Kazuyuki Noguchi

Prostaglandin production via induction of cyclooxygenase-2 by human gingival fibroblasts stimulated with lipopolysaccharides

The purpose of the present study was to investigate the involvement of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in PGE2 production by human gingival fibroblasts stimulated with lipopolysaccharides (LPS) from periodondopathogenic bacteria. LPS were isolated fromPorphyromonas gingivalis (P. gingivalis), Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) andEschericia coli (E. coli) by the phenol-water procedure. The three LPS preparations produced PGE2 up to 48 h in a time-dependent manner in human gingival fibroblasts.P. gingivalis-LPS was the most potent stimulator of PGE2 production and, to a lesser extent,A. actinomycetemcomitans- andE. coli-LPS. Treatment of the cells with indomethacin, a non selective COX-1/COX-2 inhibitor and NS-398, a selective COX-2 inhibitor, completely depressed PGE2 production. Treatment of dexamethasone, known to inhibit COX-2 expression, also significantly prevented PGE2 production. Immunohistochemical staining of COX-2 protein demonstrated that expression of COX-2 protein was increased at 24 h afterP. gingivalis-LPS stimulation, while expression of COX-1 protein was not affected byP. gingivalis-LPS. In order to investigate the regulation of PGE2 production,P. gingivalis-LPS-stimulated cells were treated with herbimycin A and genistein, both inhibitors of tyrosine kinases. Both the inhibitors significantly inhibited PGE2 production. Herbimycin A treatment depressed expression of COX-2 protein. These data suggest that human gingival fibroblasts stimulated with LPS from periodontopathogenic bacteria mainly produce PGE2 not by COX-1, but by COX-2, induction of which may be regulated by tyrosine kinase and that the produced PGE2 may be involved in the pathogenesis of periodontal diseases.

Twist negatively regulates osteoblastic differentiation in human periodontal ligament cells

Periodontal ligament (PDL) is a thin fibrous connective tissue located between two mineralized tissues, alveolar bone and cementum, which maintains a constant width physiologically. The mechanisms by which PDL resists mineralization are not well understood. Twist is a basic helix loop helix protein that plays a central role in regulation of early osteogenesis. We investigated the localization of Twist in PDL and compared the expression of Twist and osteoblast‐related genes in PDL cells with those in osteoblast‐like cells in the presence or absence of recombinant human bone morphogenetic protein (BMP)‐2. Histochemical analysis showed that Twist was expressed along alveolar bone surface in PDL. PDL cells constitutively expressed Twist gene and the expression level was higher than that in osteoblast‐like cells. In osteoblast‐like cell culture, BMP‐2 enhanced osteoblast‐related gene expression, while Twist expression was slightly decreased. In contrast, BMP‐2 increased runt‐related transcription factor (Runx)‐2, but failed to enhance alkaline phosphatase (ALP) and osteocalcin (OCN) gene expression in PDL cells. Interestingly, unlike in osteoblast‐like cells, Twist expression was upregulated by BMP‐2 in PDL cells. We transiently knocked down Twist gene in PDL cells using a short interference RNA expression vector (siTwist) and found that ALP, osteopontin (OPN), bone sialoprotein (BSP) genes expression and basal level of ALP activity were slightly increased, whereas Runx2 and OCN genes were not affected. Collectively, these results suggest that Twist may act as a negative regulator of osteoblastic differentiation in PDL cells. J. Cell. Biochem. 100: 303–314, 2007.

Prostaglandin E2 Regulates Interleukin-1β-induced Matrix Metalloproteinase-3 Production in Human Gingival Fibroblasts

Prostaglandin E2 (PGE2) exerts its biological actions via EP receptors (EP1, EP2, EP3, and EP4). In the present study, we investigated whether PGE2 regulated interleukin (IL)-1β-induced matrix metalloproteinase (MMP)-3 production in human gingival fibroblasts (HGF) derived from periodontally healthy subjects and diseased patients. In HGF from healthy gingiva, PGE2 down-regulated IL-1β-induced MMP-3 production, whereas in HGF from periodontitis patients, PGE2 enhanced it. Butaprost (an EP2 agonist) and ONO-AE1-329 (an EP4 agonist) suppressed IL-1β-induced MMP-3 production, and 17-phenyl-ω-trinor PGE2 (an EP1 agonist) mimicked the PGE2 effect in HGF from healthy and periodontally diseased tissues, respectively. Analysis of these data suggests that, in HGF from healthy tissue, IL-1β-induced MMP-3 production is down-regulated by PGE2 via EP2 and EP4 receptors, whereas in cells from periodontally diseased tissue, IL-1β-induced MMP-3 production is up-regulated via EP1 receptors. Different regulation of IL-1β-induced MMP-3 production by PGE2 between healthy and periodontally diseased tissues may be involved in the pathogenesis of periodontal disease.

The detection of platelet-activating factor in inflamed human gingival tissue

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a biologically active lipid, like the prostaglandins, which mediates allergic and inflammatory reactions. Aggregation of washed rabbit platelets was induced by a lipid prepared from inflamed gingiva. The mobility of the active lipid was coincident with that of authentic PAF on thin-layer chromatography. The aggregation was dose-dependent and inhibited by pretreatment with a specific PAF antagonist, ONO 6240, but not by indomethacin or creatine phosphate/creatine phosphokinase, which inhibit the platelet aggregation due to arachidonic acid or ADP, respectively. Thus the active lipid was identified as PAF; the amount of PAF detected was 118.1 +/- 79.7 pg/50 mg tissue (n = 6, mean +/- SD), the amount in normal tissue being 13.0 +/- 11.3 pg/50 mg tissue (n = 6). There was therefore a significant difference between the tissues. Lyso PAF, the metabolite of PAF with acetylhydrolase, was not detectable in either gingival tissue. Thus PAF was produced more in inflamed gingival tissue than in normal tissue; PAF may be involved in the occurrence and maintenance of periodontal disease.

Regenerative effect of basic fibroblast growth factor on periodontal healing in two‐wall intrabony defects in dogs

AIM The aim of the present study was to evaluate the effect of a basic fibroblast growth factor (bFGF) candidate treatment on periodontal healing in two-wall intrabony defects in dogs. MATERIALS AND METHODS Two-wall intrabony defects (5 x 5 x 5 mm) were created surgically on the distal and mesial sides of bilateral mandibular second and fourth premolars in four Beagle dogs. bFGF, enamel matrix derivative (EMD) and platelet-derived growth factor with beta-tricalcium phosphate (PDGF/beta-TCP) treatments, and sham-surgery (OFD) were rotated among the four defects in each animal, EMD and PDGF/beta-TCP serving as benchmark controls. The animals were euthanized for radiographic and histologic evaluation at 8 weeks. RESULTS Bone formation was significantly greater in the bFGF group (4.11 +/- 0.77 mm) than in the EMD (3.32 +/- 0.71 mm; p<0.05) and OFD (3.09 +/- 0.52 mm; p<0.01) groups. The EMD (4.59 +/- 1.19 mm) and PDGF/beta-TCP (4.66 +/- 0.7 mm) groups exhibited significantly greater cementum regeneration with periodontal ligament-like tissue than the OFD group (2.96 +/- 0.69 mm; p<0.01). No significant differences were observed between the bFGF and the PDGF/beta-TCP groups in any of the histometric parameters. CONCLUSIONS The candidate bFGF treatment supported periodontal regeneration comparable with that of established benchmarks: EMD and PDGF/beta-TCP.

PGE2 Activates Cementoclastogenesis by Cementoblasts via EP4

Destruction of cementum and alveolar bone is the main causative event for the exfoliation of teeth as a consequence of periodontitis. Prostaglandin E2 (PGE2) and PGE receptor subtypes (EPs) play an important role in modulating osteoblast-mediated osteoclastogenesis; however, no information is available on the role of PGE2 and EPs in regulating cementoblast-mediated cementoclastogenesis. We hypothesized that the PGE2-EPs pathway also regulates cementoblasts’ ability to activate cementoclasts. For these studies, OCCM-30 cells (a mouse cementoblast cell line) were exposed to PGE2 and specific EP agonists. PGE2 (100 ng/mL) and EP4 agonist (1 μM) up-regulated RANKL and IL-6 mRNA levels, while they down-regulated OPG mRNA expression. The EP4 antagonist (1 μM) eliminated these effects of PGE2. PGE2 treatment of co-cultures of OCCM-30 cells with bone marrow cells induced TRAP-positive cells via the EP4 pathway. These findings suggest that PGE2 promotes cementoblast-mediated cementoclastogenesis by regulating the expression of RANKL and OPG via the EP4 pathway.

The possible mechanism of preterm birth associated with periodontopathic Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE Previous studies have shown that Porphyromonas gingivalis is found in the amniotic fluid and placentae of pregnant women with some obstetric diseases. However, the biological effects of P. gingivalis on intrauterine tissues remain unclear. The aim of this study was to investigate the presence of P. gingivalis in chorionic tissues from hospitalized high-risk pregnant women, and the effects of P. gingivalis lipopolysaccharide on the production of proinflammatory molecules in human chorion-derived cells. MATERIAL AND METHODS Twenty-three subjects were selected from Japanese hospitalized high-risk pregnant women. The presence of P. gingivalis in chorionic tissues was analyzed by PCR. Cultured chorion-derived cells or Toll-like receptor-2 (TLR-2) gene-silenced chorion-derived cells were stimulated with P. gingivalis lipopolysaccharide. Real-time PCR was performed to evaluate TLR-2 and Toll-like receptor-4 (TLR-4) mRNA expression in the cells. Levels of interleukin-6 and interleukin-8 in culture supernatants of the chorion-derived cells were measured by ELISA. RESULTS P. gingivalis DNA was detected in chorionic tissues from two women with threatened preterm labor, two with multiple pregnancy and two with placenta previa. Stimulation of chorion-derived cells with P. gingivalis lipopolysaccharide significantly increased TLR-2 mRNA expression, whereas TLR-4 mRNA expression was not changed. P. gingivalis lipopolysaccharide induced interleukin-6 and interleukin-8 production in chorion-derived cells, but the P. gingivalis lipopolysaccharide-induced interleukin-6 and interleukin-8 production was reduced in TLR-2 gene-silenced chorion-derived cells. CONCLUSION Our results suggest that P. gingivalis can be detected in chorionic tissues of hospitalized high-risk pregnant women, and that P. gingivalis lipopolysaccharide induces interleukin-6 and interleukin-8 production via TLR-2 in chorion-derived cells.

Protein kinase-A-dependent osteoprotegerin production on interleukin-1 stimulation in human gingival fibroblasts is distinct from periodontal ligament fibroblasts

Periodontitis, a chronic inflammatory disease, is characterized by increased expression of interleukin (IL)‐1 and other inflammatory mediators resulting in extensive osteoclast formation and bone loss. Expression of receptor activator of nuclear factor kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), by osteoblasts is important to regulate osteoclast differentiation. The aim of the present study was to investigate the regulatory effects of IL‐1 on RANKL and OPG production by mesenchymal fibroblasts in periodontal tissue. Human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (PDL) were stimulated with IL‐1α with or without protein synthesis inhibitor cycloheximide (CHX), protein kinase A (PKA) inhibitors, protein kinase C (PKC) inhibitors and prostaglandin E2 (PGE2) inhibitor. In some experiments, the cultured cells were directly stimulated with either PKA or PKC activators. In HGF, IL‐1α‐stimulated OPG mRNA expression was high and could be reduced by CHX. PKA inhibitor completely abrogated IL‐1α‐induced OPG mRNA expression and OPG production. Endogenous PGE2 further enhanced IL‐1α‐induced OPG production in HGF. In PDL, RANKL mRNA expression was greatly augmented by IL‐1α. IL‐1α induced OPG mRNA expression and protein production. PKC inhibitor partially reduced IL‐1α‐induced OPG production and PKC activator enhanced OPG production in PDL. The IL‐1α‐stimulated OPG mRNA expression in HGF was greater than PDL. These results provide new evidence for the possible osteoclastogenesis‐inhibitory function of HGF through PKA activity pathway. PDL utilized PKC for OPG production. Thus, we emphasize that HGF and PDL have different characteristics of host defence mechanism against inflammatory process.

Involvement of the endocannabinoid system in periodontal healing.

Endocannabinoids including anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are important lipid mediators for immunosuppressive effects and for appropriate homeostasis via their G-protein-coupled cannabinoid (CB) receptors in mammalian organs and tissues, and may be involved in wound healing in some organs. The physiological roles of endocannabinoids in periodontal healing remain unknown. We observed upregulation of the expression of CB1/CB2 receptors localized on fibroblasts and macrophage-like cells in granulation tissue during wound healing in a wound-healing model in rats, as well as an increase in AEA levels in gingival crevicular fluid after periodontal surgery in human patients with periodontitis. In-vitro, the proliferation of human gingival fibroblasts (HGFs) by AEA was significantly attenuated by AM251 and AM630, which are selective antagonists of CB1 and CB2, respectively. CP55940 (CB1/CB2 agonist) induced phosphorylation of the extracellular-regulated kinases (ERK) 1/2, p38 mitogen-activated protein kinase (p38MAPK), and Akt in HGFs. Wound closure by CP55940 in an in-vitro scratch assay was significantly suppressed by inhibitors of MAP kinase kinase (MEK), p38MAPK, and phosphoinositol 3-kinase (PI3-K). These findings suggest that endocannabinoid system may have an important role in periodontal healing.

Effectable application of vascular endothelial growth factor to critical sized rat calvaria defects.

OBJECTIVE An early vascular response for angiogenesis is essential for the normal progression of bone defect healing. Vascular endothelial growth factor (VEGF) is a potent inducer of angiogenesis. The aim of this study was to evaluate the effects of a poly (L,D-lactic-co-glycolic acid) (PLGA) membrane with VEGF encapsulated into PLGA microspheres on bone regeneration at bone defects in rat calvaria. STUDY DESIGN Microspheres of PLGA incorporating VEGF(165) (VEGF microspheres) were prepared, and critical-size bone defects were created in rat calvaria. The VEGF microspheres, PLGA microspheres, or VEGF microspheres plus PLGA membrane were applied to the defects. Bone regeneration was evaluated using image analysis based on soft radiographic and histologic examination. RESULTS Mature thick bone regeneration was observed in selected sites at bone defects that had been applied with VEGF microspheres/PLGA membrane compared with those that had been applied with the other treatments. CONCLUSION A combination of VEGF microspheres and a PLGA membrane effectively enhances bone regeneration.