Marina Korotkova

Characterization of Microsomal Prostaglandin E Synthase 1 Inhibitors

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible terminal synthase in PGE2 biosynthesis by inflammatory and cancer cells. Clinical and experimental data emphasize that mPGES-1 might be a valuable target, with improved selectivity and safety compared to traditional NSAIDs or selective COX-2 inhibitors, in the treatment of inflammatory diseases, different types of cancer as well as central symptoms elicited by peripheral inflammation. Since the first characterization of mPGES-1, the numbers of publications on mPGES-1 structure, pathogenic role and inhibitor development have increased exponentially; however, there are currently no selective mPGES-1 inhibitors available for clinical use. In this MiniReview, we focus on recent advances in the development of selective inhibitors of mPGES-1 activity, with the aim to discuss the effects of targeting mPGES-1 in different inflammatory models in vitro and in vivo.

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.

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-1β, 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.

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

Significance Cancer-related inflammation promotes progression and therapy resistance in tumors of adulthood. Knowledge concerning the significance of inflammation in childhood malignancies has been limited. Neuroblastoma is an embryonal tumor of early childhood with poor prognosis despite intensified therapy, and biological understanding is necessary to develop novel therapies. We found high-risk neuroblastoma, in particular the therapy-resistant subset with chromosome 11q-deletion, to be inflammatory driven and characterized by high expression of the COX/microsomal prostaglandin E synthase-1 (mPGES-1)/prostaglandin E2 (PGE2) pathway that correlates with metastatic stage and poor clinical outcome. We further detected infiltrating cancer-associated fibroblasts expressing mPGES-1, the essential enzyme for synthesis of PGE2, promoting tumor growth, angiogenesis, and metastatic spread. Treatment targeting this inflammatory pathway provides a therapeutic option for neuroblastoma and other cancers. 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 β. 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.

IL-1β/HMGB1 Complexes Promote The PGE2 Biosynthesis Pathway in Synovial Fibroblasts

PGE2 is a potent lipid mediator of pain and oedema found elevated in RA. Microsomal prostaglandin E synthase‐1 (mPGES‐1) is a terminal enzyme of the PGE2 pathway inducible by proinflammatory cytokines. mPGES‐1 is markedly upregulated in RA synovial tissue despite antirheumatic treatments, suggesting that multiple inflammatory stimuli contribute to its induction. High‐mobility group box chromosomal protein 1 (HMGB1) is known to induce inflammation both by direct interaction with TLR4 and by enhancement of other proinflammatory molecules signalling, through complex formation. The high expression of extracellular HMGB1 within the inflamed synovium, implies its pro‐arthritogenic role in RA. We aimed to investigate the effects of IL‐1β/HMGB1 complexes on mPGES‐1 and other enzymes of the PGE2 pathway in synovial fibroblasts (SFs) from patients with arthritis. Furthermore, we studied the effect of COX‐2 inhibition and IL‐1RI antagonism on prostanoid and cytokine production by SFs. Stimulation of SFs with HMGB1 in complex with suboptimal amounts of IL‐1β significantly increased mPGES‐1 and COX‐2 expressions as well as PGE2 production, as compared to treatment with HMGB1 or IL‐1β alone. Furthermore, NS‐398 reduced the production of IL‐6 and IL‐8, thus indicating that IL‐1β/HMGB1 complexes modulate cytokine production in part through prostanoid synthesis. Treatment with IL‐1RA completely abolished the induced PGE2 and cytokine production, suggesting an effect mediated through IL‐1RI. IL‐1β/HMGB1 complexes promote the induction of mPGES‐1, COX‐2 and PGE2 in SF. The amplification of the PGE2 biosynthesis pathway by HMGB1 might constitute an important pathogenic mechanism perpetuating inflammatory and destructive activities in rheumatoid arthritis.

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.

Variants of gene for microsomal prostaglandin E2 synthase show association with disease and severe inflammation in rheumatoid arthritis.

Microsomal PGE synthase 1 (mPGES-1) is the terminal enzyme in the induced state of prostaglandin E2 (PGE2) synthesis and constitutes a therapeutic target for rheumatoid arthritis (RA) treatment. We examined the role of the prostaglandin E synthase (PTGES) gene polymorphism in susceptibility to and severity of RA and related variations in the gene to its function. The PTGES gene polymorphism was analyzed in 3081 RA patients and 1900 controls from two study populations: Swedish Epidemiological Investigation of Rheumatoid Arthritis (EIRA) and the Leiden Early Arthritis Clinic (Leiden EAC). Baseline disease activity score (DAS28) was employed as a disease severity measure. mPGES-1 expression was analyzed in synovial tissue from RA patients with known genotypes using immunohistochemistry. In the Swedish study population, among women a significant association with risk for RA was observed for PTGES single-nucleotide polymorphisms (SNPs) in univariate analysis and for the distinct haplotype. These results were substantiated by meta-analysis of data from EIRA and Leiden EAC studies with overall OR 1.31 (95% confidence interval 1.11–1.56). Several PTGES SNPs were associated with earlier onset of disease or with higher DAS28 in women with RA. Patients with the genotype associated with higher DAS28 exhibited significantly higher mPGES-1 expression in synovial tissue. Our data reveal a possible influence of PTGES polymorphism on the pathogenesis of RA and on disease severity through upregulation of mPGES-1 at the sites of inflammation. Genetically predisposed individuals may develop earlier and more active disease owing to this mechanism.

Limited effect of anti-rheumatic treatment on 15-prostaglandin dehydrogenase in rheumatoid arthritis synovial tissue.

IntroductionRheumatoid arthritis (RA) is a chronic inflammatory disease in which prostaglandin E2 (PGE2) displays an important pathogenic role. The enzymes involved in its synthesis are highly expressed in the inflamed synovium, while little is known about 15- prostaglandin dehydrogenase (15-PGDH) that metabolizes PGE2. Here we aimed to evaluate the localization of 15-PGDH in the synovial tissue of healthy individuals or patients with inflammatory arthritis and determine the influence of common RA therapy on its expression.MethodsSynovial tissue specimens from healthy individuals, psoriatic arthritis, ostheoarthritis and RA patients were immunohistochemically stained to describe the expression pattern of 15-PGDH. In addition, the degree of enzyme staining was evaluated by computer analysis on stained synovial biopsies from two groups of RA patients, before and after RA specific treatment with either intra-articular glucocorticoids or oral methotrexate therapy. Prostaglandins derived from the cyclooxygenase (COX) pathway were determined by liquid-chromatography mass spectrometry in supernatants from interleukin (IL) 1β-activated fibroblast-like synoviocytes (FLS) treated with methotrexate.Results15-PGDH was present in healthy and inflamed synovial tissue, mainly in lining macrophages, fibroblasts and vessels. Intra-articular glucocorticoids showed a trend towards reduced 15-PGDH expression in RA synovium (p = 0.08) while methotrexate treatment left the PGE2 pathway unaltered both in biopsies ex vivo and in cultured FLS.ConclusionsEarly methotrexate therapy has little influence on the expression of 15-PGDH and on any of the PGE2 synthesizing enzymes or COX-derived metabolites. Thus therapeutic strategies involving blocking induced PGE2 synthesis may find a rationale in additionally reducing local inflammatory mediators.

A1.27 MPGES-1 deletion increases prostacyclin and evades the elevated systemic ADMA associated with COX-2 inhibitors: relevance to cardiovascular safety of mpges-1 inhibitors

Background and objective Cyclooxygenase-2 (COX-2) is involved in the generation of prostanoids such as prostaglandin (PG)E2, prostacyclin (PGI2) and thromboxane (TXA2); PGE2is involved in inflammation/pain, whereas the second two are involved in cardiovascular homeostasis. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit COX-2 and are used to treat inflammation, however they cause cardiovascular side effects. The mechanisms for this include reduced vascular and/or renal PGI2 and, by effects on the renal medulla, increased dimethylarginine (ADMA), an endogenous inhibitor of endothelial nitric oxide synthase (eNOS). Microsomal PGE2 synthase 1 (mPGES-1) has been intensively investigated as a safer, alternative anti-inflammatory drug-target. We aimed to investigate how mPGES-1 versus COX-2 influences pathways associated with PGI2, ADMAand eNOS in order to assess the potential cardiovascular safety of mPGES-1 as a drug-target. Materialsxa0andxa0Methods Experiments were performed on mPGES-1+/+ (WT) and mPGES-1-/- (KO) mice as well as PGI2 synthase (PGIS-/-) KO mice. In some experiments WT mice were treated orally with the selective COX-2 inhibitor, parecoxib (100mg/kg; 5 days). Plasma was analysed by immunoassay for ADMA and PGI2, or by biochemical assay for creatinine. Renal medulla was stimulated with Ca2+ionophore and PGE2 release measured by immunoassay. Gene expression levels were determined using TaqMan assays. Vascular function (eNOS response) was assessed in isolated aorta stimulated with acetylcholine by wire myography. Results WT mice treated with parecoxib displayed no change in plasma PGI2 levels but, in contrast, mPGES-1 KO mice had significantly higher plasma levels of PGI2. In the kidney, mPGES-1 gene expression was lower in the renal medullathan the cortex, and in agreement; mPGES-1 deletion did not alter renal medulla PGE2 production. mPGES-1deletion had no effect on genes responsible for the production/breakdown of ADMA in the kidney (PRMT1, AGXT2, DDAH1/2). Plasma creatinine/ADMA was elevated in mice treated with parecoxib or PGIS KO mice but unaffected in mPGES-1 KO mice. Furthermore, mPGES-1 deletion significantly improved the eNOS-driven dilator response to acetylcholine in aorta. Conclusions Targeting mPGES-1 KO avoids the effects of COX-2 inhibition on renal PGI2/ADMA pathway and therefore spares vascular eNOS responses. This supports the development of selective inhibitors of mPGES-1 to more safely treat inflammation.