartan. Alveolar bone loss was calculated and is represented as described in Materials and Methods. Each value is represented as 104 per μm2. Data of WT and MgΔ/+ mice are represented by gray and stippled columns, respectively. Data represent the mean values determined for eight mice, and error bars indicated the standard errors of the means. a, significantly different (P < 0.05) from infected WT mice without the telmisartan application; b, not significantly different from infected WT mice without the telmisartan application; c; significantly different (P < 0.05) from infected MgΔ/+ mice without the telmisartan application; d, not significantly different from infected WT mice with the application of telmisartan.
Interleukin-17 (IL-17) (A) and tumor necrosis factor alpha (TNF-α) (B) in the serum of infected heterozygous MgΔ (MgΔ/+) and wild-type (WT) mice with (denoted as Telmisartan) and without the application of telmisartan. Each column represents the value (in picograms per milliliter) at 8 weeks after Porphyromonas gingivalis infection. Values for WT (open columns) and MgΔ/+ (black columns) mice with Porphyromonas gingivalis infection are shown. Data represent the mean values determined for eight mice, and error bars indicate the standard errors of the means. a, significantly different (P < 0.05) from IL-17 levels of infected WT mice without telmisartan application; b, not significantly different from IL-17 levels of infected WT mice without telmisartan application; c, significantly different (P < 0.01) from IL-17 levels of infected MgΔ/+ mice without telmisartan application; d, not significantly different from IL-17 levels of infected WT mice with the application of telmisartan, e, significantly different (P < 0.05) from TNF-α levels of infected WT mice without telmisartan application; f, not significantly different from TNF-α level of infected WT mice without telmisartan application; g, significantly different (P < 0.01) from TNF-α levels of infected MgΔ/+ mice without telmisartan application; h, not significantly different from TNF-α level of infected WT mice with the application of telmisartan.
ARB reduced TGF-β levels.It was previously reported that active TGF-β levels were higher in MgΔ mice than in WT mice (21). This is consistent with our study results showing the significant higher level of active TGF-β level in uninfected MgΔ mice than in uninfected WT mice (Fig. 5). Interestingly, active TGF-β levels were significantly higher in Porphyromonas gingivalis-infected MgΔ mice than in uninfected MgΔ mice and infected WT mice (Fig. 5). Application of telmisartan significantly reduced the active TGF-β level in infected MgΔ mice to that in infected WT mice. These results suggest that TGF-β is related to the pathogenesis of increased alveolar resorption in infected MgΔ mice.
Active form of TGF-β in the serum of infected heterozygous MgΔ (MgΔ/+) and wild-type (WT) mice with and without Porphyromonas gingivalis infection (denoted as Infection). Infected mice were treated with (denoted as Telmisartan) and without telmisartan. Two columns represent values (in picograms per milliliter) without the infection, and four columns represent values (in picograms per milliliter) at 8 weeks after the Porphyromonas gingivalis infection (denoted as Infection). Values of MgΔ/+ (black columns) and WT (open columns) mice with Porphyromonas gingivalis infection are shown. Data represent the mean values determined for eight mice, and error bars indicated standard errors of the means. a, significantly different (P < 0.01) from the TGF-β level of uninfected WT mice without telmisartan application; b, not significantly different from the TGF-β level of uninfected WT mice without telmisartan application; c, significantly different (P < 0.01) from the TGF-β level of uninfected MgΔ/+ mice without telmisartan application; d, significantly different (P < 0.01) from the TGF-β level of infected WT mice without telmisartan application; e, not significantly different from the TGF-β level of infected WT mice without the application of telmisartan; f, significantly different (P < 0.01) from the TGF-β level of infected MgΔ/+ mice without the application of telmisartan; g, not significantly different from the TGF-β level of infected WT mice with the application of telmisartan,.
ARB inhibited osteoclast appearance.The appearance of osteoclasts was examined by immunostaining of cathepsin K (Fig. 6). Porphyromonas gingivalis infection increased the osteoclast appearance in both WT and MgΔ/+ mice (Fig. 6A, B, D, E and G). Infected MgΔ/+ mice showed a significant higher Oc.S/BS percentage than infected WT mice (Fig. 6B, E and G). Application of telmisartan reduced the Oc.S/BS percentage in infected MgΔ mice (Fig. 6F and G) to the level in infected WT mice (Fig. 6C and G).
(A to F) Immunohistochemical localization of cathepsin K in the alveolar bone of heterozygous MgΔ (MgΔ/+) (D to F) and wild-type (WT) (A to C) mice. Sections from mice with (B, C, E, and F) and without (A and D) Porphyromonas gingivalis infection are shown. (C and F) Application of telmisartan was performed in mice. Arrowheads, cathepsin K-positive cells; ab, alveolar bone; de, dentin. The areas represented by dotted lines represent periodontal ligaments. (G) Osteoclast surface [Os.S/BS (%)] of WT and MgΔ/+ mice. The evaluation of Os.S/BS was described in Materials and Methods. Values determined for WT (open columns) and MgΔ/+ (black columns) mice with Porphyromonas gingivalis infection and/or telmisartan application are shown. Data represent the mean values determined for eight mice, and error bars indicate the standard errors of the means. a, not significantly different from uninfected WT mice without telmisartan application; b, significantly different (P < 0.01) from uninfected WT mice without telmisartan application; c, significantly different (P < 0.01) from uninfected MgΔ/+ mice without telmisartan application; d, significantly different (P < 0.01) from infected WT mice without telmisartan application; e, not significantly different from infected WT mice without telmisartan application; f, significantly different (P < 0.01) from infected MgΔ/+ mice without telmisartan application; g, not significantly different from the infected WT mice with the application of telmisartan.
DISCUSSION
Periodontitis is initiated by chronic inflammation and immune reactions to bacterial pathogens (29). This is characterized by bone loss and the destruction of gingiva and PDL. It is well established that several bacteria play important roles in the pathogenesis of periodontitis. Porphyromonas gingivalis, playing a central role in pathogenesis of periodontitis (30), was used in this study. Many animal models of periodontitis have been reported so far (31–35). Among them, a murine model using gavage of Porphyromonas gingivalis has been widely used in this field (31, 35). This is because the genetics and immune system are well characterized in mice (36), and the periodontal anatomy and histopathology of periodontitis are similar to those in humans (33). Using this system, alveolar bone resorption was reproducibly induced in both WT and MgΔ mice by gavage with Porphyromonas gingivalis (Fig. 1). The resorption was significantly greater in MgΔ mice than in WT mice. Examining blood samples from infected heterozygous MgΔ mice, there was a dramatic and significant elevation in levels of IL-17 and TNF-α (Fig. 2) compared with infected WT mice. These two cytokines are related to immune-mediated diseases such as rheumatoid arthritis, allergy, and also periodontitis (37, 38). Both cytokines are reported to induce osteoclastogenesis (39, 40). Considering the increased values of Oc.S/BS in infected heterozygous MgΔ mice (Fig. 6), the increased bone resorption in the experimental periodontitis can be explained by elevated serum IL-17 and TNF-α levels.
Telmisartan is an ARB used in the management of hypertension (41), and it is anticipated to be an effective drug for the vascular therapy of Marfan syndrome (17). It has a high affinity for angiotensin II receptor type 1 (AT1), with a binding affinity 3,000 times higher for AT1 than receptor type 2 (AT2) (20). In Marfan syndrome, symptoms such as aneurysms and emphysema reflect excessive TGF-β signaling (13). Clinical (12, 17) and animal (19, 42) studies indicated that the ARBs telmisartan and losartan protected against the progression of aortic aneurysms. Telmisartan was reported to decrease TGF-β mRNA levels (43). In addition, ARBs can attenuate canonical SMAD and noncanonical extracellular signal-regulated kinase (ERK) with respect to TGF-β signaling (42). In this study, severe alveolar bone resorption induced by Porphyromonas gingivalis infection was associated with a significant elevation of active TGF-β levels in the serum of MgΔ mice (Fig. 5). Telmisartan treatment significantly decreased the active TGF-β level in MgΔ mice (Fig. 5). The treatment also decreased alveolar bone resorption (Fig. 3) and the OcS/BS percentage in MgΔ mice (Fig. 6). All these findings strongly suggest that TGF-β signaling is responsible for the severe periodontitis induced by Porphyromonas gingivalis infection in MgΔ mice. Further clarification of whether canonical SMAD or noncanonical ERK is the intracellular target of telmisartan in the present mouse periodontitis model is required.
In human studies, it was reported that TNF-α is expressed in the gingiva of periodontal tissues and that its expression increased after Porphyromonas gingivalis infection (44). The amount of IL-17 in gingiva in cases of chronic human periodontitis was also higher than in healthy gingiva (45). TGF-β signaling is activated in periodontitis-affected gingiva (46). These studies are consistent with the present findings that the levels of TNF-α, IL-17, and activated TGF-β observed in infected MgΔ mice were increased in the mice with severe periodontitis. The exact relationship between these cytokines (TNF-α and IL-17) and TGF-β signal is not known. To clarify the pharmacologic effect of telmisartan, it is necessary to understand the mechanism by which TGF-β regulates TNF-α and IL-17 expression and/or signaling.
ACKNOWLEDGMENTS
This work was supported in part by Grants-in-aid (21390546 and 24659916) for the Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
We are grateful to Francesco Ramirez (Mount Sinai School of Medicine) for providing MgΔ mice. We appreciate the late Yusuke Takahashi, Kanagawa Dental College, for his technical advice. We appreciate Masahiro Saito, Tokyo University of Science, for his helpful suggestion.
FOOTNOTES
- Received 21 August 2012.
- Returned for modification 9 September 2012.
- Accepted 18 October 2012.
- Accepted manuscript posted online 31 October 2012.
- Copyright © 2013, American Society for Microbiology. All Rights Reserved.
