5-Methoxyflavone-induced AMPKα activation inhibits NF-κB and P38 MAPK signaling to attenuate influenza A virus-mediated inflammation and lung injury in vitro and in vivo.

Influenza-related acute lung injury (ALI) is a life-threatening condition that results mostly from uncontrolled replication of influenza virus (IV) and severe proinflammatory responses. The methoxy flavonoid compound 5-methoxyflavone (5-MF) is believed to have superior biological activity in the treatment of cancer. However, the effects and underlying mechanism of 5-MF on IV-mediated ALI are still unclear. Here, we showed that 5-MF significantly improved the survival of mice with lethal IV infection and ameliorated IV-mediated lung edema, lung histological changes, and inflammatory cell lung recruitment. We found that 5-MF has antiviral activity against influenza A virus (IAV), which was probably associated with increased expression of radical S-adenosyl methionine domain containing 2 (RSAD2) and suppression of endosomal acidification. Moreover, IV-infected A549 cells with 5-MF treatment markedly reduced proinflammatory mediator expression (IL-6, CXCL8, TNF-α, CXCL10, CCL2, CCL3, CCL4, GM-CSF, COX-2, and PGE2) and prevented P-IKBα, P-P65, and P-P38 activation. Interestingly, we demonstrated that 5-MF treatment could trigger activation of AMP-activated protein kinase (AMPK)α in IV-infected A549 cells, as evidenced by activation of the AMPKα downstream molecule P53. Importantly, the addition of AMPKα blocker compound C dramatically abolished 5-MF-mediated increased levels of RSAD2, the inhibitory effects on H1N1 virus-elicited endosomal acidification, and the suppression expression of proinflammatory mediators (IL-6, TNF-α, CXCL10, COX-2 and PGE2), as well as the inactivation of P-IKBα, P-P65, and P-P38 MAPK signaling pathways. Furthermore, inhibition of AMPKα abrogated the protective effects of 5-MF on H1N1 virus-mediated lung injury and excessive inflammation in vivo. Taken together, these results indicate that 5-MF alleviated IV-mediated ALI and suppressed excessive inflammatory responses through activation of AMPKα signaling.

The Effects of High Energy Capsulotomy on Aqueous Cytokine Profiles and Pupil Size During Femtosecond Laser-Assisted Cataract Surgery.

PURPOSE: To assess whether aqueous cytokine profiles and pupil size are altered when high capsulotomy energy is used in eyes undergoing femtosecond laser-assisted cataract surgery (FLACS), and if preoperative use of a topical non-steroidal anti-inflammatory drug (NSAID) has an effect on this. METHODS: This prospective study recruited 83 eyes (63 patients) that were allocated to four treatment groups: conventional phacoemulsification (n = 20 eyes); FLACS with 90% capsulotomy energy without NSAID pretreatment (n = 20 eyes); FLACS with 90% capsulotomy energy with NSAID pre-treatment (n = 21 eyes); and FLACS with 150% capsulotomy energy with NSAID pretreatment (n = 22 eyes). Aqueous humor was collected before and after phacoemulsification to assess cytokine profiles. Pupil size was measured before and after laser capsulotomy. RESULTS: FLACS increased aqueous concentrations of pros-taglandin E2 (PGE2), interferon γ (IFN-γ), and interleukin 6 (IL-6) compared to conventional phacoemulsification. However, when increasing capsulotomy energy from 90% to 150% (with topical NSAID pretreatment), there was no significant increase in aqueous concentrations of PGE2 (37.7 ± 21.7 vs 33.6 ± 27.6 pg/mL, P = .99), IFN-γ (3.6 ± 1.1 vs 3.6 ± 0.8 pg/mL, P = .99), or IL-6 (7.1 ± 2.9 vs 6.3 ± 2.4 pg/mL, P = .99). For 90% and 150% capsulotomy energy, there was significant miosis following laser capsulotomy. Increased PGE2 concentration was significantly correlated with a reduction in pupil area (r = -0.58, P < .001) and pupil diameter (r = -0.57, P < .001). However, when a topical NSAID was given preoperatively, there was no difference in the degree of miosis between the 90% and 150% capsulotomy energy groups. CONCLUSIONS: Pretreatment with a topical NSAID prevented a rise in PGE2, IFN-γ, and IL-6 levels and excessive miosis when a higher capsulotomy energy was used. When a topical NSAID is used preoperatively, it is safe to use higher capsulotomy energy settings (with a low pulse energy femtosecond laser system) to achieve a satisfactory capsulotomy. [J Refract Surg. 2022;38(9):587-594.].

Factors secreted by monosodium urate crystal-stimulated macrophages promote a proinflammatory state in osteoblasts: a potential indirect mechanism of bone erosion in gout.

BACKGROUND: Tophi are lesions commonly present at sites of bone erosion in gout-affected joints. The tophus comprises a core of monosodium urate (MSU) crystals surrounded by soft tissue that contains macrophages and other immune cells. Previous studies found that MSU crystals directly reduce osteoblast viability and function. The aim of the current study was to determine the indirect, macrophage-mediated effects of MSU crystals on osteoblasts. METHODS: Conditioned medium from the RAW264.7 mouse macrophage cell line cultured with MSU crystals was added to the MC3T3-E1 mouse osteoblastic cell line. Conditioned medium from the THP-1 human monocytic cell line cultured with MSU crystals was added to primary human osteoblasts (HOBs). Matrix mineralization was assessed by von Kossa staining. Gene expression was determined by real-time PCR, and concentrations of secreted factors were determined by enzyme-linked immunosorbent assay. RESULTS: In MC3T3-E1 cells cultured for 13 days in an osteogenic medium, the expression of the osteoblast marker genes Col1a1, Runx2, Sp7, Bglap, Ibsp, and Dmp1 was inhibited by a conditioned medium from MSU crystal-stimulated RAW264.7 macrophages. Mineral staining of MC3T3-E1 cultures on day 21 confirmed the inhibition of osteoblast differentiation. In HOB cultures, the effect of 20 h incubation with a conditioned medium from MSU crystal-stimulated THP-1 monocytes on osteoblast gene expression was less consistent. Expression of the genes encoding cyclooxygenase-2 and IL-6 and secretion of the proinflammatory mediators PGE2 and IL-6 were induced in MC3T3-E1 and HOBs incubated with conditioned medium from MSU crystal-stimulated macrophages/monocytes. However, inhibition of cyclooxygenase-2 activity and PGE2 secretion from HOBs indicated that this pathway does not play a major role in mediating the indirect effects of MSU crystals in HOBs. CONCLUSIONS: Factors secreted from macrophages stimulated by MSU crystals attenuate osteoblast differentiation and induce the expression and secretion of proinflammatory mediators from osteoblasts. We suggest that bone erosion in joints affected by gout results from a combination of direct and indirect effects of MSU crystals.

Pueraria montana (Kudzu vine) Ameliorate the Inflammation and Oxidative Stress against Fe-NTA Induced Renal Cancer.

Renal tissue plays a crucial function in maintaining homeostasis, making it vulnerable to xenobiotic toxicity. Pueraria montana has more beneficial potential against the various diseases and has long history used as a traditional Chinese medicine. But its effect against the renal cancer not scrutinize. The goal of this study is to see if Pueraria montana can protect rats from developing kidney tumors caused by diethylnitrosamine (DEN) and ferric nitrite (Fe-NTA). Wistar rats was selected for the current study and DEN (use as an inducer) and Fe-NTA (promoter) for induction the renal cancer. For 22 weeks, the rats were given orally Pueraria montana (12.5, 25, and 50 mg/kg) treatment. At regular intervals, the body weight and food intake were calculated. The rats were macroscopically evaluated for identification of cancer in the renal tissue. The renal tumor makers, renal parameters, antioxidant enzymes, phase I and II enzymes, inflammatory cytokines and mediators were estimated at end of the experimental study. Pueraria montana treated rats displayed the suppression of renal tumors, incidence of the tumors along with suppression of tumor percentage. Pueraria montana treated rats significantly (p < 0.001) increased body weight and suppressed the renal weight and food intake. It also reduced the level of renal tumor marker ornithine decarboxylase (ODC) and [3H] thymidine incorporation along with suppression of renal parameter such as uric acid, blood urea nitrogen (BUN), urea and creatinine. Pueraria montana treatment significantly (p < 0.001) altered the level of phase enzymes and antioxidant. Pueraria montana treatment significantly (p < 0.001) repressed the level of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and improved the level of interleukin-10 (IL-10). Pueraria montana treatment suppressed the level of prostaglandin (PGE2), cyclooxygenase-2 (COX-2), nuclear kappa B factor (NF-κB) and transforming growth factor beta 1 (TGF-ß1). Pueraria montana suppressed the inflammatory necrosis, size the bowman capsules in the renal histopathology. Pueraria montana exhibited the chemoprotective effect via dual mechanism such as suppression of inflammatory reaction and oxidative stress.

Immunomodulatory kits generating leukaemia derived dendritic cells do not induce blast proliferation ex vivo: IPO-38 as a novel marker to quantify proliferating blasts in acute myeloid leukaemia.

(Leukaemia derived) dendritic cells (DC, DCleu) are potent stimulators of anti-leukaemic activity in acute myeloid leukaemia (AML) and can be generated with immunomodulatory kits containing granulocyte-macrophage-colony-stimulating-factor (GM-CSF), prostaglandin-E1 (PGE1), prostaglandin-E2 (PGE2) and/or picibanil (OK-321). Potential adverse effects initiated through kits, especially the proliferation of blasts, must be ruled out to ensure treatment safety. We quantified proliferating blasts with the proliferation markers CD71 and Ki-67 and the novel proliferation marker IPO-38 before and after kit treatment ex vivo. IPO-38 hereby appeared to be the most sensitive marker; a combination with CD71 may add value when assessing proliferation kinetics. Kit treatment did not or only slightly (<5%) induce blast proliferation in most cases. An induction of blast proliferation was only found in single cases and could be compensated by DCleu-induced anti-leukaemic activity in most times. Overall, we appraise kit treatment to be safe in vivo.

Imbalanced prostanoid release mediates cigarette smoke-induced human pulmonary artery cell proliferation.

BACKGROUND: Pulmonary hypertension is a common and serious complication of chronic obstructive pulmonary disease (COPD). Studies suggest that cigarette smoke can initiate pulmonary vascular remodelling by stimulating cell proliferation; however, the underlying cause, particularly the role of vasoactive prostanoids, is unclear. We hypothesize that cigarette smoke extract (CSE) can induce imbalanced vasoactive prostanoid release by differentially modulating the expression of respective synthase genes in human pulmonary artery smooth muscle cells (PASMCs) and endothelial cells (PAECs), thereby contributing to cell proliferation. METHODS: Aqueous CSE was prepared from 3R4F research-grade cigarettes. Human PASMCs and PAECs were treated with or without CSE. Quantitative real-time RT-PCR and Western blotting were used to analyse the mRNA and protein expression of vasoactive prostanoid syhthases. Prostanoid concentration in the medium was measured using ELISA kits. Cell proliferation was assessed using the cell proliferation reagent WST-1. RESULTS: We demonstrated that CSE induced the expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostanoid synthesis, in both cell types. In PASMCs, CSE reduced the downstream prostaglandin (PG) I synthase (PGIS) mRNA and protein expression and PGI2 production, whereas in PAECs, CSE downregulated PGIS mRNA expression, but PGIS protein was undetectable and CSE had no effect on PGI2 production. CSE increased thromboxane (TX) A synthase (TXAS) mRNA expression and TXA2 production, despite undetectable TXAS protein in both cell types. CSE also reduced microsomal PGE synthase-1 (mPGES-1) protein expression and PGE2 production in PASMCs, but increased PGE2 production despite unchanged mPGES-1 protein expression in PAECs. Furthermore, CSE stimulated proliferation of both cell types, which was significantly inhibited by the selective COX-2 inhibitor celecoxib, the PGI2 analogue beraprost and the TXA2 receptor antagonist daltroban. CONCLUSIONS: These findings provide the first evidence that cigarette smoke can induce imbalanced prostanoid mediator release characterized by the reduced PGI2/TXA2 ratio and contribute to pulmonary vascular remodelling and suggest that TXA2 may represent a novel therapeutic target for pulmonary hypertension in COPD.

Salicylic acid-based hypoxia-responsive chemodynamic nanomedicines boost antitumor immunotherapy by modulating immunosuppressive tumor microenvironment.

The delivery of salicylic acid or its derivatives to tumor tissue in the form of nanomedicine is critical for the studies on their potential synergistic mechanism in tumor therapy and chemoprevention considering the dangerous bleeding in the high-dose oral administration. To deepen the understanding of their role in adjusting immunosuppressive tumor microenvironment (ITM), herein, we firstly developed a hypoxia-sensitive Fe-5,5'-azosalicylic acid nanoscale coordination polymer nanomedicines (FeNCPs) via a "old drugs new tricks" strategy for synergistic chemodynamic therapy (CDT) and remodulation of ITM to elevate antitumor immunotherapy effect. PEGylated FeNCPs could be reductively cleaved to release 5-aminosalicylic acid (5-ASA) and ferric ions by azo-reductase under hypoxic conditions, which could induce tumor cell death by Fenton reaction-catalysis enhanced CDT and 5-ASA-converted carboxylquinone to promote the production of •OH. Meanwhile, cyclooxygenase-2 (COX-2) and its enzymatic product prostaglandin E2 (PGE2), as immune negative regulatory molecules, can promote tumor progression and immune tolerance. The released 5-ASA as a COX inhibitor could suppress the expression of PGE2, and Fe3+ was employed to reeducate M2-like tumor-associated macrophages (TAMs) to M1-like phenotype, which could initiate antitumor immune response to reach better antitumor immunotherapy. This work broadens the application of salicylic acid derivatives in antitumor immunotherapy, and provides a new strategy for their "old drugs new tricks". STATEMENT OF SIGNIFICANCE: Cyclooxygenase-2 (COX-2) and its enzymatic product prostaglandin E2 (PGE2), as immune negative regulatory molecules, facilitate the differentiation of immune cells into immunosuppressive cells to build the immunosuppressive tumor microenvironment, which can promote tumor progression and immune tolerance. Thus, down-regulation of COX-2/PGE2 expression may be a key approach to tumor treatments. Meanwhile, as a class of inhibitors of COX-2/PGE2, the potential mechanism of aspirin or 5-aminosalicylic acid has been a mystery in tumor therapy and chemoprevention. To expand the application of aspirin family nanomedicine in biomedicine, herein, we firstly developed a hypoxia-sensitive Fe-5,5'-azosalicylic acid nanoscale coordination polymer nanomedicines via a "old drugs new tricks" strategy for synergistic chemodynamic therapy and remodulation of immunosuppressive tumor microenvironment to elevate antitumor immunotherapy effect.

Immune activation of the monocyte-derived dendritic cells using patients own circulating tumor cells.

BACKGROUND: Dendritic cell (DC) therapy counts to the promising strategies how to weaken and eradicate cancer disease. We aimed to develop a good manufacturing practice (GMP) protocol for monocyte-derived DC (Mo-DC) maturation using circulating tumor cells lysates with subsequent experimental T-cell priming in vitro. METHODS: DC differentiation was induced from a population of immunomagnetically enriched CD14 + monocytes out of the leukapheresis samples (n = 6). The separation was provided automatically, in a closed bag system, using CliniMACS Prodigy® separation protocols (Miltenyi Biotec). For differentiation and maturation of CD14 + cells, DendriMACs® growing medium with supplements (GM-CSF, IL-4, IL-6, IL-1B, TNFa, PGE) was used. Immature Mo-DCs were loaded with autologous circulating tumor cell (CTCs) lysates. Autologous CTCs were sorted out by size-based filtration (MetaCell®) of the leukapheresis CD14-negative fraction. A mixture of mature Mo-DCs and autologous non-target blood cells (NTBCs) was co-cultured and the activation effect of mature Mo-DCs on T-cell activation was monitored by means of multimarker gene expression profiling. RESULTS: New protocols for mMo-DC production using automatization and CTC lysates were introduced including a feasible in vitro assay for mMo-DC efficacy evaluation. Gene expression analysis revealed elevation for following genes in NTBC (T cells) subset primed by mMo-DCs: CD8A, CD4, MKI67, MIF, TNFA, CD86, and CD80 (p ≤ 0.01). CONCLUSION: Summarizing the presented data, we might conclude mMo-DCs were generated using CliniMACS Prodigy® machine and CTC lysates in a homogenous manner showing a potential to generate NTBC activation in co-cultures. Identification of the activation signals in T-cell population by simple multimarker-qPCRs could fasten the process of effective mMo-DC production.

The effects of oil sands process-affected water naphthenic acid fraction components on GDF15 secretion in extravillous trophoblast cells.

Exposure to compounds present in petroleum and wastewaters from oil and gas extraction sites in the Alberta Oil Sands Region can impair reproductive health. It has been established that acid extractable organics found in oil sands process-affected water (OSPW) such as naphthenic acids (NA-fraction components; NAFC) can adversely affect reproductive outcomes. We have shown that NAFC exposure results in a significant upregulation of GDF15 in placental trophoblasts, a cellular stress marker known to be involved in human embryonic development and necessary for the maintenance of pregnancy. However, little is known regarding the mechanism(s) underlying NAFC-induced increases in GDF15 production during early placentation. The goal of this study was to examine the effects of NAFC exposure on the regulation of critical transcription factors of GDF15 in extravillous trophoblast cells. Of these transcription factors, inflammatory mediators including prostaglandins have been reported to inhibit proliferation and migration of trophoblast cells in vitro. Hence, the secondary goal of this study was to determine whether inflammation mediated through prostaglandin production is critical to GDF15 secretion. HTR-8/SVneo cells were exposed to an NAFC for 6 and 24 h to assess the expression of key transcriptional regulators, GDF15 secretion, and prostaglandin (PGE2) output. Treatment with NAFC (125 mg/L only) significantly increased GDF15 expression and secretion in association with upregulation of the transcription factors KLF4, EGR1, ATF3 and TP53. Similarly, PTGS2 (i.e. COX2) expression and PGE2 output were significantly increased at the same concentration. However, co-treatment with a COX2 selective antagonist (SC236) only partially blocked the NAFC-induced increase in PGE2 output and did not block GDF15 expression or secretion. These findings suggest that while NAFC may affect GDF15 production, it is not exclusively a result of prostaglandin-mediated inflammation. This study provides new insights into the mechanisms by which NAFC may adversely affect placental trophoblast cell function in mammals.

15-Hydroxyprostaglandin dehydrogenase inhibitor prevents contrast-induced acute kidney injury.

The two primary mechanisms by which iodinated contrast media (CM) causes contrast-induced acute kidney injury (CIAKI) are the hemodynamic effect causing intrarenal vasoconstriction and the tubular toxic effect causing acute tubular necrosis. Inhibition of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which degrades prostaglandin E2 (PGE2), promotes tissue repair and regeneration in many organs. PGE2 causes intrarenal arterial vasodilation. In this study, we investigated whether a 15-PGDH inhibitor can act as a candidate for blocking these two major mechanisms of CIAKI. We established a CIAKI mouse model by injecting a 10 gram of iodine per body weight (gI/kg) dose of iodixanol into each mouse tail vein. A 15-PGDH inhibitor (SW033291), PGE1, or PGE2 were administered to compare the renal functional parameters, histologic injury, vasoconstriction, and renal blood flow changes. In addition, human renal proximal tubular epithelial cells were cultured in a CM-treated medium. SW033291, PGE1, or PGE2 were added to compare any changes in cell viability and apoptosis rate. CIAKI mice that received SW033291 had lower serum levels of creatinine, neutrophil gelatinase-associated lipocalin, and kidney injury molecule 1 (p < 0.001); lower histologic injury score and TUNEL positive rates (p < 0.001); and higher medullary arteriolar area (p < 0.05) and renal blood flow (p < 0.001) than CM + vehicle group. In cell culture experiments, Adding SW033291 increased the viability rate (p < 0.05) and decreased the apoptosis rate of the tubular epithelial cells (p < 0.001). This 15-PGDH inhibitor blocks the two primary mechanisms of CIAKI, intrarenal vasoconstriction and tubular cell toxicity, and thus has the potential to be a novel prophylaxis for CIAKI. Abbreviations: 15-PGDH: 15-hydroxyprostaglandin dehydrogenase; AMP: adenosine monophosphate; CIAKI: contrast-induced acute kidney injury; CM: contrast media; EP: prostaglandin E2 receptor; hRPTECs: human-derived renal proximal tubule epithelial cells; KIM-1: kidney injury molecule-1; MTT: 3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NGAL: neutrophil gelatinase-associated lipocalin; PBS: phosphate-buffered saline; PGE1: prostaglandin E1; PGE2: prostaglandin E2; RBF: renal blood flow; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; α-SMA: α-Smooth muscle actin.

Prostaglandylinositol cyclic phosphate, the natural antagonist of cyclic AMP.

While searching for a counterpart to cyclic AMP, a new compound was found to inhibit adenylate cyclase. It was identified as prostaglandyl-(15-4')-myo-inositol (1':2'-cyclic)-phosphate (cyclic PIP). The substrates for its biosynthesis are prostaglandin E (PGE) and the novel inositol phosphate, guanosine diphospho-4-myo-inositol 1:2-cyclic phosphate (n-IP). The basic regulatory properties of cyclic PIP are to inhibit dose-dependently protein kinase A (PKA) and to seven-fold activate protein ser/thr phosphatase holoenzyme. These regulations occur as rapidly as the activation of PKA by cyclic AMP. Such regulatory properties are essential for the meticulous regulation of the equilibrium between the phospho- and de-phospho-form of interconvertible enzymes. The synthesis of cyclic PIP is stimulated by insulin and noradrenaline (α-receptor action). The insulin-stimulated cyclic PIP synthase is active in a tyrosine-phosphorylated state. A comparable characterization of the adrenaline-stimulated cyclic PIP synthase is still incomplete. In streptozotocin-diabetic rats, the hormonal stimulation of cyclic PIP synthesis decreases with time. Cyclic PIP synthesis is activated by biguanides as metformin two to four-fold and by antihypertensive drugs two-fold. Inhibition of cyclic PIP synthesis leads to a metabolic state as observed in early-stage type-2 diabetes. In summary, all living cells synthesize cyclic PIP, which switches on anabolism, whereas cyclic AMP triggers catabolism.

Prostaglandin (PTG) E and F receptors in the porcine corpus luteum; effect of tumor necrosis factor-α.

The porcine corpus luteum (CL) is NOT sensitive to the luteolytic effects of PGF-2α until days 12-13 of cycle. The control of "luteolytic sensitivity" (LS) of the pig CL to PGF-2α is unknown, but it is temporally associated with macrophage infiltration into the CL. Since macrophages are the predominant source of TNF-α in the porcine CL, in other studies we examined the effects of TNF-α on porcine luteal cells in culture and showed that TNF-α induces LS in vitro. In Experiment 1 of this study possible mechanisms involved in the control of LS were examined, and involved measurement of the protein levels of PTGER2/EP-2, and PTGER3/EP-3 in porcine CL collected before (days 7-10), versus after (day 13), the onset of the LS. In Experiment 2, an examination of potential mechanisms involved in the control of LS by TNF-α, was carried out in which the effects of TNF-α on mRNA and protein expression of EP-2, EP-3 and FP in cultured luteal cells, were examined. The results of Experiment 1 showed that PTGER-3/EP-3 (but not PTGER-2/EP-2) levels decreased in porcine CLs after (day 13) compared to before (day 7-10) LS. In Experiment 2, the data obtained showed that TNF-α decreased PTGER-3/EP-3 and increased PTGFR/FP protein (in EARLY stage CL). In conclusion, these studies suggest a role for PTGER-3/EP-3 in the acquisition of LS, and support the hypothesis that TNF-α from CL macrophages plays a critical role in the control of LS in the porcine CL, by increasing PTGFR/FP, and decreasing PTGER-3/EP-3 protein.

Loss of luteotropic prostaglandin E plays an important role in the regulation of luteolysis in women.

STUDY QUESTION: Do intraluteal prostaglandins (PG) contribute to luteal regulation in women? SUMMARY ANSWER: Prostaglandin E (PGE), which is produced in human granulosa-lutein cells stimulated with luteotropic hCG, exerts similar luteotropic effects to hCG, and the expression of PG synthetic and metabolic enzymes in the human CL is driven toward less PGE but more prostaglandin F (PGF) during luteolysis. WHAT IS KNOWN ALREADY: Uterine PGF is a major luteolysin in many non-primate species but not in women. Increases in the PGF synthase, aldo-ketoreductase family one member C3 (AKR1C3), have been observed in the CL of marmoset monkeys during luteolysis. PGE prevents spontaneous or induced luteolysis in domestic animals. STUDY DESIGN, SIZE, DURATION: Human CL tissues staged as the early-luteal (n = 6), mid-luteal (n = 6), late-luteal (n = 5) and menstrual (n = 3) phases were obtained at the time of hysterectomy for benign gynecological conditions. Luteinized granulosa cells (LGCs) were purified from follicular fluids obtained from patients undergoing assisted conception. PARTICIPANTS/MATERIALS, SETTING, METHODS: Upon collection, one half of the CL was snap-frozen and the other was fixed with formalin and processed for immunohistochemical analysis of a PGE synthase (PTGES). Quantitative RT-PCR was employed to examine changes in the mRNA abundance of PG synthetic and metabolic enzymes, steroidogenic enzymes, and luteolytic molecules in the staged human CL and in human LGCs in vitro treated with hCG, PGE and PGF. A PGE withdrawal experiment was also conducted in order to reveal the effects of the loss of PGE in LGCs. Progesterone concentrations in the culture medium were measured. MAIN RESULTS AND THE ROLE OF CHANCE: The key enzyme for PGE synthesis, PTGES mRNA was abundant in the functional CL during the mid-luteal phase (P < 0.01), while mRNA abundance for genes involved in PGF synthesis (AKR1B1 and AKR1C1-3) increased in the CL during the late-luteal phase and menstruation (P < 0.05-0.001). PTGES mRNA expression positively correlated with that of 3ß-hydroxysteroid dehydrogenase (HSD3B1; r = 0.7836, P < 0.001), while AKR1C3 expression inversely correlated with that of HSD3B1 (r = -0.7514, P = 0.0012) and PTGES (r = -0.6923, P = 0.0042). PGE exerted similar effects to hCG-promoting genes, such as steroidogenic acute regulatory protein (STAR) and HSD3B1, to produce progesterone and luteotropic PGE, suppress PGF synthetic enzymes and down-regulate luteolytic molecules such as ßA- and ßB-inhibin subunits (INHBA and INHBB) and bone morphogenetic proteins (BMP2, BMP4 and BMP6). PGE withdrawal resulted in reductions in the enzymes that produce progesterone (STAR; P < 0.001) and PGE (PTGES; P < 0.001), and the capacity to produce PGE decreased, while the capacity to produce PGF increased during the culture. The addition of PGF did not recapitulate the luteolytic effects of PGE withdrawal. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: Changes in mRNA expression of PG synthetic and metabolic enzymes may not represent actual increases in PGF during luteolysis in the CL. The effects of PGF on luteal cells currently remain unclear and the mechanisms responsible for decreases in the synthesis of PGE in vitro and at luteolysis have not been elucidated in detail. WIDER IMPLICATIONS OF THE FINDINGS: The results obtained strongly support a luteotropic function of PGE in regulation of the human CL. They suggest that the main PG produced in human luteal tissue changes from PGE to PGF during the maturation and regression of the CL, and the loss of PGE is more important than the effects of PGF during luteolysis in women. This may be accompanied by reduced effects of LH/hCG in luteal cells, particularly decreased activation of cAMP/protein kinase A; however, the underlying mechanisms remain unknown. STUDY FUNDING AND COMPETING INTEREST(S): This study was supported by the Cunningham Trust to WCD, a Postdoctoral Fellowship for Research Abroad from the Japan Society for the Promotion of Science and the Suntory Foundation for Life Sciences to J.N.-K.; W.C.D. is supported by an MRC Centre Grant G1002033 and a Scottish Senior Clinical Fellowship. The authors have nothing to disclose.

Osteoblasts stimulate osteoclastogenesis via RANKL expression more strongly than periodontal ligament cells do in response to PGE(2).

OBJECTIVE: Periodontal ligament cells (PDLs) produce prostaglandin E(2) (PGE(2)) in response to orthodontic force. PGE(2) is a potent osteoclast-inducing factor that induces the receptor activator of nuclear factor-κB ligand (RANKL). Some studies reported that PDLs express RANKL in response to mechanical stress, whereas another study reported that they do not. Based on an immunohistochemical study, RANKL expression is localized around the alveolar bone surface 3 days after tooth movement. However, ankylosed teeth cannot be moved by therapeutic mechanical stress, suggesting that PDLs play a major role in alveolar bone resorption. In this study, we compared the functional difference in osteoclastogenesis between human PDLs (HPDLs) and normal human osteoblasts (HOBs) as a direct effect of PGE(2) exposure. DESIGN: We examined the expression of RANKL, osteoprotegerin, and macrophage colony-stimulating factor after 48-h culture with or without PGE(2) (10(-11) to 10(-5)M) in HPDLs and HOBs. Then to confirm whether RANKL produced by PGE(2) treatment induces osteoclastogenesis or not, RAW264.7 cells were co-cultured on HPDLs or HOBs pretreated with 10(-6)M of PGE(2). RESULT: PGE(2) exposure increased significantly RANKL expression in HOBs compared with HPDLs. PGE(2) exposure significantly decreased osteoprotegerin expression in HPDLs compared with HOBs. The number of tartrate-resistant acid phosphatase staining osteoclast-like cells from RAW264.7 cells increased significantly by PGE(2) pretreatment in HOBs and was reduced by small interfering RNA knockdown of RANKL. CONCLUSION: These results suggest that osteoblasts strongly influence the stimulation of osteoclastogenesis via RANKL, induced by PGE(2) in periodontal tissues, compared with PDLs.

Prostaglandin EP1 receptor down-regulates expression of cyclooxygenase-2 by facilitating its proteasomal degradation.

The enzyme cyclooxygenase-2 (COX-2) is rapidly and transiently up-regulated by a large variety of signals and implicated in pathologies such as inflammation and tumorigenesis. Although many signals cause COX-2 up-regulation, much less is known about mechanisms that actively down-regulate its expression. Here we show that the G protein-coupled receptor prostaglandin E(1) (EP(1)) reduces the expression of COX-2 in a concentration-dependent manner through a mechanism that does not require receptor activation. The reduction in COX-2 protein is not due to decreased protein synthesis and occurs because of enhancement of substrate-independent COX-2 proteolysis. Although EP(1) does not interfere with the entry of COX-2 into the endoplasmic reticulum-associated degradation cascade, it facilitates COX-2 ubiquitination through complex formation. Blockade of proteasomal activity results in degradation of the receptor and concomitant recovery in the expression of COX-2, suggesting that EP(1) may scaffold an unknown E3 ligase that ubiquitinates COX-2. These findings propose a new role for the EP(1) receptor in resolving inflammation through down-regulation of COX-2.

Identification of a novel class of anti-inflammatory compounds with anti-tumor activity in colorectal and lung cancers.

Chronic inflammation is associated with 25% of all cancers. In the inflammation-cancer axis, prostaglandin E(2) (PGE(2)) is one of the major players. PGE(2) synthases (PGES) are the enzymes downstream of the cyclooxygenases (COXs) in the PGE(2) biosynthesis pathway. Microsomal prostaglandin E(2) synthase 1 (mPGES-1) is inducible by pro-inflammatory stimuli and constitutively expressed in a variety of cancers. The potential role for this enzyme in tumorigenesis has been reported and mPGES-1 represents a novel therapeutic target for cancers. In order to identify novel small molecule inhibitors of mPGES-1, we screened the ChemBridge library and identified 13 compounds as potential hits. These compounds were tested for their ability to bind directly to the enzyme using surface plasmon resonance spectroscopy and to decrease cytokine-stimulated PGE(2) production in various cancer cell lines. We demonstrate that the compound PGE0001 (ChemBridge ID number 5654455) binds to human mPGES-1 recombinant protein with good affinity (K(D) = 21.3 ± 7.8 µM). PGE0001 reduces IL-1ß-induced PGE(2) release in human HCA-7 colon and A549 lung cancer cell lines with EC(50) in the sub-micromolar range. Although PGE0001 may have alternative targets based on the results from in vitro assays, it shows promising effects in vivo. PGE0001 exhibits significant anti-tumor activity in SW837 rectum and A549 lung cancer xenografts in SCID mice. Single injection i.p. of PGE0001 at 100 mg/kg decreases serum PGE(2) levels in mice within 5 h. In summary, our data suggest that the identified compound PGE0001 exerts anti-tumor activity via the inhibition of the PGE(2) synthesis pathway.

P2Y12 and EP3 antagonists promote the inhibitory effects of natural modulators of platelet aggregation that act via cAMP.

Several antiplatelet drugs that are used or in development as antithrombotic agents, such as antagonists of P2Y12 and EP3 receptors, act as antagonists at G(i)-coupled receptors, thus preventing a reduction in intracellular cyclic adenosine monophosphate (cAMP) in platelets. Other antiplatelet agents, including vascular prostaglandins, inhibit platelet function by raising intracellular cAMP. Agents that act as antagonists at G(i)-coupled receptors might be expected to promote the inhibitory effects of agents that raise cAMP. Here, we investigate the ability of the P2Y12 antagonists cangrelor, ticagrelor and prasugrel active metabolite (PAM), and the EP3 antagonist DG-041 to promote the inhibitory effects of modulators of platelet aggregation that act via cAMP. Platelet aggregation was measured by platelet counting in whole blood in response to the TXA2 mimetic U46619, thrombin receptor activating peptide and the combination of these. Vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) was measured using a cytometric bead assay. Cangrelor always increased the potency of inhibitory agents that act by raising cAMP (PGI2, iloprost, PGD2, adenosine and forskolin). Ticagrelor and PAM acted similarly to cangrelor. DG-041 increased the potency of PGE1 and PGE2 as inhibitors of aggregation, and cangrelor and DG-041 together had more effect than either agent alone. Cangrelor and DG-041 were able to increase the ability of agents to raise cAMP in platelets as measured by increases in VASP-P. Thus, P2Y12 antagonists and the EP3 antagonist DG-041 are able to promote inhibition of platelet aggregation brought about by natural and other agents that raise intracellular cAMP. This action is likely to contribute to the overall clinical effects of such antagonists after administration to man.

COX-2-dependent PGE(2) acts as a growth factor in mycosis fungoides (MF).

Cancer often originates from a site of persistent inflammation, and the mechanisms turning chronic inflammation into a driving force of carcinogenesis are intensely investigated. Cyclooxygenase-2 (COX-2) is an inducible key modulator of inflammation that carries out the rate-limiting step in prostaglandin synthesis. Aberrant COX-2 expression and prostaglandin E(2) (PGE(2)) production have been implicated in tumorigenesis. In this study we show that COX-2 is ectopically expressed in malignant T-cell lines from patients with cutaneous T-cell lymphoma (CTCL) as well as in situ in lymphocytic cells in 21 out of 22 patients suffering from mycosis fungoides (MF) in plaque or tumor stage. COX-2 is not expressed in lymphocytes of 11 patients with patch-stage MF, whereas sporadic COX-2 staining of stromal cells is observed in the majority of patients. COX-2 expression correlates with a constitutive production of PGE(2) in malignant T cells in vitro. These cells express prostaglandin receptors EP3 and EP4 and the receptor antagonist as well as small interfering RNA (siRNA) directed against COX-2, and specific COX-2 inhibitors strongly reduce their spontaneous proliferation. In conclusion, our data indicate that COX-2 mediated PGE(2) exerts an effect as a tumor growth factor in MF.

Effects of (-)-epigallocatechin-3-gallate on cyclooxygenase 2, PGE(2), and IL-8 expression induced by IL-1beta in human synovial fibroblasts.

The objective of this study was to examine the effects of (-)-epigallocatechin-3-gallate (EGCG) on cyclooxygenase 2 (COX-2), prostaglandin E(2) (PGE(2)), and interleukin 8 (IL-8) expression induced by IL-1beta in human synovial fibroblasts. Cells were enzymatically isolated from synovial tissue taken from patients undergoing joint replacement surgery for osteoarthritis. Reverse transcriptase-polymerase chain reaction, immunocytochemistry, and western blotting were used to assess the COX-2 gene and protein expression with the associated mechanisms. PGE(2) and IL-8 secretion into the culture medium was assayed by enzyme-linked immunosorbent assay. COX-2 upregulation in synovial fibroblasts induced by IL-1beta was significantly suppressed by EGCG in a dose-dependent manner. PGE(2) and IL-8 secretion was also induced by IL-1beta stimulation and significantly suppressed by EGCG. The mechanism was associated with the phosphorylation of IKKbeta. EGCG may inhibit the expression of inflammatory mediators, such as COX-2, PGE(2), and IL-8, induced by IL-1beta in human synovial fibroblasts. EGCG may be of value in the treatment of synovial inflammation.

Low concentrations of reactive gamma-ketoaldehydes prime thromboxane-dependent human platelet aggregation via p38-MAPK activation.

Oxidative stress has been strongly implicated in pathological processes. Isoketals are highly reactive gamma-ketoaldehydes of the isoprostanes pathway of free radical-induced peroxidation of arachidonic acid that are analogous to cyclooxygenase-derived levuglandins. Because aldehydes, that are much less reactive than isoketals, have been shown to trigger platelet activation, we investigated the effect of one isoketal (E(2)-IsoK) on platelet aggregation. Isoketal potentiated aggregation and the formation of thromboxane B(2) in platelets challenged with collagen at a concentration as low as 1 nM. Moreover, the potentiating effect of 1 nM isoketal on collagen-induced platelet aggregation was prevented by pyridoxamine, an effective scavenger of gamma-ketoaldehydes. Furthermore, we provide evidence for the involvement of p38 mitogen-activated protein kinase in isoketal-mediated platelet priming, suggesting that isoketals may act upstream the activation of collagen-induced cytosolic phospholipase A(2). Additionally, the incubation of platelets with 1 nM isoketal led to the phosphorylation of cytosolic phospholipase A(2). The cytosolic phopholipase A(2) inhibitors AACOCF3 and MAFP both fully prevented the increase in isoketal-mediated platelet aggregation challenged with collagen. These results indicate that isoketals could play an important role in platelet hyperfunction observed in pathological states such as atherosclerosis and thrombosis through the activation of the endogenous arachidonic acid cascade.