Prostaglandin Receptor Agonists Research Articles

Steroid-Induced Ocular Hypertension in Mice Is Differentially Reduced by Selective EP2, EP3, EP4, and IP Prostanoid Receptor Agonists.

We tested five chemically and metabolically stable prostaglandin (PG) receptor agonists in a mouse model of dexamethasone-induced ocular hypertension (OHT). Whilst all compounds significantly (p < 0.05, ANOVA) lowered intraocular pressure (IOP) after twice-daily bilateral topical ocular dosing (5 µg/dose) over three weeks, the time course and magnitude of the responses varied. The onset of action of NS-304 (IP-PG receptor agonist) and rivenprost (EP4-PG receptor agonist) was slower than that of misoprostol (mixed EP2/EP3/EP4-PG receptor agonist), PF-04217329 (EP2-PG receptor agonist), and butaprost (EP2-PG receptor agonist). The rank order of IOP-lowering efficacies aligned with the onset of actions of these compounds. Peak IOP reductions relative to vehicle controls were as follows: misoprostol (74.52%) = PF-04217329 (74.32%) > butaprost (65.2%) > rivenprost (58.4%) > NS-304 (55.3%). A literature survey indicated that few previously evaluated compounds (e.g., latanoprost, timolol, pilocarpine, brimonidine, dorzolamide, cromakalim analog (CKLP1), losartan, tissue plasminogen activator, trans-resveratrol, sodium 4-phenyl acetic acid, etc.) in various animal models of steroid-induced OHT were able to match the effectiveness of misoprostol, PF-04217329 or butaprost. Since a common feature of the latter compounds is their relatively high affinity and potency at the EP2-PG receptor sub-type, which activates the production of intracellular cAMP in target cells, our studies suggest that drugs selective for the EP2-PG receptor may be suited to treat corticosteroid-induced OHT.

The oxylipin analog CS585 prevents platelet activation and thrombosis through activation of the prostacyclin receptor

AbstractCardiovascular disease remains the primary cause of morbidity and mortality globally. Platelet activation is critical for maintaining hemostasis and preventing the leakage of blood cells from the vessel. There has been a paucity in the development of new drugs to target platelet reactivity. Recently, the oxylipin 12(S)-hydroxy-eicosatrienoic acid (12-HETrE), which is produced in platelets, was shown to limit platelet reactivity by activating the prostacyclin receptor. Here, we demonstrated the synthesis of a novel analog of 12-HETrE, known as CS585. Human blood and mouse models of hemostasis and thrombosis were assessed for the ability of CS585 to attenuate platelet activation and thrombosis without increasing the risk of bleeding. Human platelet activation was assessed using aggregometry, flow cytometry, western blot analysis, total thrombus formation analysis system, microfluidic perfusion chamber, and thromboelastography. Hemostasis, thrombosis, and bleeding assays were performed in mice. CS585 was shown to potently target the prostacyclin receptor on the human platelet, resulting in a highly selective and effective mechanism for the prevention of platelet activation. Furthermore, CS585 was shown to inhibit platelet function in human whole blood ex vivo, prevent thrombosis in both small and large vessels in mouse models, and exhibit long-lasting prevention of clot formation. Finally, CS585 was not observed to perturb coagulation or increase the risk of bleeding in the mouse model. Hence, CS585 represents a new validated target for the treatment of thrombotic diseases without the risk of bleeding or off-target activation observed with other prostaglandin receptor agonists.

Ocular Treatments Targeting Separate Prostaglandin Receptors in Mice Exhibit Alterations in Intraocular Pressure and Optic Nerve Lipidome.

Background: Prostaglandin (PG) receptor agonists are the first-line eyedrop medication treatment for glaucoma. The pathophysiology of this disease is not completely known, and elevated intraocular pressure (IOP) is the key risk factor. The membranes of the axons (of the retinal ganglion cells) passing through the optic nerve (ON) head experience significant damage. Lipids are an essential component of the cell's membranes, and their profile changes owing to neurodegeneration. In this investigation, three agonists for distinct PG receptors were used to lower IOP and to determine their effect on the ON lipids. We utilized DBA/2J mice as a model of progressive IOP increase and C57BL/6J mice as a model of ON crush. Methods: DBA/2J and C57BL/6J mice were treated daily for 2 weeks with Latanoprost, PF-04217329, or Rivenprost. The IOP was measured every 2 days and pattern electroretinogram was conducted for DBA/2J throughout the study. Lipidomics of ONs were performed for each model and treatment group. Results: Of the tested compounds, Latanoprost and Rivenprost were the most effective agents decreasing IOP in DBA/2J mice. Triglyceride levels increased in the ONs of DBA/2J mouse model, but phosphatidylethanolamine levels underwent highest level changes in the C57BL/6J mouse model when treated with Latanoprost. Conclusions: Topical ocular FP- and EP4-receptor agonists appreciably lowered IOP in the DBA/2J mice representing pigmentary glaucoma. The observed changes in ON lipidomics in the different models of neurodegeneration suggest possible use of such measures in the development of more effective medicines for both IOP reduction and ON protection.

Current status of diagnosis and treatment of pulmonary hypertension in Chinese tertiary hospitals: A nationwide survey.

We intended to evaluate the diagnosis and treatment status of pulmonary hypertension (PH) in China and provide the basis for the design of the Chinese PH centers system.A questionnaire survey was conducted by sampling from Chinese Class A tertiary hospitals that have carried out the clinical work of PH, including the composition of PH clinical team, the current application of examinations related to PH diagnosis, the availability of PAH-specific medicine and the implementation of PH-related intervention and surgery.A total of 44 valid questionnaires from 20 provinces were collected in this survey. In the vast majority of centers (83.33%, n = 35), pulmonary artery catheterization was routinely performed under X-ray guidance. In 19.05% (n = 8) of centers, pressure measurements were determined at the right time (the end of normal expiration). Only 73.81% (n = 31) centers have carried out acute vasoreactivity testing. Prostacyclin analogues and prostaglandin receptor agonists were just prescribed in 45.45% (n = 20) of the centers. 19 centers (43.18%) were capable of performing balloon pulmonary angioplasty (BPA) and pulmonary endarterectomy (PEA), while 25% (n = 11) were able to perform BPA, PEA, and lung transplantation. There was no significant difference in the diagnosis and treatment of PH between economic regions.The majority of Chinese tertiary hospitals were well equipped with the corresponding personnel, examinations and medicines related to PH, but the standardization and specialization of the management of PH need to be strengthened.

A Canadian, retrospective, multicenter experience with selexipag for a heterogeneous group of pediatric pulmonary hypertension patients.

Selexipag, an oral nonprostanoid prostaglandin receptor agonist, has led to reduced morbidity and mortality in adults with pulmonary arterial hypertension (PAH). While the adult literature has been extrapolated to suggest selexipag as an oral treatment for severe pediatric pulmonary hypertension (PH), longitudinal, multicenter data on the benefits of selexipag in this population are lacking. The purpose of this study is to present a longitudinal, multicentre experience with selexipag in a relatively large cohort of pediatric PH patients and add to the existing selexipag literature. We performed a retrospective, multicenter review describing the clinical outcomes of pediatric PH patients receiving selexipag in addition to standard oral pulmonary vasodilator therapy across three Canadian centers between January 2005 and June 2021. Twenty-four pediatric patients (fifteen female) with a mean age of 9.7 (range 2.0-15.5) years were included. Of this cohort, eighteen (75.0%) were in group 1, one (4.2%) was in group 2, four (16.7%) were in group 3, and one (4.2%) was in group 4. Twenty-two (91.7%) patients were on dual PH therapy after six months. Dosing was targeted to achieve 20-30 mcg/kg/dose orally every twelve hours. Median dose after twelve months was 30 mcg/kg/dose. Twelve months following selexipag initiation, median decreases of 0.2 cm in tricuspid annular plane systolic excursion, 3.5 mmHg in right-ventricular systolic pressure, and 6.1 mmHg in mean pulmonary arterial pressure were observed; none of these changes were statistically significant. Three patients died, one clinically deteriorated and required admission to a pediatric intensive care unit, ten had gastrointestinal symptoms, and three had flushing. Selexipag appears to be a safe and effective adjunctive therapy for pediatric PH patients and has a tolerable adverse effect profile aside from gastrointestinal disturbances. Additional prospective studies of changes in hemodynamics and functional classification over a longer period and with a larger sample are needed. Future research should aim to identify subgroups that stand to benefit from the addition of selexipag as well as optimal timing and dosing for the pediatric population.

Translational profiling of mouse dopaminoceptive neurons reveals region-specific gene expression, exon usage, and striatal prostaglandin E2 modulatory effects.

Forebrain dopamine-sensitive (dopaminoceptive) neurons play a key role in movement, action selection, motivation, and working memory. Their activity is altered in Parkinson's disease, addiction, schizophrenia, and other conditions, and drugs that stimulate or antagonize dopamine receptors have major therapeutic applications. Yet, similarities and differences between the various neuronal populations sensitive to dopamine have not been systematically explored. To characterize them, we compared translating mRNAs in the dorsal striatum and nucleus accumbens neurons expressing D1 or D2 dopamine receptor and prefrontal cortex neurons expressing D1 receptor. We identified genome-wide cortico-striatal, striatal D1/D2 and dorso/ventral differences in the translating mRNA and isoform landscapes, which characterize dopaminoceptive neuronal populations. Expression patterns and network analyses identified novel transcription factors with presumptive roles in these differences. Prostaglandin E2 (PGE2)was a candidate upstream regulator in the dorsal striatum. We pharmacologically explored this hypothesis and showed that misoprostol, a PGE2 receptor agonist, decreased the excitability of D2 striatal projection neurons in slices, and diminished their activity in vivo during novel environment exploration. We found that misoprostol also modulates mouse behavior including by facilitating reversal learning. Our study provides powerful resources for characterizing dopamine target neurons, new information about striatal gene expression patterns and regulation. It also reveals the unforeseen role of PGE2 in the striatum as a potential neuromodulator and an attractive therapeutic target.

Dose-Dependent Effects of a Novel Selective EP4 Prostaglandin Receptor Agonist on Treatment of Critical Size Femoral Bone Defects in a Rat Model.

Difficulties in treating pseudarthrosis and critical bone defects are still evident in physicians’ clinical routines. Bone morphogenetic protein 2 (BMP-2) has shown promising osteoinductive results but also considerable side effects, not unexpected given that it is a morphogen. Thus, the bone regenerative potential of the novel selective, non-morphogenic EP4 prostaglandin receptor agonist KMN-159 was investigated in this study. Therefore, mineralized collagen type-1 matrices were loaded with different amounts of BMP-2 or KMN-159 and implanted into a 5 mm critical-sized femoral defect in rats. After 12 weeks of observation, micro-computed tomography scans were performed to analyze the newly formed bone volume (BV) and bone mineral density (BMD). Histological analysis was performed to evaluate the degree of defect healing and the number of vessels, osteoclasts, and osteoblasts. Data were evaluated using Kruskal-Wallis followed by Dunn’s post hoc test. As expected, animals treated with BMP-2, the positive control for this model, showed a high amount of newly formed BV as well as bone healing. For KMN-159, a dose-dependent effect on bone regeneration could be observed up to a dose optimum, demonstrating that this non-morphogenic mechanism of action can stimulate bone formation in this model system.

Optimizing and Profiling Prostaglandin E2 as a Medical Countermeasure for the Hematopoietic Acute Radiation Syndrome.

Identification of medical countermeasures (MCM) to mitigate radiation damage and/or protect first responders is a compelling unmet medical need. The prostaglandin E2 (PGE2) analog, 16,16 dimethyl-PGE2 (dmPGE2), has shown efficacy as a radioprotectant and radiomitigator that can enhance hematopoiesis and ameliorate intestinal mucosal cell damage. In this study, we optimized the time of administration of dmPGE2 for protection and mitigation against mortality from the hematopoietic acute radiation syndrome (H-ARS) in young adult mice, evaluated its activity in pediatric and geriatric populations, and investigated potential mechanisms of action. Windows of 30-day survival efficacy for single administration of dmPGE2 were defined as within 3 h prior to and 6-30 h after total-body γ irradiation (TBI). Radioprotective and radio-mitigating efficacy was also observed in 2-year-old geriatric mice and 6-week-old pediatric mice. PGE2 receptor agonist studies suggest that signaling through EP4 is primarily responsible for the radioprotective effects. DmPGE2 administration prior to TBI attenuated the drop in red blood cells and platelets, accelerated recovery of all peripheral blood cell types, and resulted in higher hematopoietic and mesenchymal stem cells in survivor bone marrow. Multiplex analysis of bone marrow cytokines together with RNA sequencing of hematopoietic stem cells indicated a pro-hematopoiesis cytokine milieu induced by dmPGE2, with IL-6 and G-CSF strongly implicated in dmPGE2-mediated radioprotective activity. In summary, we have identified windows of administration for significant radio-mitigation and radioprotection by dmPGE2 in H-ARS, demonstrated survival efficacy in special populations, and gained insight into radioprotective mechanisms, information useful towards development of dmPGE2 as a MCM for first responders, military personnel, and civilians facing radiation threats.

Utilizing Designed Receptors Exclusively Activated by Designer Drug Chemogenetic Tools to Identify Beneficial G Protein-Coupled Receptor Signaling for Fibrosis.

Fibrosis or accumulation of extracellular matrix is an evolutionarily conserved mechanism adopted by an organism as a response to chronic injury. Excessive fibrosis, however, leads to disruption of organ homeostasis and is a common feature of many chronic diseases. G protein-coupled receptors (GPCRs) are important cell signaling mediators and represent molecular targets for many Food and Drug Administration-approved drugs. To identify new targets for fibrosis, we used a synthetic GPCR system named designed receptors exclusively activated by designer drugs (DREADDs) to probe signaling pathways essential for fibrotic response. We found that upon expression in human lung fibroblasts, activation of Gq- and Gs-DREADDs abrogated the induction of TGFβ-induced fibrosis marker genes. Genome-wide transcriptome analysis identified dysregulation of multiple GPCRs in lung fibroblasts treated with TGFβ To investigate endogenous GPCR modulating TGFβ signaling, we selected 13 GPCRs that signal through Gq or Gs and activated them by using specific agonists. We examined the impact of each agonist and how activation of endogenous GPCR affects TGFβ signaling. Among the agonists examined, prostaglandin receptor agonists demonstrated the strongest inhibitory effect on fibrosis. Together, we have demonstrated that the DREADDs system is a valuable tool to identify beneficial GPCR signaling for fibrosis. This study in fibroblasts has served as a proof of concept and allowed us to further develop in vivo models for fibrosis GPCR discovery. SIGNIFICANCE STATEMENT: Fibrosis is the hallmark of many end-stage cardiometabolic diseases, and there is an unmet medical need to discover new antifibrotic therapies, reduce disease progression, and bring clinically meaningful efficacy to patients. Our work utilizes designed receptors exclusively activated by designer drug chemogenetic tools to identify beneficial GPCR signaling for fibrosis, providing new insights into GPCR drug discovery.

Regulatory effect of PGE2 on microbicidal activity and inflammatory cytokines in canine leishmaniasis.

Canine leishmaniasis (CanL) is caused by the intracellular parasite Leishmania infantum. Prostaglandin E2 (PGE2 ) exerts potent regulatory effects on the immune system in experimental model Leishmania infection, but this influence has not yet been studied in CanL. In this study, PGE2 and PGE2 receptor levels and the regulatory effect of PGE2 on arginase activity, NO2 , IL-10, IL-17, IFN-γ, TNF-α and parasite load were evaluated in cultures of splenic leucocytes obtained from dogs with CanL in the presence of agonists and inhibitors. Our results showed that splenic leucocytes from dogs with CanL had lower EP2 receptor levels than those of splenic leucocytes from healthy animals. We observed that NO2 levels decreased when the cells were treated with a PGE2 receptor agonist (EP1/EP2/EP3) or COX-2 inhibitor (NS-398) and that TNF-α, IL-17 and IFN-γ cytokine levels decreased when the cells were treated with a PGE2 receptor agonist (EP2) or PGE2 itself. The parasite load in splenic leucocyte cell cultures from dogs with CanL decreased after stimulation of the cells with PGE2 . We conclude that Leishmania infection of dogs modulates PGE2 receptors and speculate that the binding of PGE2 to its receptors may activate the microbicidal capacity of cells.

Oral treprostinil in the treatment of pulmonary arterial hypertension

ABSTRACTIntroduction: Pulmonary arterial hypertension (PAH) is a rare disease resulting in progressive remodeling of the pulmonary vasculature and eventual right ventricular failure. Despite the development of 13 therapies for PAH since 2000, the use of continuously infused prostanoids retains a special role. Infused medications present unique challenges, and the search for an efficacious oral prostanoid culminated in the FDA approval of oral treprostinil – a first in class oral prostanoid medication approved to treat pulmonary arterial hypertension (PAH).Areas covered: In this discussion, we review the pharmacologic properties of oral treprostinil, and discuss three original major registration studies that resulted in the approval and widespread use of the drug. We also review several post-approval analyses and transitional studies. We discuss administration issues including side effects, transitioning, cost, and comparative analysis with selexipag.Expert opinion: Though the prospects of harnessing the benefits of continuously infused prostanoid therapy in a pill form are tantalizing, the gap in efficacy between oral and infused treatment is substantial. Major side effects and exorbitant cost are further barriers to broad uptake. Competition from oral prostaglandin receptor agonist selexipag challenges the commercial success of oral treprostinil. The long-term viability of oral treprostinil rests largely on the outcome of the long-term event-driven study of the molecule added to background approved ERA or PDE5 inhibitor monotherapy.

Mitigation of Hypoxic‐Ischemic Damage in Newborn Rats by Misoprostol, a Prostaglandin E2 Receptor Agonist

Hypoxic‐Ischemic (HI) episodes occur in approximately 50% of preterm infants and result in significant morbidity and mortality. HI activates the nuclear transcription factor HIF1α, which in turn up‐regulates a variety of antioxidant response element‐driven genes including Bnip3, whose protein product has a pivotal role in hypoxia‐induced apoptosis and/or necrosis. Increased Bnip3 expression has been implicated in necrotizing enterocolistis (NEC), an inflammatory bowel disease of premature infants. Previously our laboratory has shown that Misoprostol, a prostaglandin E2 receptor agonist, mitigates the effects of Bnip3 in an enterocyte cell model. We hypothesized that misoprostol regulates intestinal Bnip3 activity in a rat model of HI. Dams and their pups were exposed to environmental hypoxia (10% O2) or normoxia (21% O2) from post‐natal days (PND) 3–10. Experimental animals were injected subcutaneously with 0.1mg/kg Misoprostol each day between PND 3–10, while control animals received saline injections over the same period. PND10 pups were sacrificed, and transverse colon regions were isolated and analyzed for Bnip3 protein expression via western blotting. HI induced a 17‐fold increase in Bnip3 protein expression (p&lt;0.001); in contrast, Misoprostol induced a 72% decrease in protein expression (p&lt;0.05) in hypoxic rats. To explore this effect mechanistically, we examined mitochondrial permeability transition pore (mPTP) opening in HEK293 cells. As expected, Bnip3 transfection increased mPTP opening, whereas Misoprostol inhibited mPTP opening in cells transfected with Bnip3. Furthermore, cotransfection with both Bnip3 and protein kinase A (PKA) mitigated Bnip3‐induced cell death in 3T3 fibroblasts. Finally, we present data showing that PKA phosphorylates Bnip3 protein. We suggest that misoprostol may attenuate hypoxia‐induced cell death via cAMP/PKA signaling pathways.Support or Funding InformationAthabasca UniversityNatural Sciences and Engineering Research Council of CanadaChildren's Hospital Research Institute of Manitoba

Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis

Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/TxA2 receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G2/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cell delay at G2/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy.

Tu1876 Chronic Stress-Induced Visceral Hyperalgesia: Evidence for Differential Effects of Lubiprostone on Tight Junction Protein Expression in the Colon

Introduction: In humans, chronic psychological stress is associated with increased intestinal permeability and visceral hyperalgesia, which is recapitulated in the chronic intermittent water avoidance stress (WAS) rat model. Previously, we reported that WAS rats demonstrated increased intestinal permeability for macromolecules specifically in the colon, which correlated with reduced epithelial tight junction protein expression in the colon epithelium. Lubiprostone (Lub), a prostaglandin receptor agonist used to treat chronic constipation and IBS-C, has been shown to restore barrier function and epithelial tight junction protein expression in the ischemic porcine intestine. Hypothesis: Lub treatment ameliorates increased colon permeability and visceral hyperalgesia in the WAS rat model. Methods: Male Sprague Dawley rats were subjected to one hour of WAS for 10 consecutive days with or without oral administration of Lub. A 20 μg/kg/day dose of Lub was chosen based on a dose response study. On day 11, visceral motor response (VMR) to colorectal distention (CRD) or colon permeability were measured. Permeability was assayed by measuring plasma levels of the non-ionic molecule FITC-Dextran (4KDa), which was given by gavage at 400 mg/Kg. Distal colon tissue was used for Western blot analysis. Results: WAS was associated with increased colon permeability compared to controls in 7 of 14 rats (p = 0.0036) and a decrease in the level of the epithelial tight junction protein occludin (p < 0.05). Lub prevented the stress-induced increase in colon permeability (p = 0.01), as well as the decrease in occludin. WAS rats had a significant increase in pellet output over controls (p < 0.0001), however there were no significant difference in pellet output between Lub treated and untreated WAS rats. All WAS rats showed visceral hyperalgesia in response to CRD (60 mmHg, p < 0.0001) in comparison to controls. Lub treatment of WAS rats prevented visceral hyperalgesia in response to CRD for 7 out of 14 rats (p < 0.0001), whereas the other 7 had VMR to CRD levels equivalent to untreated WAS rats. Conclusion: Lub prevents WASassociated: 1) decrease in epithelial tight junction protein expression in a subset of rats that showed an increase in colon permeability and 2) hyperalgesia in a subset of rats, despite genetic homogeneity. We hypothesize that increased gut permeability and hyperalgesia in response to WAS are linked, and the differential effects of WAS on colon permeability and Lub treatment on visceral hyperalgesia reflects differences in epigenetic regulation of relevant target genes.

Tu1875 BDNF Regulates NMDA Receptor Phosphorylation via the PLC/PKC Pathway in the Spinal Cord During Colitis

Introduction: In humans, chronic psychological stress is associated with increased intestinal permeability and visceral hyperalgesia, which is recapitulated in the chronic intermittent water avoidance stress (WAS) rat model. Previously, we reported that WAS rats demonstrated increased intestinal permeability for macromolecules specifically in the colon, which correlated with reduced epithelial tight junction protein expression in the colon epithelium. Lubiprostone (Lub), a prostaglandin receptor agonist used to treat chronic constipation and IBS-C, has been shown to restore barrier function and epithelial tight junction protein expression in the ischemic porcine intestine. Hypothesis: Lub treatment ameliorates increased colon permeability and visceral hyperalgesia in the WAS rat model. Methods: Male Sprague Dawley rats were subjected to one hour of WAS for 10 consecutive days with or without oral administration of Lub. A 20 μg/kg/day dose of Lub was chosen based on a dose response study. On day 11, visceral motor response (VMR) to colorectal distention (CRD) or colon permeability were measured. Permeability was assayed by measuring plasma levels of the non-ionic molecule FITC-Dextran (4KDa), which was given by gavage at 400 mg/Kg. Distal colon tissue was used for Western blot analysis. Results: WAS was associated with increased colon permeability compared to controls in 7 of 14 rats (p = 0.0036) and a decrease in the level of the epithelial tight junction protein occludin (p < 0.05). Lub prevented the stress-induced increase in colon permeability (p = 0.01), as well as the decrease in occludin. WAS rats had a significant increase in pellet output over controls (p < 0.0001), however there were no significant difference in pellet output between Lub treated and untreated WAS rats. All WAS rats showed visceral hyperalgesia in response to CRD (60 mmHg, p < 0.0001) in comparison to controls. Lub treatment of WAS rats prevented visceral hyperalgesia in response to CRD for 7 out of 14 rats (p < 0.0001), whereas the other 7 had VMR to CRD levels equivalent to untreated WAS rats. Conclusion: Lub prevents WASassociated: 1) decrease in epithelial tight junction protein expression in a subset of rats that showed an increase in colon permeability and 2) hyperalgesia in a subset of rats, despite genetic homogeneity. We hypothesize that increased gut permeability and hyperalgesia in response to WAS are linked, and the differential effects of WAS on colon permeability and Lub treatment on visceral hyperalgesia reflects differences in epigenetic regulation of relevant target genes.

Prostaglandin E2 Signaling via EP4 Receptor is Important for Cell Cycle Progression and the Regulation of Reactive Oxygen Species Production in Primary Myoblast

Our previous data have shown that conditioned media from osteocytes and prostaglandin E2 (PGE2), a major secreted factor by osteocytes, promotes C2C12 myogenesis. PGE2 and EP4 receptor agonist CAY10598 enhance, but EP4 receptor antagonist L161,982 inhibits primary myoblast proliferation. Since the activation of other EP receptors did not show any noticeable effect, EP4 receptor may play a major role in the regulation of primary myoblast proliferation. To further study the mechanisms behind these effects of PGE2/EP4 signaling, we determined the gene expression of cell cycle regulators, such as cyclin E, p21, and myostatin, following the treatments with PGE2, CAY10598 or L161,982. Consistent with our findings in cell cycle analysis, the results indicated that PGE2 and CAY 10598 upregulate cylin E expression, but downregulate p21 and myostatin expression. In contrast, L161,982 decreases cylin E expression, but increases p21 and myostatin expression. Moreover, treatment with L161,982 significantly increased reactive oxygen species (ROS) levels by 250%. Cotreatment with antioxidant N‐acetyl cysteine or sodium ascorbate successfully reverses the inhibition of myoblast proliferation and excessive ROS production caused by L161,982. These results suggest that PGE2 signaling via EP4 receptor could regulate myogenesis by promoting myoblast proliferation via a ROS‐dependent modulation of cell cycle.

Lipopolysaccharide induces inflammation and facilitates lung metastasis in a breast cancer model via the prostaglandin E2-EP2 pathway

Inflammation is a potent promoter of tumor metastasis. The aim of the present study was to explore the function of systemic inflammation in the formation of lung metastasis of breast cancer cells in a mouse model. BALB/c mice were injected intraperitoneally with lipopolysaccharide (LPS) in order to establish an inflammatory animal model and 4T1 murine breast cancer cells were injected through the tail vein to induce lung metastasis. The levels of proinflammatory cytokines were evaluated by ELISA. Metastases on the surface of the lungs were counted and histologically analyzed by hematoxylin and eosin staining. Angiogenesis in the lungs was examined by CD31 immunofluorescence. Mouse pulmonary endothelial cells (MPVECs) were isolated and used to assay endothelial tube formation and determine the protein expression levels of vascular endothelial growth factor (VEGF) invitro. Serum levels of VEGF and prostaglandinE2 (PGE2), the number and size of metastatic lesions, and the expression levels of cyclooxygenase‑2 were significantly greater in the lungs of LPS‑treated mice, as compared with those in control mice threated with phosphate‑buffered saline. Blood vessel density was also markedly increased in the LPS‑treated mice. These increases were reversed by treatment with celecoxib. Invitro, the protein expression levels of VEGF produced by the PGE2‑treated cells were significantly increased in a concentration‑dependent manner. In addition, the production of VEGF was increased in response to treatment with the PGE2 receptor (EP2) agonist ONO‑AE1‑259‑01; however, this increase was abrogated by treatment with AH6809, an EP2 receptor antagonist. Treatment with PGE2 or VEGF alone promoted the tube formation of MPVECs and this effect was reversed by treatment with celecoxib. These results demonstrated that PGE2 may regulate the release of VEGF by MPVECs through the EP2 receptor pathway and thereby promoted pulmonary angiogenesis and breast cancer metastasis in a mouse model.

Study on Physiological Roles of Stimulation of Prostaglandin E2 Receptor Subtype EP2 in Urethral Function in Rats.

We investigated the relaxant effect of stimulation of prostaglandin E2 (PGE2 ) receptor subtype EP2 as well as the involvement of a cyclic AMP (cAMP)-dependent pathway related to stimulation of EP2 receptors in urethral function in rats by evaluating effects of PGE2 and selective EP2 receptor agonist CP-533,536. Effects of PGE2 and CP-533,536 on cAMP accumulation were assessed in Chinese hamster ovary (CHO)-K1 cells expressing rat EP2 or EP4 receptors. Relaxant responses to PGE2 and CP-533,536 (0.01-10 µmol/L) in rat urethral tissue pre-contracted with 10 µmol/L phenylephrine were evaluated, and cAMP levels in isolated rat urethral tissue treated with these compounds were determined as well. The effects of PGE2 and CP-533,536 (0.003-0.3 mg/kg intravenously) on urethral perfusion pressure (UPP) in anesthetized rats were also evaluated. PGE2 concentration-dependently increased the accumulation of cAMP in cells expressing rat EP2 (EC50 value = 1.3 nmol/L) and EP4 receptors (EC50 value = 17 nmol/L). While CP-533,536 similarly increased the accumulation of cAMP in cells expressing rat EP2 receptors (EC50 value = 3.0 nmol/L), no such effects were noted in cells expressing rat EP4 receptors up to 10 µmol/L. Both PGE2 and CP-533,536 produced relaxation and increased cAMP levels in urethral tissues in a concentration-dependent manner. PGE2 and CP-533,536 both dose-dependently decreased UPP in anesthetized rats. Taken together, these results suggest that stimulation of EP2 receptors induces relaxation likely via activation of cAMP-dependent mechanisms in rat urethral tissue, leading to a reduction of UPP.

TGF-β1 Downregulates COX-2 Expression Leading to Decrease of PGE2 Production in Human Lung Cancer A549 Cells, Which Is Involved in Fibrotic Response to TGF-β1

Transforming growth factor-ß1 (TGF-β1) is a multifunctional cytokine that is involved in various pathophysiological processes, including cancer progression and fibrotic disorders. Here, we show that treatment with TGF-β1 (5 ng/mL) induced downregulation of cyclooxygenase-2 (COX-2), leading to reduced synthesis of prostaglandin E2 (PGE2), in human lung cancer A549 cells. Treatment of cells with specific inhibitors of COX-2 or PGE2 receptor resulted in growth inhibition, indicating that the COX-2/PGE2 pathway contributes to proliferation in an autocrine manner. TGF-β1 treatment induced growth inhibition, which was attenuated by exogenous PGE2. TGF-β1 is also a potent inducer of epithelial mesenchymal transition (EMT), a phenotype change in which epithelial cells differentiate into fibroblastoid cells. Supplementation with PGE2 or PGE2 receptor EP4 agonist PGE1-alcohol, as compared with EP1/3 agonist sulprostone, inhibited TGF-β1-induced expression of fibronectin and collagen I (extracellular matrix components). Exogenous PGE2 or PGE2 receptor agonists also suppressed actin remodeling induced by TGF-β1. These results suggest that PGE2 has an anti-fibrotic effect. We conclude that TGF-β1-induced downregulation of COX-2/PGE2 signaling is involved in facilitation of fibrotic EMT response in A549 cells.

Prostaglandin E2 receptor EP1 phosphorylate CREB and mediates MMP2 expression in human cholangiocarcinoma cells

Cyclooxygenase-2 (COX-2) and COX-2-induced prostaglandin E2 (PGE2) have been implicated in all stages of malignant tumorigenesis. Although many aspects of matrix metalloproteinase (MMP2) on tumor invasion have been studied, the exact mechanism of PGE2-induced MMP2 overproduction has not been clearly defined. We have previously demonstrated that PGE2-enhanced extracellular signal-regulated kinase (Erk) phosphorylation via EP1 signaling pathway involved in PGE2-induced cell proliferation. Based on the identification of the transcription factor cyclic AMP response element-binding protein (CREB) as an important regulator of MMP2 and Erk phosphorylate CREB at ser133, we hypothesize that CREB may be implicated in the signaling of PGE2 stimulation to MMP2 overproduction via EP1 receptor. In the study, we investigated the role of EP1 receptor on PGE2-induced MMP2 expression and delineated the signaling pathway that contributes to EP1 receptor modulation of MMP2 in human cholangiocarcinoma cells. We found PGE2 or selective EP1 receptor agonist 17-P-T-PGE2-stimulated MMP2 expression and selective EP1 receptor antagonist SC-51322 or EP1 receptor siRNA abrogated PGE2-induced MMP2 expression. Intracellular calcium chelator BAPTA-AM, the selective inhibitor of EGFR AG1478 and the selective inhibitor of Erk PD98059 blocked EP1 receptor activation-induced CREB phosphorylation and MMP2 expression. A novel dominant-negative (D-N) inhibitor protein of the CREB, termed A-CREB, attenuated EP1 receptor activation-induced MMP2 expression. Our findings suggest that PGE2-enhanced MMP2 expression is, at least in part, mediated through EP1 receptors and calcium signaling pathway-induced CREB phosphorylation in human cholangiocarcinoma cells.