Administration Of PGE2 Research Articles

Follicle Diameter and Systemic Hormone Interrelationships during Induction of Follicle Collapse with Intrafollicular Prostaglandin E2 and F2α in Mares.

The objectives were to determine: (i) whether intrafollicular administration of PGE2 and PGF2α to mares would hasten follicle collapse and (ii) the differences in reproductive hormone characteristics in mares with spontaneous and prostaglandin-induced follicle collapses. Six mares were followed for two oestrous cycles each: when the mares reached a follicle diameter of 30-35 mm and showed mild-to-moderate endometrial oedema, mares were administered a single 0.5 ml dose containing 500 μg PGE2 and 125 μg PGF2α (treatment cycle) or a placebo (0.5 ml of water for injection; control cycle) into the preovulatory follicle (Hour 0). Blood samples were collected, and serial ultrasound examinations were performed until follicle collapse. Treated mares showed follicle collapse significantly earlier (20.0 ± 5.9 h) than the control mares (72.0 ± 10.7 h). The LH, progesterone, total oestrogens and oestradiol concentrations did not differ between groups; however, the progesterone concentration increased more between 48 and 72 h after follicle injection in the treatment compared to the control cycles (P < 0.05). In conclusion, intrafollicular treatment with PGE2 and PGF2α hastened follicle collapse in mares without the simultaneous use of an inductor of ovulation; despite the early induction of follicle collapse, the profiles of LH and oestradiol were not altered. This study provides information on the role of prostaglandins (PGs) in the process of follicle wall rupture and collapse and suggests that this may happen even before the beginning of the sharp rise in circulating LH at the final stage of the ovulatory surge.

Prostaglandin E2 Signaling through EP4 Receptor Promotes Hematopoietic Stem Cell Niche Regeneration and Enhances Hematopoietic Recovery

High-dose radiation treatment for hematological malignancies such as leukemia, lymphoma or multiple myeloma induces damage to the bone marrow microenvironment that limits hematopoietic regeneration. We previously demonstrated that single administration of dimethyl prostaglandin E2 (dmPGE2) at 24 hrs post lethal irradiation (796 cGys; LD70/30) in mice increases survival and accelerates hematopoietic recovery. Since bone marrow niches regulate hematopoiesis, we investigated whether the effects of PGE2 administration on hematopoietic recovery and survival were dependent on PGE2 signaling in bone marrow stromal cells. Total body irradiation (TBI) severely disrupts bone marrow niche components including MSC, osteoblasts and endothelial cells. Multivariate flow cytometry revealed that exposure of mice to 650 cGy TBI reduced the total number of bone marrow MSC defined as CD45- Ter119- CD31- PDGFR+ CD51+ MSC by 8 fold and reduced functional fibroblast colony formation (CFU-F) by 8.5 fold. Osteoblast (OB) and endothelial cell (EC) counts were reduced by 6.5 fold and 6 fold respectively. Treatment of mice with dmPGE2 at 24 hours post-irradiation substantially rescued MSC and EC, which were 3.5 and 2.2-fold higher compared to un-treated irradiated mice. Histological and flow cytometric analysis indicated that total OB, OB precursors and mesenchymal progenitor cells (MPC) in bone marrow were also enhanced by PGE2 administration. Interestingly, dmPGE2 failed to rescue MSC and OB from aged (24-27 mo/old) mice compared to young (4-6 mo/old) mice. Since PGE2 signals through four receptors (EP1-4), each with unique signaling pathways, we hypothesized that the hematopoietic niche regeneration was due to activation of PGE2 signaling via one or more EP receptors. Treatment of mice with the EP4 receptor agonist L-902,688 following irradiation enhanced the recovery of bone marrow MSC by 3.1 fold and EC by 2.0 fold compared to untreated irradiated control mice. EP1, EP2 and EP3 receptor agonists failed to enhance hematopoietic niche regeneration in irradiated mice. To further investigate the role of EP4 signaling in bone marrow niche reconstruction and hematopoietic recovery, we transplanted BM cells from wild-type mice into syngeneic wild-type or conditional inducible EP4-/- recipients after lethal irradiation, and analyzed stromal cells recovery and hematopoietic reconstitution. At 15 days post-transplantation, chimeric mice with EP4-/- stroma displayed attenuated niche recovery and hematopoietic reconstitution compared to mice with wild-type stroma. PGE2 or EP4 agonist administration increased the expression of the endogenous anti-apoptotic protein Survivin, and enhanced survival of MSC and EC. In conclusion, our study suggests that PGE2 signaling through the EP4 receptor increases hemotopoietic recovery after TBI by enhancing the survival and expansion of MSC and endothelial cells. Furthermore, our data suggest the modulation/regulation PGE2 signaling in hematopoietic niche components could be beneficial to enhance HSC recovery following clinical hematopoietic transplantation. Disclosures No relevant conflicts of interest to declare.

Prostaglandin E2 increases proximal tubule fluid reabsorption, and modulates cultured proximal tubule cell responses via EP1 and EP4 receptors

Renal prostaglandin (PG) E2 regulates salt and water transport, and affects disease processes via EP1–4 receptors, but its role in the proximal tubule (PT) is unknown. Our study investigates the effects of PGE2 on mouse PT fluid reabsorption, and its role in growth, sodium transporter expression, fibrosis, and oxidative stress in a mouse PT cell line (MCT). To determine which PGE2 EP receptors are expressed in MCT, qPCR for EP1–4 was performed on cells stimulated for 24 h with PGE2 or transforming growth factor beta (TGFβ), a known mediator of PT injury in kidney disease. EP1 and EP4 were detected in MCT, but EP2 and EP3 are not expressed. EP1 was increased by PGE2 and TGFβ, but EP4 was unchanged. To confirm the involvement of EP1 and EP4, sulprostone (SLP, EP1/3 agonist), ONO8711 (EP1 antagonist), and EP1 and EP4 siRNA were used. We first show that PGE2, SLP, and TGFβ reduced H3-thymidine and H3-leucine incorporation. The effects on cell-cycle regulators were examined by western blot. PGE2 increased p27 via EP1 and EP4, but TGFβ increased p21; PGE2-induced p27 was attenuated by TGFβ. PGE2 and SLP reduced cyclinE, while TGFβ increased cyclinD1, an effect attenuated by PGE2 administration. Na-K-ATPase α1 (NaK) was increased by PGE2 via EP1 and EP4. TGFβ had no effect on NaK. Additionally, PGE2 and TGFβ increased fibronectin levels, reaching 12-fold upon co-stimulation. EP1 siRNA abrogated PGE2-fibronectin. PGE2 also increased ROS generation, and ONO-8711 blocked PGE2-ROS. Finally, PGE2 significantly increased fluid reabsorption by 31 and 46% in isolated perfused mouse PT from C57BL/6 and FVB mice, respectively, and this was attenuated in FVB-EP1 null mice. Altogether PGE2 acting on EP1 and EP4 receptors may prove to be important mediators of PT injury, and salt and water transport.

Catherine Pasquier Memorial Award Lecture (shared): Critical role of oxidative and nitrosative stress in pathophysiology: Inflammation, cell injury and cancer

The presence of chronic inflammation associated with sustained cell death and proliferation turnover is a potential tissue environment for cancer development. In this situation, oxidative and nitrosative stress are the major driving force playing a key role either as a preventing or inducer/promoter of tumorigenesis. During inflammatory response, activated macrophages release a great variety of protease that degraded extracellular matrix, inflammatory mediators that plays a relevant role in chemotaxis and cell signaling, as well as oxygen and nitrogen reactive species (ROS and RNS, respectively) actively involved in cell defense through oxidation of essential biological molecules such as DNA, carbohydrates, proteins and lipids. Iron-loosely bound plays a relevant role in promoting oxidative-dependent reactions leading to cell damage. In this sense, we have shown that locally iron overload induced an accumulation of neutrophils with increased NADPH-derived superoxide anion generation in exudates from rats submitted to carrageenan-induced granuloma model. The increase of lipid peroxidation products was associated with the presence of iron-loosely bound, reduction of antioxidant status in inflammatory exudates and blood, as well as induction of hepatic acute phase response. A similar pro-inflammatory and oxidant reaction was observed by intraperitoneal iron-dextran administration in animals with Mycobacterium butyricum-induced adjuvant arthritis. The administration of a diet devoid of polyunsaturated fatty acids reduced the substrate for lipid peroxidation, and consequently a decrease of conjugated diennes and thiobarbituric acid-reactive substances in plasma and hepatic microsomal fraction were observed in animals with adjuvant arthritis. The acute phase response induced by different inflammatory mediators in hepatocytes has been studied in detail. The induction of carrageenan-induced granuloma drastically reduced the expression of drug metabolizing enzymes (DME) (CYP3A1, CYP4A, CYP2B1, CYP2D and CYP2E1) and their related-activities in rat livers. Cytokines (IL-1α, IL-6 and TNF-α) exerted a specific timely-dependent regulation of DME expression (CYP1A1, CYP1A2 and CYP3A4) and secretion of acute phase reactants (albumin, ferritin, fibrinogen and ceruloplasmin) in cultured primary human hepatocytes. Although TNF-α induces acute phase response and liver injury in different experimental settings such as sepsis, we have also shown that TNF-α-dependent induction of nitric oxide synthase type II (NOS-2) hepatic expression during PGE1 pre-administration drastically reduces D-galactosamine (D-GalN)-induced liver injury in rats. The pre-administration of PGE1 also reduced cell death induced by D-GalN in primary culture of human and rat hepatocytes. However, the cytoprotective properties of PGE1 do not involve a reduction of D-GalN-induced ROS production. Nitric oxide (NO) has demonstrated to exert pro- and anti-apoptotic properties according to local concentration, target cell, as well as the presence of other ROS such as superoxide anion generating high reactive peroxynitrite. We have observed that moderate increase of PGE1-dependent NO prevented further potent NF-kB-dependent NOS-2 expression and apoptosis induced by D-GalN in cultured rat hepatocytes. However, the administration of PGE1 at advanced stages of cell injury or high doses of NO donor increased cell death in D-GalN-treated human and rat hepatocytes. The induction of apoptosis by D-GalN was associated with ROS production, mitochondrial dysfunction and post-translation (S-nitrosylation and Tyrosine nitration) modifications of proteins involved in unfolded protein response, enzymatic antioxidants, cell metabolism and death in cultured human hepatocytes. In consequences, the administration of antioxidants such as N-acetylcysteine, Q10 and MnTBAB reduced ROS generation, mitochondrial dysfunction and apoptosis induced by D-GalN and glycochenodeoxycholate (GCDCA) in cultured human hepatocytes. The cytoprotective properties of α-tocopherol appears to mostly related to gene regulation (NOS-2, PPAR-α and carnitine palmitoyl transferase) rather than to its antioxidant activity in D-GalN-induced cell death in hepatocytes. The reduction of cell death by α-tocopherol was also associated with reduction of CYP7A1, Na+-taurocholate co-transporting polypeptide (NTCP), and heme oxygenase-1 expression, as well as increased NOS-2 expression and NO production in GCDCA-treated hepatocytes. α-Tocopherol and NO donors increased NTCP cysteine S-nitrosylation and Tyrosine nitration, and reduced Taurocholic acid uptake in hepatocytes. The pro-apoptotic properties of high NO donor concentration have been successfully applied to exert antitumoral activity in liver cancer cells. In this sense, the administration of NO donor or NOS-3 overexpression induces p53-dependent cell death receptor expression and apoptosis in hepatoma cell lines. The first generation adenovirus specifically designed to induce NOS-3 overexpression in liver cancer cells induced oxidative and nitrosative stress, DNA damage, p53, cell death receptors expression and their S-nitrosylation, and apoptosis that was correlated to a reduction of cell proliferation and growth of tumors orthotopically implanted in fibrotic livers. The S-nitrosylation of cell death receptors (TNF-α, CD95 and TRAIL-R1) plays a critical role on the shift from extrinsic to intrinsic cell death pathways during antitumoral molecular therapy (Sorafenib Tosylate) in liver cancer cells. In conclusion, new data are emerging that elucidate the extraordinary role that plays ROS- and RNS-dependent posttranslational target modifications in regulating intracellular cell signaling.

Intrafollicular treatment with prostaglandins PGE2 and PGF2α inhibits the formation of luteinised unruptured follicles and restores normal ovulation in mares treated with flunixin-meglumine.

Haemorrhagic anovulatory follicle is the most common pathological anovulatory condition in the mare, but its cause remains unknown. An experimental model to induce luteinised unruptured follicles (LUF) with flunixin-meglumine (FM) has been developed. Luteinised unruptured follicles share similar morphological and hormonal characteristics with haemorrhagic anovulatory follicles. To test the effect of intrafollicular administration of prostaglandins PGE2 and PGF2α during the periovulatory period on ovulation and pregnancy in FM-treated mares. In vivo experiment in a crossover design. Five mares were followed during 2 oestrous cycles each. All mares were given FM at 1.7 mg/kg bwt i.v. every 12 h from Hour 0 (Hour 0 = human chorionic gonadotrophin treatment) to Hour 36. In treatment cycles (n = 5), at Hour 32 the preovulatory follicle was punctured and 0.5 ml of a solution containing 500 μg of PGE2 and 125 μg of PGF2α was deposited within the follicle. In control cycles, water for injection was administered into the follicle at the same time. In 3 control and 3 treatment cycles, mares were also inseminated at Hour 24. Diagnosis of ovulation/LUF formation and pregnancy was performed by ultrasound examination between Hours 36 and 72 and 14 days after ovulation/LUF formation, respectively. During the treatment cycles, all mares ovulated normally (100% ovulation rate) 36-48 h after human chorionic gonadotrophin, while in 4 of 5 control cycles the mares developed an LUF (80%, P<0.05). All 3 inseminated mares became pregnant in the treatment cycles, but not in the control cycles. Intrafollicular treatment with PGE2 and PGF2α overcame the anovulatory effect of FM. This sheds new insights into the knowledge on the possible therapeutic options for ovulatory failure in the mare.

Vasodilating prostaglandin E1 does not reproduce interleukin-1β-induced oxygen metabolism abnormalities in rabbits.

We have reported that administration of recombinant human interleukin (IL)-1β induced circulatory shock in rabbits by causing overproduction of vasodilating prostaglandin(s) and simultaneously impaired oxygen metabolism by causing an abnormal dependence of oxygen consumption (VO2) on oxygen delivery (DO2). The present study was carried out to compare the effect of administration of exogenous PGE1 with that of IL-1β on oxygen metabolism. Sixteen rabbits were assigned to one of three groups and given a single injection of 10 μg/kg IL-1β (IL-1β group, n = 5), continuous infusion of 1 μg/kg/min PGE1 (PGE1 group, n = 6), or saline (control group, n = 5). All rabbits were subjected to stepwise cardiac tamponade to decrease DO2 by inflating a balloon placed into the pericardial sac. The VO2/DO2 relation was analyzed by the dual-line method. Both IL-1β and PGE1 decreased the baseline value of mean arterial pressure by approximately 25% without inducing significant alteration of the cardiac index. With respect to the VO2/DO2 relation, the slope of the supply-independent line was significantly increased in the IL-1β group (y = 0.13x + 6.4), but not in the PGE1 group (y = 0.01x + 10.0) compared to that in the control group (y = 0.05x + 8.7). These results indicate that simple vasodilation and hypotension induced by administration of PGE1 are insufficient to account for the abnormal oxygen metabolism induced by IL-1β.

Multiple roles of the PGE2 -EP receptor signal in vascular permeability.

PGE2 is a major prostanoid that regulates inflammation by stimulating EP1-4 receptors. However, how PGE2 induces an initial inflammatory response to vascular hyper-permeability remains unknown. Here we investigated the role of the PGE2 -EP receptor signal in modulating vascular permeability both in vivo and in vitro. We used a modified Miles assay and intravital microscopy to examine vascular permeability in vivo. Endothelial barrier property was assessed by measuring transendothelial electrical resistance (TER) in vitro. Local administration of PGE2 , an EP2 or EP4 receptor agonist into FVB/NJcl mouse ear skin caused vascular leakage, indicated by dye extravasation. Intravital microscopy and laser Doppler blood-flow imaging revealed that these treatments dilated peripheral vessels and increased local blood flow. Pretreatment with the vasoconstrictor phenylephrine inhibited the PGE2 -induced blood flow increase and vascular leakage. In contrast to the EP2 and EP4 receptor agonists, administration of an EP3 receptor agonist suppressed vascular leakage without altering vascular diameter or blood flow. In isolated HUVECs, the EP3 receptor agonist elevated TER and blocked thrombin-induced dextran passage. Inhibiting PKA restored the hypo-permeability induced by the EP3 receptor agonist. Activation of the PGE2 -EP2 or -EP4 receptor signal induces vasodilatation in mural cells, resulting in increased local blood flow and hyper-permeability. In contrast, activation of the PGE2 -EP3 receptor signal induces a cAMP-dependent enhancement of the endothelial barrier, leading to hypo-permeability. We provide the first evidence that endothelial cells and mural cells cooperate to modulate vascular permeability.

Application of prostaglandin E2 improves ileal blood flow in NEC

PurposeIndomethacin, a nonselective prostaglandin inhibitor used to treat patent ductus arteriosus, is associated with intestinal perforation inducing an NEC-like illness. We sought to define the contribution of prostaglandin E2 (PG E2) and its receptor EP4 to intestinal blood flow regulation in premature neonates with NEC. MethodsNewborn Sprague-Dawley rats were randomized by litter to undergo experimental NEC induction or to serve as a CONTROL. At 48hours of age, intestinal laser Doppler blood flow was assessed at baseline and after intraperitoneal administration of indomethacin, PG E2, EP4 antagonist, or EP4 agonist. Data were analyzed using a 2-way ANOVA with post hoc Tukey-Kramer correction. ResultsAt baseline, NEC animals had lower intestinal blood flow than controls. Indomethacin, PG E2 and EP4 agonist all increased ileal blood flow, but PG E2 and EP4 agonist increased blood flow the most in NEC pups. EP4 antagonist decreased intestinal perfusion in both groups. ConclusionThe above evidence suggests the importance of PG E2 and EP4 in regulation of neonatal intestinal blood flow. Since indomethacin treatment of patent ductus arteriosus in the premature infant is associated with an increased risk of intestinal perforation owing to compromised blood flow, PG E2 supplementation might provide intestinal protection if administered simultaneously with indomethacin.

Nonsteroidal Anti-Inflammatory Drugs and the Heart

Aspirin has been on the market for 115 years. Beginning with the marketing of indomethacin for the treatment of rheumatoid arthritis in 1963, at least 20 other nonsteroidal anti-inflammatory drugs (NSAIDs) with aspirin-like actions have been developed over the past 50 years,1 culminating with the introduction of a new class of selective inhibitors of cyclooxygenase (COX)-2, the coxibs, approximately 15 years ago.2 The NSAIDs represent the single most crowded family of drugs sharing the same therapeutic activities and mechanism of action, perhaps reflecting the unmet therapeutic need in the area of pain management and the large interindividual variability in response to these agents. NSAIDs provide symptomatic relief of pain and inflammation associated with a variety of human disorders, including the rheumatic diseases. Their shared therapeutic actions (ie, analgesic, anti-inflammatory, and antipyretic) are usually accompanied by mechanism-based adverse effects that can, at least in part, be attenuated as a function of individual pharmacokinetic or pharmacodynamic properties.1 Nuances in the tolerability of different NSAIDs had been described in the precoxib era on the basis of clinical trials of a few hundred patients treated for up to a few months with soft end points. More recently, however, important differences in safety have been demonstrated in head-to-head randomized comparisons of individual coxibs and 1 or more traditional NSAIDs that involved tens of thousands of patients treated for up to a few years with hard end points. Even more significantly, the interpretation of the effects of NSAIDs has been greatly enhanced by the availability, for the first time, of large, placebo-controlled trials that aimed to assess the potential for chemoprevention of colorectal cancer by rofecoxib or celecoxib. As a result of these developments, the whole field of NSAIDs has been illuminated during the past 15 years. Although epidemiological studies had previously associated …

Hematopoietic Stem and Progenitor Cells Locally Produce Neutrophils Necessary To Resolve S. Aureus-Infected Wounds

Staphylococcus aureus is a frequent cause of chronic nosocomial skin and soft tissue infections that can spread to muscle, bone and connective tissue. During the normal inflammatory response to infection, a heterogeneous population of circulating and bone marrow (BM)-derived cells are recruited to the site of injury that collectively contributes to host defense and tissue repair. Included among these are polymorphonuclear leukocytes (PMN), which serve a critical role as the first-line of defense in the innate immune response to S. aureus. Through detection and intervention of the number of PMN accessing the wound in a transgenic mouse model of S. aureus infected skin wounds, we discovered that a subset of hematopoietic stem and progenitor cells (HSPC) that are lineage negative, Sca-1+ and c-kit+ (LSK cells) traffic to the site of infection, where they undergo local granulopoiesis and account for up to 30% of total PMN found within the abscess. Blocking trafficking of LSK revealed that local expansion into PMN is critical for clearance of bacteria and efficient wound closure. To determine if toll like receptor (TLR) signaling contributed to local granulopoiesis, we compared the capacity of multi-potent LSK cells from WT, TLR2-deficient or MyD88-deficient mice transferred into wounds functioned in a similar manner to those trafficking into WT wounds in terms of PMN expansion and resolution of infection. LSK isolated and adoptively transferred from BM of WT mice into WT, TLR2-deficient or MyD88-deficient mice produced equivalent numbers of PMN and contributed to wound resolution. In contrast, LSK derived from TLR2- or MyD88-deficient mice that were transferred into WT wounds produced few PMN, indicating that TLR2 and MyD88 are both necessary for local granulopoiesis in response to S. aureus infection. The signaling pathway involved downstream production of PGE2 that acts in an autocrine manner to signal LSK cell expansion and myeloid proliferation. Local administration of PGE2 to infected wounds of TLR2- or MyD88-deficient mice restored PMN production and rescued resolution of infection in knockout mice. These findings shed light on a novel innate immune mechanism by which stem and progenitor cells traffic to S. aureus-infected wounds and produce PMN following direct stimulation in a manner dependent on TLR2/MyD88 signaling. Current studies are focused on the process of local granulopoiesis in terms of: The local granulopoietic niche and comparing it with myeloid proliferation in BM; The antibacterial capacity of PMN locally produced versus systemically recruited to the wound; Identification of molecular pathways parallel or downstream of PGE2 that are critical for on demand production of PMN within infection. An overall goal is to strategically manipulate the process of on demand production of PMN to more efficiently battle chronic and persistent infections as may occur in immune compromised patients. Disclosures: No relevant conflicts of interest to declare.

Induction of Cyclooxygenase-2 Signaling by Stomatococcus mucilaginosus Highlights the Pathogenic Potential of an Oral Commensal

Stomatococcus mucilaginosus is an oral commensal that has been occasionally reported to cause severe infections in immunocompromised patients. There is no information about the pathogenic role of S. mucilaginosus in airway infections. In a cohort of 182 subjects with bronchiectasis, we found that 9% were colonized with S. mucilaginosus in their lower airways by culture growth from bronchoalveolar lavage. To address the pathogenic potential of S.mucilaginosus, we developed a murine model of S. mucilaginosus lung infection. Intratracheal injection of S. mucilaginosus in C57BL/6 mice resulted in a neutrophilic influx with production of proinflammatory cytokines, chemokines, and lipid mediators, mainly PGE₂ with induction of cyclooxygenase-2 (COX-2) in the lungs. Presence of TLR2 was necessary for induction of COX-2 and production of PGE₂ by S. mucilaginosus. TLR2-deficient mice showed an enhanced clearance of S. mucilaginosus compared with wild-type mice. Administration of PGE₂ to TLR2(-/-) mice resulted in impaired clearance of S. mucilaginosus, suggesting a key role for COX-2-induced PGE₂ production in immune response to S. mucilaginosus. Mechanistically, induction of COX-2 in macrophages was dependent on the p38-ERK/MAPK signaling pathway. Furthermore, mice treated with S. mucilaginosus and Pseudomonas aeruginosa showed an increased mortality compared with mice treated with PA103 or S. mucilaginosus alone. Inhibition of COX-2 significantly improved survival in mice infected with PA103 and S. mucilaginosus. These data provide novel insights into the bacteriology and personalized microbiome in patients with bronchiectasis and suggest a pathogenic role for S. mucilaginosus in patients with bronchiectasis.

Intrauterine Group A Streptococcal Infections Are Exacerbated by Prostaglandin E2

Streptococcus pyogenes (Group A Streptococcus; GAS) is a major cause of severe postpartum sepsis, a re-emerging cause of maternal morbidity and mortality worldwide. Immunological alterations occur during pregnancy to promote maternofetal tolerance, which may increase the risk for puerperal infection. PGE2 is an immunomodulatory lipid that regulates maternofetal tolerance, parturition, and innate immunity. The extent to which PGE2 regulates host immune responses to GAS infections in the context of endometritis is unknown. To address this, both an in vivo mouse intrauterine (i.u.) GAS infection model and an in vitro human macrophage-GAS interaction model were used. In C57BL/6 mice, i.u. GAS inoculation resulted in local and systemic inflammatory responses and triggered extensive changes in the expression of eicosanoid pathway genes. The i.u. administration of PGE2 increased the mortality of infected mice, suppressed local IL-6 and IL-17A levels, enhanced neutrophilic inflammation, reduced uterine macrophage populations, and increased bacterial dissemination. A role for endogenous PGE2 in the modulation of antistreptococcal host defense was suggested, because mice lacking the genes encoding the microsomal PGE2 synthase-1 or the EP2 receptor were protected from death, as were mice treated with the EP4 receptor antagonist, GW627368X. PGE2 also regulated GAS-macrophage interactions. In GAS-infected human THP-1 (macrophage-like) cells, PGE2 inhibited the production of MCP-1 and TNF-α while augmenting IL-10 expression. PGE2 also impaired the phagocytic ability of human placental macrophages, THP-1 cells, and mouse peritoneal macrophages in vitro. Exploring the targeted disruption of PGE2 synthesis and signaling to optimize existing antimicrobial therapies against GAS may be warranted.

A new and automated method for objective analysis of detrusor rhythm during the filling phase

There is growing acceptance that the detrusor muscle is not silent during the filling phase of the micturition cycle but displays low-amplitude phasic contractions that have been associated with urinary urgency. Unfortunately, there is currently no standardized methodology to quantify detrusor rhythm during the filling phase. Therefore, the purpose of this study was to develop an automated computer algorithm to analyze rat detrusor rhythm in a quick, accurate, and reproducible manner. Strips of detrusor smooth muscle from rats (n = 17) were placed on force transducers and subjected to escalating doses of PGE2 to generate contractile rhythm tracings. An automated computer algorithm was developed to analyze contractile frequency, amplitude, and tone on the generated rhythm tracings. Results of the automated computerized analysis were compared to human (n = 3) interpretations. Human interpreters manually counted contractions and then recounted the same data two weeks later. Intra-observer, inter-observer, and human-to-computer comparisons were performed. The computer algorithm quantified concentration-dependent changes in contractile frequency, amplitude, and tone after administration of PGE2 (10(-9)-10(-6)M). Concentration-response curves were similar for all contractile components with increases in frequency identified mainly at physiologic concentrations of PGE2 and increases in amplitude at supra-physiologic concentrations. The computer algorithm consistently over-counted the human interpreters, but with less variability. Differences in inter-observer consistency were statistically significant. Our computerized algorithm accurately and consistently identified changes in detrusor muscle contractile frequency, amplitude, and tone with varying doses of PGE2. Frequency counts were consistently higher than those obtained by human interpreters but without variability or bias. Refinements of this method may allow for more standardized approach in the study of pharmacologic agents on filling phase rhythmic activity.

Prostaglandin E2 regulates murine hematopoietic stem/progenitor cells directly via EP4 receptor and indirectly through mesenchymal progenitor cells

AbstractProstaglandin E2 (PGE2) regulates hematopoietic stem/progenitor cell (HSPC) activity. However, the receptor(s) responsible for PGE2 signaling remains unclear. Here, we identified EP4 as a receptor activated by PGE2 to regulate HSPCs. Knockdown of Ep4 in HSPCs reduced long-term reconstitution capacity, whereas an EP4-selective agonist induced phosphorylation of GSK3β and β-catenin and enhanced long-term reconstitution capacity. Next, we analyzed the niche-mediated effect of PGE2 in HSPC regulation. Bone marrow mesenchymal progenitor cells (MPCs) expressed EP receptors, and stimulation of MPCs with PGE2 significantly increased their ability to support HSPC colony formation. Among the EP receptor agonists, only an EP4 agonist facilitated the formation of HSPC colonies after the coculture with MPCs. PGE2 up-regulated the expression of cytokine-, cell adhesion-, extracellular matrix-, and protease-related genes in MPCs. We also examined the function of PGE2/EP4 signaling in the recovery of the HSPCs after myelosuppression. The administration of PGE2 or an EP4 agonist facilitated the recovery of HSPCs from 5-fluorouracil (5-FU)–induced myelosuppression, indicating a role for PGE2/EP4 signaling in this process. Altogether, these data suggest that EP4 is a key receptor for PGE2-mediated direct and indirect regulation of HSPCs.

Locally administered prostaglandin E2 prevents aeroallergen-induced airway sensitization in mice through immunomodulatory mechanisms

Prostaglandin E2 attenuates airway pathology in asthmatic patients and exerts a protective effect in antigen-sensitized mice when administered systemically. We aimed to establish the consequences of intranasal PGE2 administration on airway reactivity to aeroallergens in mice and reveal the underlying immunoinflammatory mechanisms.PGE2 was administered either daily during a 10-day exposure to house dust mite (HDM) extracts or for limited intervals. Airway hyperreactivity was measured by whole-body and invasive plethysmography. The phenotypes of lung immune cells and cytokine production were analysed by flow cytometry and ELISA, respectively.Airway hyperreactivity was sustainably reduced only when PGE2 administration was restricted to the initial 5 days of exposure to HDM. Lung inflammation, IL-4 production, and airway mast cell activity were also prevented under this early short-term treatment with PGE2. Interestingly, a Th2 response was already committed on day 5 of exposure to HDM. This was paralleled by GM-CSF and osteopontin upregulation and a decreased number of plasmacytoid dendritic and T regulatory cells, as well as a trend towards reduced IL-10 expression. Local PGE2 administration prevented the increase of airway IL-13 and osteopontin and kept lung plasmacytoid dendritic cell counts close to baseline. GM-CSF and Tregs were unaffected by the treatment.These findings suggest that the protection provided by PGE2 is a result of the modulation of early lung immunomodulatory mechanisms, and possibly a shift in the balance of dendritic cells towards a tolerogenic profile.

E-prostanoid 2 receptors dampen mast cell degranulation via cAMP/PKA-mediated suppression of IgE-dependent signaling.

The experimental administration of PGE(2) for the treatment of asthma dampens clinical symptoms, and similar efficacy has been found in dust mite-induced hypersensitivity reactions in animal models. Here, we investigate the mechanism by which PGE(2) mediates suppression of MC degranulation. We find that the effect of PGE(2) on FcεRI-dependent MC degranulation varies from activating to suppressing, depending on the relative ratio of EP(2) to EP(3) expression on these cells with suppression evident only in cells having increased EP(2) to EP(3) expression. Consistent with a role for EP(2) in suppressing MC responses in vitro, we found that a selective EP(2) agonist, Butaprost, inhibited MC-mediated FcεRI-induced immediate hypersensitivity in a model of PCA. EP(2) engagement on MCs increased cAMP production and inhibited FcεRI-mediated calcium influx. In addition, it also decreased the extent of FcεRI-induced Fyn kinase activity, leading to decreased phosphorylation of key signaling molecules such as Gab2 and Akt. Treatment with an antagonist of cAMP or shRNA down-regulation of PKA (the principal intracellular target of cAMP) reversed the EP(2)-mediated inhibitory effect on MC degranulation and restored calcium influx and phosphorylation of Akt. Collectively, the findings demonstrate that EP(2) suppresses the Fyn-mediated signals that are central to FcεRI-dependent MC degranulation, suggesting that engagement of the EP(2) on MCs may be beneficial in dampening allergic responses.

Prostaglandin E2 Stimulates Estradiol Synthesis in the Cerebellum Postnatally with Associated Effects on Purkinje Neuron Dendritic Arbor and Electrophysiological Properties

Prostaglandins (PGs) are ubiquitous membrane-derived, lipid-signaling molecules with wide ranging effects throughout the body. In the brain, PGE(2) is the key regulator of fever after inflammation but is also implicated in neural development and synaptic plasticity. The steroid hormone estradiol is also a key regulator of neural development and synaptic plasticity. Recently, we showed that administering cyclooxygenase (COX) inhibitors to block PGE(2) production increased the total length of Purkinje cell dendrites, the number of dendritic spines, and the level of spinophilin protein, which is enriched in dendritic spines. Correspondingly, PGE(2) administration into the cerebellum decreased spinophilin protein content. We now report that PGE(2) stimulates estradiol synthesis in the immature rat cerebellum via enhanced activity of the aromatase enzyme. Treatment with cyclooxygenase inhibitors reduced cerebellar aromatase activity and estradiol content whereas PGE(2) administration increased both. Treatment with either PGE(2) or estradiol stunted Purkinje neuron dendritic length and complexity and produced a corresponding reduction in spinophilin content. Treatment with formestane to inhibit aromatase activity led to excessive sprouting of the dendritic tree, whereas elevated estradiol had the opposite effect. Electrophysiological measurements from Purkinje neurons revealed novel sex differences in input resistance and membrane capacitance that were abolished by estradiol exposure, whereas a sex difference in the amplitude of the afterhyperpolarization after an action potential was not. Correlated changes in action potential threshold suggest that prolonged alterations in neuronal firing activity could be a consequence of increased estradiol content during the second week of life. These findings reveal a previously unappreciated role for PG-stimulated steroidogenesis in the developing brain and a new potential route for inflammation-mediated disruption of neuronal maturation.

Cytochrome P450 2S1 Depletion Enhances Cell Proliferation and Migration in Bronchial Epithelial Cells, in Part, through Modulation of Prostaglandin E2 Synthesis

Cytochromes P450 (P450s) contribute to the metabolic activation and inactivation of various endogenous substrates. Despite years of research, the physiological role of CYP2S1 remains unknown. CYP2S1 has demonstrated NADPH P450-reductase-independent metabolism of cyclooxygenase (COX)-derived prostaglandins [e.g., prostaglandin G(2) (PGG(2))] at nanomolar concentrations. Arachidonic acid is converted to prostaglandin precursors [PGG(2) and prostaglandin H(2) (PGH(2))] through COX. These precursors are used to synthesize numerous prostanoids, including PGE(2). Prostaglandin E(2) (PGE(2)) promotes cell proliferation and cell migration and inhibits apoptosis. CYP2S1 metabolism of PGG(2) presumably sequesters PGG(2) and PGH(2), making them unavailable for synthesis of prostanoids such as PGE(2). Whether CYP2S1 contributes to prostaglandin metabolism and influences cell physiological remains to be determined. The purpose of this study was to evaluate the physiological role of CYP2S1, if any, in human bronchial epithelial cells [SV40-derived bronchial epithelial cell line (BEAS-2B)]. To do this, we used small interfering RNA to deplete CYP2S1 mRNA and protein by approximately 75% and evaluated the impact of CYP2S1 depletion on cell proliferation and migration. CYP2S1 depletion enhanced both cell proliferation and migration in BEAS-2B cells. Consistent with the proposed role of CYP2S1 in PGE(2) synthesis, the reduction in CYP2S1 expression doubled intracellular PGE(2) levels. Pharmacological administration of PGE(2) enhanced cell proliferation in BEAS-2B cells but failed to promote migration. Our data reveal an important role for CYP2S1 in the regulation of cell proliferation and migration, occurring in part through modulation of prostaglandin synthesis.

Cryptorchidism-induced CFTR down-regulation results in disruption of testicular tight junctions through up-regulation of NF- B/COX-2/PGE2

Does elevated temperature-induced cystic fibrosis transmembrane conductance regulator (CFTR) down-regulation in Sertoli cells in cryptorchid testis disrupt testicular tight junctions (TJs) through the nuclear factor kappa B (NF-κB)/cyclooxygenase-2 (COX-2)/prostaglandin E(2) (PGE(2)) pathway? Our results suggest that CFTR may be involved in regulating testicular TJs and the blood-testis barrier (BTB) through its negative regulation of the NF-κB/COX-2/PGE(2) pathway in Sertoli cells, a defect of which may result in the spermatogenesis defect in cryptorchidism. Cryptorchidism, or undescended testes, is known to result in defective spermatogenesis. Although an elevated testicular temperature is regarded as an important factor affecting spermatogenesis in cryptorchidism, the exact mechanism remains elusive. It is known that the expression of functional CFTR is temperature sensitive. Our previous study has demonstrated that CFTR negatively regulates NF-κB/COX-2/PGE(2) in bronchial epithelial cells. Disruption of TJs by COX-2/PGE(2) has been found in tumour cells. Expression of CFTR, NF-κB, COX-2 and TJ proteins was examined in the testes of a surgical-induced cryptorchidism mouse model and a testicular hyperthermia mouse model, as well as in control or CFTR-inhibited/knocked down primary rat Sertoli cells. PGE(2) production was measured by ELISA. Sertoli cell barrier function was determined by transepethelial resistance (TER) measurements in rat Sertoli cell primary cultures. BTB integrity in the cryptorchidism model was monitored by examining tracker dye injected into seminiferous tubules. Down-regulation of CFTR accompanied by activation of NF-κB, up-regulation of COX-2 and down-regulation of TJ proteins, including ZO-1 and occludin, was observed in a cryptorchidism mouse model. BTB leakage revealed impaired BTB integrity in cryptorchid testes, confirming the destruction of TJs. The inverse correlation of CFTR and COX-2 was further confirmed in a mouse testis hyperthermia model and CFTR knockout mouse model. Culturing primary Sertoli cells at 37°C, which mimics the pathological condition of cryptorchidism, led to a significant decrease in CFTR and increase in COX-2 expression and PGE(2) production compared with the culture at the physiological 32°C. Inhibition or knockdown of CFTR led to increased COX-2 but decreased ZO-1 and occludin expression in Sertoli cells, which could be mimicked by PGE(2), but reversed by NF-κB or COX-2 inhibitor, suggesting that the regulation of TJs by CFTR is mediated by a NF-κB/COX-2/PGE(2) pathway. Inhibition of CFTR or administration of PGE(2) significantly decreased Sertoli cell TER. This study has tested only the CFTR/NF-κB/COX-2/PGE(2) pathway in mouse testes in vivo and in rat Sertoli cells in vitro, and thus, it has some limitations. Further investigations in other species, especially humans, are needed. Our study may shed more light on one of the aspects of the complicated underlying mechanisms of defective spermatogenesis induced by cryptorchidism.

Distinct Roles of Central and Peripheral Prostaglandin E2 and EP Subtypes in Blood Pressure Regulation

Prostaglandin E(2) (PGE(2)) is a major prostanoid with a wide variety of biological activities. PGE(2) can influence blood pressure (BP) both positively and negatively. In particular, centrally administered PGE(2) induces hypertension whereas systemic administration of PGE(2) produces a hypotensive effect. These physiologically opposing effects are generated by the existence of multiple EP receptors, namely EP(1-4), which are G protein-coupled receptors with distinct signaling properties. This review highlights the distinct roles of PGE(2) in BP regulation and the involvement of specific EP receptor subtypes.