Effects of pulpotomy using mineral trioxide aggregate on prostaglandin transporter and receptors in rat molars.
ABSTRACT: Mineral trioxide aggregate (MTA) is a commonly used dental pulp-capping material with known effects in promoting reparative dentinogenesis. However, the mechanism by which MTA induces dentine repair remains unclear. The aim of the present study was to investigate the role of prostaglandin E2 (PGE2) in dentine repair by examining the localisation and mRNA expression levels of its transporter (Pgt) and two of its receptors (Ep2 and Ep4) in a rat model of pulpotomy with MTA capping. Ep2 expression was detected in odontoblasts, endothelial cells, and nerve fibres in normal and pulpotomised tissues, whereas Pgt and Ep4 were immunolocalised only in the odontoblasts. Moreover, mRNA expression of Slco2a1 (encoding Pgt), Ptger2 (encoding Ep2), and Ptger4 (encoding Ep4) was significantly upregulated in pulpotomised dental pulp and trigeminal ganglia after MTA capping. Our results provide insights into the functions of PGE2 via Pgt and Ep receptors in the healing dentine/pulp complex and may be helpful in developing new therapeutic targets for dental disease.
Project description:Regenerative endodontic procedures (REPs) are a new option for the treatment of dental pulp or periapical diseases in permanent teeth with open apices. Histologically, the new tissues formed in the root canal after REPs are mainly cementum- or bone-like mineralised tissues, but not the real dentine-pulp complex. Therefore, how to promote dentine-pulp complex regeneration and improve the clinical effects of REPs has become a prominent research topic. Stem cells from apical papilla (SCAP) are derived from the dental papilla that can differentiate into primary odontoblasts and dental pulp cells that produce root dentine and dental pulp. Exosomes are the key regulator for the paracrine activity of stem cells and can influence the function of recipient cells. In this study, SCAP-derived exosomes (SCAP-Exo) were introduced into the root fragment containing bone marrow mesenchymal stem cells (BMMSCs) and transplanted subcutaneously into immunodeficient mice. We observed that dental pulp-like tissues were present and the newly formed dentine was deposited onto the existing dentine in the root canal. Afterwards, the effects of SCAP-Exo on the dentinogenesis of BMMSCs were elucidated in vitro. We found that the gene and protein expression of dentine sialophosphoprotein and mineralised nodule formation in BMMSCs treated with SCAP-Exo were significantly increased. In summary, SCAP-Exo were endocytosed by BMMSCs and obviously improved their specific dentinogenesis. The use of exosomes derived from dental stem cells could comprise a potential therapeutic approach for dentine-pulp complex regeneration in REPs.
Project description:Cyclooxygenase-2 (COX-2) is induced under inflammatory conditions, and prostaglandin E2 (PGE2) is one of the products of COX activity. PGE2 has pleiotropic actions depending on the activation of specific E-type prostanoid EP1-4 receptors. We investigated the involvement of PGE2 and EP receptors in glial activation in response to an inflammatory challenge induced by LPS.Cultures of mouse microglia or astroglia cells were treated with LPS in the presence or absence of COX-2 inhibitors, and the production of PGE2 was measured by ELISA. Cells were treated with PGE2, and the effect on LPS-induced expression of TNF-α messenger RNA (mRNA) and protein was studied in the presence or absence of drug antagonists of the four EP receptors. EP receptor expression and the effects of EP2 and EP4 agonists and antagonists were studied at different time points after LPS.PGE2 production after LPS was COX-2-dependent. PGE2 reduced the glial production of TNF-α after LPS. Microglia expressed higher levels of EP4 and EP2 mRNA than astroglia. Activation of EP4 or EP2 receptors with selective drug agonists attenuated LPS-induced TNF-α in microglia. However, only antagonizing EP4 prevented the PGE2 effect demonstrating that EP4 was the main target of PGE2 in naïve microglia. Moreover, the relative expression of EP receptors changed during the course of classical microglial activation since EP4 expression was strongly depressed while EP2 increased 24 h after LPS and was detected in nuclear/peri-nuclear locations. EP2 regulated the expression of iNOS, NADPH oxidase-2, and vascular endothelial growth factor. NADPH oxidase-2 and iNOS activities require the oxidation of NADPH, and the pentose phosphate pathway is a main source of NADPH. LPS increased the mRNA expression of the rate-limiting enzyme of the pentose pathway glucose-6-phosphate dehydrogenase, and EP2 activity was involved in this effect.These results show that while selective activation of EP4 or EP2 exerts anti-inflammatory actions, EP4 is the main target of PGE2 in naïve microglia. The level of EP receptor expression changes from naïve to primed microglia where the COX-2/PGE2/EP2 axis modulates important adaptive metabolic changes.
Project description:In the non-pregnant dog, ovarian cyclicity is independent of a uterine luteolysin. This is in contrast to pregnant animals where a prepartum increase of luteolytic PGF2? occurs, apparently originating in the pregnant uterus. Recently, the placenta as a source of prepartum prostaglandins (PGs) was investigated, indicating fetal trophoblast cells as the likely main source. However, the possible contribution of uterine interplacental tissues to the production of these hormones has not yet been thoroughly examined in the dog.Several key factors involved in the production and/or actions of PGs were studied: cyclooxygenase 2 (COX2, PTGS2), PGF2?-synthase (PGFS/AKR1C3), PGE2-synthase (PGES), and the respective receptors FP (PTGFR), EP2 (PTGER2) and EP4 (PGTER4), 15-hydroxyprostaglandin dehydrogenase (HPGD), PG-transporter (PGT, SLCO2A1) and progesterone receptor. Their expression and localization patterns were assessed by Real Time PCR and immunohistology in the interplacental uterine sites from pregnant dogs during the pre-implantation period (days 8-12), post-implantation (days 18-25), mid-gestation (days 35-40) and during antigestagen-induced luteolysis/abortion.Whereas only low COX2 expression was observed in uterine samples at all the selected time points, expression of PGFS/AKR1C3 strongly increased post-implantation. A gradual increase in PGES-mRNA expression was noted towards mid-gestation. FP-mRNA expression decreased significantly with the progression of pregnancy until mid-gestation. This was associated with clearly detectable expression of HPGD, which did not change significantly over time. The expression of FP and EP2-mRNA decreased significantly over time while EP4-mRNA expression remained unaffected. The antigestagen-treatment led to a significant increase in expression of COX2, PGES, EP2 and PGT (SLCO2A1) mRNA. COX2 was localized predominantly in the myometrium. The expression of PGFS/AKR1C3, which was unchanged, was localized mostly to the surface luminal epithelium. The expression of EP4, PGT and HPGH did not change during treatment, they were co-localized with PGES and EP2 in all uterine compartments.The data clearly demonstrate the basic capability of the canine pregnant uterus to produce and respond to PGs and suggests their functions both as local regulatory factors involved in the establishment and maintenance of pregnancy, as well as potential contributors to the process of parturition, supporting the myometrial contractility associated with fetal expulsion.
Project description:PGE2 inhibits cytokine generation from human lung macrophages. However, the EP receptor that mediates this beneficial anti-inflammatory effect of PGE2 has not been defined. The aim of this study was to identify the EP receptor by which PGE2 inhibits cytokine generation from human lung macrophages. This was determined by using recently developed EP receptor ligands.The effects of PGE2 and EP-selective agonists on LPS-induced generation of TNF-? and IL-6 from macrophages were evaluated. The effects of EP2 -selective (PF-04852946, PF-04418948) and EP4 -selective (L-161,982, CJ-042794) receptor antagonists on PGE2 responses were studied. The expression of EP receptor subtypes by human lung macrophages was determined by RT-PCR.PGE2 inhibited LPS-induced and Streptococcus pneumoniae-induced cytokine generation from human lung macrophages. Analysis of mRNA levels indicated that macrophages expressed EP2 and EP4 receptors. L-902,688 (EP4 receptor-selective agonist) was considerably more potent than butaprost (EP2 receptor-selective agonist) as an inhibitor of TNF-? generation from macrophages. EP2 receptor-selective antagonists had marginal effects on the PGE2 inhibition of TNF-? generation, whereas EP4 receptor-selective antagonists caused rightward shifts in the PGE2 concentration-response curves.These studies demonstrate that the EP4 receptor is the principal receptor that mediates the anti-inflammatory effects of PGE2 on human lung macrophages. This suggests that EP4 receptor agonists could be effective anti-inflammatory agents in human lung disease.
Project description:Prostaglandin E2 (PGE2) is an important biological mediator involved in the defense against Mycobacterium tuberculosis (Mtb) infection. Previously, we reported that in macrophages (M?s), infection with avirulent Mtb H37Ra resulted in inhibition of necrosis by an inhibitory effect on mitochondrial permeability transition via the PGE2 receptor EP2. However, human M?s also express EP4, a PGE2 receptor functionally closely related to EP2 that also couples to stimulatory guanine nucleotide binding protein, but the functional differences between EP2 and EP4 in Mtb-infected M?s have been unclear. EP4 antagonist addition to H37Ra-infected M?s inhibited the expression of cyclooxygenase 2 (COX2) and microsomal prostaglandin E synthase-1 (mPGES-1), which are involved in PGE2 production. Moreover, H37Ra infection induced PGE2 production through the Toll-like receptor (TLR) 2/p38 mitogen-activated protein kinase (MAPK) signaling pathway. Induction of COX2 and mPGES-1 expression by TLR2 stimulation or Mtb infection was increased after additional stimulation with EP4 agonist. Hence, in Mtb-infected M?s, PGE2 production induced by pathogen recognition receptors/p38 MAPK signaling is up-regulated by EP4-triggered signaling to maintain an effective PGE2 concentration.
Project description:Preservation of a vital dental pulp is a central goal of restorative dentistry. Currently, there is significant interest in the development of tissue engineering scaffolds that can serve as biocompatible and bioactive pulp-capping materials, driving dentin bridge formation without causing cytotoxic effects. Our earlier in vitro studies described the biocompatibility of multidomain peptide (MDP) hydrogel scaffolds with dental pulp-derived cells but were limited in their ability to model contact with intact 3-dimensional pulp tissues. Here, we utilize an established ex vivo mandible organ culture model to model these complex interactions. MDP hydrogel scaffolds were injected either at the interface of the odontoblasts and the dentin or into the pulp core of mandible slices and subsequently cultured for up to 10 d. Histology reveals minimal disruption of tissue architecture adjacent to MDP scaffolds injected into the pulp core or odontoblast space. Additionally, the odontoblast layer is structurally preserved in apposition to the MDP scaffold, despite being separated from the dentin. Alizarin red staining suggests mineralization at the periphery of MDP scaffolds injected into the odontoblast space. Immunohistochemistry reveals deposition of dentin sialophosphoprotein by odontoblasts into the adjacent MDP hydrogel, indicating continued functionality. In contrast, no mineralization or dentin sialophosphoprotein deposition is evident around MDP scaffolds injected into the pulp core. Collagen III expression is seen in apposition to gels at all experimental time points. Matrix metalloproteinase 2 expression is observed associated with centrally injected MDP scaffolds at early time points, indicating proteolytic digestion of scaffolds. Thus, MDP scaffolds delivered centrally and peripherally within whole dental pulp tissue are shown to be biocompatible, preserving local tissue architecture. Additionally, odontoblast function and pulp vitality are sustained when MDP scaffolds are intercalated between dentin and the odontoblast region, a finding that has significant implications when considering these materials as pulp-capping agents.
Project description:BACKGROUND: The regulatory mechanisms of the expression of connective tissue growth factor/CCN family member 2 (CTGF/CCN2) in human articular chondrocytes have not been clarified. We investigated the effect of prostaglandin E2 (PGE2) on CTGF/CCN2 expression in chondrocytes. FINDINGS: Articular cartilage samples were obtained from patients with osteoarthritis (OA) and chondrocytes were isolated and cultured in vitro. Chondrocytes were stimulated with PGE2, PGE receptor (EP)-specific agonists, or interleukin (IL)-1. CTGF expression was analyzed using quantitative polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. The inhibitory effects of EP receptor antagonists (for EP2 and EP4) against PGE2 stimulation were also investigated. Stimulation of chondrocytes with PGE2 or IL-1 significantly suppressed CTGF expression. The suppressive effect of PGE2 was reproduced by EP2/EP4 receptor agonists but not by EP1/EP3 receptor agonists, and was partially blocked by an EP4 receptor antagonist, suggesting that the EP4 receptor has a dominant role. CONCLUSIONS: PGE2 may be involved in the regulation of CTGF/CCN2 expression in human articular chondrocytes via the EP4 receptor. Elucidation of EP4-mediated signaling in chondrocytes may contribute to a better understanding of the effects of PGE2 in arthritis.
Project description:We have shown that calcium (Ca2+) oscillations in human pulmonary fibroblasts (HPFs) contribute to profibrotic effects of transforming growth factor-? (TGF-?) and that disruption of these oscillations blunts features of pulmonary fibrosis. Prostaglandin E2 (PGE2) exerts antifibrotic effects in the lung, but the mechanisms for this action are not well defined. We thus sought to explore interactions between PGE2 and the profibrotic agent TGF-? in pulmonary fibroblasts (PFs) isolated from patients with or without idiopathic pulmonary fibrosis (IPF). PGE2 inhibited TGF-?-promoted [Ca2+] oscillations and prevented the activation of Akt and Ca2+/calmodulin-dependent protein kinase-II (CaMK-II) but did not prevent activation of Smad-2 or ERK. PGE2 also eliminated TGF-?-stimulated expression of collagen A1, fibronectin, and ?-smooth muscle actin and reduced stress fiber formation in the HPFs. RNA sequencing revealed that HPFs preferentially express EP2 receptors relative to other prostanoid receptor subtypes: EP2 expression is ~10-fold higher than that of EP4 receptors; EP1 and EP3 receptors are barely detectable; and EP2-receptor expression is ~3.5-fold lower in PFs from IPF patients than in normal HPFs. The inhibitory effects of PGE2 on synthetic function and stress fiber formation were blocked by selective EP2 or EP4 antagonists and mimicked by selective EP2 or EP4 agonists, the phosphodiesterase inhibitor isobutylmethylxanthine and forskolin, all of which elevate cellular cAMP concentrations. We conclude that PGE2, likely predominantly via EP2 receptors, interferes with Ca2+ signaling, CaMK-II activation, and Akt activation in IPF-HPFs and HPFs treated with TGF-?. Moreover, a decreased expression of EP2 receptors in pulmonary fibroblasts from IPF patients may contribute to the pathophysiology of this disease.
Project description:BACKGROUND:IL-23 is the key cytokine for generation of pathogenic IL-17-producing helper T (TH17) cells, which contribute critically to autoimmune diseases. However, how IL-23 generates pathogenic TH17 cells remains to be elucidated. OBJECTIVES:We sought to examine the involvement, molecular mechanisms, and clinical implications of prostaglandin (PG) E2-EP2/EP4 signaling in induction of IL-23-driven pathogenic TH17 cells. METHODS:The role of PGE2 in induction of pathogenic TH17 cells was investigated in mouse TH17 cells in culture in vitro and in an IL-23-induced psoriasis mouse model in vivo. Clinical relevance of the findings in mice was examined by using gene expression profiling of IL-23 and PGE2-EP2/EP4 signaling in psoriatic skin from patients. RESULTS:IL-23 induces Ptgs2, encoding COX2 in TH17 cells, and produces PGE2, which acts back on the PGE receptors EP2 and EP4 in these cells and enhances IL-23-induced expression of an IL-23 receptor subunit gene, Il23r, by activating signal transducer and activator of transcription (STAT) 3, cAMP-responsive element binding protein 1, and nuclear factor ? light chain enhancer of activated B cells (NF-?B) through cyclic AMP-protein kinase A signaling. This PGE2 signaling also induces expression of various inflammation-related genes, which possibly function in TH17 cell-mediated pathology. Combined deletion of EP2 and EP4 selectively in T cells suppressed accumulation of IL-17A+ and IL-17A+IFN-?+ pathogenic Th17 cells and abolished skin inflammation in an IL-23-induced psoriasis mouse model. Analysis of human psoriatic skin biopsy specimens shows positive correlation between PGE2 signaling and the IL-23/TH17 pathway. CONCLUSIONS:T cell-intrinsic EP2/EP4 signaling is critical in IL-23-driven generation of pathogenic TH17 cells and consequent pathogenesis in the skin.
Project description:Glomerular hyperfiltration is an important mechanism in the development of albuminuria. During hyperfiltration, podocytes are exposed to increased fluid flow shear stress (FFSS) in Bowman's space. Elevated Prostaglandin E2 (PGE2) synthesis and upregulated cyclooxygenase 2 (Cox2) are associated with podocyte injury by FFSS. We aimed to elucidate a PGE2 autocrine/paracrine pathway in human podocytes (hPC). We developed a modified liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) protocol to quantify cellular PGE2, 15-keto-PGE2, and 13,14-dihydro-15-keto-PGE2 levels. hPC were treated with PGE2 with or without separate or combined blockade of prostaglandin E receptors (EP), EP2, and EP4. Furthermore, the effect of FFSS on COX2, PTGER2, and PTGER4 expression in hPC was quantified. In hPC, stimulation with PGE2 led to an EP2- and EP4-dependent increase in cyclic adenosine monophosphate (cAMP) and COX2, and induced cellular PGE2. PTGER4 was downregulated after PGE2 stimulation in hPC. In the corresponding LC/ESI-MS/MS in vivo analysis at the tissue level, increased PGE2 and 15-keto-PGE2 levels were observed in isolated glomeruli obtained from a well-established rat model with glomerular hyperfiltration, the Munich Wistar Frömter rat. COX2 and PTGER2 were upregulated by FFSS. Our data thus support an autocrine/paracrine COX2/PGE2 pathway in hPC linked to concerted EP2 and EP4 signaling.