RATIONAL DESIGN, SYNTHESIS, AND CHARACTERIZATION OF NOVEL mPGES-1 INHIBITORS AS NEXT GENERATION OF ANTI-INFLAMMATORY DRUGS

Aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) are currently widely used as fever and pain relief in patients with arthritis and other inflammatory symptoms. NSAIDs effect by inhibiting cyclooxygenase-1 (COX-1) and/or cyclooxygenase-2 (COX-2). COX isozymes (COXs) are key enzymes in the biosynthesis of prostaglandin H2 (PGH2) from arachidonic acid (AA). It is now clear that prostaglandin E2 (PGE2), one of the downstream products of PGH2, is the main mediator in both chronic and acute inflammation. Microsomal prostaglandin E synthase (mPGES-1) is the terminal enzyme of COX-2 in the PGE2 biosynthesis pathway. Different from other two constitutively expressed PGE2 synthase (PGES), mPGES-2 and cPGES, mPGES-1 is induced by pro-inflammatory stimuli and responsible for the production of PGE2 related to inflammation, fever and pain. For these reasons, selective inhibition of mPGES-1 is expected to suppress inflammation induced PGE2 production and, therefore, will exert anti-inflammatory activity while avoid the side effects of COXs inhibitors, such as gastrointestinal (GI) toxicity, and cardiovascular events. A combination of computational and experimental approaches was used to discovery mPGES-1 inhibitors with new scaffolds. The methods used include molecular docking, molecular dynamic simulation, molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculation, and in vitro activity assays. Our large-scale structure-based virtual screening was performed on compounds in the NCI libraries, containing a total of ~260,000 compounds. 7 compounds have been determined for their IC50 values (about 300 nM to 8000 nM). What’s more, these new inhibitors of mPGES-1 identified from virtual screening did not shown significant inhibition against COX isozymes even at substantially high concentrations (e.g. 100 µM). Rational methodology for drug design and organic synthesis were applied to generate three series of mPGES-1 inhibitors with different scaffolds. In total, about 200 compounds were synthesized and tested for their in vitro inhibition against human mPGES-1. Compounds with high potency against human mPGES-1 were further screened for their inhibition against mouse mPGES-1 and selectivity of human mPGES-1 over COXs. Several compounds were identified as submicromolar inhibitors against human mPGES-1 with high selectivity over COXs. In general, we have successfully identified a library of compounds as potent mPGES-1 inhibitors without significant inhibition against COXs. Structure information and in vitro activity evaluation data generated from the virtual screening and the library of compounds will be used to guide future design and synthesis of the mPGES-1 inhibitors

Characterization of the PGE2 pathway in arthritis and inflammation : mPGES-1 as a therapeutic target

The inducible prostaglandin (PG) E2 pathway is defined by the concerted activities of the enzymes cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase (mPGES)-1 in producing PGE2. PGE2 has pro-inflammatory and immunomodulatory functions and is involved in an array of diseases with an autoimmune and chronic inflammatory component, including rheumatoid arthritis (RA). In RA, mPGES-1 and COX-2 are up-regulated in the inflamed synovium of patients. COX inhibitors like non-steroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors (COXibs) relieve inflammation and pain in RA, but their respective GI tract and cardiovascular side effects preclude long-term use. Moreover, other effective RA therapies like TNF blockade and B-cell depletion therapy leave the inducible PGE2 pathway unaffected, suggesting that targeting this pathway could have additional benefits in a combinatorial approach. mPGES-1 is currently investigated as a target that could dissociate the anti-inflammatory benefits of COX inhibitors from their detrimental side effects. However, most inhibitors of human mPGES-1 activity generated so far failed to inhibit the murine enzyme ortholog, which complicates their characterization in vivo in relevant disease models. The first objective of this thesis was to extend the characterization of the PGE2 pathway in arthritis. The second objective was to investigate mPGES-1 as a therapeutic target. The latter was accomplished in part through the development and evaluation of pharmacological inhibitors active on both human and murine mPGES-1. To fulfill the first objective, we characterized a new mechanism for the induction of PGE2 production in RA synovial fibroblasts (RASFs), whereby complexes of the alarmin high mobility group box protein-1 (HMGB1) and the cytokine IL-1β cause an up-regulation of COX-2 and mPGES-1 expression. We also characterized 15- prostaglandin dehydrogenase (15-PGDH) to co-localize with mPGES-1 and the COX enzymes in the synovium of RA patients, suggesting a concerted activity of the anabolic and catabolic parts of the PGE2 cascade in the joint. In the same patients, we determined that methotrexate therapy did not interfere with the expression of COXs, mPGES-1 or 15-PGDH, adding that therapy to the list of approaches leaving the PGE2 pathway unaffected. Lastly, we could not confirm an association between the expression of COXs, mPGES-1 and 15-PGDH and quantitative pain assessment or arthritis development in arthralgic individuals at risk of developing arthritis or in early arthritis patients. In line with the second objective, we developed compounds II and III, which inhibit PGE2 synthesis in vitro in different human and murine cell assays and in vivo in the air pouch model of acute inflammation. Compound II also reduced edema in the rat adjuvant-induced arthritis (AIA) model. When the PG profile elicited by mPGES-1 inhibition with compound III was compared to that resulting from mPGES- 1 gene deletion in the air pouch model, different results were observed suggesting the two modes of inhibition might not have the exact same outcome. While inhibition of PGE2 synthesis with both compound II and III did not result in the shunting of PGH2 to other prostanoids, a shunt to thromboxane (TX) B2 was observed in the mPGES-1 knockout mouse. We also used the mPGES-1 knockout mouse to investigate the impact of mPGES-1 gene deletion on the eicosanoid and fatty acid profiles in inflammation. We discovered that it resulted in macrophages producing more 15- deoxy-Δ 12,14 PGJ2 and the spleen containing more eicosadienoic acid (EDA). This suggests mPGES-1 inhibition could not only inhibit the synthesis of the pro- inflammatory PGE2, but also cause the up-regulation of anti-inflammatory pathways. In conclusion, this thesis further advances the knowledge about the PGE2 synthesis cascade in arthritis and describes two new mPGES-1 inhibitors with an in vivo activity in native rodent models of disease. The latter constitute new valuable tools for the study of mPGES-1 in whichever pathology it has an involvement

Studies on anti-inflammatory and vasoactive effects of mPGES-1 inhibition

Inflammation is the basis for various serious illnesses such as rheumatic diseases, cardiovascular diseases, and cancer. Prostaglandin E2 (PGE2) is a pro-inflammatory lipid mediator produced by cyclooxygenases (COX1/2) and the microsomal prostaglandin E synthase 1 (mPGES-1). Nonsteroidal anti-inflammatory drugs (NSAIDS) targeting COX successfully reduce pain and inflammation. However, gastrointestinal and cardiovascular side effects associated with blockade of all prostaglandins limit their use. Selective inhibition of mPGES-1 is an alternative therapeutic strategy to impede PGE2 production while sparing or even upregulating other lipid mediators. When and where the shunting of PGH2 to other prostanoids occurs and whether it interferes with or contributes to the therapeutic effects of mPGES-1 inhibition is not fully understood. The overall aim of this thesis was to study the anti-inflammatory and vasoactive effects of mPGES-1 inhibition in models of inflammation and cardiovascular disease and to investigate the possible shunting of PGH2 to PGD2 and PGI2. The methodological approach was principally based on liquid chromatography-tandem mass spectrometry, biochemical assays as well as wire-myography. In Paper I five new mPGES-1 inhibitors were characterized. The inhibitors selectively suppressed PGE2 formation in in-vitro and in-vivo assays, reduced acute paw swelling in rats, and reduced adrenergic vasoconstriction. The results of this study serve as a basis for the application of these inhibitors in pre-clinical research. Depletion of mPGES-1 may lead to the re-direction of PGH2 into the PGD2/15-deoxy-Δ12,14-PGJ2 (15dPGJ2) pathway, which has been described to be anti-inflammatory and pro-resolving. In Paper II, we studied the biosynthesis and metabolism of 15dPGJ2 via conjugation to glutathione in immune cells and upon inhibition of mPGES-1. The results of this study demonstrate the formation of 15dPGJ2-glutathione and 15dPGJ2-cysteine conjugates in immune cells, the involvement of MGST3 in this pathway and the preservation of the PGD2/15dPGJ2 pathway upon inhibition of mPGES-1. Another important aspect of mPGES-1 inhibition is the redirection of excess PGH2 into the PGI2 pathway. A decrease in PGI2 levels as a result of COX-2 inhibition has been associated with increased cardiovascular risk in patients treated with selective NSAIDs. In Paper III we aimed to study the effects of mPGES-1 inhibition on human resistance arteries. Inhibition of mPGES- 1 significantly reduced adrenergic vasoconstriction and enhanced relaxation. Our results suggest that multiple pathways in addition to shunting to PGI2 may be involved in the vasoactive effects of mPGES-1 inhibition in human microcirculation. In Paper IV we studied the effects of mPGES-1 inhibition in a mouse model of MI. The results of this study indicate that pharmacological inhibition of mPGES-1 could improve cardiac function after MI and increase the PGI2/PGE2 metabolite ratio in urine compared with controls. The results from this thesis contribute to a better understanding of the mechanisms underlying the effects seen after inhibition of mPGES-1 in models of inflammation and cardiovascular disease

Regulation of microsomal prostaglandin E2 synthase by cyclopentenone prostaglandins in colon cancer cells

Prostaglandin E2 is the major prostaglandin involved in colorectal carcinogenesis. The biosynthesis of prostaglandin E2 is accomplished by several terminal prostaglandin E synthases through catalytical conversion of the cyclooxygenase product prostaglandin H2. Among the known terminal prostaglandin E synthases, microsomal prostaglandin E synthase type 1 and type 2 were found to be overexpressed in colorectal cancer, however the role and regulation of these enzymes in this tumor entity are yet not fully understood. Here we report that the cyclopentenone prostaglandins 15-deoxy-D12,14-prostaglandin J2 and prostaglandin A2, which have been shown to modulate cell growth and neoplasia, selectively down-regulate microsomal prostaglandin E synthase type 2 mRNA and protein expression in the human colorectal carcinoma cell lines Caco-2 and HCT 116. This effect appeared to be PPARgamma independent and was not found to require G-protein-coupled receptor activation. Instead, inhibition of microsomal prostaglandin E synthase type 2 by cyclopentenone prostaglandins may be mediated by covalent binding of the cyclopentenone ring to cysteine residues on signalling molecules or via a redox-dependent mechanism. Inhibition of microsomal prostaglandin E synthase type 2 was subsequently followed by decreased prostaglandin E synthase activity, which in turn contributed at least in part to the anti-proliferative action of cyclopentenone prostaglandins in HCT 116 cells. Collectively, these data unravel a novel mechanism for the growth-inhibitory effects of cyclopentenone prostaglandins and expose microsomal prostaglandin E synthase type 2 as a new potential target for pharmacological intervention in the treatment of colorectal cancer.Zahlreiche Untersuchungen von Prostaglandinen (PG) unterstreichen die herausragende Bedeutung von PGE2 in der Ätiopathogenese des kolorektalen Karzinoms. Drei terminale PGE2 Synthasen (PGES) sind jetzt bekannt. Die Expression von ein perinukleäres membrangebundenes Enzym, mPGES-1, wird durch proinflammatorische Stimuli gesteigert und ist dabei zumeist an die der COX-2 gekoppelt. Die cytosolische Form von PGES (cPGES) in einer Vielzahl von Zelltypen konstitutiv exprimiert. Ein zweites membran-assoziiertes PGES (mPGES-2) wird in einer Vielzahl von Zellen konstitutiv exprimiert. Beide mikrosomale PGES beim humanen kolorektalen Karzinom überexprimiert. Cyclopentenon-Prostaglandine sind durch eine Cyclopentenon-Ringstruktur mit chemisch reaktionsfähiger ungesätigten Carbonyl-Gruppe charakterisiert. Verschiedene Vertreter dieser Prostaglandin-Familie weisen antineoplastische, antiinflammatorische und antivirale Aktivitäten auf. In der vorliegenden Arbeit sollte die mögliche Wirkung von ein Cyclopentenon-Protaglandin 15-deoxy-D12,14-Prostaglandin J2 (15d-PGJ2) auf die PGE2-vermittelte Entstehung des kolorektalen Karzinoms untersucht werden. Die Wirkung von 15d-PGJ2 auf die Gen- und Proteinexpression von cPGES, mPGES-1 und mPGES-2 sowie COX-1 und COX-2, wie auch die PGES-Aktivität wurde in den beiden kolorektalen Tumorzelllinine HCT 116 und Caco-2 ( Caco-2-Zelllinie exprimiert keine COX-1, HCT 116-Zellen exprimiert keine COX-2 ) untersucht. Dabei zeigte sich eine selektive Hemmung der Expression von mPGES-2 durch 15d-PGJ2 an beiden Zelllinine ohne Effekt auf andere, an der PGES beteiligten Gene. Parallel zur Reduktion der mRNA-Expression fand sich eine zeitlich verzögerte Abnahme der Proteinkonzentration von mPGES-2 und konnte auch eine signifikante Abnahme der enzymatischen Aktivität beobachtet werden. Darüber hinaus wiesen sowohl 15d-PGJ2 einen wachstumshemmenden. Um den Mechanismus der 15d-PGJ2-induzierten mPGES-2-Hemmung verstehen zu können, wurden einige bekannte Reaktionsmechanismen dieses Cyclopentenon untersucht. 15d-PGJ2 ist ein natürlicher Ligand des Peroxisome Proliferator-Activated Receptor gamma (PPARgamma). Die Wirkung eines weiteren PPARgamma-Agonisten, des Thiazolidinedionhomologs MCC555, auf die mPGES-2-mRNA-Expression untersucht wurde. Nach Behandlung von Caco-2- und HCT 116-Zellen mit MCC555 fand sich jedoch keine Veränderungen in der Genexpression von mPGES-2. Dieses Ergebnis wurde durch die Behandlung von Caco-2-Zellen, bei welchen die PPARgamma-Aktivität durch eine dominant negative Rezeptor gehemmt wurde, bestätigt. Eine Kontrolle der Regulation von mPGES-2 durch 15d-PGJ2 über den PPARγ-Signaltransduktionsweg kann somit ausgeschlossen werden. Als weitere Möglichkeit der Vermittlung 15d-PGJ2–spezifischer Zellefekte ist die Bindung an PGD2-Rezeptoren, wie dem DP1 und dem CRTH2 Rezeptor beschrieben. Die Aktivierung von DP1 führt über eine Stimulierung der Adenylcyclase-Aktivität, mit nachfolgender Steigerung der intrazellulären cAMP-Konzentration. Die Aktivierung von CRTH2 verstärkt die intrazellulare Kalziumfreisetzung. 15d-PGJ2 zeigte jedoch weder Effekte auf die intrazelluläre Kalziumkonzentration, noch übte cAMP eine regulatorische Wirkung auf die mPGES-2-Expression aus. Somit konnte eine Beteiligung dieser Rezeptoren an der Regulation von mPGES-2 durch 15d-PGJ2 ausgeschlossen werden. Zusammengefaßt deuten diese Ergebnisse darauf hin, daß die 15d-PGJ2-vermittelte Kontrolle von mPGES-2 möglicherweise nicht auf einem Mechanismus beruht, der spezifisch für dieses Cyclopentenon-Protaglandin ist, sondern vielmehr auf einem generellen Wirkprinzip von Cyclopentenon-Prostaglandinen beruht. So wird ein Teil der biologische Aktivität von Cyclopentenon-Prostaglandinen über oxidativen Stress ausgelöst. Um eine potentielle Beteiligung von oxidativem Stress an der durch 15d-PGJ2 vermittelten Expressionshemmung von mPGES-2 zu untersuchen, wurden Caco-2-Zellen und HCT 116-Zellen vor der 15d-PGJ2-Stimulation mit verschiedenen Antioxidantien behandelt. Die Hemmwirkung von 15d-PGJ2 auf die mPGES-2-Proteinexpression konnte sowohl mit DTT wie auch NAC komplett aufgehoben werden. Letztlich sind Cyclopentenon-Prostaglandine aufgrund der chemischen Eigenschaften ihrer typischen Ringstruktur zur direkten Interaktion mit zellulären Zielproteinen in der Lage. In der Tat war die Regulation von mPGES-2 nicht auf 15d-PGJ2 begrenzt. Auch PGA2, ein weiteres Cyclopentenon-Prostaglandin, übte eine der von 15d-PGJ2 vergleichbare biologische Aktivität auf die Expression von mPGES-2 in HCT 116 und Caco-2 aus. Andere Eikosanoide, die über jeweils keine Cyclopentenon-Struktur verfügen, zeigten keine regulatorischen Effekte auf Expression von mPGES-2. Mit den hier vorgestellten Resultaten wird das Spektrum der antiproliferativen Mechanismen von Cyclopentenon-Prostaglandine erweitert. Darüber hinaus wurde mit der PGES-2 ein neues interessantes Zielprotein in der Theraopie des kolorektalen Karzinoms etabliert

In Silico Approaches for the Identification of Novel Inhibitors for Mitochondrial Prostaglandin E Synthase (Mpges) – 1

In the present study, an in silico approach using similarity search and molecular docking methods are utilized to identify new inhibitors of mPGES-1. The initial study revealed from these two approaches top 5 molecules found to occupy the binding pocket of mPGES-1 and form electrostatic interaction with amino acids and water molecules. The selected 5 molecules also showed favourable docking score and interaction with the enzyme. These compounds can be further considered in-vitro testing. The approach opens up new scaffolds as inhibitors of mPGES-1

Endogenous and exogenous modulation of 5-lipoxygenase: impact of pregnancy, menstrual cycle and pharmacological inhibitors

This thesis comprises two parts both dealing with the modulation of 5-lipoxygenase (5-LO), the enzyme responsible for the synthesis of pro-inflammatory leukotrienes (LT). First, the impact of pregnancy was studied. LT formation was higher in blood from pregnant compared to non-pregnant females. This higher LT synthesis in blood of pregnant females is influenced by synergistic and opposite effects: (I) higher numbers of LT forming cells (“plus”), (II) lower LT formation capacity of isolated granulocytes (“minus”) and (III) upregulating effects of plasma from pregnant donors on LT formation from isolated enzyme and cells (“plus”). These results suggest that LTs might be involved in the immune regulation during pregnancy. In the second part dealing with benzoquinones as exogenous modulators of 5-LO new inhibitors were identified and their molecular mode of inhibition characterized. The natural compound embelin acted as potent inhibitor of 5-LO and microsomal prostaglandin E2 synthase-1 (IC50 = 0.06 and 0.2 µM). New targets of embelin are presented for which lower concentrations are needed for inhibition than for others described before. Besides embelin, the benzoquinone RF-Id was studied in cell-free, cellular and blood assays for its interference with LT synthesis and identified as potent 5-LO inhibitor. Mechanistic studies showed that embelin and RF-Id, though similarly structured, interfered with 5-LO in different modes. Interestingly, both inhibitors do not inhibit via redox-type 5-LO inhibition as often supposed for benzoquinones in literature. After being activated by reduction in the cell RF-Id interacts with 5-LO in a nonredox-type fashion. Structure activity relationships of 31 embelin derived benzoquinones all modified in backbone position 3 with alkyl or prenyl chains revealed distinct features for potent inhibition of 5-LO. The most potent compounds were ortho-quinones (IC50 = 0.03 to 0.06 µM) which are valuable for future pharmacological studies

Microcirculation in chronic kidney disease : from injury targets to potential therapeutics

Chronic kidney disease (CKD) is a major public health problem worldwide, and patients with end-stage kidney disease (ESKD) are at increased risk of developing cardiovascular complications. Small artery dysfunction is a common feature of CKD, which contributes to the development of early vascular ageing (EVA) and cardiovascular complications. The exact mechanisms of how small artery dysfunction contributes to these complications are not fully understood. The ultimate goal of this PhD thesis is to gain a better understanding on how small artery dysfunction contributes to EVA and cardiovascular risk in the uremic environment, and to define specific targets for potential therapeutic benefit. The methodological approaches involve both ex vivo and in vivo investigations, including biochemical marker measurements, immuno staining, as well as isolated small artery bioassays and wire myography technique together with EndoPAT to assess peripheral arterial tone. In Paper I we investigated both in vivo and ex vivo functional properties (reactive hyperemia index [RHI], contractility, vasodilatory, stiffness) of small resistance arteries from ESKD patients and non-CKD controls. We also investigated ex vivo effects of trimethylamine Noxide (TMAO), phenylacetyl glutamine (PAG) and extracellular vesicles (EVs) from CKD-5 patients, as well as pharmacological interventions using senolytics. We assessed markers of senescence, calcification, endothelial function, and oxidative stress; these data were also correlated with functional and structural properties of resistance arteries. We observed that the uremic environment influences vascular function by changing the contribution of endotheliumderived factors (i.e. reduced nitric oxide and increased endothelium derived hyperpolarization factor) and increasing vascular stiffness in patients with ESKD; these events were further modulated by inflammation, TMAO, PAG and EVs. Moreover, the vasculature of ESKD patients was characterized by hallmarks of EVA –presence of the senescence signature, microcalcification, reduced anti-oxidant control, and decreased contractile markers which might confer the development of cardiovascular complications in this specific patient group. We also showed that senolytics could be used to target senescent cells. As Paper I comprehensively phenotypes the microcirculation from ESKD patients, this study serves as a backbone for the overall thesis. Paper II, a complementary study of Paper I, adds more insight into endothelial function and vascular structure biology in respect to different amino acids (AA) and their metabolites. New findings include impaired AA metabolism with decreased biopterin BH4/BH2 ratio in CKD, as well as elevated asymmetric dimethylarginine levels that were associated with higher vascular stiffness and reduced NO contribution. In Paper III, we investigated differences in the expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) receptors in resistance arteries and subcutaneous adipose tissue, alongside circulating soluble ACE2 levels in female and male ESKD patients versus non-CKD controls. Our results demonstrated that soluble ACE2 levels were higher in ESKD patients. In addition, ACE2 tissue expression was higher in ESKD patients with a higher prevalence in male subjects and was present in both the endothelium and VSMCs from arteries in peripheral microcirculation. The aim of Paper IV was to better understand the role of prostaglandin contribution to vasoactive properties and characterize the effects of microsomal prostaglandin E synthase-1 (mPGES-1) inhibition in the microvasculature of CKD patients. A significant reduction in adrenergic vasoconstriction and improvement in relaxation was observed following mPGES-1 inhibition. Based on our findings, it can be inferred that mPGES-1 inhibition has additional vasoactive effects in the human microcirculation beyond the shunting to prostacyclin (PGI2) pathway, i.e. a reduction in the levels of local prostaglandin E2 (PGE2), as well as influencing other vascular factors. This indicates the interaction of several pathways after mPGES-1 inhibition. The findings of this thesis provide valuable insights into the mechanisms underlying small artery maintenance and dysfunction in ESKD patients and identify potential therapeutic targets for improving vascular function in this patient population

PGE2 and other lipids in rheumatic diseases

Despite numerous options for treatment of rheumatic diseases, there is an unfulfilled clinical need for therapeutic strategies that can reduce inflammation and prevent tissue destruction. Lipid mediators (eicosanoids and fatty acids (FA)) are involved in the regulation of inflammatory processes and contribute to the pathogenesis of rheumatic diseases. Thus, selective targeting of the lipid mediators might enable improved antiinflammatory treatment. Microsomal prostaglandin synthase (mPGES) -1 produces prostaglandin E2 (PGE2) at sites of inflammation in rheumatic diseases. Inhibitors of mPGES-1 have been proposed as a more selective anti-inflammatory treatment retaining the therapeutic potential of non-steroidal anti-inflammatory drugs (NSAIDs) but with less severe side effects associated with NSAIDs. However, the impact of mPGES-1 inhibition on different pathological and physiological processes is not completely elucidated. Moreover, chronic inflammation might cause dysregulation of lipid and FA metabolism that may contribute to skeletal muscle weakness in patients with polymyositis (PM) and dermatomyositis (DM). The major aim of this thesis was to gain better understanding of the regulation of PGE2 and other lipid mediators in RA, PM and in DM to improve treatment of patients. First, we have determined the catalytic mechanism of mPGES-1 activity by site-directed mutagenesis (Paper I). The amino acid residues arginine (Arg) 126 and aspartate (Asp) 49 were identified as essential for the catalytic activity of mPGES-1, as when exchanged, the enzyme variants lost their enzymatic activity. Previous high-resolution structural studies predicted a role for serine (Ser) 127 in the enzymatic activity of mPGES-1. In contrast, we have demonstrated that Ser127, as well as Arg73, do not seem to be significant to the catalytic mechanism because when exchanged, their variants retained considerable activity. These results are of relevance for the development of the new generation of mPGES-1 inhibitors. Further, we studied whether mPGES-1 deletion might be beneficial for reducing inflammation via the suppression of platelet functions (Paper II). Platelet activation, the formation of platelet-leukocyte aggregates, and release of platelet-derived microparticles (PMP) were significantly reduced in mPGES-1 KO mice compared to WT after lipopolysaccharide (LPS) treatment. In addition, KO mice displayed a significant decrease in platelet aggregation ex vivo. The reduced activation of platelets may contribute to antiinflammatory effect and cardiovascular safety of mPGES-1 inhibitors. In Paper III, we investigated effects of mPGES-1, PGIS, and cyclooxygenase (COX) -2 on vascular and renal pathways associated with asymmetric dimethylarginine (ADMA) and endothelial nitric oxide synthase (eNOS). WT mice treated with COX-2 inhibitor displayed no change in the plasma levels of cardioprotective prostacyclin (PGI2), while mPGES-1 KO mice showed significantly higher PGI2 levels in the plasma. In contrast to COX-2 inhibition, mPGES-1 deletion had no effect on genes responsible for the production or breakdown of ADMA in the kidney. Plasma creatinine and ADMA were elevated in mice treated with COX-2 inhibitor or PGIS KO mice but unaltered in mPGES-1 KO mice. Furthermore, the deletion of mPGES-1 significantly improved the eNOS-driven dilator response to acetylcholine in the aorta. These data further confirmed the cardioprotective effects of mPGES-1 deletion suggesting selective inhibitors of mPGES-1 as a safer alternative to NSAIDs. To clarify mechanisms involved in muscle weakness, we examined effects of the conventional immunosuppressive treatment on global gene expression profiles in skeletal muscle from PM and DM patients (Paper IV). The genes related to immune response and inflammation including the interferon and the inflammasome pathways were downregulated by treatment. The genes involved in muscle tissue remodeling and growth were negatively affected by treatment. The immunosuppressive treatment caused an induction of gene markers of fast type II fibers. Furthermore, the fiber composition of the muscle tissue from patients was switched towards type II fibers after treatment. Importantly, the expression of genes involved in lipid metabolism was altered, signifying a probable lipotoxic effect on muscles, that at least partly might explain the persistent muscle weakness and fatigue observed in PM and DM patients despite treatment. To confirm dysregulated lipid metabolism in myositis patients, we analyzed lipid and FA profiles in serum from patients with PM and DM in comparison to healthy individuals and response to immunosuppressive treatment (Paper V). FA composition of total serum lipids was changed in myositis patients compared to healthy individuals. In myositis patients, the levels of palmitic 16:0 acid was significantly higher while the levels of arachidonic 20:4(n- 6) acid was significantly lower. The levels of serum lipid species within phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and triglycerides (TG) were also significantly changed in myositis patients compared to healthy individuals. Immunosuppressive treatment resulted in increased serum levels of C20:2(n-6) acid and C20:5(n-3) acids as well as in the changed serum PC, phosphatidylethanolamine (PE) and LPC profiles in myositis patients. In conclusion, in this thesis, we have provided new knowledge on the catalytic mechanism and the impact of mPGES-1 on inflammation and cardiovascular safety. Furthermore, we have demonstrated that lipid metabolism is altered in PM and DM patients and might contribute to disease pathogenesis

Microsomal prostaglandin E synthase-1 is involved in the metabolic and cardiovascular alterations associated with obesity

Background and Purpose: Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible isomerase responsible for prostaglandin E2 production in inflammatory con ditions. We evaluated the role of mPGES-1 in the development and the metabolic and cardiovascular alterations of obesity. Experimental Approach: mPGES-1+/+ and mPGES-1 / mice were fed with normal or high fat diet (HFD, 60% fat). The glycaemic and lipid profile was evaluated by glu cose and insulin tolerance tests and colorimetric assays. Vascular function, structure and mechanics were assessed by myography. Histological studies, q-RT-PCR, and western blot analyses were performed in adipose tissue depots and cardiovascular tissues. Gene expression in abdominal fat and perivascular adipose tissue (PVAT) from patients was correlated with vascular damage. Key Results: Male mPGES-1 / mice fed with HFD were protected against body weight gain and showed reduced adiposity, better glucose tolerance and insulin sensi tivity, lipid levels and less white adipose tissue and PVAT inflammation and fibrosis, compared with mPGES-1+/+ mice. mPGES-1 knockdown prevented cardiomyocyte hypertrophy, cardiac fibrosis, endothelial dysfunction, aortic insulin resistance, and vascular inflammation and remodelling, induced by HFD. Obesity-induced weight gain and endothelial dysfunction of resistance arteries were ameliorated in female mPGES-1 / mice. In humans, we found a positive correlation between mPGES-1 expression in abdominal fat and vascular remodelling, vessel stiffness, and systolic blood pressure. In human PVAT, there was a positive correlation between mPGES-1 expression and inflammatory markers. Conclusions and Implications: mPGES-1 inhibition might be a novel therapeutic approach to the management of obesity and the associated cardiovascular and meta bolic alterations

Prostaglandins in Cancer Cell Adhesion, Migration, and Invasion

Prostaglandins exert a profound influence over the adhesive, migratory, and invasive behavior of cells during the development and progression of cancer. Cyclooxygenase-2 (COX-2) and microsomal prostaglandin E2 synthase-1 (mPGES-1) are upregulated in inflammation and cancer. This results in the production of prostaglandin E2 (PGE2), which binds to and activates G-protein-coupled prostaglandin E1–4 receptors (EP1–4). Selectively targeting the COX-2/mPGES-1/PGE2/EP1–4 axis of the prostaglandin pathway can reduce the adhesion, migration, invasion, and angiogenesis. Once stimulated by prostaglandins, cadherin adhesive connections between epithelial or endothelial cells are lost. This enables cells to invade through the underlying basement membrane and extracellular matrix (ECM). Interactions with the ECM are mediated by cell surface integrins by “outside-in signaling” through Src and focal adhesion kinase (FAK) and/or “inside-out signaling” through talins and kindlins. Combining the use of COX-2/mPGES-1/PGE2/EP1–4 axis-targeted molecules with those targeting cell surface adhesion receptors or their downstream signaling molecules may enhance cancer therapy