Constitutive internalisation of EP2 differentially regulates G protein signalling.

The prostanoid G protein-coupled receptor (GPCR) EP2 is widely expressed and implicated in endometriosis, osteoporosis, obesity, pre-term labour and cancer. Internalisation and intracellular trafficking are critical for shaping GPCR activity, yet little is known regarding the spatial programming of EP2 signalling and whether this can be exploited pharmacologically. Using three EP2-selective ligands that favour activation of different EP2 pathways, we show that EP2 undergoes limited agonist-driven internalisation but is constitutively internalised via dynamin-dependent, ß-arrestin-independent pathways. EP2 was constitutively trafficked to early and very early endosomes (VEE), which was not altered by ligand activation. APPL1, a key adaptor and regulatory protein of the VEE, did not impact EP2 agonist-mediated cAMP. Internalisation was required for ~70% of the acute butaprost- and AH13205-mediated cAMP signalling, yet PGN9856i, a Gαs-biased agonist, was less dependent on receptor internalisation for its cAMP signalling, particularly in human term pregnant myometrial cells that endogenously express EP2. Inhibition of EP2 internalisation partially reduced calcium signalling activated by butaprost or AH13205 and had no effect on PGE2 secretion. This indicates an agonist-dependent differential spatial requirement for Gαs and Gαq/11 signalling and a role for plasma membrane-initiated Gαq/11-Ca2+-mediated PGE2 secretion. These findings reveal a key role for EP2 constitutive internalisation in its signalling and potential spatial bias in mediating its downstream functions. This, in turn, could highlight important considerations for future selective targeting of EP2 signalling pathways.

Risk factors of retained products of conception after miscarriage or termination with gemeprost in the second trimester of pregnancy: a retrospective case-controlled study in Japanese population.

Retained products of conception (RPOC) is a complication that occurs in the second trimester of pregnancy. We enrolled 98 women who had a miscarriage or termination with gemeprost in the second trimester of pregnancy. Eighteen cases (18.4%) were RPOC-positive. The gestational week at miscarriage or termination was earlier in the RPOC-positive group than those in the RPOC-negative group (p = .003). The period of the third stage of labour was longer in the RPOC-positive group than in RPOC-negative group (p = .040). The proportion of placental forceps use was higher in the RPOC-positive group than in RPOC-negative group (p = .003). Multivariate logistic regression analysis showed that gestational week (OR: 3.53; p = .04) and use of placental forceps at delivery (OR: 2.21; p = .012) were independent risk factors for RPOC. Earlier gestational weeks at miscarriage or termination and use of placental forceps at delivery were predictive factors for RPOC after second trimester miscarriage or termination with gemeprost.Impact StatementWhat is already known on this subject? There have been some reports on risk factors of RPOC. A previous report showed that the termination of pregnancy with misoprostol at earlier periods was associated with an increased risk of RPOC.What the results of this study add? There have been few studies on the risk factors of RPOC after miscarriage or termination with gemeprost. In this study, we evaluated the risk factors of RPOC after miscarriage or termination of pregnancy with gemeprost in the second trimester. We found that an earlier gestational age (between 12 and 17 weeks) at delivery and using placental forceps to remove placenta were significant risk factors of RPOC after miscarriage or termination of pregnancy with gemeprost in the second trimester.What the implications are of these findings for clinical practice and/or further research? An earlier gestational age and using forceps to remove placenta may be significant risk factors for RPOC. The accurate evaluation and treatment for RPOC is important for maternal life-saving efforts and subsequent pregnancies. Further research is needed to draft a standardised protocol for RPOC.

Proliferation of bovine endometrial epithelial cells is promoted by prostaglandin E2-PTGER2 signaling through cell cycle regulation.

It is known that prostaglandin E2 (PGE2) induces proliferation of epithelia in bovine endometrial explants, however, the detailed mechanism of regulation of PGE2 in inducing bovine endometrial epithelial cell (bEEC) proliferation is unclear. In this study, we determined whether proliferation of bEECs is promoted by PGE2-prostaglandin E receptor 2 (PTGER2) signaling activation through cell cycle regulation. The results demonstrated that bEECs proliferation was induced by treatment of PGE2 and PTGER2 agonist butaprost. These processes were down-regulated by PTGER2 antagonist AH6809 and CDK inhibitors (LEE011, CDK2 Inhibitor II and Ro 3306). PGE2 and butaprost induced cyclins (A, B1, D1, D3 and E2), cyclin-dependent kinases (CDKs, 1, 2, 4 and 6), and epidermal growth factor (EGF) expression were inhibited by AH6809 treatment in bEECs. Moreover, proliferating cell nuclear antigen (PCNA), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and PTGER2 expression in bEECs were up-regulated by PGE2 and butaprost treatment. Our data demonstrate that PGE2-PTGER2 signaling activation has a direct molecular association with cell cycle regulation and cell proliferation in bEECs. Collectively, these findings will improve our understanding of the roles for PGE2-PTGER2 signaling activation in the physiological and pharmacological processes of bovine endometrium.

Effects of Sodium Selenite Injection on Serum Metabolic Profiles in Women Diagnosed with Breast Cancer-Related Lymphedema-Secondary Analysis of a Randomized Placebo-Controlled Trial Using Global Metabolomics.

In our previous study, intravenous (IV) injection of selenium alleviated breast cancer-related lymphedema (BCRL). This secondary analysis aimed to explore the metabolic effects of selenium on patients with BCRL. Serum samples of the selenium-treated (SE, n = 15) or the placebo-controlled (CTRL, n = 14) groups were analyzed by ultra-high-performance liquid chromatography with Q-Exactive Orbitrap tandem mass spectrometry (UHPLC-Q-Exactive Orbitrap/MS). The SE group showed a lower ratio of extracellular water to segmental water (ECW/SW) in the affected arm to ECW/SW in the unaffected arm (arm ECW/SW ratio) than the CTRL group. Metabolomics analysis showed a valid classification at 2-weeks and 107 differential metabolites were identified. Among them, the levels of corticosterone, LTB4-DMA, and PGE3-which are known anti-inflammatory compounds-were elevated in the SE group. Pathway analysis demonstrated that lipid metabolism (glycerophospholipid metabolism, steroid hormone biosynthesis, or arachidonic acid metabolism), nucleotide metabolism (pyrimidine or purine metabolism), and vitamin metabolism (pantothenate and CoA biosynthesis, vitamin B6 metabolism, ascorbate and aldarate metabolism) were altered in the SE group compared to the CTRL group. In addition, xanthurenic acid levels were negatively associated with whole blood selenium level (WBSe) and positively associated with the arm ECW/SW. In conclusion, selenium IV injection improved the arm ECW/SW ratio and altered the serum metabolic profiles in patients with BCRL, and improved the anti-inflammatory process in lipid, nucleotide and vitamin pathways, which might alleviate the symptoms of BCRL.

Addition of EP2 agonists to an FP agonist additively and synergistically modulates adipogenesis and the physical properties of 3D 3T3-L1 sphenoids.

The additive effects of prostaglandin (PG)-EP2 agonists on a PG-FP agonist toward adipogenesis in two- or three-dimension (2D or 3D) cultures of 3T3-L1 cells was examined by lipid staining, the mRNA expression of adipogenesis related genes, and extracellular matrixes (ECMs) including collagen molecules (Col) -1, -4 and -6, and fibronectin (Fn), and the sizes and physical properties of 3D sphenoids, as measured by a micro-squeezer. The results indicate that adipogenesis induced 1) an enlargement in the sizes of 3D sphenoids, 2) a substantial enhancement in lipid staining, the expression of the PParγ, Ap2 and Leptin genes, and 3) a significant decrease in the stiffness of the 3D sphenoids. These effects were inhibited by bimatoprost acid (BIM-A), but 4) adipogenesis induced significant down-regulation of Col1 and Fn, and the significant up-regulation of the Col4 and Col6 genes were unchanged by BIM-A. On the addition of an EP2 agonist, such as omidenepag (OMD) or butaprost (Buta), to BIM-A, 1) the sizes of the 3D sphenoids were further decreased, 2) lipid staining was decreased (2D; OMD, 3D; Buta) 3) the stiffness of the 3D sphenoids was increased by Buta, 4) the expression of PParγ was up-regulated (2D; Buta) or unchanged (3D), the expression of Ap2 was down-regulated (2D; OMD) or up-regulated (3D; Buta), and the expression of Leptin was increased (2D), 5) the expression of all four (OMD) or all except Col4 (buta) in 2D, and Col1and Col4 (OMD) in 3D were up-regulated. These collective findings indicate that the addition of an EP2 agonist, OMD or Buta significantly modulated the BIM-A induced suppression of adipogenesis as well as physical properties of 2D and 3D cultured 3T3-L1 cells in different manners.

Relationship between the n-3 index, serum metabolites and breast cancer risk.

The present study aimed to investigate the relationship between the n-3 index, serum metabolites and breast cancer risk. A total of 104 newly diagnosed breast cancer patients and 70 healthy controls were recruited. The erythrocyte phospholipid fatty acid composition was determined by gas-liquid chromatography, and the n-3 index was calculated with the percentage of eicosapentaenoic acid plus docosahexaenoic acid in total fatty acids. Serum metabolomic profiles were analyzed by UHPLC-Q-Exactive Orbitrap/MS. The results showed that the erythrocyte phospholipid n-3 index was significantly lower in breast cancer patients than in healthy controls, and it was inversely associated with breast cancer risk (OR = 0.60; 95% CI: 0.36-0.84). Metabolomics analyses showed that serum 16α-hydroxy dehydroepiandrosterone (DHEA) 3-sulfate, lysophatidylethanolamines (LPE) 22:0/0:0 and hexanoylcarnitine were significantly higher, while thromboxane B3, prostaglandin E3 (PGE3) and 18ß-glycyrrhetinic acid were significantly lower in breast cancer patients than those in healthy controls. In addition, serum 16α-hydroxy DHEA 3-sulfate was inversely correlated with the n-3 index (r = -0.412, p = 0.036). In conclusion, our findings suggest that the lack of n-3 PUFAs might be a potential risk factor for breast cancer, and the serum metabolite 16α-hydroxy DHEA 3-sulfate may play an important role in linking n-3 PUFA deficiency and breast disease etiology.

Prostaglandin E3 attenuates macrophage-associated inflammation and prostate tumour growth by modulating polarization.

Alternative polarization of macrophages regulates multiple biological processes. While M1-polarized macrophages generally mediate rapid immune responses, M2-polarized macrophages induce chronic and mild immune responses. In either case, polyunsaturated fatty acid (PUFA)-derived lipid mediators act as both products and regulators of macrophages. Prostaglandin E3 (PGE3 ) is an eicosanoid derived from eicosapentaenoic acid, which is converted by cyclooxygenase, followed by prostaglandin E synthase successively. We found that PGE3 played an anti-inflammatory role by inhibiting LPS and interferon-γ-induced M1 polarization and promoting interleukin-4-mediated M2 polarization (M2a). Further, we found that although PGE3 had no direct effect on the growth of prostate cancer cells in vitro, PGE3 could inhibit prostate cancer in vivo in a nude mouse model of neoplasia. Notably, we found that PGE3 significantly inhibited prostate cancer cell growth in a cancer cell-macrophage co-culture system. Experimental results showed that PGE3 inhibited the polarization of tumour-associated M2 macrophages (TAM), consequently producing indirect anti-tumour activity. Mechanistically, we identified that PGE3 regulated the expression and activation of protein kinase A, which is critical for macrophage polarization. In summary, this study indicates that PGE3 can selectively promote M2a polarization, while inhibiting M1 and TAM polarization, thus exerting an anti-inflammatory effect and anti-tumour effect in prostate cancer.

Simultaneous Use of ROCK Inhibitors and EP2 Agonists Induces Unexpected Effects on Adipogenesis and the Physical Properties of 3T3-L1 Preadipocytes.

To elucidate the additive effects of an EP2 agonist, omidenepag (OMD) or butaprost (Buta) on the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) on adipose tissue, two- or three-dimension (2D or 3D) cultures of 3T3-L1 cells were analyzed by lipid staining, the mRNA expression of adipogenesis-related genes, extracellular matrix (ECM) molecules including collagen (Col) -1, -4 and -6, and fibronectin (Fn), and the sizes and physical properties of 3D organoids, as measured by a micro-squeezer. The results indicate that adipogenesis induced (1) an enlargement of the 3D organoids; (2) a substantial enhancement in lipid staining as well as the expression of the Pparγ, Ap2 and Leptin genes; (3) a significant softening of the 3D organoids, the effects of which were all enhanced by Rip except for Pparγ expression; and (4) a significant downregulation in Col1 and Fn, and a significant upregulation in Col4, Col6, the effects of which were unchanged by Rip. When adding the EP2 agonist to Rip, (1) the sizes of the 3D organoids were reduced substantially; (2) lipid staining was increased (OMD), or decreased (Buta); (3) the stiffness of the 3D organoids was substantially increased in Buta; (4-1) the expression of Pparγ was suppressed (2D, OMD) or increased (2D, Buta), and the expressions of Ap2 were downregulated (2D, 3D) and Leptin was increased (2D) or decreased (3D), (4-2) all the expressions of four ECM molecules were upregulated in 2D (2D), and in 3D, the expression of Col1, Col4 was upregulated. The collective findings reported herein indicate that the addition of an EP2 agonist, OMD or Buta significantly but differently modulate the Rip-induced effects on adipogenesis and the physical properties of 2D and 3D cultured 3T3-L1 cells.

Leucoselect Phytosome Modulates Serum Eicosapentaenoic Acid, Docosahexaenoic Acid, and Prostaglandin E3 in a Phase I Lung Cancer Chemoprevention Study.

Grape seed procyanidin extract (GSE) has been shown to exert antineoplastic properties in preclinical studies. Recently, we reported findings from a modified phase I, open-label, dose escalation clinical study conducted to evaluate the safety, tolerability, MTD, and potential chemopreventive effects of leucoselect phytosome, a standardized GSE complexed with soy phospholipids to enhance bioavailability, in heavy active and former smokers. Three months of leucoselect phytosome treatment significantly decreased bronchial Ki-67 labeling index (LI), a marker of cell proliferation on the bronchial epithelium. Because GSE is widely used as a supplement to support cardiovascular health, we evaluate the impact of oral leucoselect phytosome on the fasting serum complex lipid metabolomics profiles in our participants. One month of leucoselect phytosome treatment significantly increased eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the omega-3 polyunsaturated fatty acids (n-3 PUFA) with well-established anticancer properties. Leucoselect phytosome also significantly increased unsaturated phosphatidylcholines (PC), likely from soy phospolipids in the phytosome and functioning as transporters for these PUFAs. Furthermore, 3-month leucoselect phytosome treatment significantly increased serum prostaglandin (PG) E3 (PGE3), a metabolite of EPA with anti-inflammatory and antineoplastic properties. Such increases in PGE3 correlated with reductions of bronchial Ki-67 LI (r = -0.9; P = 0.0374). Moreover, posttreatment plasma samples from trial participants significantly inhibited proliferation of human lung cancer cell lines A549 (adenocarcinoma), H520 (squamous cell carcinoma), DMS114 (small cell carcinoma), and 1198 (preneoplastic cell line). Our findings further support the potential utility of leucoselect phytosome in reducing cardiovascular and neoplastic risks in heavy former and active smokers. PREVENTION RELEVANCE: In this correlative study of leucoselect phytosome for lung cancer chemoprevention in heavy active and former smokers, we demonstrate for the first time, favorable modulations of n-3PUFA and downstream PGE3 in fasting serum, further supporting the chemopreventive potential of leucoselect phytosome against lung cancer.

Lumbar Spinal Stenosis and Potential Management With Prostaglandin E1 Analogs.

ABSTRACT: Lumbar spinal stenosis is one of the most commonly diagnosed spinal disorders worldwide and remains a major cause for surgery in older adults. Lumbar spinal stenosis is clinically defined as a progressive degenerative disorder with low back pain and associated neurogenic intermittent claudication. Conservative and surgical management of lumbar spinal stenosis has been shown to be minimally effective on its symptoms. A treatment option that has not been investigated in the United States is the utilization of prostaglandin E1 analogs, which have been used primarily in Japan for the treatment of lumbar spinal stenosis since the 1980s. The vasodilatory and antiplatelet aggregation effects of prostaglandin E1 presumably improve symptoms of lumbar spinal stenosis by increasing blood flow to the spinal nerve roots. This brief report examines the potential vascular pathology of lumbar spinal stenosis, reviews evidence on the use of prostaglandin E1 analog limaprost in Japan for lumbar spinal stenosis, and briefly discusses misoprostol as a possible alternative in the United States. The studies summarized in this report suggest that prostaglandin E1 analogs may provide benefit as a conservative treatment option for patients with lumbar spinal stenosis. However, higher-quality studies conducted in the United States and comparison with other currently used conservative treatments are required before it can be recommended for routine clinical use.

In Vivo Targeted Metabolomic Profiling of Prostanit, a Novel Anti-PAD NO-Donating Alprostadil-Based Drug.

Prostanit is a novel drug developed for the treatment of peripheral arterial diseases. It consists of a prostaglandin E1 (PGE1) moiety with two nitric oxide (NO) donor fragments, which provide a combined vasodilation effect on smooth muscles and vascular spastic reaction. Prostanit pharmacokinetics, however, remains poorly investigated. Thus, the object of this study was to investigate the pharmacokinetics of Prostanit-related and -affected metabolites in rabbit plasma using the liquid chromatography-mass spectrometry (LC-MS) approach. Besides, NO generation from Prostanit in isolated rat aorta and human smooth muscle cells was studied using the Griess method. In plasma, Prostanit was rapidly metabolized to 1,3-dinitroglycerol (1,3-DNG), PGE1, and 13,14-dihydro-15-keto-PGE1. Simultaneously, the constant growth of amino acid (proline, 4-hydroxyproline, alanine, phenylalanine, etc.), steroid (androsterone and corticosterone), and purine (adenosine, adenosine-5 monophosphate, and guanosine) levels was observed. Glycine, aspartate, cortisol, and testosterone levels were decreased. Ex vivo Prostanit induced both NO synthase-dependent and -independent NO generation. The observed pharmacokinetic properties suggested some novel beneficial activities (i.e., effect prolongation and anti-inflammation). These properties may provide a basis for future research of the effectiveness and safety of Prostanit, as well as for its characterization from a clinical perspective.

Crosstalk between EP2 and PPARα Modulates Hypoxic Signaling and Myopia Development in Guinea Pigs.

Purpose: Cyclic adenosine monophosphate (cAMP) and peroxisome proliferator-activated receptor alpha (PPARα) levels mediate extracellular matrix (ECM) changes by altering the levels of hypoxia-inducible factor 1-alpha (HIF-1α) in various tissues. We aimed to determine, in the sclera of guinea pigs, whether a prostanoid receptor (EP2)-linked cAMP modulation affects PPARα and HIF-1α signaling during myopia. Methods: Three-week-old guinea pigs (n = 20 in each group), were monocularly injected with either an EP2 agonist (butaprost 1 µmol/L/10 µmol/L), an antagonist (AH6809 10 µmol/L/30 µmol/L) or a vehicle solution for two weeks during normal ocular growth. Separate sets of animals received these injections and underwent form deprivation (FD) simultaneously. Refraction and axial length (AL) were measured at two weeks, followed by scleral tissue isolation for quantitative PCR (qPCR) analysis (n = 10) and cAMP detection (n = 10) using a radioimmunoassay. Results: Butaprost induced myopia development during normal ocular growth, with proportional increases in AL and cAMP levels. FD did not augment the magnitude of myopia or cAMP elevations in these agonist-injected eyes. AH6809 suppressed cAMP increases and myopia progression during FD, but had no effect in a normal visual environment. Of the diverse set of 27 genes related to cAMP, PPARα and HIF-1α signaling and ECM remodeling, butaprost differentially regulated 15 of them during myopia development. AH6809 injections during FD negated such differential gene expressions. Conclusion: EP2 agonism increased cAMP and HIF-1α signaling subsequent to declines in PPARα and RXR mRNA levels, which in turn decreased scleral fibrosis and promoted myopia. EP2 antagonism instead inhibited each of these responses. Our data suggest that EP2 suppression may sustain scleral ECM structure and inhibit myopia development.

cAMP signaling primes lung endothelial cells to activate caspase-1 during Pseudomonas aeruginosa infection.

Activation of the inflammasome-caspase-1 axis in lung endothelial cells is emerging as a novel arm of the innate immune response to pneumonia and sepsis caused by Pseudomonas aeruginosa. Increased levels of circulating autacoids are hallmarks of pneumonia and sepsis and induce physiological responses via cAMP signaling in targeted cells. However, it is unknown whether cAMP affects other functions, such as P. aeruginosa-induced caspase-1 activation. Herein, we describe the effects of cAMP signaling on caspase-1 activation using a single cell flow cytometry-based assay. P. aeruginosa infection of cultured lung endothelial cells caused caspase-1 activation in a distinct population of cells. Unexpectedly, pharmacological cAMP elevation increased the total number of lung endothelial cells with activated caspase-1. Interestingly, addition of cAMP agonists augmented P. aeruginosa infection of lung endothelial cells as a partial explanation underlying cAMP priming of caspase-1 activation. The cAMP effect(s) appeared to function as a priming signal because addition of cAMP agonists was required either before or early during the onset of infection. However, absolute cAMP levels measured by ELISA were not predictive of cAMP-priming effects. Importantly, inhibition of de novo cAMP synthesis decreased the number of lung endothelial cells with activated caspase-1 during infection. Collectively, our data suggest that lung endothelial cells rely on cAMP signaling to prime caspase-1 activation during P. aeruginosa infection.

A cross-sectional analysis of mifepristone, misoprostol, and combination mifepristone-misoprostol package inserts obtained in 20 countries.

OBJECTIVES: To evaluate the characteristics, clinical information, and storage instructions contained in package inserts from medical abortion commodities collected in low- and middle-income countries. STUDY DESIGN: From November 2017 to February 2018 mifepristone, misoprostol, and combined mifepristone-misoprostol (combipack) products were collected to populate the Medical Abortion Commodities Database. We extracted stated indications for use, storage instructions, and date of last revision from each package insert obtained. For those inserts listing medical abortion as an indication, we also extracted eligibility criteria, recommended regimens, side effects, and contraindications. RESULTS: We identified 41 package inserts from 20 countries; 19 (46%) listed medical abortion as an indication including all 7 combipacks, all 7 mifepristone products, and 5/27 (19%) misoprostol products. Date of last insert revision ranged from 1991 to 2016. Gestational age limits for early medical abortion ranged from 49 days to "first trimester." Three (43%) mifepristone products recommended a 600 mg oral dose and two (29%) recommended regimens with gemeprost. Eighteen (67%) misoprostol and one (14%) combipack inserts recommended protection from moisture. CONCLUSIONS: The characteristics, clinical information, and storage instructions in medical abortion product package inserts from a variety of field settings in low- and middle-income countries included inadequate storage instructions and outdated gestational age limits and regimens. IMPLICATIONS: There is an urgent need to revisit approved inserts for medical abortion products in low- and middle-income countries to ensure information is accurate and reflects the current evidence base. Simultaneously, providing supplemental instructions targeted at users may fill some gaps. People have a right to accurate information to ensure a safe and effective medical abortion experience.

Synergetic Effect of EP1 Receptor Antagonist and (-)-Epigallocatechin-3-gallate in Hepatocellular Carcinoma.

Epigallocatechin-3-gallate (EGCG), the principal catechin of green tea, modulates different molecular mechanisms underlying hepatocellular carcinoma (HCC). Accumulating studies showed that the activation of prostaglandin (PG) receptor EP1 promotes cell migration and invasion in different cancers, which could be inverted by blocking the EP1 receptor. This study investigated the synergetic effects of EP1-selective antagonist ONO-8711 and EGCG treatment on HCC to better understand the potential strategy to treat HCC. We found that EGCG significantly inhibited PGE2 and EP1-selective agonist induced migration of HCC cells and increased the ratio of Bax/Bcl-2 even in the presence of ONO-DI-004 or PGE2. ONO-8711 significantly inhibited PGE2-induced HCC proliferation while increased the inhibitory effect of EGCG on HCC cell viability and migration ability compared with EGCG alone. These findings suggest that a combination of ONO-8711 and EGCG is a potential treatment for HCC therapy.

Activation of the prostaglandin E2 EP2 receptor attenuates renal fibrosis in unilateral ureteral obstructed mice and human kidney slices.

AIM: Renal fibrosis plays a pivotal role in the development and progression of chronic kidney disease, which affects 10% of the adult population. Previously, it has been demonstrated that the cyclooxygenase-2 (COX-2)/prostaglandin (PG) system influences the progression of renal injury. Here, we evaluated the impact of butaprost, a selective EP2 receptor agonist, on renal fibrosis in several models of kidney injury, including human tissue slices. METHODS: We studied the anti-fibrotic efficacy of butaprost using Madin-Darby Canine Kidney (MDCK) cells, mice that underwent unilateral ureteral obstruction and human precision-cut kidney slices. Fibrogenesis was evaluated on a gene and protein level by qPCR and Western blotting. RESULTS: Butaprost (50 µM) reduced TGF-ß-induced fibronectin (FN) expression, Smad2 phosphorylation and epithelial-mesenchymal transition in MDCK cells. In addition, treatment with 4 mg/kg/day butaprost attenuated the development of fibrosis in mice that underwent unilateral ureteral obstruction surgery, as illustrated by a reduction in the gene and protein expression of α-smooth muscle actin, FN and collagen 1A1. More importantly, a similar anti-fibrotic effect of butaprost was observed in human precision-cut kidney slices exposed to TGF-ß. The mechanism of action of butaprost appeared to be a direct effect on TGF-ß/Smad signalling, which was independent of the cAMP/PKA pathway. CONCLUSION: In conclusion, this study demonstrates that stimulation of the EP2 receptor effectively mitigates renal fibrogenesis in various fibrosis models. These findings warrant further research into the clinical application of butaprost, or other EP2 agonists, for the inhibition of renal fibrosis.

Difluoromethylene at the γ-Lactam α-Position Improves 11-Deoxy-8-aza-PGE1 Series EP4 Receptor Binding and Activity: 11-Deoxy-10,10-difluoro-8-aza-PGE1 Analog (KMN-159) as a Potent EP4 Agonist.

A series of small-molecule full agonists of the prostaglandin E2 type 4 (EP4) receptor have been generated and evaluated for binding affinity and cellular potency. KMN-80 and its gem-difluoro analog KMN-159 possess high selectivity relative to other prostanoid receptors. Difluoro substitution is positioned alpha to the lactam ring carbonyl and results in KMN-159's fivefold increase in potency versus KMN-80. The two analogs exhibit electronic and conformational variations, including altered nitrogen hybridization and lactam ring puckering, that may drive the observed difluoro-associated increased potency within this four-compound series.

Involvement of EP2 and EP4 Receptors in Eosinophilic Esophagitis: A Pilot Study.

BACKGROUND: The prostaglandin D2 receptor DP2 has been implicated in eosinophil infiltration and the development of eosinophilic esophagitis (EoE). AIMS AND METHODS: In this study, we investigated an involvement of PGE2 (EP1-EP4) and PGD2 (DP1) receptors in EoE by measuring their expression in peripheral blood eosinophils and esophageal mucosal biopsies of EoE patients and by performing migration and adhesion assays with eosinophils from healthy donors. RESULTS: Expression of EP2 and EP4, but not EP1 and EP3, was decreased in blood eosinophils of patients with EoE vs. control subjects. Adhesion of eosinophils to esophageal epithelial cells was decreased by EP2 receptor agonist butaprost and EP4 agonist ONO-AE1-329, whereas DP1 agonist BW245C increased adhesion. In chemotaxis assays with supernatant from human esophageal epithelial cells, only ONO-AE1-329 but not butaprost or BW245C inhibited the migration of eosinophils. Expression of EP and DP receptors in epithelial cells and eosinophils was detected in sections of esophageal biopsies from EoE patients by immunohistochemistry. qPCR of biopsies from EoE patients revealed that gene expression of EP4 and DP1 was the highest among PGE2 and PGD2 receptors. Esophageal epithelial cells in culture showed high gene expression for EP2 and EP4. Activation of EP2 and EP4 receptors decreased barrier integrity of esophageal epithelial cells in impedance assays. CONCLUSIONS: Activation of EP2 and EP4 receptors may inhibit eosinophil recruitment to the esophageal mucosa. However, their activation could negatively affect esophageal barrier integrity suggesting that eosinophilic rather than epithelial EP2 and EP4 have a protective role in EoE.

Single versus multiple cervical dilation by osmotic dilator before induction of labor for second-trimester abortion.

AIM: In this study, we aimed to investigate whether there was a significant prognostic difference between single and multiple cervical dilations when inducing second-trimester abortion. METHODS: We conducted a retrospective review of 238 pregnant women who underwent termination of pregnancy at 12-21 weeks of gestation at Osaka University Hospital in Osaka, Japan, between January 2010 and May 2018. Termination of pregnancy was performed by vaginal administration of 1 mg gemeprost every 3 h for up to five doses per day after uterine cervical dilation using lamicel. RESULTS: The women were categorized into two groups: 191 women had a delivery time of <24 h, whereas 47 had delivery times >24 h. Contrasting the groups, there were significant differences with regard to numbers of primiparas (88 [46.1%] and 32 [68.1%], respectively) and lamicel exchanges ± SD (1.9 ± 0.67 for <24 h and 2.4 ± 0.87 for >24 h, respectively). Additionally, we compared the prognosis of primiparas that received just a single lamicel with that of primiparas that had ≥2 exchanged, but no significant differences were noted in the number of patients with a delivery time of >24 h and the number of used gemeprost. CONCLUSION: Primipara is a risk factor for delayed delivery time of induced abortion. However, increasing the number of exchanged lamicel did not significantly reduce the delivery time; therefore, it should be performed as minimally as possible.

Prostaglandin E2/EP2 receptor signalling pathway promotes diabetic retinopathy in a rat model of diabetes.

AIMS/HYPOTHESIS: Diabetic retinopathy is a common microvascular complication of diabetes mellitus and is initiated by inflammation and apoptosis-associated retinal endothelial cell damage. Prostaglandin E2 (PGE2) has emerged as a critical regulator of these biological processes. We hypothesised that modulating PGE2 and its E-prostanoid receptor (EP2R) would prevent diabetes mellitus-induced inflammation and microvascular dysfunction. METHODS: In a streptozotocin (STZ)-induced rat model of diabetes, rats received intravitreal injection of PGE2, butaprost (a PGE2/EP2R agonist) or AH6809 (an EP2R antagonist). Retinal histology, optical coherence tomography, ultrastructure of the retinal vascular and biochemical markers were assessed. RESULTS: Intravitreal injection of PGE2 and butaprost significantly accelerated retinal vascular leakage, leucostasis and endothelial cell apoptosis in STZ-induced diabetic rats. This response was ameliorated in diabetic rats pre-treated with AH6809. In addition, pre-treatment of human retinal microvascular endothelial cells with AH6809 attenuated PGE2- and butaprost-induced activation of caspase 1, activation of the complex containing nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) and apoptosis-associated speck-like protein containing a C-terminal caspase-activation and recruitment domain (ASC), and activation of the EP2R-coupled cAMP/protein kinase A/cAMP response element-binding protein signalling pathway. CONCLUSIONS/INTERPRETATION: The PGE2/EP2R signalling pathway is involved in STZ-induced diabetic retinopathy and could be considered as a potential target for diabetic retinopathy prevention and treatment.