Microsomal prostaglandin E synthase-1 inhibition promotes shunting in arachidonic acid metabolism during inflammatory responses in vitro.

Microsomal Prostaglandin E Synthase 1 (mPGES-1) is the key enzyme for the generation of the pro-inflammatory lipid mediator prostaglandin E2 (PGE2), which contributes to several pathological features of many diseases. Inhibition of mPGES-1 has been shown to be a safe and effective therapeutic strategy in various pre-clinical studies. In addition to reduced PGE2 formation, it is also suggested that the potential shunting into other protective and pro-resolving prostanoids may play an important role in resolution of inflammation. In the present study, we analysed the eicosanoid profiles in four in vitro inflammation models and compared the effects of mPGES-1 inhibition with those of cyclooxygenase-2 (Cox-2) inhibition. Our results showed a marked shift to the PGD2 pathway under mPGES-1 inhibition in A549 cells, RAW264.7 cells and mouse bone marrow-derived macrophages (BMDMs), whereas enhanced prostacyclin production was observed in rheumatoid arthritis synovial fibroblasts (RASFs) treated with an mPGES-1 inhibitor. As expected, Cox-2 inhibition completely suppressed all prostanoids. This study suggests that the therapeutic effects of mPGES-1 inhibition may be mediated by modulation of other prostanoids in addition to PGE2 reduction.

Biosynthesis of prostaglandin 15dPGJ2 -glutathione and 15dPGJ2-cysteine conjugates in macrophages and mast cells via MGST3.

Inhibition of microsomal prostaglandin E synthase-1 (mPGES-1) results in decreased production of proinflammatory PGE2 and can lead to shunting of PGH2 into the prostaglandin D2 (PGD2)/15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) pathway. 15dPGJ2 forms Michael adducts with thiol-containing biomolecules such as GSH or cysteine residues on target proteins and is thought to promote resolution of inflammation. We aimed to elucidate the biosynthesis and metabolism of 15dPGJ2 via conjugation with GSH, to form 15dPGJ2-glutathione (15dPGJ2-GS) and 15dPGJ2-cysteine (15dPGJ2-Cys) conjugates and to characterize the effects of mPGES-1 inhibition on the PGD2/15dPGJ2 pathway in mouse and human immune cells. Our results demonstrate the formation of PGD2, 15dPGJ2, 15dPGJ2-GS, and 15dPGJ2-Cys in RAW264.7 cells after lipopolysaccharide stimulation. Moreover, 15dPGJ2-Cys was found in lipopolysaccharide-activated primary murine macrophages as well as in human mast cells following stimulation of the IgE-receptor. Our results also suggest that the microsomal glutathione S-transferase 3 is essential for the formation of 15dPGJ2 conjugates. In contrast to inhibition of cyclooxygenase, which leads to blockage of the PGD2/15dPGJ2 pathway, we found that inhibition of mPGES-1 preserves PGD2 and its metabolites. Collectively, this study highlights the formation of 15dPGJ2-GS and 15dPGJ2-Cys in mouse and human immune cells, the involvement of microsomal glutathione S-transferase 3 in their biosynthesis, and their unchanged formation following inhibition of mPGES-1. The results encourage further research on their roles as bioactive lipid mediators.

Where traditional Chinese medicine meets Western medicine in the prevention of rheumatoid arthritis.

Chinese medicine has a long tradition of use against rheumatoid arthritis (RA). The formulations are based on combinations of typically 5-10 plants, which are usually boiled and administered as a decoction or tea. There are few clinical trials performed so the clinical evidence is sparse. One fundamental of traditional medicine is to prevent disease. RA is an autoimmune, inflammatory and chronic disease that primarily affects the joints of 0.5%-1% of the population. In two out of three of the cases, the patients are characterised by the presence of autoantibodies such as the rheumatoid factor and the more disease-specific autoantibody against citrullinated proteins, so-called 'ACPA' (anticitrullinated protein/peptide antibodies). ACPA positivity is also strongly associated with specific variations in the HLA-DRB1 gene, the shared epitope alleles. Together with smoking, these factors account for the major risks of developing RA. In this review, we will summarise the background using certain plant-based formulations based on Chinese traditional medicine for the treatment and prevention of RA and the strategy we have taken to explore the mechanisms of action. We also summarise the major pathophysiological pathways related to RA and how these could be analysed. Finally, we summarise our ideas on how a clinical trial using Chinese herbal medicine to prevent RA could be conducted.

Establishment of an in vitro 3D model for neuroblastoma enables preclinical investigation of combined tumor-stroma drug targeting.

The majority of anti-cancer therapies target the proliferating tumor cells, while the tumor stroma, principally unaffected, survives, and provide a niche for surviving tumor cells. Combining tumor cell and stroma-targeting therapies thus have a potential to improve patient outcome. The neuroblastoma stroma contains cancer-associated fibroblasts expressing microsomal prostaglandin E synthase-1 (mPGES-1). mPGES-1-derived prostaglandin E2 (PGE2 ) is known to promote tumor growth through increased proliferation and survival of tumor cells, immune suppression, angiogenesis, and therapy resistance, and we, therefore, hypothesize that mPGES-1 constitutes an interesting stromal target. Here, we aimed to develop a relevant in vitro model to study combination therapies. Co-culturing of neuroblastoma and fibroblast cells in 3D tumor spheroids mimic neuroblastoma tumors with regard to the cyclooxygenase/mPGES-1/PGE2 pathway. Using the spheroid model, we show that the inhibition of fibroblast-derived mPGES-1 enhanced the cytotoxic effect of doxorubicin and vincristine and significantly reduced tumor cell viability and spheroid growth. Cyclic treatment with vincristine in combination with an mPGES-1 inhibitor abrogated cell repopulation. Moreover, inhibition of mPGES-1 potentiated the cytotoxic effect of vincristine on established neuroblastoma allografts in mice. In conclusion, we established a 3D neuroblastoma model, highlighting the potential of combining stromal targeting of mPGES-1 with tumor cell targeting drugs like vincristine.

miR-574-5p as RNA decoy for CUGBP1 stimulates human lung tumor growth by mPGES-1 induction.

MicroRNAs (miRs) are important posttranscriptional regulators of gene expression. Besides their well-characterized inhibitory effects on mRNA stability and translation, miRs can also activate gene expression. In this study, we identified a novel noncanonical function of miR-574-5p. We found that miR-574-5p acts as an RNA decoy to CUG RNA-binding protein 1 (CUGBP1) and antagonizes its function. MiR-574-5p induces microsomal prostaglandin E synthase-1 (mPGES-1) expression by preventing CUGBP1 binding to its 3'UTR, leading to an enhanced alternative splicing and generation of an mPGES-1 3'UTR isoform, increased mPGES-1 protein expression, PGE2 formation, and tumor growth in vivo. miR-574-5p-induced tumor growth in mice could be completely inhibited with the mPGES-1 inhibitor CIII. Moreover, miR-574-5p is induced by IL-1ß and is strongly overexpressed in human nonsmall cell lung cancer where high mPGES-1 expression correlates with a low survival rate. The discovered function of miR-574-5p as a CUGBP1 decoy opens up new therapeutic opportunities. It might serve as a stratification marker to select lung tumor patients who respond to the pharmacological inhibition of PGE2 formation.-Saul, M. J., Baumann, I., Bruno, A., Emmerich, A. C., Wellstein, J., Ottinger, S. M., Contursi, A., Dovizio, M., Donnini, S., Tacconelli, S., Raouf, J., Idborg, H., Stein, S., Korotkova, M., Savai, R., Terzuoli, E., Sala, G., Seeger, W., Jakobsson, P.-J., Patrignani, P., Suess, B., Steinhilber, D. miR-574-5p as RNA decoy for CUGBP1 stimulates human lung tumor growth by mPGES-1 induction.

COX/mPGES-1/PGE2 pathway depicts an inflammatory-dependent high-risk neuroblastoma subset.

The majority of solid tumors are presented with an inflammatory microenvironment. Proinflammatory lipid mediators including prostaglandin E2 (PGE2) contribute to the establishment of inflammation and have been linked to tumor growth and aggressiveness. Here we show that high-risk neuroblastoma with deletion of chromosome 11q represents an inflammatory subset of neuroblastomas. Analysis of enzymes involved in the production of proinflammatory lipid mediators showed that 11q-deleted neuroblastoma tumors express high levels of microsomal prostaglandin E synthase-1 (mPGES-1) and elevated levels of PGE2. High mPGES-1 expression also corresponded to poor survival of neuroblastoma patients. Investigation of the tumor microenvironment showed high infiltration of tumor-promoting macrophages with high expression of the M2-polarization markers CD163 and CD206. mPGES-1-expressing cells in tumors from different subtypes of neuroblastoma showed differential expression of one or several cancer-associated fibroblast markers such as vimentin, fibroblast activation protein α, α smooth muscle actin, and PDGF receptor ß. Importantly, inhibition of PGE2 production with diclofenac, a nonselective COX inhibitor, resulted in reduced tumor growth in an in vivo model of 11q-deleted neuroblastoma. Collectively, these results suggest that PGE2 is involved in the tumor microenvironment of specific neuroblastoma subgroups and indicate that therapeutic strategies using existing anti-inflammatory drugs in combination with current treatment should be considered for certain neuroblastomas.

Immunomodulatory effects of nicotine on interleukin 1ß activated human astrocytes and the role of cyclooxygenase 2 in the underlying mechanism.

BACKGROUND: The cholinergic anti-inflammatory pathway (CAP) primarily functions through acetylcholine (ACh)-alpha7 nicotinic acetylcholine receptor (α7nAChR) interaction on macrophages to control peripheral inflammation. Interestingly, ACh can also bind α7nAChRs on microglia resulting in neuroprotective effects. However, ACh effects on astrocytes remain elusive. Here, we investigated the effects of nicotine, an ACh receptor agonist, on the cytokine and cholinesterase production of immunocompetent human astrocytes stimulated with interleukin 1ß (IL-1ß) in vitro. In addition, the potential involvement of prostaglandins as mediators of nicotine was studied using cyclooxygenase 2 (COX-2) inhibition. METHODS: Cultured human fetal astrocytes were stimulated with human recombinant IL-1ß and treated simultaneously with nicotine at different concentrations (1, 10, and 100 µM). Cell supernatants were collected for cytokine and cholinesterase profiling using ELISA and MesoScale multiplex assay. α7nAChR expression on activated human astrocytes was studied using immunofluorescence. For the COX-2 inhibition studies, enzyme activity was inhibited using NS-398. One-way ANOVA was used to perform statistical analyses. RESULTS: Nicotine treatment dose dependently limits the production of critical proinflammatory cytokines such as IL-6 (60.5 ± 3.3, %inhibition), IL-1ß (42.4 ± 1.7, %inhibition), and TNF-α (68.9 ± 7.7, %inhibition) by activated human astrocytes. Interestingly, it also inhibits IL-8 chemokine (31.4 ± 8.5, %inhibition), IL-13 (34.243 ± 4.9, %inhibition), and butyrylcholinesterase (20.8 ± 2.8, %inhibition) production at 100 µM. Expression of α7nAChR was detected on the activated human astrocytes. Importantly, nicotine's inhibitory effect on IL-6 production was reversed with the specific COX-2 inhibitor NS-398. CONCLUSIONS: Activation of the cholinergic system through α7nAChR agonists has been known to suppress inflammation both in the CNS and periphery. In the CNS, earlier experimental data shows that cholinergic activation through nicotine inhibits microglial activation and proinflammatory cytokine release. Here, we report similar anti-inflammatory effects of cholinergic activation on human astrocytes, at least partly mediated through the COX-2 pathway. These results confirm the potential for cholinergic neuroprotection, which is looked upon as a promising therapy for neuroinflammation as well as neurodegenerative diseases and stroke. Our data implicates an important role for the prostaglandin system in cholinergic regulatory effects.

Deletion of mPGES-1 affects platelet functions in mice.

Microsomal prostaglandin E2 synthase-1 (mPGES-1) constitutes an essential player in inflammation and is involved in the pathogenesis of rheumatoid arthritis. Platelets participate in the regulation of inflammatory processes by the release of proinflammatory mediators and platelet-derived microparticles (PMPs). However, the role of the inducible mPGES-1/PGE2 pathway in platelet functions has not been investigated. In the present study we report a significant impact of mPGES-1 on platelet functions during inflammation. Wild-type (WT) and mPGES-1-/- knockout (KO) mice were stimulated with lipopolysaccharide (LPS) for 24 h. Platelet counts and activation were assessed by flow cytometry analysing CD62P-CD154 expression, PMP numbers, platelet-leukocyte aggregates and platelet aggregation. The accumulation of platelets and fibrinogen in the liver was analysed by immunofluorescent staining. In native platelets from WT and mPGES-1 KO mice, there were no differences among the investigated functions. After LPS treatment, the number of platelets was significantly decreased in WT, but not in KO mice. Platelet activation, platelet-leukocyte aggregates and PMP numbers were all significantly lower in KO mice compared with WT mice after LPS treatment. In addition, KO mice displayed a significant reduction in platelet aggregation ex vivo In the liver of LPS-stimulated WT and KO mice, there were no differences in platelet accumulation, although the percentage of total vessel area in the KO liver was significantly lower compared with the WT one. Our results demonstrate that systemic inhibition of mPGES-1 prevents platelet activation, which should have important implications with regard to the cardiovascular safety of mPGES-1 inhibitors.

Impaired vagus-mediated immunosuppression in microsomal prostaglandin E synthase-1 deficient mice.

The cholinergic anti-inflammatory pathway controls innate immune responses and inflammation. The prostaglandin (PG) system is involved in several neuro-processes and associated with inflammatory activation of cells in vagal nuclei. Here we aimed to investigate the potential role of PG in cholinergic neuro-regulation. The effect of vagus nerve stimulation (VNS) has been evaluated in microsomal prostaglandin E synthase-1 (mPGES-1) knockout (-/-) and wild-type (+/+) mice regarding cytokine and PG levels after lipopolysaccharides (LPS) challenge. As expected, VNS decreased the release of pro-inflammatory cytokines both in serum and spleen extracts of mPGES-1 (+/+)animals. However, the immune suppressive effect of VNS was completely abolished in mPGES-1 (-/-) mice. The PG content was not affected by VNS in the spleen of mPGES-1 (+/+) and mPGES-1 (-/-) mice but interestingly, acetylcholine (ACh) release in spleen induced by VNS confirmed an intact cholinergic pathway in mPGES-1 (+/+) whereas no VNS-induced ACh release was found in mPGES-1 (-/-) animals. Our data show that mPGES-1 and consequently PGE2 are crucial in the cholinergic anti-inflammatory pathway. Moreover, the mechanisms involved do not affect PG content in the spleen, but lack of mPGES-1 was found to strongly affect cholinergic mechanisms in the inflamed spleen. These findings illustrate previously unrecognized associations between the cholinergic and prostaglandin systems, and may be of importance for further development of therapeutic strategies directed at modulation of the inflammatory reflex, and immunosuppression in chronic inflammatory diseases.

NG2 glia protect against prion neurotoxicity by inhibiting microglia-to-neuron prostaglandin E2 signaling.

Oligodendrocyte-lineage cells, including NG2 glia, undergo prominent changes in various neurodegenerative disorders. Here, we identify a neuroprotective role for NG2 glia against prion toxicity. NG2 glia were activated after prion infection in cerebellar organotypic cultured slices (COCS) and in brains of prion-inoculated mice. In both model systems, depletion of NG2 glia exacerbated prion-induced neurodegeneration and accelerated prion pathology. Loss of NG2 glia enhanced the biosynthesis of prostaglandin E2 (PGE2) by microglia, which augmented prion neurotoxicity through binding to the EP4 receptor. Pharmacological or genetic inhibition of PGE2 biosynthesis attenuated prion-induced neurodegeneration in COCS and mice, reduced the enhanced neurodegeneration in NG2-glia-depleted COCS after prion infection, and dampened the acceleration of prion disease in NG2-glia-depleted mice. These data unveil a non-cell-autonomous interaction between NG2 glia and microglia in prion disease and suggest that PGE2 signaling may represent an actionable target against prion diseases.

High-content screening of drug combinations of an mPGES-1 inhibitor in multicellular tumor spheroids leads to mechanistic insights into neuroblastoma chemoresistance.

High-throughput drug screening enables the discovery of new anticancer drugs. Although monolayer cell cultures are commonly used for screening, their limited complexity and translational efficiency require alternative models. Three-dimensional cell cultures, such as multicellular tumor spheroids (MCTS), mimic tumor architecture and offer promising opportunities for drug discovery. In this study, we developed a neuroblastoma MCTS model for high-content drug screening. We also aimed to decipher the mechanisms underlying synergistic drug combinations in this disease model. Several agents from different therapeutic categories and with different mechanisms of action were tested alone or in combination with selective inhibition of prostaglandin E2 by pharmacological inhibition of microsomal prostaglandin E synthase-1 (mPGES-1). After a systematic investigation of the sensitivity of individual agents and the effects of pairwise combinations, GFP-transfected MCTS were used in a confirmatory screen to validate the hits. Finally, inhibitory effects on multidrug resistance proteins were examined. In summary, we demonstrate how MCTS-based high-throughput drug screening has the potential to uncover effective drug combinations and provide insights into the mechanism of synergy between an mPGES-1 inhibitor and chemotherapeutic agents.

Urinary prostanoids are elevated by anti-TNF and anti-IL6 receptor disease-modifying antirheumatic drugs but are not predictive of response to treatment in early rheumatoid arthritis.

BACKGROUND: Disease-modifying antirheumatic drugs (DMARDs) are widely used for treating rheumatoid arthritis (RA). However, there are no established biomarkers to predict a patient's response to these therapies. Prostanoids, encompassing prostaglandins, prostacyclins, and thromboxanes, are potent lipid mediators implicated in RA progression. Nevertheless, the influence of DMARDs on prostanoid biosynthesis in RA patients remains poorly understood. This study aims to assess the impact of various DMARDs on urinary prostanoids levels and to explore whether urinary prostanoid profiles correlate with disease activity or response to therapy. METHODS: This study included 152 Swedish female patients with early RA, all rheumatoid factor (RF) positive, enrolled in the NORD-STAR trial (registration number: NCT01491815). Participants were randomized into four therapeutic regimes: methotrexate (MTX) combined with (i) prednisolone (arm ACT), (ii) TNF-α blocker certolizumab pegol (arm CZP), (iii) CTLA-4Ig abatacept (arm ABA), or (iv) IL-6R blocker tocilizumab (arm TCZ). Urine samples, collected before start of treatment and at 24 weeks post-treatment, were analyzed for tetranor-prostaglandin E metabolite (tPGEM), tetranor-prostaglandin D metabolite (tPGDM), 2,3-dinor thromboxane B2 (TXBM), 2,3-dinor-6-keto prostaglandin F1a (PGIM), leukotriene E4 (LTE4) and 12-hydroxyeicosatetraenoic acid (12-HETE) using liquid chromatography-mass spectrometry (LC-MS). Generalized estimating equation (GEE) models were used to analyze the change in urinary eicosanoids and their correlations to clinical outcomes. RESULTS: Patients receiving MTX combined with CZP or TCZ exhibited significant elevations in urinary tPGEM and TXBM levels after 24 weeks of treatment. Other eicosanoids did not show significant alterations in response to any treatment. Baseline urinary eicosanoid levels did not correlate with baseline clinical disease activity index (CDAI) levels, nor with changes in CDAI from baseline to week 24. Their levels were also similar between patients who achieved CDAI remission and those with active disease at week 24. CONCLUSIONS: Treatment with anti-TNF or anti-IL6R agents in early RA patients leads to an increased systemic production of proinflammatory and prothrombotic prostanoids. However, urinary eicosanoid levels do not appear to be predictive of the response to DMARDs therapy.

High-mannose glycans from Schistosoma mansoni eggs are important for priming of Th2 responses via Dectin-2 and prostaglandin E2.

The parasitic helminth Schistosoma mansoni is a potent inducer of type 2 immune responses by stimulating dendritic cells (DCs) to prime T helper 2 (Th2) responses. We previously found that S. mansoni soluble egg antigens (SEA) promote the synthesis of Prostaglandin E2 (PGE2) by DCs through ERK-dependent signaling via Dectin-1 and Dectin-2 that subsequently induces OX40L expression, licensing them for Th2 priming, yet the ligands present in SEA involved in driving this response and whether specific targeting of PGE2 synthesis by DCs could affect Th2 polarization are unknown. We here show that the ability of SEA to bind Dectin-2 and drive ERK phosphorylation, PGE2 synthesis, OX40L expression, and Th2 polarization is impaired upon cleavage of high-mannose glycans by Endoglycosidase H treatment. This identifies high-mannose glycans present on glycoproteins in SEA as important drivers of this signaling axis. Moreover, we find that OX40L expression and Th2 induction are abrogated when microsomal prostaglandin E synthase-1 (mPGES) is selectively inhibited, but not when a general COX-1/2 inhibitor is used. This shows that the de novo synthesis of PGE2 is vital for the Th2 priming function of SEA-stimulated DCs as well as points to the potential existence of other COX-dependent lipid mediators that antagonize PGE2-driven Th2 polarization. Lastly, specific PGE2 inhibition following immunization with S. mansoni eggs dampened the egg-specific Th cell response. In summary, our findings provide new insights in the molecular mechanisms underpinning Th2 induction by S. mansoni and identify druggable targets for potential control of helminth driven-Th2 responses.

Lack of microsomal prostaglandin E(2) synthase-1 in bone marrow-derived myeloid cells impairs left ventricular function and increases mortality after acute myocardial infarction.

BACKGROUND: Microsomal prostaglandin E(2) synthase-1 (mPGES-1), encoded by the Ptges gene, catalyzes prostaglandin E(2) biosynthesis and is expressed by leukocytes, cardiac myocytes, and cardiac fibroblasts. Ptges(-/-) mice develop more left ventricle (LV) dilation, worse LV contractile function, and higher LV end-diastolic pressure than Ptges(+/+) mice after myocardial infarction. In this study, we define the role of mPGES-1 in bone marrow-derived leukocytes in the recovery of LV function after coronary ligation. METHODS AND RESULTS: Cardiac structure and function in Ptges(+/+) mice with Ptges(+/+) bone marrow (BM(+/+)) and Ptges(+/+) mice with Ptges(-/-) BM (BM(-/-)) were assessed by morphometric analysis, echocardiography, and invasive hemodynamics before and 7 and 28 days after myocardial infarction. Prostaglandin levels and prostaglandin biosynthetic enzyme gene expression were measured by liquid chromatography-tandem mass spectrometry and real-time polymerase chain reaction, immunoblotting, immunohistochemistry, and immunofluorescence microscopy, respectively. After myocardial infarction, BM(-/-) mice had more LV dilation, worse LV systolic and diastolic function, higher LV end-diastolic pressure, more cardiomyocyte hypertrophy, and higher mortality but similar infarct size and pulmonary edema compared with BM(+/+) mice. BM(-/-) mice also had higher levels of COX-1 protein and more leukocytes in the infarct, but not the viable LV, than BM(+/+) mice. Levels of prostaglandin E(2) were higher in the infarct and viable myocardium of BM(-/-) mice than in BM(+/+) mice. CONCLUSIONS: Lack of mPGES-1 in bone marrow-derived leukocytes negatively regulates COX-1 expression, prostaglandin E(2) biosynthesis, and inflammation in the infarct and leads to impaired LV function, adverse LV remodeling, and decreased survival after acute myocardial infarction.

Activated LTB4 pathway in muscle tissue of patients with polymyositis or dermatomyositis.

OBJECTIVE: To investigate the involvement of the leukotriene B4 (LTB4) pathway in polymyositis (PM) and dermatomyositis (DM) and the effect of immunosuppressive treatment on the LTB4 pathway. METHODS: 5-lipoxygenase (5-LO), 5-LO activating protein (FLAP) and LTB4 receptor-1 (BLT1) expression was analysed by immunohistochemistry in muscle tissue from patients with PM/DM before and after immunosuppressive treatment and from healthy individuals. In vivo LTB4 in thigh muscle was measured by microdialysis at rest and after acute exercise in another cohort of patients and healthy controls. RESULTS: The number of 5-LO-positive cells and BLT1-positive capillaries was higher in patients with PM/DM than in healthy individuals. The number of FLAP-expressing cells divided the patients into two groups (high/low expression). Treatment reduced the number of FLAP-positive cells in the group with initial high levels, however the expression remained high compared with healthy individuals. The number of BLT1-positive cells was also reduced while staining for 5-LO was unchanged. An inverse correlation was observed between the number of 5-LO or FLAP-positive cells in muscle tissue and muscle performance. LTB4 could be detected in dialysate of muscle tissue in vivo in both patients and healthy controls and was significantly increased after exercise in patients. CONCLUSION: The LTB4 pathway is upregulated in muscle tissue from patients with PM/DM and this upregulation correlated negatively to muscle performance, suggesting a role for LTB4 in myositis muscle weakness. The immunosuppressive treatment was insufficient on the LTB4 pathway and, for patients with high expression of FLAP, FLAP inhibitors may be considered as possible therapy.

Effects of mPGES-1 deletion on eicosanoid and fatty acid profiles in mice.

mPGES-1 is considered an alternative target for anti-inflammatory treatment with improved selectivity and safety compared to NSAIDs. mPGES-1 depletion not only suppresses inflammation via absence of inducible PGE2 but might also cause an activation of anti-inflammatory pathways. We studied effects of mPGES-1 deletion on the eicosanoid and fatty acid (FA) profiles in mice. In LPS-induced peritoneal macrophages from mPGES-1 knock-out (mPGES-1-/-, KO) mice PGE2 production was markedly attenuated, whereas levels of PGD2 metabolites (15-deoxy-Δ(12,14) PGJ2 and 15-deoxy-Δ(12,14) PGD2) were increased compared to wild type mice. The levels of oxidized fatty acid 13-HODE were also significantly up-regulated in KO macrophages. Significant differences in the total lipid FA composition (decrease in monounsaturated FA and increase in eicosadienoic acid) were detected in spleen of KO and WT mice. These effects of mPGES-1 deletion on eicosanoid and fatty acid metabolism have important implications for future mPGES-1 inhibitors and deserve further investigation.

Characterization of a new mPGES-1 inhibitor in rat models of inflammation.

Microsomal prostaglandin E synthase (mPGES)-1 inhibition has been proposed as an alternative to cyclooxygenase (COX) inhibition in the treatment of pain and inflammation. This novel approach could potentially mitigate the gastro-intestinal and cardiovascular side effects seen after long-term treatment with traditional non-steroidal anti-inflammatory drugs (NSAIDs) and Coxibs respectively. Several human mPGES-1 inhibitors have been developed in the recent years. However, they were all shown to be considerably less active on rodent mPGES-1, precluding the study of mPGES-1 inhibition in rodent models of inflammation and pain. The aim of this study was to characterize the new mPGES-1 inhibitor compound II, a pyrazolone that has similar potency on rat and human recombinant mPGES-1, in experimental models of inflammation. In cell culture, compound II inhibited PGE2 production in synovial fibroblasts from patients with rheumatoid arthritis (RASF) and in rat peritoneal macrophages. In vivo, compound II was first characterized in the rat air pouch model of inflammation where treatment inhibited intra-pouch PGE2 production. Compound II was also investigated in a rat adjuvant-induced arthritis model where it attenuated both the acute and delayed inflammatory responses. In conclusion, compound II represents a valuable pharmacological tool for the study of mPGES-1 inhibition in rat models.

Characterization of a human and murine mPGES-1 inhibitor and comparison to mPGES-1 genetic deletion in mouse models of inflammation.

Microsomal prostaglandin E synthase-1 (mPGES-1) inhibition has been suggested as an alternative to cyclooxygenase (COX) inhibition in the treatment of pain and inflammation. We characterized a selective inhibitor of mPGES-1 activity (compound III) and studied its impact on the prostanoid profile in various models of inflammation. Compound III is a benzoimidazole, which has a submicromolar IC50 in both human and rat recombinant mPGES-1. In cellular assays, it reduced PGE2 production in A549 cells, mouse macrophages and blood, causing a shunt to the prostacyclin pathway in the former two systems. Lastly, we assayed compound III in the air pouch model to verify its impact on the prostanoid profile and compare it to the profile obtained in mPGES-1 k.o. mice. As opposed to mPGES-1 genetic deletion, which attenuated PGE2 production and caused a shunt to the thromboxane pathway, mPGES-1 inhibition with compound III reduced PGE2 production and tended to decrease the levels of other prostanoids.

Microsomal prostaglandin E synthase-1 inhibition prevents adverse cardiac remodelling after myocardial infarction in mice.

BACKGROUND AND PURPOSE: Heart failure with reduced ejection fraction (HFrEF) is a major consequence of myocardial infarction (MI). The microsomal prostaglandin E synthase-1 (mPGES-1)/PGE2 pathway has been shown to constrain reperfusion injury after acute myocardial ischaemia. However, it is unknown whether pharmacological inhibition of mPGES-1, a target with lower risk of thrombosis compared with selective inhibition of cyclooxygenase-2, affects chronic cardiac remodelling after MI. EXPERIMENTAL APPROACH: Mice were subjected to left anterior descending coronary artery ligation, followed by intraperitoneal treatment with the mPGES-1 inhibitor compound III (CIII) or 118, celecoxib (cyclooxygenase-2 inhibitor) or vehicle, once daily for 28 days. Urinary prostanoid metabolites were measured by liquid chromatography-tandem mass spectrometry. KEY RESULTS: Chronic administration of CIII improved cardiac function in mice after MI compared with vehicle or celecoxib. CIII did not affect thrombogenesis or blood pressure. In addition, CIII reduced infarct area, augmented scar thickness, decreased collagen I/III ratio, decreased the expression of fibrosis-related genes and increased capillary density in the ischaemic area. Shunting to urinary metabolites of PGI2 , not thromboxane B2 or PGD2 , after inhibition of mPGES-1 was positively correlated with cardiac function after MI. CIII administration significantly increased urinary PGI2 /PGE2 metabolite ratio compared to vehicle or celecoxib. The PGI2 /PGE2 metabolite ratio correlated positively with ejection fraction, fractional shortening and scar thickness. Treatment with 118 also improved cardiac function. CONCLUSION AND IMPLICATIONS: Inhibition of mPGES-1 prevented chronic adverse cardiac remodelling via an augmented PGI2 /PGE2 metabolite ratio and therefore represents a potential therapeutic strategy for development of HFrEF after MI.

Expression of prostaglandin E synthases in periodontitis immunolocalization and cellular regulation.

The inflammatory mediator prostaglandin E(2) (PGE(2)) is implicated in the pathogenesis of chronic inflammatory diseases including periodontitis; it is synthesized by cyclooxygenases (COX) and the prostaglandin E synthases mPGES-1, mPGES-2, and cPGES. The distribution of PGES in gingival tissue of patients with periodontitis and the contribution of these enzymes to inflammation-induced PGE(2) synthesis in different cell types was investigated. In gingival biopsies, positive staining for PGES was observed in fibroblasts and endothelial, smooth muscle, epithelial, and immune cells. To further explore the contribution of PGES to inflammation-induced PGE(2) production, in vitro cell culture experiments were performed using fibroblasts and endothelial, smooth muscle, and mast cells. All cell types expressed PGES and COX-2, resulting in basal levels of PGE(2) synthesis. In response to tumor necrosis factor (TNF-α), IL-1ß, and cocultured lymphocytes, however, mPGES-1 and COX-2 protein expression increased in fibroblasts and smooth muscle cells, accompanied by increased PGE(2), whereas mPGES-2 and cPGES were unaffected. In endothelial cells, TNF-α increased PGE(2) production only via COX-2 expression, whereas in mast cells the cytokines did not affect PGE(2) enzyme expression or PGE(2) production. Furthermore, PGE(2) production was diminished in gingival fibroblasts derived from mPGES-1 knockout mice, compared with wild-type fibroblasts. These results suggest that fibroblasts and smooth muscle cells are important sources of mPGES-1, which may contribute to increased PGE(2) production in the inflammatory condition periodontitis.