Synergistic prostaglandin E synthesis by myeloid and endothelial cells promotes fetal hematopoietic stem cell expansion in vertebrates.

During development, hematopoietic stem cells (HSCs) are produced from the hemogenic endothelium and will expand in a transient hematopoietic niche. Prostaglandin E2 (PGE2) is essential during vertebrate development and HSC specification, but its precise source in the embryo remains elusive. Here, we show that in the zebrafish embryo, PGE2 synthesis genes are expressed by distinct stromal cell populations, myeloid (neutrophils, macrophages), and endothelial cells of the caudal hematopoietic tissue. Ablation of myeloid cells, which produce the PGE2 precursor prostaglandin H2 (PGH2), results in loss of HSCs in the caudal hematopoietic tissue, which could be rescued by exogeneous PGE2 or PGH2 supplementation. Endothelial cells contribute by expressing the PGH2 import transporter slco2b1 and ptges3, the enzyme converting PGH2 into PGE2. Of note, differential niche cell expression of PGE2 biosynthesis enzymes is also observed in the mouse fetal liver. Taken altogether, our data suggest that the triad composed of neutrophils, macrophages, and endothelial cells sequentially and synergistically contributes to blood stem cell expansion during vertebrate development.

Nonsteroidal anti-inflammatory drugs as a targeted therapy for bone marrow failure in Ghosal hematodiaphyseal dysplasia.

Advances in genomic diagnostics hold promise for improved care of rare hematologic diseases. Here, we describe a novel targeted therapeutic approach for Ghosal hematodiaphyseal dysplasia, an autosomal recessive disease characterized by severe normocytic anemia and bone abnormalities due to loss-of-function mutations in thromboxane A synthase 1 (TBXAS1). TBXAS1 metabolizes prostaglandin H2 (PGH2), a cyclooxygenase (COX) product of arachidonic acid, into thromboxane A2. Loss-of-function mutations in TBXAS result in an increase in PGH2 availability for other PG synthases. The current treatment for Ghosal hematodiaphyseal dysplasia syndrome consists of corticosteroids. We hypothesize that nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit COX-1 and COX-2, could ameliorate the effects of TBXAS1 loss and improve hematologic function by reducing prostaglandin formation. We treated 2 patients with Ghosal hematodiaphyseal dysplasia syndrome, an adult and a child, with standard doses of NSAIDs (aspirin or ibuprofen). Both patients had rapid improvements concerning hematologic parameters and inflammatory markers without adverse events. Mass spectrometry analysis demonstrated that urinary PG metabolites were increased along with proinflammatory lipoxygenase (LOX) products 5-hydroxyeicosatetraenoic acid and leukotriene E4. Our data show that NSAIDs at standard doses surprisingly reduced both COX and LOX products, leading to the resolution of cytopenia, and should be considered for first-line treatment for Ghosal hematodiaphyseal dysplasia syndrome.

Substrate-induced product-release mechanism of lipocalin-type prostaglandin D synthase.

Prostaglandin D2 (PGD2), an endogenous somnogen, is a unique PG that is secreted into the cerebrospinal fluid. PGD2 is a relatively fragile molecule and should be transported to receptors localized in the basal forebrain without degradation. However, it remains unclear how PGD2 is stably carried to such remote receptors. Here, we demonstrate that the PGD2-synthesizing enzyme, Lipocalin-type prostaglandin D synthase (L-PGDS), binds not only its substrate PGH2 but also its product PGD2 at two distinct binding sites for both ligands. This behaviour implys its PGD2 carrier function. Nevertheless, since the high affinity (Kd = âˆ¼0.6 µM) of PGD2 in the catalytic binding site is comparable to that of PGH2, it may act as a competitive inhibitor, while our binding assay exhibits only weak inhibition (Ki = 189 µM) of the catalytic reaction. To clarify this enigmatic behavior, we determined the solution structure of L-PGDS bound to one substrate analog by NMR and compared it with the two structures: one in the apo form and the other in substrate analogue complex with 1:2 stoichiometry. The structural comparisons showed clearly that open or closed forms of loops at the entrance of ligand binding cavity are regulated by substrate binding to two sites, and that the binding to a second non-catalytic binding site, which apparently substrate concentration dependent, induces opening of the cavity that releases the product. From these results, we propose that L-PGDS is a unique enzyme having a carrier function and a substrate-induced product-release mechanism.

Prostaglandin Endoperoxide H Synthase-2 (PGHS-2) Variants and Risk of Obesity and Microvascular Dysfunction Among Tunisians: Relevance of rs5277 (306G/C) and rs5275 (8473T/C) Genetic Markers.

The purpose of this study was to determine the impact of six PGHS-2 genetic variants on obesity development and microvascular dysfunction. The study included 305 Tunisian subjects (186 normal weights, 35 overweights and 84 obeses). PCR analyses were used for allelic discrimination between polymorphisms. Prostaglandin (PGE2, PGI2), leptin, and matrix metalloproteinase (MMP1, 2, 3, 9) levels were evaluated by ELISA. Fatty acid composition was performed by gas chromatography-mass spectrometry. Our results revealed that subjects carrying the PGHS-2 306CC (rs5277) and 8473CC (rs5275) genotypes present higher anthropometric values compared to wild-type genotypes (306GG, BMI (Kg/m2): 27.11 ± 0.58; WC (cm): 93.09 ± 1.58; 306CC, BMI: 33.83 ± 2.46; WC: 109.93 ± 5.41; 8473TT, BMI: 27.75 ± 0.68; WC: 93.96 ± 1.75; 8473CC, BMI: 33.72 ± 2.2; WC: 117.89 ± 2.94). A reduced microvascular reactivity and a higher PGE2 level were also found in individuals with the 306CC and 8473CC genotypes in comparison to 306GG and 8473TT carriers (306GG, Peak Ach-CVC (PU/mmHg): 0.46 ± 0.03; PGE2 (pg/ml): 7933.1 ± 702; 306CC, Peak Ach-CVC: 0.24 ± 0.01; PGE2: 13,380.3 ± 966.2; 8473TT, Peak Ach-CVC: 0.48 ± 0.05; PGE2: 7086.41 ± 700.31; 8473CC, Peak Ach-CVC: 0.23 ± 0.01; PGE2: 13,175.7 ± 1165.8). Fatty acid analysis showed a significant increase of palmitic acid (PA) (34.2 ± 2.09 vs. 16.82% ± 1.76, P < 0.001), stearic acid (SA) (25.76 ± 3.29 vs. 9.05% ± 2.53, P < 0.001), and linoleic acid (LA) (5.25 ± 1.18 vs. 0.5% ± 0.09, P < 0.001) levels in individuals carrying the PGHS-2 306CC genotype when compared to GG genotype individuals. Subjects with the 8473CC genotype showed also a significant increase of PA, SA ,and LA levels when compared to TT genotype carriers (PA: 38.02 ± 1.51 vs. 12.65% ± 1.54, P < 0.001; SA: 32.96 ± 1.87 vs. 1.38% ± 0.56, P < 0.001; LA: 26.84 ± 2.09 vs. 3.7% ± 1.54, P < 0.001). Logistic regression analysis revealed that PGHS-2 306CC and 8473CC variants are significantly associated with obesity status (OR 6.25, CI (1.8-21.6), P = 0.004; OR 3.01, CI (1.13-8.52), P = 0.03, respectively). Haplotypes containing the C306:T8473 (OR 2.91; P = 0.01) and G306:C8473 (OR 5.25; P = 0.002) combinations were associated with an enhanced risk for obesity development in the studied population. In conclusion, our results highlight that PGHS-2 306G/C and 8473T/C variants could be useful indicators of obesity development, inflammation, and microvascular dysfunction among Tunisians.

A seven-step plan for becoming a moderately rich and famous biochemist.

Omega-6 polyunsaturated fatty acids were identified as essential nutrients in 1930. Their essentiality is largely due to their function as prostaglandin (PG) precursors. I spent most of my career in biochemistry determining how PG biosynthesis is regulated. PGs are lipid mediators formed in response to certain circulating hormones and cytokines. PGs act near their sites of synthesis to signal neighboring cells to coordinate their responses (e.g. when platelets interact with blood vessels). The committed step in PG synthesis is the conversion of a 20-carbon omega-6 fatty acid called arachidonic acid to prostaglandin endoperoxide H2 (PGH2). Depending on the tissue and the hormone or cytokine stimulus, this reaction is catalyzed by either cyclooxygenase-1 or cyclooxygenase-2 (COX-1 or COX-2). Once formed, PGH2 is converted, again depending on the context, to one of several downstream PG subtypes that act via specific G protein-coupled receptors. Nonsteroidal anti-inflammatory drugs (e.g. aspirin, ibuprofen, and naproxen) block PG synthesis by inhibiting COX-1 and COX-2. COX-2 is also inhibited by COX-2-selective inhibitors. Inhibition of COX-1 by low-dose aspirin prevents thrombosis. COX-2 inhibition reduces inflammation and pain. Investigating the mysteries of COXs anchored my scientific career. I attribute my successes to the great good fortune of having been surrounded by people who helped me make the most of my talents. I have written this reflection in a light-hearted fashion as a self-help essay, while highlighting the people and factors that most impacted me during my upbringing and then during my maturation and evolution as a biochemist.

A novel single-chain enzyme complex with chain reaction properties rapidly producing thromboxane A2 and exhibiting powerful anti-bleeding functions.

Uncontrollable bleeding is still a worldwide killer. In this study, we aimed to investigate a novel approach to exhibit effective haemostatic properties, which could possibly save lives in various bleeding emergencies. According to the structure-based enzymatic design, we have engineered a novel single-chain hybrid enzyme complex (SCHEC), COX-1-10aa-TXAS. We linked the C-terminus of cyclooxygenase-1 (COX-1) to the N-terminus of the thromboxane A2 (TXA2 ) synthase (TXAS), through a 10-amino acid residue linker. This recombinant COX-1-10aa-TXAS can effectively pass COX-1-derived intermediate prostaglandin (PG) H2 (PGH2 ) to the active site of TXAS, resulting in an effective chain reaction property to produce the haemostatic prostanoid, TXA2 , rapidly. Advantageously, COX-1-10aa-TXAS constrains the production of other pro-bleeding prostanoids, such as prostacyclin (PGI2 ) and prostaglandin E2 (PGE2 ), through reducing the common substrate, PGH2 being passed to synthases which produce aforementioned prostanoids. Therefore, based on these multiple properties, this novel COX-1-10aa-TXAS indicated a powerful anti-bleeding ability, which could be used to treat a variety of bleeding situations and could even be useful for bleeding prone situations, including nonsteroidal anti-inflammatory drugs (NSAIDs)-resulted TXA2 -deficient and PGI2 -mediated bleeding disorders. This novel SCHEC has a great potential to be developed into a biological haemostatic agent to treat severe haemorrhage emergencies, which will prevent the complications of blood loss and save lives.

Different Fatty Acids Compete with Arachidonic Acid for Binding to the Allosteric or Catalytic Subunits of Cyclooxygenases to Regulate Prostanoid Synthesis.

Prostaglandin endoperoxide H synthases (PGHSs), also called cyclooxygenases (COXs), convert arachidonic acid (AA) to PGH2. PGHS-1 and PGHS-2 are conformational heterodimers, each composed of an (Eallo) and a catalytic (Ecat) monomer. Previous studies suggested that the binding to Eallo of saturated or monounsaturated fatty acids (FAs) that are not COX substrates differentially regulate PGHS-1 versus PGHS-2. Here, we substantiate and expand this concept to include polyunsaturated FAs known to modulate COX activities. Non-substrate FAs like palmitic acid bind Eallo of PGHSs stimulating human (hu) PGHS-2 but inhibiting huPGHS-1. We find the maximal effects of non-substrate FAs on both huPGHSs occurring at the same physiologically relevant FA/AA ratio of ∼20. This inverse allosteric regulation likely underlies the ability of PGHS-2 to operate at low AA concentrations, when PGHS-1 is effectively latent. Unlike FAs tested previously, we observe that C-22 FAs, including ω-3 fish oil FAs, have higher affinities for Ecat than Eallo subunits of PGHSs. Curiously, C-20 ω-3 eicosapentaenoate preferentially binds Ecat of huPGHS-1 but Eallo of huPGHS-2. PGE2 production decreases 50% when fish oil consumption produces tissue EPA/AA ratios of ≥0.2. However, 50% inhibition of huPGHS-1 itself is only seen with ω-3 FA/AA ratios of ≥5.0. This suggests that fish oil-enriched diets disfavor AA oxygenation by altering the composition of the FA pool in which PGHS-1 functions. The distinctive binding specificities of PGHS subunits permit different combinations of non-esterified FAs, which can be manipulated dietarily, to regulate AA binding to Eallo and/or Ecat thereby controlling COX activities.

Interactions of 2-O-arachidonylglycerol ether and ibuprofen with the allosteric and catalytic subunits of human COX-2.

Prostaglandin (PG) endoperoxide H synthase (PGHS)-2, also known as cyclooxygenase (COX)-2, can convert arachidonic acid (AA) to PGH2 in the committed step of PG synthesis. PGHS-2 functions as a conformational heterodimer composed of an allosteric (Eallo) and a catalytic (Ecat) monomer. Here we investigated the interplay between human (hu)PGHS-2 and an alternative COX substrate, the endocannabinoid, 2-arachidonoylglycerol (2-AG), as well as a stable analog, 2-O-arachidonylglycerol ether (2-AG ether). We also compared the inhibition of huPGHS-2-mediated oxygenation of AA, 2-AG, and 2-AG ether by the well-known COX inhibitor, ibuprofen. When tested with huPGHS-2, 2-AG and 2-AG ether exhibit very similar kinetic parameters, responses to stimulation by FAs that are not COX substrates, and modes of inhibition by ibuprofen. The 2-AG ether binds Ecat more tightly than Eallo and, thus, can be used as a stable Ecat-specific substrate to examine certain Eallo-dependent responses. Ibuprofen binding to Eallo of huPGHS-2 completely blocks 2-AG or 2-AG ether oxygenation; however, inhibition by ibuprofen of huPGHS-2-mediated oxygenation of AA engages a combination of both allosteric and competitive mechanisms.

Identification of the two-phase mechanism of arachidonic acid regulating inflammatory prostaglandin E2 biosynthesis by targeting COX-2 and mPGES-1.

Through linking inducible cyclooxygenase (COX)-2 with microsomal prostaglandin E2 (PGE2) synthase-1 (mPGES-1), a Single-Chain Enzyme Complex (SCEC, COX-2-10aa-mPGES-1) was engineered to mimic a specific inflammatory PGE2 biosynthesis from omega-6 fatty acid, arachidonic acid (AA), by eliminating involvements of non-inducible COX-1 and other PGE2 synthases. Using the SCEC, we characterized coupling reactions between COX-2 and mPGES-1 at 1:1 ratio of inflammatory PGE2 production. AA demonstrated two phase activities to regulate inflammatory PGE2 production. In the first phase (<2 µM), AA was a COX-2 substrate and converted to increasing production of PGE2. In the second phase with a further increased AA level (2-10 µM), AA bound to mPGES-1 and inhibited the PGE2 production. The SCEC study was identical to the co-expression of COX-2 and mPGES-1. This was further confirmed by using mPGES-1 and PGH2 as a direct enzyme target and substrate, respectively. Furthermore, the carboxylic acid group of AA binding to R67 and R70 of mPGES-1 was identified by X-ray structure-based docking and mutagenesis. mPGES-1 mutants, R70A, R70K, R67A and R67K, lost 40-100% binding to [(14)C]-AA. To conclude, a cellular model, in which AA is involved in self-controlling initial initiating and later resolving inflammation by its two phase activities, was discussed.

Development of a scintillation proximity binding assay for high-throughput screening of hematopoietic prostaglandin D2 synthase.

Prostaglandin D2 synthase (PGDS) catalyzes the isomerization of prostaglandin H2 (PGH2) to prostaglandin D2 (PGD2). PGD2 produced by hematopoietic prostaglandin D2 synthase (H-PGDS) in mast cells and Th2 cells is proposed to be a mediator of allergic and inflammatory responses. Consequently, inhibitors of H-PGDS represent potential therapeutic agents for the treatment of inflammatory diseases such as asthma. Due to the instability of the PGDS substrate PGH2, an in-vitro enzymatic assay is not feasible for large-scale screening of H-PGDS inhibitors. Herein, we report the development of a competition binding assay amenable to high-throughput screening (HTS) in a scintillation proximity assay (SPA) format. This assay was used to screen an in-house compound library of approximately 280,000 compounds for novel H-PGDS inhibitors. The hit rate of the H-PGDS primary screen was found to be 4%. This high hit rate suggests that the active site of H-PGDS can accommodate a large diversity of chemical scaffolds. For hit prioritization, these initial hits were rescreened at a lower concentration in SPA and tested in the LAD2 cell assay. 116 compounds were active in both assays with IC50s ranging from 6 to 807 nM in SPA and 82 nM to 10 µM in the LAD2 cell assay.

Relationship of the Topological Distances and Activities between mPGES-1 and COX-2 versus COX-1: Implications of the Different Post-Translational Endoplasmic Reticulum Organizations of COX-1 and COX-2.

In vascular inflammation, prostaglandin E2 (PGE2) is largely biosynthesized by microsomal PGE2 synthase-1 (mPGES-1), competing with other downstream eicosanoid-synthesizing enzymes, such as PGIS, a synthase of a vascular protector prostacyclin (PGI2), to isomerize the cyclooxygenase (COX)-2-derived prostaglandin H2 (PGH2). In this study, we found that a majority of the product from the cells co-expressing human COX-2, mPGES-1, and PGIS was PGE2. We hypothesize that the molecular and cellular mechanisms are related to the post-translational endoplasmic reticulum (ER) arrangement of those enzymes. A set of fusion enzymes, COX-2-linker [10 amino acids (aa)]-PGIS and COX-2-linker (22 amino acids)-PGIS, were created as "The Bioruler", in which the 10 and 22 amino acids are defined linkers with known helical structures and distances (14.4 and 30.8 Å, respectively). Our experiments have shown that the efficiency of PGI2 biosynthesis was reduced when the separation distance increased from 10 to 22 amino acids. When COX-2-10aa-PGIS (with a 14.4 Å separation) was co-expressed with mPGES-1 on the ER membrane, a major product was PGE2, but not PGI2. However, expression of COX-2-10aa-PGIS and mPGES-1 on a separated ER with a distance of ≫30.8 Å reduced the level of PGE2 production. These data indicated that the mPGES-1 is "complex-likely" colocalized with COX-2 within a distance of 14.4 Å. In addition, the cells co-expressing COX-1-10aa-PGIS and mPGES-1 produced PGI2 mainly, but not PGE2. This indicates that mPGES-1 is expressed much farther from COX-1. These findings have led to proposed models showing the different post-translational ER organization between COX-2 and COX-1 with respect to the topological arrangement of the mPGES-1 during vascular inflammation.

Prostaglandin synthase activity of sigma- and mu-class glutathione transferases in a parasitic trematode, Clonorchis sinensis.

Sigma-class glutathione transferase (GST) proteins with dual GST and prostaglandin synthase (PGS) activities play a crucial role in the establishment of Clonorchis sinensis infection. Herein, we analyzed the structural and enzymatic properties of sigma-class GST (CsGST-σ) proteins to obtain insight into their antioxidant and immunomodulatory functions in comparison with mu-class GST (CsGST-µ) proteins. CsGST-σ proteins conserved characteristic structures, which had been described in mammalian hematopoietic prostaglandin D2 synthases. Recombinant forms of these CsGST-σ and CsGST-µ proteins expressed in Escherichia coli exhibited considerable degrees of GST and PGS activities with substantially different specific activities. All recombinant proteins displayed higher affinities toward prostaglandin H2 (PGS substrate; average Km of 30.7 and 3.0 µm for prostaglandin D2 [PGDS] and E2 synthase [PGES], respectively) than those toward CDNB (GST substrate; average Km of 1,205.1 µm). Furthermore, the catalytic efficiency (Kcat/Km) of the PGDS/PGES activity was higher than that of GST activity (average Kcat/Km of 3.1, 0.7, and 7.0×10-3 s-1µm-1 for PGDS, PGES, and GST, respectively). Our data strongly suggest that the C. sinensis sigma- and mu-class GST proteins are deeply involved in regulating host immune responses by generating PGD2 and PGE2 in addition to their roles in general detoxification.

Thromboxane A synthase-independent production of 12-hydroxyheptadecatrienoic acid, a BLT2 ligand.

12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) has long been considered a by-product of thromboxane A2 (TxA2) biosynthesis with no biological activity. Recently, we reported 12-HHT to be an endogenous ligand for BLT2, a low-affinity leukotriene B4 receptor. To delineate the biosynthetic pathway of 12-HHT, we established a method that enables us to quantify various eicosanoids and 12-HHT using LC-MS/MS analysis. During blood coagulation, 12-HHT levels increased in a time-dependent manner and were relatively higher than those of TxB2, a stable metabolite of TxA2. TxB2 production was almost completely inhibited by treatment with ozagrel, an inhibitor of TxA synthase (TxAS), while 12-HHT production was inhibited by 80-90%. Ozagrel-treated blood also exhibited accumulation of PGD2 and PGE2, possibly resulting from the shunting of PGH2 into synthetic pathways for these prostaglandins. In TxAS-deficient mice, TxB2 production during blood coagulation was completely lost, but 12-HHT production was reduced by 80-85%. HEK293 cells transiently expressing TxAS together with cyclooxygenase (COX)-1 or COX-2 produced both TxB2 and 12-HHT from arachidonic acid, while HEK293 cells expressing only COX-1 or COX-2 produced significant amounts of 12-HHT but no TxB2. These results clearly demonstrate that 12-HHT is produced by both TxAS-dependent and TxAS-independent pathways 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.

[Cyclooxygenases, lipoxygenases, their targeted drugs and the prevention of Alzheimer's disease].

Many studies have shown that chronic inflammation occurs in the brain of patients with Alzheimer's disease (AD). It is well known that long-term administration of non-steroidal anti-inflammatory drugs (NSAIDs) can alleviate the cognitive decline of AD patient and elderly. Several inflammatory cytokines produced in the metabolism of arachidonic acid (AA) are closely related to inflammatory diseases. Lipoxygenases (LOXs) and cyclooxygenases (COXs) play a crucial role in the AA network, the products eicosanoids have an important impact on the progression of AD. Although there are many arguments and conflicting evidence, currently LOXs and COXs are still the hot topics in the research on AD pathogenesis and drug development. Here, we review the progress in research on COXs and LOXs, including their actions on CNS and their association with AD, and explore the feasibility of LOXs and COXs as targets for the drugs to prevent and/or treat AD.

Cyclooxygenases and platelet functions.

Cyclooxygenase (COX) isozymes, i.e., COX-1 and COX-2, are encoded by separate genes and are involved in the generation of the same products, prostaglandin (PG)G2 and PGH2 from arachidonic acid (AA) by the COX and peroxidase activities of the enzymes, respectively. PGH2 is then transformed into prostanoids in a tissue-dependent fashion due to the different expression of downstream synthases. Platelets present almost exclusively COX-1, which generates large amounts of thromboxane (TX)A2, a proaggregatory and vasoconstrictor mediator. This prostanoid plays a central role in atherothrombosis, as shown by the benefit of the antiplatelet agent low-dose aspirin, a preferential inhibitor of platelet COX-1. Recent findings have shown the relevant role played by platelets and TXA2 in developing chronic inflammation associated with several diseases, including tissue fibrosis and cancer. COX-2 is induced in response to inflammatory and mitogenic stimuli to generate PGE2 and PGI2 (prostacyclin), in inflammatory cells. However, PGI2 is constitutively expressed in vascular cells in vivo and plays a crucial role in protecting the cardiovascular systems due to its antiplatelet and vasodilator effects. Here, platelets' role in regulating COX-2 expression in cells of the inflammatory microenvironment is described. Thus, the selective inhibition of platelet COX-1-dependent TXA2 by low-dose aspirin prevents COX-2 induction in stromal cells leading to antifibrotic and antitumor effects. The biosynthesis and functions of other prostanoids, such as PGD2, and isoprostanes, are reported. In addition to aspirin, which inhibits platelet COX-1 activity, possible strategies to affect platelet functions by influencing platelet prostanoid receptors or synthases are discussed.

LDL-Dependent Regulation of TNFα/PGE2 Induced COX-2/mPGES-1 Expression in Human Macrophage Cell Lines.

Inflammation is a hallmark in severe diseases such as atherosclerosis and non-alcohol-induced steatohepatitis (NASH). In the development of inflammation, prostaglandins, especially prostaglandin E2 (PGE2), are major players alongside with chemo- and cytokines, like tumor-necrosis-factor alpha (TNFα) and interleukin-1 beta (IL-1ß). During inflammation, PGE2 synthesis can be increased by the transcriptional induction of the two key enzymes: cyclooxygenase 2 (COX-2), which converts arachidonic acid to PGH2, and microsomal prostaglandin E2 synthase 1 (mPGES-1), which synthesizes PGE2 from PGH2. Both COX-2 and mPGES-2 were induced by a dietary intervention where mice were fed a fatty acid-rich and, more importantly, cholesterol-rich diet, leading to the development of NASH. Since macrophages are the main source of PGE2 synthesis and cholesterol is predominantly transported as LDL, the regulation of COX-2 and mPGES-1 expression by native LDL was analyzed in human macrophage cell lines. THP-1 and U937 monocytes were differentiated into macrophages, through which TNFα and PGE-2 induced COX-2 and mPGES-1 expression by LDL could be analyzed on both mRNA and protein levels. In addition, the interaction of LDL- and EP receptor signal chains in COX-2/mPGES-1 expression and PGE2-synthesis were analyzed in more detail using EP receptor specific agonists. Furthermore, the LDL-mediated signal transduction in THP-1 macrophages was analyzed by measuring ERK and Akt phosphorylation as well as transcriptional regulation of transcription factor Egr-1. COX-2 and mPGES-1 were induced in both THP-1 and U937 macrophages by the combination of TNFα and PGE2. Surprisingly, LDL dose-dependently increased the expression of mPGES-1 but repressed the expression of COX-2 on mRNA and protein levels in both cell lines. The interaction of LDL and PGE2 signal chains in mPGES-1 induction as well as PGE2-synthesis could be mimicked by through simultaneous stimulation with EP2 and EP4 agonists. In THP-1 macrophages, LDL induced Akt-phosphorylation, which could be blocked by a PI3 kinase inhibitor. Alongside blocking Akt-phosphorylation, the PI3K inhibitor inhibited LDL-mediated mPGES-1 induction; however, it did not attenuate the repression of COX-2 expression. LDL repressed basal ERK phosphorylation and expression of downstream transcription factor Egr-1, which might lead to inhibition of COX-2 expression. These findings suggest that simultaneous stimulation with a combination of TNFα, PGE2, and native LDL-activated signal chains in macrophage cell lines leads to maximal mPGES-1 activity, as well repression of COX-2 expression, by activating PI3K as well as repression of ERK/Egr-1 signal chains.

Microsomal prostaglandin e2 synthase-1 modulates the response to vascular injury.

BACKGROUND: Microsomal (m) prostaglandin (PG) E2 synthase (S)-1 catalyzes the formation of PGE2 from PGH2, a cyclooxygenase product that is derived from arachidonic acid. Previous studies in mice suggest that targeting mPGES-1 may be less likely to cause hypertension or thrombosis than cyclooxygenase-2-selective inhibition or deletion in vivo. Indeed, deletion of mPGES-1 retards atherogenesis and angiotensin II-induced aortic aneurysm formation. The role of mPGES-1 in the response to vascular injury is unknown. METHODS AND RESULTS: Mice were subjected to wire injury of the femoral artery. Both neointimal area and vascular stenosis were significantly reduced 4 weeks after injury in mPGES-1 knockout mice compared with wild-type controls (65.6 ± 5.7 versus 37.7 ± 5.1 × 10³ pixel area and 70.5 ± 13.4% versus 47.7 ± 17.4%, respectively; P < 0.01). Induction of tenascin-C, a proproliferative and promigratory extracellular matrix protein, after injury was attenuated in the knockouts. Consistent with in vivo rediversion of PG biosynthesis, mPGES-1-deleted vascular smooth muscle cells generated less PGE2 but more PGI2 and expressed reduced tenascin-C compared with wild-type cells. Both suppression of PGE2 and augmentation of PGI2 attenuate tenascin-C expression and vascular smooth muscle cell proliferation and migration in vitro. CONCLUSIONS: Deletion of mPGES-1 in mice attenuates neointimal hyperplasia after vascular injury, in part by regulating tenascin-C expression. This raises for consideration the therapeutic potential of mPGES-1 inhibitors as adjuvant therapy for percutaneous coronary intervention.

Functional analysis of human thromboxane synthase polymorphic variants.

BACKGROUND: Thromboxane A synthase (TXAS) metabolizes the cyclooxygenase product prostaglandin (PG) H2 into thromboxane H2 (TXA2), a potent inducer of blood vessel constriction and platelet aggregation. Nonsynonymous polymorphisms in the TXAS gene have the potential to alter TXAS activity and affect TXA2 generation. OBJECTIVES: The aim of this study was to assess the functional effects of genetic variants in the TXAS protein, including K258E, L357V, Q417E, E450K, and T451N. METHODS: Wild-type TXAS and the variant proteins were expressed in a bacterial system and purified by affinity and hydroxyapatite chromatography. The two characteristic catalytic activities of TXAS were assayed in each of the purified recombinant proteins: isomerization of PGH2 to TXA2 and fragmentation of PGH2 to 12-hydroxyheptadecatrienoic acid and malondialdehyde. RESULTS: All of the variants showed both isomerization and fragmentation activities. The Km values of the variants ranged from 27 to 52 µmol/l PGH2 (wild-type value: 32 µmol/l PGH2); the Vmax values of the variants ranged from 18 to 40 U/mg (wild-type value: 41 U/mg). The kinetic differences were largest for the L357V variant, whose Vmax/Km ratio was just 27% of the wild-type value. CONCLUSION: The increased Km and decreased Vmax values observed with L357V suggest that this variant may generate less TXA2 at the low levels of PGH2 expected in vivo, raising the possibility of attenuated signaling through the thromboxane pathway.

NMR resonance assignments of mouse lipocalin-type prostaglandin D synthase/prostaglandin J2 complex.

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH2 to produce PGD2, an endogenous somenogen, in the brains of various mammalians. We recently reported that various other PGs also bind to L-PGDS, suggesting that it could serve as an extracellular carrier for PGs. Although the solution and crystal structure of L-PGDS has been determined, as has the structure of L-PGDS complexed PGH2 analog, a structural analysis of L-PGDS complexed with other PGs is needed in order to understand the mechanism responsible for the PG trapping. Here, we report the nearly complete 1H, 13C, and 15N backbone and side chain resonance assignments of the L-PGDS/PGJ2 complex and the binding site for PGJ2 on L-PGDS.