Low Levels Of Prostaglandin E2 Research Articles

EVA NEXUS-Phaco performance study.

To investigate a novel phacoemulsification system "EVA NEXUS" (D.O.R.C., Dutch Opthalmic Research Center) in comparison to the existing system "EVA" in clinical use. And to compare both phacoemulsification systems in terms of efficiency, safety and postoperative inflammatory activity. In this study standardized cataract surgery was performed on both eyes of the study participant, using the "EVA system" (control group, n=20) on one eye and the "EVA NEXUS system" (intervention group, n=20) on the other eye. Only patients with cataract LOCS Grading 1-3 and no accompanying eye diseases were included in this study. A total of 20 patients were included in this study, with each treatment arm including 20 eyes. During surgery a 0.1 mL aqueous humor sample was collected 1min after phacoemulsification to measure the total prostaglanin E2 concentrations using an enzyme-linked immunosorbent assay. The endothelial cell count, visual and refractive outcomes, and anterior chamber flare were evaluated preoperatively, and 1d, 1wk, and 3mo postoperatively. There were no statistically significant differences between both groups regarding intraoperative safety parameters including effective phacoemulsification time (P=0.904), balanced saline solution flow (P=0.701) and total surgery time (P=0.565). Postoperative prostaglandin E2 levels, anterior chamber flare as well as endothelial cell loss tended to be lower in the NEXUS-Group, however not being statistically significant (P=0.718; 0.164; 0.486). Both systems provided similar clinical outcomes, regarding best corrected visual acuity and refractive parameters, showing no statistically significant differences between both groups. Both systems show a high level of safety and efficency with similar results in terms of safety parameters including postoperative inflammatory activity and endothelial cell loss as well as visual and refractive outcomes. Although statistically not significant, the EVA NEXUS system tends to cause less postoperative inflammation with lower prostaglandin E2 levels and lower anterior chamber flare values.

Label-free detection of prostaglandin transporter (SLCO2A1) function and inhibition: insights by wound healing and TRACT assays.

The prostaglandin transporter (PGT, SLCO2A1) mediates transport of prostanoids (a.o. prostaglandin E2 (PGE2)) into cells and thereby promotes their degradation. Overexpression of PGT leads to low extracellular PGE2 levels and has been linked to impaired wound healing of diabetic foot ulcers. Inhibition of PGT could thus be beneficial, however, no PGT inhibitors are currently on the market and drug discovery efforts are hampered by lack of high-through screening assays for this transporter. Here we report on a label-free impedance-based assay for PGT that measures transport activity through receptor activation (TRACT) utilizing prostaglandin E2 receptor subtype EP3 and EP4 that are activated by PGE2. We found that induction of PGT expression on HEK293-JumpIn-SLCO2A1 cells that also express EP3 and EP4 leads to an over 10-fold reduction in agonistic potency of PGE2. PGE2 potency could be recovered upon inhibition of PGT-mediated PGE2 uptake with PGT inhibitors olmesartan and T26A, the potency of which could be established as well. Moreover, the TRACT assay enabled the assessment of transport function of PGT natural variants. Lastly, HUVEC cells endogenously expressing prostanoid receptors and PGT were exploited to study wound healing properties of PGE2 and T26A in real-time using a novel impedance-based scratch-induced wound healing assay. These novel impedance-based assays will advance PGT drug discovery efforts and pave the way for the development of PGT-based therapies.

Lower serum 15-HETE level predicts nasal ILC2 accumulation during COX-1 inhibition in AERD

Aspirin-exacerbated respiratory disease (AERD) is associated with high levels of cysteinyl leukotrienes, prostaglandin D2, and low levels of prostaglandin E2. Further, 15-hydroxyeicosatetraenoic acid (15-HETE) levels may have predictive value in therapeutic outcomes of aspirin desensitization. Accumulation of nasal group 2 innate lymphoid cells (ILC2s) has been demonstrated during COX-1 inhibition in AERD, although the relationships between tissue ILC2 accumulation, reaction symptom severity, and novel lipid biomarkers are unknown. We sought to determine whether novel lipid mediators are predictive of nasal ILC2 accumulation and symptom scores during COX-1 inhibitor challenge in patients with AERD. Blood and nasal scraping samples from patients with AERD were collected at baseline and COX-1 inhibitor reaction and then processed for flow cytometry for nasal ILC2s and serum for lipidomic analysis. Eight patients with AERD who were undergoing aspirin desensitization were recruited. Of the 161 eicosanoids tested, 42 serum mediators were detected. Baseline levels of 15-HETE were negatively correlated with the change in numbers of airway ILC2s (r= -0.6667; P= .0428). Docosahexaenoic acid epoxygenase metabolite 19,20-dihydroxy-4Z,7Z,10Z,13Z,16Z-docosapentaenoic acid (19,20-diHDPA) was positively correlated with both changes in airway ILC2s (r= 0.7143; P= .0305) and clinical symptom scores (r= 0.5000; P= .0081). Low levels of baseline 15-HETE predicted a greater accumulation of airway ILC2s in patients with AERD who were receiving COX-1 inhibition. Further, increases in the cytochrome P pathway metabolite 19,20-dihydroxy-4Z,7Z,10Z,13Z,16Z-docosapentaenoic acid (19,20-diHDPA) were associated with increased symptoms and nasal ILC2 accumulation. Future studies to assess how these mediators might control ILC2s may improve the understanding of AERD pathogenesis.

D-dimer and CoV-2 spike-immune complexes contribute to the production of PGE2 and proinflammatory cytokines in monocytes.

An overreactive inflammatory response and coagulopathy are observed in patients with severe form of COVID-19. Since increased levels of D-dimer (DD) are associated with coagulopathy in COVID-19, we explored whether DD contributes to the aberrant cytokine responses. Here we show that treatment of healthy human monocytes with DD induced a dose dependent increase in production of pyrogenic mediator, Prostaglandin E2 (PGE2) and inflammatory cytokines, IL-6 and IL-8. The DD-induced PGE2 and inflammatory cytokines were enhanced significantly by co-treatment with immune complexes (IC) of SARS CoV-2 recombinant S protein or of pseudovirus containing SARS CoV-2 S protein (PVCoV-2) coated with spike-specific chimeric monoclonal antibody (MAb) containing mouse variable and human Fc regions. The production of PGE2 and cytokines in monocytes activated with DD and ICs was sensitive to the inhibitors of β2 integrin and FcγRIIa, and to the inhibitors of calcium signaling, Mitogen-Activated Protein Kinase (MAPK) pathway, and tyrosine-protein kinase. Importantly, strong increase in PGE2 and in IL-6/IL-8/IL-1β cytokines was observed in monocytes activated with DD in the presence of IC of PVCoV-2 coated with plasma from hospitalized COVID-19 patients but not from healthy donors. The IC of PVCoV-2 with convalescent plasma induced much lower levels of PGE2 and cytokines compared with plasma from hospitalized COVID-19 patients. PGE2 and IL-6/IL-8 cytokines produced in monocytes activated with plasma-containing IC, correlated well with the levels of spike binding antibodies and not with neutralizing antibody titers. Our study suggests that a combination of high levels of DD and high titers of spike-binding antibodies that can form IC with SARS CoV-2 viral particles might accelerate the inflammatory status of lung infiltrating monocytes leading to increased lung pathology in patients with severe form of COVID-19.

Adipose-Derived Stem Cells From Patients With Ulcerative Colitis Exhibit Impaired Immunosuppressive Function.

Adipose-derived stem cells (ADSCs) are able to modulate the immune response and are used for treating ulcerative colitis (UC). However, it is possible that ADSCs from patients with inflammatory or autoimmune disorders may show defective immunosuppression. We investigated the use of ADSCs from UC patients for autologous cell treatment, specifically, ADSCs from healthy donors (H-ADSCs) and UC patients (P-ADSCs) in terms of various functions, including differentiation, proliferation, secretion, and immunosuppression. The efficacy of P-ADSCs for treating UC was examined in mouse models of acute or chronic colitis. Both H-ADSCs and P-ADSCs were similar in cell morphology, size, adipogenic differentiation capabilities, and cell surface markers. We found that P-ADSCs had lower proliferative capacity, cloning ability, and osteogenic and chondrogenic differentiation potential than H-ADSCs. P-ADSCs exhibited a diminished capacity to inhibit peripheral blood mononuclear cell proliferation, suppress CD25 and CD69 marker expression, decrease the production of inflammation-associated cytokines interferon-γ and tumor necrosis factor-α, and reduce their cytotoxic effect on A549 cells. When primed with inflammatory cytokines, P-ADSCs secreted lower levels of prostaglandin E2, indoleamine 2, 3-dioxygenase, and tumor necrosis factor-α–induced protein 6, which mediated their reduced immunopotency. Moreover, P-ADSCs exhibited weaker therapeutic effects than H-ADSCs, determined by disease activity, histology, myeloperoxidase activity, and body weight. These findings indicate that the immunosuppressive properties of ASCs are affected by donor metabolic characteristics. This study shows, for the first time, the presence of defective ADSC immunosuppression in UC, indicating that autologous transplantation of ADSCs may be inappropriate for patients with UC.

Physiologically relevant aspirin concentrations trigger immunostimulatory cytokine production by human leukocytes.

Acetylsalicylic acid is a globally used non-steroidal anti-inflammatory drug (NSAID) with diverse pharmacological properties, although its mechanism of immune regulation during inflammation (especially at in vivo relevant doses) remains largely speculative. Given the increase in clinical perspective of Acetylsalicylic acid in various diseases and cancer prevention, this study aimed to investigate the immunomodulatory role of physiological Acetylsalicylic acid concentrations (0.005, 0.02 and 0.2 mg/ml) in a human whole blood of infection-induced inflammation. We describe a simple, highly reliable whole blood assay using an array of toll-like receptor (TLR) ligands 1-9 in order to systematically explore the immunomodulatory activity of Acetylsalicylic acid plasma concentrations in physiologically relevant conditions. Release of inflammatory cytokines and production of prostaglandin E2 (PGE2) were determined directly in plasma supernatant. Experiments demonstrate for the first time that plasma concentrations of Acetylsalicylic acid significantly increased TLR ligand-triggered IL-1β, IL-10, and IL-6 production in a dose-dependent manner. In contrast, indomethacin did not exhibit this capacity, whereas cyclooxygenase (COX)-2 selective NSAID, celecoxib, induced a similar pattern like Acetylsalicylic acid, suggesting a possible relevance of COX-2. Accordingly, we found that exogenous addition of COX downstream product, PGE2, attenuates the TLR ligand-mediated cytokine secretion by augmenting production of anti-inflammatory cytokines and inhibiting release of pro-inflammatory cytokines. Low PGE2 levels were at least involved in the enhanced IL-1β production by Acetylsalicylic acid.

Gut microbiota modulation and anti-inflammatory properties of mixed lactobacilli in dextran sodium sulfate-induced colitis in mice.

Correlations between gut microbiota activities and inflammatory bowel disease (IBD) treatment are gaining research interest. In our previous study, Lactobacillus acidophilus KLDS 1.0901, Lactobacillus helveticus KLDS 1.8701, and Lactobacillus plantarum KLDS 1.0318 showed antibacterial, antioxidant, and immunomodulatory activities. In the current study, we evaluated the effects of three tested strains and their mixture on dextran sulfate sodium (DSS)-induced colitis in C57BL/6J mice. The three tested strains and their mixture significantly decreased the disease activity index (DAI), colon shortening, and myeloperoxidase (MPO) activity. Additionally, the three tested strains and their mixture improved the histological damage, increased the colonic mucous layer integrity, and exhibited lower levels of prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), while up-regulating colonic anti-inflammatory cytokine IL-10 levels, tight junction proteins (E-cadherin, zonulae occludens (ZO)-1, occludin and claudin-1) and mucin (MUC1 and MUC2) mRNA expressions to some extent. In addition, mixed lactobacilli showed better anti-inflammatory effects than single-strain treatment. Our study further revealed that mixed lactobacilli increased bacterial diversity and improved gut microbiota composition, increasing short-chain fatty acid (SCFA) production. These results indicated that mixed lactobacilli supplementation could attenuate DSS-induced colitis by modulating the gut microbiota and repairing the intestinal barrier, which provided a scientific basis for its clinical application in the future.

PGE2 deficiency predisposes to anaphylaxis by causing mast cell hyperresponsiveness

Reduced levels of prostaglandin E2 (PGE2) contribute to aspirin-induced hypersensitivity. COX inhibitors are also frequent cofactors in anaphylaxis. Whether alterations in the PGE2 system contribute to anaphylaxis independently of COX inhibitor intake is unclear. Our aim was to test the hypothesis that relative PGE2 deficiency predisposes to anaphylaxis. Sera from 48 patients with anaphylaxis and 27 healthy subjects were analyzed for PGE2 levels and correlated against severity; 9α,11β-PGF2 and PGI2 metabolites were measured for control purposes. PGE2 stabilization by 15-hydroxyprostaglandin dehydrogenase inhibitor or EP2 or EP4 receptor agonists were used in a murine model of passive systemic anaphylaxis. FcεRI-triggered mediator release was determined in bone marrow-derived cultured mast cells (MCs) and human skin-derived MCs. Signaling was studied by Western blot analysis. Patients with anaphylaxis were characterized by markedly reduced PGE2 levels vis-à-vis healthy subjects, whereas prostacyclin metabolite levels were diminished only weakly, and 9α,11β-PGF2 levels conversely increased. PGE2 was negatively correlated with severity. Lower PGE2 levels and higher susceptibility to anaphylaxis were also found in C57BL/6 mice vis-à-vis in Balb/c mice. Stabilization of PGE2 level by 15-hydroxyprostaglandin dehydrogenase inhibitor protected mice against anaphylaxis. Exogenous PGE2 attenuated bone marrow-derived cultured MC activation through EP2 and EP4 receptors. EP2 and EP4 agonism also curbed FcεRI-mediated degranulation of human MCs. Mechanistically, PGE2 interfered with the phosphorylation of phospholipase C gamma-1 and extracellular signal-regulated kinase. Homeostatic levels of PGE2 attenuate MC activation via EP2/EP4 and protect against anaphylaxis. Relative deficiency of PGE2 predisposes to anaphylaxis in humans and mice, whereas PGE2 stabilization protects against anaphylactic reactions.

Suppression of aspirin-mediated eosinophil activation by prostaglandin E2: Relevance to aspirin and nonsteroidal anti-inflammatory drug hypersensitivity

BackgroundAspirin-exacerbated respiratory disease (AERD) is characterized by severe, sometimes life-threatening reactions to nonsteroidal anti-inflammatory drugs (NSAIDs). Mechanisms driving the disease include overproduction of leukotrienes and loss of anti-inflammatory prostaglandin E2 (PGE2) production. Many cell types contribute to the disease; however, eosinophils are markedly elevated and are important drivers of pathologic findings. ObjectiveTo investigate the capacity of aspirin and NSAIDs to drive eosinophil activation and the ability of PGE2 to inhibit this activation. MethodsEosinophils were purified from blood of healthy individuals without AERD and stimulated with lysine aspirin, ketorolac, or sodium salicylate. The role of PGE2 in altering activation was determined by incubating eosinophils with increasing doses of PGE2 before lysine aspirin stimulation. Specific PGE2 receptor use was determined by incubating eosinophils with receptor agonists and antagonists before aspirin stimulation. Cysteinyl leukotrienes (CysLTs), leukotriene B4 (LTB4), and eosinophil-derived neurotoxin (EDN) were quantified by enzyme-linked immunosorbent assay. ResultsStimulation of eosinophils with lysine aspirin, ketorolac, or sodium salicylate resulted in secretion of CysLTs and LTB4 in the absence of EDN release. Low doses of PGE2 inhibited LTB4 and CysLT release, an effect lost at higher PGE2 concentrations. Use of butaprost, an EP2 receptor agonist, suppressed lysine aspirin stimulation. This mechanism was supported by blocking activity of the EP1 and EP3 receptors. ConclusionEosinophils can be directly activated by NSAIDs via cyclooxygenase-independent pathways to produce CysLTs and LTB4. This effect can be inhibited by PGE2 acting through the EP2 receptor. The recognized loss of EP2 receptor expression combined with low PGE2 levels explains in part the sensitivity to NSAIDs.

Urinary PGE2 metabolite levels in hospitalised infants with infections compared to age-matched controls.

To determine the urinary tetranor-prostaglandin E2 metabolite in healthy infants and in hospitalised infants with upper and lower respiratory tract as well as gastrointestinal infections. A prospective cross-sectional study to determine baseline concentrations of urinary tetranor-prostaglandin E2 metabolite was conducted in 81 healthy infants aged one week to one year and in 142 hospitalised infants with infections. Prostaglandin metabolite levels were measured by liquid chromatography tandem mass spectrometry. In healthy infants, urinary prostaglandin E2 metabolite levels decreased with age and did not differ between girls and boys. Infections of the lower respiratory (n=78) and gastrointestinal tract (n=12) correlated with increased levels of the prostaglandin E2 metabolite. In contrast, infants hospitalised with upper respiratory tract infections (n=23) exhibited similar levels as healthy, age-matched controls. Lower prostaglandin E2 levels were found after treatment with acetaminophen in hospitalised children. Prostaglandin E2 metabolite levels did not correlate with length of hospitalisation or need for respiratory support. This study first provides normal levels of urinary prostaglandin E2 metabolite in infants and secondly demonstrates elevated levels in hospitalised children with lower respiratory tract and gastrointestinal infections.

The cAMP-EPAC Pathway Mediates PGE2-Induced FGF2 in Bovine Granulosa Cells.

During the periovulatory period, the profile of fibroblast growth factor 2 (FGF2) coincides with elevated prostaglandin E2 (PGE2) levels. We investigated whether PGE2 can directly stimulate FGF2 production in bovine granulosa cells and, if so, which prostaglandin E2 receptor (PTGER) type and signaling cascades are involved. PGE2 temporally stimulated FGF2. Accordingly, endoperoxide-synthase2-silenced cells, exhibiting low endogenous PGE2 levels, had reduced FGF2. Furthermore, elevation of viable granulosa cell numbers by PGE2 was abolished with FGF2 receptor 1 inhibitor, suggesting that FGF2 mediates this action of PGE2. Epiregulin (EREG), a known PGE2-inducible gene, was studied alongside FGF2. PTGER2 agonist elevated cAMP as well as FGF2 and EREG levels. However, a marked difference between cAMP-induced downstream signaling was observed for FGF2 and EREG. Whereas FGF2 upregulated by PGE2, PTGER2 agonist, or forskolin was unaffected by the protein kinase A (PKA) inhibitor H89, EREG was significantly inhibited. FGF2 was dose-dependently stimulated by the exchange protein directly activated by cAMP (EPAC) activator; a similar induction was observed for EREG. However, forskolin-stimulated FGF2, but not EREG, was inhibited in EPAC1-silenced cells. These findings ascribe a novel autocrine role for PGE2, namely, elevating FGF2 production in granulosa cells. This study also reveals that cAMP-activated EPAC1, rather than PKA, mediates the effect of PGE2/PTGER2 on the expression of FGF2. Stimulation of EREG by PGE2 is also mediated by PTGER2 but, in contrast to FGF2, EREG was found to be PKA sensitive. PGE2-stimulated FGF2 can act to maintain granulosa cell survival; it can also act on ovarian endothelial cells to promote angiogenesis.

Proximal signaling responses in peripheral T cells from colorectal cancer patients are affected by high concentrations of circulating prostaglandin E2

Patients with colorectal cancer (CRC) have been shown to have elevated levels of circulating prostaglandin E2 (PGE2) which promotes cancer progression and suppresses T cell immune responses. In this study we evaluated whether signaling responses in T lymphocytes obtained from peripheral blood of CRC patients were affected by the sustained exposure to increased levels of PGE2. The phosphorylation status of an extended panel of proteins involved in downstream signaling cascades in T cells was profiled at a single cell level both in naïve and antigen-experienced cells after triggering T cell-, prostaglandin- and interleukin-2 receptors. Peripheral T cells from patients with elevated PGE2 levels displayed aberrant T cell signaling responses downstream of the T cell receptor (assessed by reduced phosphorylation of CD3ζ and SLP76), and after triggering the IL-2 receptor (assessed by reduced phosphorylation of STAT5) when compared to T cells from CRC patients with lower levels of PGE2 and T cells from healthy blood donors. This signaling study of circulating T cells from CRC patients indicates that increased systemic PGE2 levels affect proximal T cell responses and confirms phospho-specific flow cytometry to be a valuable tool for revealing signaling signatures in immunological disorders.

Abstract 2257: Multiple drug resistance-associated protein 4 (MRP4) may contribute to breast cancer metastasis by exporting the COX-2 product PGE2

Abstract Cyclooxygenase-2 (COX-2) and its enzymatic product, prostaglandin E2 (PGE2), are elevated in breast cancer and are associated with a poor prognosis and increased metastatic potential. PGE2 initiates various signaling pathways upon binding to each of four cognate EP receptors. We have previously shown that PGE2 signaling through the EP4 receptor increases metastatic potential and supports the survival of breast cancer stem-like cells. Multiple drug resistance-associated protein 4 (MRP4) is responsible for the active export of PGE2 from cells, while the prostaglandin transporter (PGT) imports PGE2 for 15-hydroxyprostaglandin dehydrogenase (15-PGDH)-mediated degradation. The role of neither MRP4 nor PGT has been investigated in breast cancer progression. The purpose of this study is to elucidate the role of MRP4 in PGE2 signaling in breast cancer progression. We hypothesize that elevated expression of MRP4 would cause increased PGE2 export and receptor-mediated signaling and, therefore, enhance metastatic potential, tumor progression, and support breast cancer stem-like cells. We examined MRP4 gene expression data from multiple breast cancer datasets using Oncomine. We found higher expression of ABCC4 in breast cancer versus normal breast. MRP4 was elevated in invasive (IDC) versus localized (DCIS) lesions and elevated in the majority of basal-type breast cancer. To investigate the role of MRP4 further, we examined MRP4 mRNA and protein expression in cell lines representing several molecular subtypes and metastatic capacities. Normal mammary epithelium (MCF10A), luminal (MCF7, T47D), basal (MDA-MB-231, MDA-MB-468, MDA-MB-436, BT549), and Her2-enriched (SKBR3) cell lines were evaluated. As in the primary sample data, MRP4 mRNA and protein expression are elevated in basal and Her2 enriched cell lines (231, 436, BT549, SKBR3) while expression of PGT mRNA and protein is decreased in these cells when compared to cells with lower metastatic potential. This inverse relationship between MRP4 and PGT should lead to higher concentrations of extracellular PGE2 in the tumor microenvironment. We evaluated MRP4 activity by measuring PGE2 export from cells via enzyme immunoassay or resistance to the cytotoxic compound 6-mercaptopurine (6-MP), two substrates of MRP4. Pharmacologic inhibition of MRP4 with MK571 (MRP antagonist) results in decreased efflux of PGE2 and increased sensitivity to 6-MP as expected. Likewise, genetic suppression by RNAi results in lower levels of PGE2 exported from cells and increased sensitivity to 6-MP, confirming altered MRP4 activity. Conversely, ectopic MRP4 overexpression increases PGE2 export. These data support the hypothesis that MRP4 is a critical member of the PGE2 signaling pathway that leads to high extracellular PGE2 and increased PGE2 signaling, implicating MRP4 as a possible therapeutic target in this oncogenic pathway. Citation Format: Tyler J. Kochel, Namita Kundu, Xinrong Ma, Jocelyn Reader, Amy Fulton. Multiple drug resistance-associated protein 4 (MRP4) may contribute to breast cancer metastasis by exporting the COX-2 product PGE2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2257. doi:10.1158/1538-7445.AM2015-2257

Prostaglandin E2 (PGE2) Exerts Biphasic Effects on Human Tendon Stem Cells

Prostaglandin E2 (PGE2) has been reported to exert different effects on tissues at low and high levels. In the present study, cell culture experiments were performed to determine the potential biphasic effects of PGE2 on human tendon stem/progenitor cells (hTSCs). After treatment with PGE2, hTSC proliferation, stemness, and differentiation were analyzed. We found that high concentrations of PGE2 (>1 ng/ml) decreased cell proliferation and induced non-tenocyte differentiation. However, at lower concentrations (<1 ng/ml), PGE2 markedly enhanced hTSC proliferation. The expression levels of stem cell marker genes, specifically SSEA-4 and Stro-1, were more extensive in hTSCs treated with low concentrations of PGE2 than in cells treated with high levels of PGE2. Moreover, high levels of PGE2 induced hTSCs to differentiate aberrantly into non-tenocytes, which was evident by the high levels of PPARγ, collagen type II, and osteocalcin expression in hTSCs treated with PGE2 at concentrations >1 ng/ml. The findings of this study reveal that PGE2 can exhibit biphasic effects on hTSCs, indicating that while high PGE2 concentrations may be detrimental to tendons, low levels of PGE2 may play a vital role in the maintenance of tendon homeostasis in vivo.

Intratumoral COX‐2 inhibition enhances GM‐CSF immunotherapy against established mouse GL261 brain tumors

Immunotherapy has shown effectiveness against experimental malignant brain tumors, but the clinical results have been less convincing most likely due to immunosuppression. Prostaglandin E2 (PGE2 ) is the key immunosuppressive product of cyclooxygenase-2 (COX-2) and increased levels of PGE2 and COX-2 have been shown in several tumor types, including brain tumors. In the current study, we report enhanced cure rate of mice with established mouse GL261 brain tumors when immunized with granulocyte macrophage-colony stimulating factor (GM-CSF) secreting tumor cells and simultaneously treated with the selective COX-2 inhibitors parecoxib systemically (5 mg/kg/day; 69% cure rate) or valdecoxib intratumorally (5.3 µg/kg/day; 63% cure rate). Both combined therapies induced a systemic antitumor response of proliferating CD4(+) and CD8(+) T cells, and further analysis revealed T helper 1 (Th 1) cell supremacy. The GL261 tumor cell line produced low levels of PGE2 in vitro, and co-staining at the tumor site demonstrated that a large fraction of the COX-2(+) cells were derived from CD45(+) immune cells and more specifically macrophages (F4/80(+)), indicating that tumor-infiltrating immune cells constitute the primary source of COX-2 and PGE2 in this model. We conclude that intratumoral COX-2 inhibition potentiates GM-CSF immunotherapy against established brain tumors at substantially lower doses than systemic administration. These findings underscore the central role of targeting COX-2 during immunotherapy and implicate intratumoral COX-2 as the primary target.

Human Mesenchymal Stem/Stromal Cells Cultured as Spheroids are Self‐activated to Produce Prostaglandin E2 that Directs Stimulated Macrophages into an Anti‐inflammatory Phenotype

Culturing cells in three dimension (3D) provides an insight into their characteristics in vivo. We previously reported that human mesenchymal stem/stromal cells (hMSCs) cultured as 3D spheroids acquire enhanced anti-inflammatory properties. Here, we explored the effects of hMSC spheroids on macrophages that are critical cells in the regulation of inflammation. Conditioned medium (CM) from hMSC spheroids inhibited lipopolysaccharide-stimulated macrophages from secreting proinflammatory cytokines TNFα, CXCL2, IL6, IL12p40, and IL23. CM also increased the secretion of anti-inflammatory cytokines IL10 and IL1ra by the stimulated macrophages, and augmented expression of CD206, a marker of alternatively activated M2 macrophages. The principal anti-inflammatory activity in CM had a small molecular weight, and microarray data suggested that it was prostaglandin E2 (PGE2). This was confirmed by the observations that PGE2 levels were markedly elevated in hMSC spheroid-CM, and that the anti-inflammatory activity was abolished by an inhibitor of cyclooxygenase-2 (COX-2), a silencing RNA for COX-2, and an antibody to PGE2. The anti-inflammatory effects of the PGE2 on stimulated macrophages were mediated by the EP4 receptor. Spheroids formed by human adult dermal fibroblasts produced low levels of PGE2 and displayed negligible anti-inflammatory effects on stimulated macrophages, suggesting the features as unique to hMSCs. Moreover, production of PGE2 by hMSC spheroids was dependent on the activity of caspases and NFκB activation in the hMSCs. The results indicated that hMSCs in 3D-spheroid cultures are self-activated, in part by intracellular stress responses, to produce PGE2 that can change stimulated macrophages from a primarily proinflammatory M1 phenotype to a more anti-inflammatory M2 phenotype.

Elevated cerebrospinal fluid levels of prostaglandin E2 and 15‐(S)‐hydroxyeicosatetraenoic acid in multiple sclerosis

To test the hypothesis that the arachodinic acid metabolites prostaglandin E2 (PGE2) and 15-(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) in cerebrospinal fluid (CSF) are elevated and reflect neuroinflammation and degenerative changes in multiple sclerosis (MS). We measured PGE2 and 15(S)-HETE concentrations, as well as markers of axonal and astroglial injury in CSF from 46 MS patients, 46 healthy siblings and 50 controls. We found elevated levels of both PGE2 and 15(S)-HETE in MS compared with the control and sibling groups. Siblings had lower PGE2 levels and higher 15(S)-HETE levels than controls. There were no correlations between either PGE2 or 15(S)-HETE and clinical scores of MS severity or biochemical markers of axonal or astroglial injury. These data suggest no direct involvement of PGE2 and 15(S)-HETE in the MS disease process. Rather, the elevated levels reflect a general up-regulation of arachidonic acid metabolism and neuroinflammation.

On the Horizon From the ORS

On the Horizon From the ORS

Reciprocal Regulation of Cyclooxygenase (COX)‐1 and COX‐2 Expression in Murine Resident and Elicited Peritoneal Macrophages by Lipopolysaccharide

Macrophages express COX‐1 and COX‐2 that are involved in the biosynthesis of prostanoids including prostaglandin E2 (PGE2). In this study, we examined the relative expression of COX‐1 and COX‐2 in murine resident (RPM) and thioglycollate‐elicited peritoneal macrophages (EPM) and determined the influence of lipopolysaccharide (LPS) on their gene expression and functions. Freshly harvested RPM and EPM from C57Bl/6J mice were allowed to adhere on cell culture plates and incubated with LPS (0.1 to 100 ng/ml) for 4 h for measuring COX‐1 and COX‐2 mRNA expression by quantitative RT‐PCR. To monitor COX‐1 and COX‐2 protein expression, Western blot analyses were carried out after incubating macrophages with LPS for 2, 4, 8, 16 and 24 h. The production of PGE2 and TNF‐α by RPM and EPM were monitored as functional indices. The study revealed that, in comparison to EPM, RPM constitutively express markedly higher levels of COX‐1 (12‐fold) and COX‐2 (3.5‐fold) mRNA and produce substantially higher quantities of PGE2 when stimulated with LPS. In contrast, EPM generate considerably lower levels of PGE2 and higher levels of TNF‐α than RPM when activated with LPS. Incubation of RPM or EPM with LPS resulted in marked increases in COX‐2 mRNA (up to 1000 fold) and protein expression. On the other hand, LPS challenge significantly inhibited COX‐1 mRNA (60 to 90%) and protein expression in both RPM and EPM. These results demonstrate that LPS reciprocally regulate the expression of COX‐1 and COX‐2 in murine peritoneal macrophages. Furthermore, the newly recruited peritoneal macrophages seem to undergo plausible phenotype switching from high‐TNFα/low‐PGE2 producing EPM to low‐TNFα/high‐PGE2 producing RPM. (Supported by NIH R01‐HL070101 and Carey Arthritis Fund).

Selective and Nonselective Cyclooxygenase-2 Inhibitors and Experimental Fracture-Healing

Background: Cyclooxygenase-2-specific anti-inflammatory drugs (coxibs) and nonspecific nonsteroidal anti-inflammatory drugs have been shown to inhibit experimental fracture-healing. The present study tested the hypothesis that these effects are reversible after short-term treatment. Methods: With use of a standard model of fracture-healing, identical ED50 dosages of either a nonsteroidal anti-inflammatory drug (ketorolac), a coxib (valdecoxib), or vehicle (control) were orally administered to rats for either seven or twenty-one days and fracture-healing was assessed with biomechanical, histological, and biochemical analyses. Results: When healing was assessed at twenty-one days, the seven-day treatment produced only a trend for a higher rate of nonunion in valdecoxib and ketorolac-treated animals as compared with controls. No differences were observed at thirty-five days. The twenty-one-day treatment produced significantly more nonunions in valdecoxib-treated animals as compared with either ketorolac-treated or control animals (p < 0.05), but these differences disappeared by thirty-five days. The dose-specific inhibition of these drugs on prostaglandin E2 levels and the reversibility of the effects after drug withdrawal were assessed in fracture calluses and showed that ketorolac treatment led to twofold to threefold lower levels of prostaglandin E2 than did valdecoxib. Withdrawal of either drug after six days led to a twofold rebound in these levels by fourteen days. Histological analysis showed delayed remodeling of calcified cartilage and reduced bone formation in association with valdecoxib treatment. Conclusions: Cyclooxygenase-2-specific drugs inhibit fracture-healing more than nonspecific nonsteroidal anti-inflammatory drugs, and the magnitude of the effect is related to the duration of treatment. However, after the discontinuation of treatment, prostaglandin E2 levels are gradually restored and the regain of strength returns to levels similar to control. Clinical Relevance: While animal studies have suggested that both nonsteroidal anti-inflammatory drugs and coxibs inhibit bone repair, there have been no clinical reports with Level-1 evidence to support these findings. Because most patients discontinue these medications when pain has subsided, it is important to determine whether the inhibitory effects are reversible. The present animal study confirms that inhibition of cyclooxygenase 2 impairs fracture-healing and that the effects are dependent on both the dose and duration of treatment. However, the reduced prostaglandin levels in callus rebound with drug withdrawal, and the impairment in the mechanical integrity of the healing fractures is reversed after short-term treatment.