[Show abstract][Hide abstract] ABSTRACT: Epidemiological studies have revealed a link between dental infection and preterm birth or low birth weight (PTB/LBW), however, the underlying mechanisms remain unclear. Progress in understanding the associated mechanisms has been limited in part by lack of an animal model for chronic infection-induced PTB/LBW, mimicking pregnancy under conditions of periodontitis. We aimed to establish a mouse model of chronic periodontitis in order to investigate the link between periodontitis and PTB/LBW.
To establish chronic inflammation beginning with dental infection, we surgically opened mouse (female, 8 weeks old) 1st molar pulp chambers and directly infected with w83 strain Porphyromonas gingivalis (P.g.), a keystone periodontal pathogen. Mating was initiated at 6 wks post-infection, by which time dental granuloma tissue had developed and live P.g. was cultured from extracted tooth root, which serves as a persistent source of P.g. The gestational day (gd) and birth weight were recorded during for P.g.-infected and control mice, and serum and placental tissues were collected at gd 15 to evaluate the systemic and local conditions during pregnancy.
Dental infection with P.g. significantly increased circulating TNF-α (2.5-fold), IL-17 (2-fold), IL-6 (2-fold) and IL-1β (2-fold). The P.g.-infected group delivered at gd 18.25 vs. gd 20.45 in the non-infected control (NC) group (p < 0.01), and pups exhibited LBW compared to controls (p < 0.01). P.g. was localized to placental tissues by immunohistochemistry and PCR, and defects in placental tissues of P.g. infected mice included premature rupture of membrane, placental detachment, degenerative changes in trophoblasts and endothelial cells, including necrotic areas. P.g. infection caused significantly increased numbers of polymorphonuclear leukocytes (PMNLs) and macrophages in placental tissues, associated with increased local expression of pro-inflammatory mediators including TNF-α and COX-2. Further placental tissue damage was indicated in P.g. infected mice by decreased CD-31 in endothelial cells, increased expression of 8OHdG, an indicator of oxidative DNA damage, and cleaved caspase-3, a marker of apoptosis. In vitro, P.g. lipopolysaccharide significantly increased expression of COX-2, IL-8 and TNF-α, in HTR-8 trophoblasts in an NF-κB-dependent fashion.
Our novel mouse model supports previous epidemiological studies signifying dental infection as predisposing factor for PTB/LBW. We demonstrate PTB and LBW in infected mice, translocation of P.g to placental tissues, increased circulating and local pro-inflammatory markers, and the capability of P.g. LPS to directly induce cytokine production in trophoblasts, in vitro. These findings further underscore the importance of local and systemic infections and inflammation during pregnancy and suggest that prevention and/or elimination of dental infections such as marginal or periapical periodontitis before pregnancy may have a beneficial effect on PTB/LBW.
PLoS ONE 08/2015; 10(8):e0137249. DOI:10.1371/journal.pone.0137249 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Peripheral ameloblastoma (PA), a rare and unusual variant of odontogenic tumors, comprises about 1% of all ameloblastomas. PA is an exophytic growth localized to the soft tissues overlying the tooth-bearing areas of the jaws, and the initial diagnosis is often fibrous epulis. PA with histologically low-grade malignant features is extremely rare. We report a case of peripheral ameloblastoma with histologically low-grade malignant features in a 69-year-old woman that presented with a hemorrhage from a tumor on the right buccal mucosa. The tumor was surgically removed by blunt dissection, with no evidence of recurrence after two years and six months. After the case presentation, microscopic and genetic findings are discussed.
International journal of clinical and experimental pathology 05/2015; 8(2):2085-9. · 1.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nonalcoholic steatohepatitis (NASH) is associated with increased risks of developing lifestyle-related diseases including type 2 diabetes, cardiovascular disease. While the two-hit hypothesis and recently, multiple parallel hits hypothesis of NASH pathogenesis was proposed, further details have not been understood. Recently, dental infection of Porphyromonas gingivalis (P. gingivalis) has been reported as a critical risk factor for NASH progression, which acts as multiple parallel hits to induce inflammation and fibrogenic responses in steatosis. We describe here that 54-year-old woman died on sepsis and diagnosed as NASH. Briefly, her body mass index (BMI) at 35 years old was 25.6 Kg/m(2) , but she became obese owing to her withdrawal at home since 45 years old. Severe obesity continued over 19 years without diabetes mellitus. She was admitted to our hospital due to a sudden disturbance of consciousness. On admission, BMI was 48.5 Kg/m(2) . Computed tomography revealed cirrhotic liver with massive ascites, and laboratory data indicated increased inflammatory responses, renal failure, and C grade Child-Pugh classification, suggesting the diagnosis of sepsis. Also severe periodontal disease was present, because the patient's front teeth fell easily during intubation. Although the focus of infection was not specified, the oral flora Parvimonus micra, one of periodontal pathogens, was detected in venous blood. Despite of intensive cares including artificial respiration management and continuous hemodiafiltration, she died on the 43rd day after admission. Surprisingly, P. gingivalis was detected in her hepatocytes. This case might represent the significance of P. gingivalis in the progress to cirrhosis in NASH patients.
This article is protected by copyright. All rights reserved.
Hepatology Research 05/2015; DOI:10.1111/hepr.12528 · 2.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective
Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration. However, the effects of EMD on gingival epithelial cells during regeneration of periodontal tissues are unclear. In this in vitro study, we purified ameloblastin from EMD and investigated its biological effects on epithelial cells.
Material and Methods
Bioactive fractions were purified from EMD by reversed-phase high-performance liquid chromatography using hydrophobic support with a C18 column. The mouse gingival epithelial cell line GE-1 and human oral squamous cell carcinoma line SCC-25 were treated with purified EMD fraction, and cell survival was assessed with a WST-1 assay. To identify the proteins in bioactive fractions of EMD, we used proteome analysis with two-dimensional gel electrophoresis followed by identification with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.
Purified fractions from EMD suppressed proliferation of GE-1 and SCC-25. LC-MS/MS revealed that ameloblastin in EMD is the component responsible for inhibiting epithelial cell proliferation. The inhibitory effect of ameloblastin on the proliferation of GE-1 and SCC-25 was confirmed using recombinant protein.
The inhibitory effects of EMD on epithelial cell proliferation are caused by the biological activities of ameloblastin, which suggests that ameloblastin is involved in regulating epithelial downgrowth in periodontal tissues.
[Show abstract][Hide abstract] ABSTRACT: Dentinoid is an integral part of some odontogenic tumors. This article describes the clinico-pathological features of three cases of odontogenic carcinomas with dentinoid (OCD). A comparison of these with previously reported cases of dentinoid-producing epithelial odontogenic tumors allowed us to identify another six cases that may be considered as examples of OCD. Six cases occurred in the mandible and three in the maxilla, all developing behind the canines. There was no sex predilection (five men and four women; age range 14-61 years, mean 38.1). Pain or discomfort was mentioned in five cases, four of which showed tooth resorption. All cases appeared initially as well-defined radiolucencies, five of which showed variable amounts of calcified material. Recurrences were recorded in three instances, but no evidence of metastasis has been found. Seven cases were composed predominantly or entirely of clear cells, usually with minimal cellular atypia and variable mitotic activity; however, in all cases there was evidence of tumor infiltration into adjacent tissues, including the presence of perineural invasion in two tumors. Those cases in which no reference was made to the presence of clear cells exhibited evident mitotic activity and cellular pleomorphism. The epithelium in OCD does not produce buds or enamel organ-like structures such as those found in ameloblastic fibro-dentinoma and this tumor does not contain a mesenchyme-like connective tissue resembling dental papilla as observed in several mixed odontogenic tumors. Based on the existing data and the present series of cases, OCD appears to represent a distinct entity.
Head and Neck Pathology 12/2014; 8(4):421-31. DOI:10.1007/s12105-014-0586-9
[Show abstract][Hide abstract] ABSTRACT: Background
A number of studies have revealed a link between chronic periodontitis and cardiovascular disease in obese patients. However, there is little information about the influence of periodontitis-associated bacteria, Porphyromonas gingivalis (Pg), on pathogenesis of atherosclerosis in obesity.
In vivo experiment: C57BL/6J mice were fed with a high-fat diet (HFD) or normal chow diet (CD), as a control. Pg was infected from the pulp chamber. At 6 weeks post-infection, histological and immunohistochemical analysis of aortal tissues was performed. In vitro experiment: hTERT-immortalized human umbilical vein endothelial cells (HuhT1) were used to assess the effect of Pg/Pg-LPS on free fatty acid (FFA) induced endothelial cells apoptosis and regulation of cytokine gene expression.
Weaker staining of CD31 and increased numbers of TUNEL positive cells in aortal tissue of HFD mice indicated endothelial injury. Pg infection exacerbated the endothelial injury. Immunohistochemically, Pg was detected deep in the smooth muscle of the aorta, and the number of Pg cells in the aortal wall was higher in HFD mice than in CD mice. Moreover, in vitro, FFA treatment induced apoptosis in HuhT1 cells and exposure to Pg-LPS increased this effect. In addition, Pg and Pg-LPS both attenuated cytokine production in HuhT1 cells stimulated by palmitate.
Dental infection of Pg may contribute to pathogenesis of atherosclerosis by accelerating FFA-induced endothelial injury.
PLoS ONE 10/2014; 9(10):e110519. DOI:10.1371/journal.pone.0110519 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
F-spondin, known to be a secreted neuronal glycoprotein, is highly expressed on the tooth root surface. The authors previously reported that F-spondin is one of the specific markers of cementoblasts in periodontal tissue. In chronic periodontitis, significant cemental resorption rarely occurs on the root side, although alveolar bone resorption by osteoclasts is one of the major pathologic changes. Thus, it was hypothesized that secretory F-spondin from cementoblasts might be involved in differentiation of clastic cells on the root surface. The authors studied effects of secretory F-spondin from F-spondin-expressing cells and its pathway on receptor activator of nuclear factor-κB ligand (RANKL)-mediated differentiation of clastic cells.
Osteoclast precursors were used in this study. With a chamber assay, the authors examined effects of secretory molecules from F-spondin-expressing cells of transgenic mice on RANKL-induced clastic cell differentiation.
Secretory molecules from F-spondin-overexpressing cells significantly inhibited the RANKL-mediated tartrate-resistant acid phosphatase (TRAP)-positive cells from primary progenitor cells with the chamber system. F-spondin suppressed RANKL-mediated nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1); TRAP; cathepsin K; and dendritic cell-specific transmembrane protein (DC-STAMP) expression in the cells. The suppressive effect of F-spondin on RANKL-induced differentiation of clastic cells was partially blocked by knockdown of low-density lipoprotein receptor-related protein 8 (LRP8).
These findings indicate that secretory factors from F-spondin-expressing cells, including F-spondin, downregulate differentiation of clastic precursors. Moreover, F-spondin inhibits RANKL-mediated differentiation of clastic cells partially via LRP8. It is suggested that secretory F-spondin may act protectively from cemental resorption partially via LRP8 in periodontal tissue.
Journal of Periodontology 10/2014; 86(3):1-25. DOI:10.1902/jop.2014.140419 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial growth factor (VEGF) plays a critical role in tumor angiogenesis. VEGF and its receptor FLT-1 signaling may contribute to cell migration of macrophages, osteoclasts and some tumor cells. However, the direct role of VEGF-Flt-1 signaling in malignant behaviors of oral squamous cell carcinoma (OSCC) is not well understood. To clarify the effects of VEGF-Flt-1 signaling in OSCC bone invasion, VEGF expression in OSCC cells and number of Flt-1+ osteoclasts at bone invasion front in 55 gingival OSCC cases were evaluated by immunohistochemistry. Twenty-four of 55 cases (43.63%) strongly expressed VEGF. The VEGF highly expressing cases were statistically correlated with the increased number of Flt-1+ osteoclasts and the more aggressive radiographic pattern of bone invasion (p < 0.001). Proliferation, migration and invasion assays were examined in HSC2 (high Flt-1 expressing) and Ho1N1 (no Flt-1 expressing) cells. Flt-1 specific ligand-placental growth factor (PlGF) induced proliferation and promoted the migration and invasion of HSC2 only. Interestingly, PlGF up-regulated MMP1 and MMP3 expression and induced RANKL expression in HSC2 via phosphorylation of Akt, ERK1/2 and p38. In conclusion, VEGF-Flt-1 signaling promotes osteoclastogenesis, proliferation and facilitating invasion and migration of OSCC through production of MMP-1 and 3. Blocking of VEGF-Flt-1 signaling may be beneficial for OSCC treatment.
[Show abstract][Hide abstract] ABSTRACT: Brain-derived neurotrophic factor (BDNF) activates its receptor TrkB, and promotes neuronal survival, differentiation, and synaptic functions. Furthermore, we have revealed that BDNF can also regulate cementoblast differentiation and cellular survival via TrkB–ERK/Akt signaling cascade, which, in turn, results in the induction of periodontal tissue regeneration. Recently, using in silico screening with a BDNF loop-domain pharmacophore, a small molecule BDNF mimetic, called LM22A-4 that can facilitate TrkB signaling in hippocampal neurons to prevent cell death, was identified. Therefore, this study aimed to investigate the effect of LM22A-4 on cementoblast differentiation and its molecular mechanism. LM22A-4 and BDNF stimulation was found to enhance OPN, ALPase, and OC mRNA expression in immortalized human cementoblast-like (HCEM) cells, indicating cementoblast differentiation. In addition, similar to this result, both LM22A-4 and BDNF treatment facilitated TrkB phosphorylation and TrkB binding to adaptor proteins, such as Shc, GRB2, and SOS1, indicating TrkB activation. Importantly, the downstream target ERK and Akt was also phosphorylated by LM22A-4 and BDNF stimulation. Moreover, BDNF mimetic stimulation transactivated ERK from the cytoplasm into the nuclei in HCEM cells. It is noteworthy that a tyrosine kinase receptor inhibitor, K252a, an MEK–ERK inhibitor (U0126), and a PI3Kinase–Akt inhibitor (LY294002) remarkably attenuated TrkB, ERK, and Akt phosphorylation as well as increase of OPN mRNA expression in the HCEM cells, respectively. These findings suggest that the small molecule BDNF mimetic LM22A-4 regulates cementoblast differentiation via the TrkB–ERK/Akt signaling cascade. Therefore, this small compound may lead to the development of a novel therapeutic approach for periodontal tissue regeneration.
International immunopharmacology 04/2014; 19(2). DOI:10.1016/j.intimp.2014.01.028 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Control of periodontal tissue inflammation during orthodontic treatment is very important in achieving a favourable therapeutic goal. We previously demonstrated that orally applied bovine lactoferrin (bLF) inhibited LPS-induced bone resorption but not orthodontic force-induced tooth movement in vivo. This study is designed to examine the underlying mechanism of it.
We examined the inhibitory effects of bLF on the expression of RANKL, OPG, TNF-α and COX-2 in osteoblasts loaded with compressive stress (CS) in comparison with LPS stimulated osteoblasts. Formation of osteoclasts was evaluated by co-culture system.
Both CS- and LPS-applications upregulated COX-2 and RANKL but downregulated OPG. TNF-α was upregulated in LPS-stimulated osteoblasts but downregulated in CS-loaded osteoblasts. NS398 (a specific inhibitor of COX-2) significantly inhibited CS-induced RANKL-upregulation but not LPS-induced RANKL upregulation, indicating a critical role of COX-2/PGE2 pathway in CS-induced osteoclastogenesis. bLF significantly downregulated LPS-induced upregulation of RANKL and eliminated OPG suppression but not affected in CS-induced changes. Moreover, bLF significantly decreased LPS-induced osteoclast formation, whereas bLF had no effect on PGE2-induced osteoclast formation.
bLF can effectively suppress harmful bone destruction associated with periodontitis without inhibiting bone remodelling by CS-loading. Therefore, oral administration of bLF may be highly beneficial for control of periodontitis in orthodontic patients.
[Show abstract][Hide abstract] ABSTRACT: Background:
Epithelial-mesenchymal transition (EMT) is a crucial process in cancer progression that provides cancer cells with the ability to escape from the primary focus, invade stromal tissues and migrate to distant regions. Cell lines that lack E-cadherin show increased tumorigenesis and metastasis, and the expression levels of E-cadherin and Snail correlate inversely with the prognosis of patients suffering from breast cancer or oral squamous cell carcinoma (OSCC). Moreover, recent studies have shown that most EMT cases are regulated by soluble growth factors or cytokines. Among these factors, fibroblast growth factors (FGFs) execute diverse functions by binding to and activating members of the FGF receptor (FGFR) family, including FGFR1-4. Fibroblast growth factor receptor 1 is an oncoprotein that is involved in tumorigenesis, and PD173074 is known to be a selective inhibitor of FGFR1. However, the roles of FGFR1 and FGFR1 inhibitors have not yet been examined in detail.
Here, we investigated the expression of FGFR1 in head and neck squamous cell carcinoma (HNSCC) and the role of the FGFR1 inhibitor PD173074 in carcinogenesis and the EMT process.
Fibroblast growth factor receptor 1 was highly expressed in 54% of HNSCC cases and was significantly correlated with malignant behaviours. Nuclear FGFR1 expression was also observed and correlated well with histological differentiation, the pattern of invasion and abundant nuclear polymorphism. Fibroblast growth factor receptor 1 was also overexpressed in EMT cell lines compared with non-EMT cell lines. Furthermore, treatment of HOC313 cells with PD173074 suppressed cellular proliferation and invasion and reduced ERK1/2 and p38 activation. These cells also demonstrated morphological changes, transforming from spindle- to cobble stone-like in shape. In addition, the expression levels of certain matrix metalloproteinases (MMPs), whose genes contain activator protein-1 (AP-1) promoter sites, as well as Snail1 and Snail2 were reduced following PD173074 treatment.
Taken together, these data suggest that PD173074 inhibits the MAPK pathway, which regulates the activity of AP-1 and induces MET. Furthermore, this induction of MET likely suppresses cancer cell growth and invasion.
British Journal of Cancer 09/2013; 109(8). DOI:10.1038/bjc.2013.550 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective: Tooth loss is a critical risk factor for oral hypofunction. The prevalence of chronic periodontitis is still high and is the most causative factor of tooth loss leading indigestion. Therefor inhibiting tooth loss is inevitable for better quality of life. To explore new candidate molecules evaluating periodontal disease activity for early treatment intervention, proteomic analysis was applied to the pocket exudate (PE) of periodontal patient.
Method: The proteomic analysis of PEs of 2 patients with severe periodontitis was performed in comparison with gingival crevicular fluid of a healthy volunteer. The mRNA levels of some proteins specifically expressed in PE were examined in resected gingival tissues obtained from 7 periodontitis, 2 gingivitis patients and 2 healthy volunteers and were compared with those of proinflammatory cytokines like TNF-α, IL-1β and IL-8.
Result: Fifteen proteins including heat shock 70kDa protein 2 (Hspa2), heat shock 70kDa protein 5 (Hspa5), Snif2-related CREBBP activator protein (SRCAP), myeloid cell nuclear differentiation antigen (MNDA), Down syndrome cell adhesion molecule like 1 (Dscaml1), fibroblast activation protein-alpha (FAPα) were specifically detected in PE. Among these candidate molecules for periodontal disease activity Hspa2, Hspa5, SRCAP and MNDA were upregulated in 4 periodontitis and 2 gingivitis specimens, in which TNF-α, IL-1β and IL-8 were upregulated. FAPα was expressed only in periodontitis with proinflammatory cytokine-expression. In 2 healthy gingival tissues and 3 periodontitis specimens without the cytokine-expression, the levels of candidate molecules were very low. All tissue specimens examined showed no mRNA expression of Dscaml1. MNDA-immunolocalization was seen in periodontitis specimens with severe neutrophil infiltration.
Conclusion: The candidate molecules, Hspa2, Hspa5, FAPα, SRCAP and MADA, showed the positive correlation with proinflammatory cytokines-expression, which are known as indicators of periodontal disease activity. Moreover, MNDA-upregulation in PE may be related to acute exacerbation of periodontitis. These molecules may be useful makers for periodontal disease activity.
IADR Asia/Pacific Region (APR) Regional Meeting and Co-Annual Scientific Meeting of IADR Divisions 2013; 08/2013
[Show abstract][Hide abstract] ABSTRACT: Objective: To determine anti-inflammatory roles of F-spondin (SPON1), that we first determined to be expressed in cementoblasts, in periodontitis, we examined effects of SPON1 on expression of inflammatory cytokines and the destruction of periodontal tissues induced by lipopolysaccharide (LPS).
Method: We used immortalized human cementoblast-like cell lines (HCEM), human periodontal ligament cell lines (HPL) and HPL-F-spondin cells transfected with a retroviral plasmid encoding rat SPON1 cDNA into HPL (HPL-SPON1). SPON1 expression of HCEM was knock-downed by siSPON1. To investigate the effect of SPON1 on inflammation induced by LPS in these cells, we examined expression of inflammatory cytokines mRNAs by RT-PCR and phosphorylation of Erk expression by western blot. We furthermore compared inflammatory reactions of periodontal tissues induced by LPS in SPON1 transgenic mice (SPON1-TG) with those in wild type mice (WT).
Result: LPS treatment up-regulated SPON1 expression and down-regulated IL-6 expression in HCEM. Expression of TNF-α, IL-1ß and IL-8 didn’t show remarkable change with or without LPS treatment. Transfection of siSPON1 recovered down-regulation of IL-6 induced by LPS in HCEM. Furthermore, IL-6 expression was significantly decreased in HPL-SPON1. Phosphorylation of Erk induced by LPS was detected in HPL, but not in HPL with SPON1 transfection. SPON1 may down-regulate IL-6 expression through inhibiting activation of Erk pathway. In vivo, the number of mature osteoclasts and neutrophils induced by LPS in the periodontal tissue were fewer in SPON1-TG than in WT. SPON1-TG showed less bone destruction than WT.
Conclusion: We conclude that SPON1 may play anti-inflammatory action in periodontitis by down-regulation of IL-6 expression. SPON1 in the cementoblasts may protect cementum from pathologic resorption. This may explain the reason why cementum is not so common a feature of periodontitis-involved teeth comparing to bone resorption. SPON1 may be a novel molecule applicable for prevention and treatment of periodontal inflammation and destruction.
IADR Asia/Pacific Region (APR) Regional Meeting and Co-Annual Scientific Meeting of IADR Divisions 2013; 08/2013