EP3 receptor agonist 17-pt-PGE2 acts as an EP4 receptor agonist on endothelial barrier function and in a model of LPS-induced pulmonary inflammation

VPH-06363; No of Pages 10 Vascular Pharmacology xxx (2016) xxx–xxx

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The EP1/EP3 receptor agonist 17-pt-PGE2 acts as an EP4 receptor agonist on endothelial barrier function and in a model of LPS-induced pulmonary inflammation Anna Theiler, Viktoria Konya, Lisa Pasterk, Jovana Maric, Thomas Bärnthaler, Ilse Lanz, Wolfgang Platzer, Rufina Schuligoi, Akos Heinemann ⁎ Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Universitaetsplatz 4, 8010 Graz, Austria

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Article history: Received 21 October 2015 Received in revised form 16 September 2016 Accepted 20 September 2016 Available online xxxx Keywords: 17-Phenyl-trinor-prostaglandin E2 EP receptors Endothelial barrier function Vascular hyperpermeability Platelet aggregation Pulmonary inflammation

a b s t r a c t Endothelial dysfunction is a hallmark of inflammatory conditions. We recently demonstrated that prostaglandin (PG)E2 enhances the resistance of pulmonary endothelium in vitro and counteracts lipopolysaccharide (LPS)-induced pulmonary inflammation in vivo via EP4 receptors. The aim of this study was to investigate the role of the EP1/EP3 receptor agonist 17-phenyl-trinor-(pt)-PGE2 on acute lung inflammation in a mouse model. In LPS-induced pulmonary inflammation in mice, 17-pt-PGE2 reduced neutrophil infiltration and inhibited vascular leakage. These effects were unaltered by an EP1 antagonist, but reversed by EP4 receptor antagonists. 17-pt-PGE2 increased the resistance of pulmonary microvascular endothelial cells and prevented thrombin-induced disruption of endothelial junctions. Again, these effects were not mediated via EP1 or EP3 but through activation of the EP4 receptor, as demonstrated by the lack of effect of more selective EP1 and EP3 receptor agonists, prevention of these effects by EP4 antagonists and EP4 receptor knock-down by siRNA. In contrast, the aggregation enhancing effect of 17-pt-PGE2 in human platelets was mediated via EP3 receptors. Our results demonstrate that 17-ptPGE2 enhances the endothelial barrier in vitro on pulmonary microvascular endothelial cells, and accordingly ameliorates the recruitment of neutrophils, via EP4 receptors in vivo. This suggests a beneficial effect of 17-ptPGE2 on pulmonary inflammatory diseases. © 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction The vascular endothelium forms the barrier between blood circulation and the interstitial space and regulates the exchange of plasma components, adhesion and extravasation of leukocytes, and haemostasis [1]. During inflammatory processes endothelial leakage occurs resulting in plasma extravasation and edema formation [2]. The integrity of the endothelial barrier is tightly regulated by cell-to-cell contacts like adherent and tight junctions between adjacent cells and connection to the actin cytoskeleton [1,3,4]. Prostanoids and phospholipids such as sphingosin1-phosphate are involved in the regulation of endothelial barrier [5,6]. Abbreviations: 17-pt-PGE2, 17-phenyl trinor prostaglandin E2; BAL, bronchoalveolar lavage; EP, E-type prostanoid receptor; HMVEC-L, human pulmonary microvascular endothelial cells; HUVEC, human umbilical vein endothelial cells; LPS, lipopolysaccharide; MMVEC-L, murine pulmonary microvascular endothelial cells. ⁎ Corresponding author. E-mail addresses: [email protected] (A. Theiler), [email protected] (V. Konya), [email protected] (L. Pasterk), [email protected] (J. Maric), [email protected] (T. Bärnthaler), [email protected] (I. Lanz), [email protected] (W. Platzer), rufi[email protected] (R. Schuligoi), [email protected] (A. Heinemann).

Prostaglandin (PG)E2 is the most abundant prostanoid in humans [7] and exerts a variety of biological functions through four different receptors (EP1–4), which differ in tissue specific gene expression [8]. These receptors activate different signaling pathways: EP1 receptor binding leads to an increase of intracellular Ca2+ levels, assumed of being coupled to a Gαq-protein. EP2 and EP4 receptors induce cyclic AMP (cAMP) production, whereas EP3 receptors couple to a Gαi-protein and inhibit cAMP synthesis [9]. PGE2 is mainly regarded as a potent pro-inflammatory mediator due to its effects on vasodilation, vascular permeability and nociception [10]. However, the role of PGE2 in the regulation of immune responses is more complex. Notably the lung represents a privileged organ with regard to PGE2 actions [11]. In the airways, PGE2 shows an anti-inflammatory mode of action as it was demonstrated to inhibit the release of a number of cytokines and chemokines via activation of the EP4 receptor [10–12] and inhibition of mast cellinduced bronchoconstriction via the EP2 receptors [13]. However, EP1 and EP3 receptors play a minor role in regulation of inflammatory processes in the lung [12]. PGE2 was shown to enhance the endothelial barrier function of human pulmonary artery endothelial cells via PKA and Epac/Rap activation leading to Rac activation and cytoskeletal remodeling [6]. We recently revealed that PGE2 promotes barrier

http://dx.doi.org/10.1016/j.vph.2016.09.008 1537-1891/© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: A. Theiler, et al., The EP1/EP3 receptor agonist 17-pt-PGE2 acts as an EP4 receptor agonist on endothelial barrier function and in a model of LPS-induced pulmonary inflammation, Vascul. Pharmacol. (2016), http://dx.doi.org/10.1016/j.vph.2016.09.008

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A. Theiler et al. / Vascular Pharmacology xxx (2016) xxx–xxx

function in human pulmonary microvascular endothelial cells (HMVEC-L) via EP4 receptor-induced strengthening of the junction and reduces endothelial trafficking of neutrophils [14]. Moreover, we recently demonstrated that PGE2, via activation of the EP4 receptor, also shows barrier promoting effects in vivo in a mouse model of lipopolysaccharide (LPS)-induced acute lung inflammation [15]. In this study we investigated the role 17-phenyl tinor (pt)-PGE2 on endothelial barrier function and the underlying molecular mechanism in HMVEC-Ls as well as in a murine model of LPS-induced acute pulmonary inflammation. We found that 17-pt-PGE2 concentration-dependently enhanced endothelial barrier function, whereas a more specific EP1 receptor agonist ONO DI-004 [16] or the EP3 receptor agonist sulprostone did not mimic this effect. Surprisingly, the effect induced by 17-pt-PGE2 was mediated by the EP4 receptor and not by EP1 or EP3 receptors. Furthermore, we show that 17-pt-PGE2 strengthens the endothelial junctions of HMVEC-Ls and reduces stress fiber formation upon treatment with thrombin. Conversely to our findings in endothelial cells, 17-pt-PGE2 promotes platelet aggregation via EP3 receptors. In a murine model of acute pulmonary inflammation, 17-pt-PGE2 caused a decrease in pulmonary extravasation and a reduction of infiltrating neutrophils, which was mediated by EP4 receptor activation. Our results demonstrate that 17-pt-PGE2 - in addition to its described effects on EP1 and EP3 receptors - also acts as an EP4 agonist and thereby enhances vascular barrier function.

mg/kg i.p.). LPS (Escherichia coli O55:B5; 20 μg per mouse in 50 μL PBS,) or vehicle or LPS in combination with 17-pt-PGE2 (20 μg per mouse), the EP1 agonist ONO DI-004 (20 μg per mouse) or the respective vehicle were applied intranasally in a volume of 25 μL to each snare. Pretreatment with the EP1 receptor antagonist SC51089 (25 mg/kg) [18] and the EP4 receptor antagonists ONO AE3-208 (10 mg/ kg) [19] and GW627368X (10 mg/kg) [20] was performed subcutaneously, 30 min prior to intranasal application. Mice were sacrificed 4 h post intranasal treatment with an overdose of pentobarbital (100 mg/ kg i.p), followed by bronchoalveolar lavage (BAL) fluid sampling as previously described [21]. Leukocytes in BAL fluid were analyzed by flow cytometry as described below. 2.4. Leukocyte analysis by flow cytometry Leukocytes were analyzed as described [15]. Briefly, BAL fluid was centrifuged at 400 × g for 7 min at 4 °C. Following two washing steps with Ca2+ and Mg2+ free PBS, cells were incubated for 30 min at 4 °C with the following monoclonal antibodies: FITC-conjugated MHC-II PE conjugated CCR3 AB, PE-Cy5.5 conjugated CD3e Ab PE-Cy7 conjugated B220 Ab APC conjugated CD11c Ab and CD16 Block (all Ab from BD Pharmingen). Samples were washed, fixed and measured on a FACSCalibur flow cytometer. 2.5. Pulmonary vascular permeability

2. Materials and methods 2.1. Reagents Laboratory chemicals were from Sigma-Aldrich (Vienna, Austria) unless specified. The EP1 receptor agonist ONO DI-004, the EP4 receptor agonist ONO AE1-329 and the EP4 receptor antagonist ONO AE3-208 were kind gifts from ONO Pharmaceuticals (Osaka, Japan). PGE2, 17pt-PGE2, SC51089, GW627368X, L-161,982, ONO-8711, iloprost, and isobutylmethylxanthine were from Cayman Chemical (Ann Arbor, MI, USA). L-161,798 was purchased from Tocris Biosciences (Bristol, UK) and SC51322 from Biomol (Hamburg, Germany). The VE-cadherin mouse monoclonal antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and secondary fluorescently-labeled antibodies and Texas Red-X Phalloidin were purchased from Invitrogen (Invitrogen, Lofer, Austria). Antibody diluent was from Dako (Glostrup, Denmark), Ultra V Block from Fisher Scientific (Vienna, Austria). Vectashield/DAPI mounting medium was obtained from Vector Laboratories (Vector Laboratories, Burlingam, CA, USA). 2.2. Animals Male BALB/c mice, 6–8 week old (body weight 22–25 g), were obtained from Charles River (Sulzfeld, Germany). Mice were housed in individually ventilated cages (4 per cage) under controlled conditions of temperature (set point 21 °C), air humidity (set point 50%) and a 12 h light/dark cycle (lights on at 6:00 a.m.) and habituated to the environment for at least one week. Standard chow and water was provided ad libitum. Mice were randomly assigned before treatment. The experimental procedure used in this study was approved by the Austrian Federal Ministry of Science, Research and Economy (BMWF 66.010/032-II/ 10b/2012 and BMWF 66.010/0094_II/3b/2013) and performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and comply with the ARRIVE guidelines [17]. Experiments were performed as humanely as possible to minimize all suffering. 2.3. LPS-induced acute pulmonary inflammation Pulmonary inflammation was induced as described [15]. Briefly, mice were slightly anaesthetized with ketamine/xylazine (50 mg/5

Mice were intranasally treated with LPS (20 μg per mouse), vehicle or LPS in combination with 17-pt-PGE2 (20 μg or 60 μg per mouse) and in additional experiments mice were pretreated with the EP4 antagonist ONO AE3-208 (10 mg/kg) as described above and Evans blue leakage was determined [15]. Briefly, 3 h post treatment mice were injected with Evans blue (60 mg/kg in saline) in the tail vein, 1 h later mice were sacrificed with pentobarbital (100 mg/kg, i.p) and exsanguinated by cutting the abdominal aorta. The lungs were perfused via the right ventricle with 10 mL of phosphate buffered saline (PBS) containing 5 mM EDTA. Thereafter lungs were excised and weighed, then homogenized and incubated with formamide (18 h, 60 °C). After centrifugation (5000 × g for 20 min) the absorbance at 620 nm and 740 nm was measured. The Evans blue content of tissue was calculated and corrected for the presence of heme pigments as described [22]. 2.6. Culture of endothelial cells and electrical resistance measurements HMVEC-L were purchased from Lonza (Verviers, Belgium) as tertiary cultures and were cultivated in EGM-2 MV bullet kit media supplemented with 5% FCS and cultivated as previously described [14]. For resistance measurements cells were grown to confluence on 1% gelatincoated polycarbonate biochips with gold microelectrodes (ECIS 8W10E+) for 48 h. Cells were serum starved for 1 h and endothelial resistance was measured with an Electrical Cell-substrate Impedance Sensing System (ECIS; Applied Biophysics, Troy, NY, USA), as described [14]. 3–4 independent experiments were performed in duplicates. 2.7. Transfection of endothelial cells and real time RT-PCR HMVEC-L were transfected with Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Briefly, HMVEC-L were seeded in 6-well plates and transfected when reaching 50–70% confluence with PTGER4 FlexiTube – GeneSolution siRNA or a non-targeting control siRNA (Qiagen, Hilden, Germany). Knock-down of PTGER4 was achieved by application of 50 nM of specific PTGER4 siRNA. For relative quantification of mRNA real time PCR was performed (CDX Connect™ Real-Time PCR detection system with CFX Manager™ software 3.1 (Bio-Rad, Hercules, CA, USA)). 48 h after transfection, RNA isolation was performed using RNA easy kit (Qiagen) and DNA removal was performed with Ambion DNA removal

Please cite this article as: A. Theiler, et al., The EP1/EP3 receptor agonist 17-pt-PGE2 acts as an EP4 receptor agonist on endothelial barrier function and in a model of LPS-induced pulmonary inflammation, Vascul. Pharmacol. (2016), http://dx.doi.org/10.1016/j.vph.2016.09.008

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kit (Thermo Fisher Scientific, Waltham, MA, USA). 1 μg of total RNA was reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's instruction. Real-time PCR was performed using SsoAdvanced™ Universal SYBR® Green Supermix with PrimePCR™ SYBR® Green Assay primers for PTGER4 or GAPDH, human (both Bio-Rad) according manufacturer's instructions. Samples were measured in triplicates and GAPDH was used as the reference gene. Quantification of mRNA expression relative to control siRNA were calculated with the 2−ΔΔCT method and data are shown as percentage of control siRNA. For subsequent ECIS assays, transfected endothelial cells were detached using trypsin/EDTA (Lonza, Verviers, Belgium) pooled and equally divided to the ECIS array. Cells were cultured overnight, and then the ECIS experiments were performed as described above.

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2.9. Blood collection and washed platelet preparation The study was approved by the Institutional Review Board (Ethics committee of the Medical University Graz). Blood was drawn from healthy volunteers after they signed an informed consent form. Whole blood from healthy donors was collected using sodium citrate (3.8%) as anticoagulant. To obtain washed platelet preparation, platelet rich plasma was prepared by centrifugation at 400 ×g for 20 min. Thereafter, the platelets were washed twice with a low pH buffer (140 mM NaCl, 10 mM NaHCO3, 2.5 mM KCl, 0.9 mM Na2HPO4, 2.1 mM MgCl2, 22 mM C6H5Na3O7, 0.055 mM glucose and 0.35% BSA, pH = 6.5) and centrifuged for 15 min at 1000 × g. The pellet was resuspended in Tyrode buffer (10 mM HEPES, 134 mM NaCl, 1 mM CaCl2, 12 mM NaHCO3, 2.9 mM KCl, 0.34 mM Na2HPO4, 1 mM MgCl2 and 0.055 mM glucose, pH = 7.4). These washed platelets were used for functional platelet assays.

2.8. VE-cadherin and F-actin staining VE-cadherin and F-actin staining was performed as described previously [14]. Briefly, HMVEC-L were grown on gelatin-coated chamber slides for 2 days. When reaching confluence, endothelial cells were incubated with 17-pt-PGE2 or vehicle for 10 min followed by thrombin stimulation for 15 min. Endothelial cells were fixed with formaldehyde and permeabilized using Triton-X-100 (0.1%), unspecific binding sites were blocked with Ultra V blocking solution. Slides were incubated with a specific VE-cadherin antibody (1 μg/mL) or isotype control antibody, followed by staining with Alexa Fluor 488-conjugated secondary antibody (4 μg/mL) and Texas Red-X Phalloidin conjugate (5 U/mL). Slides were mounted with Vectashield/DAPI mounting medium and images were taken utilizing the Olympus IX70 fluorescence microscope and an Olympus UPlanApo-60×/14.2 oil immersion lens.

2.10. Platelet aggregation Platelet aggregation was recorded at 37 °C with constant stirring using the 4-channel platelet aggregometer APACT4004 (LABiTec, Ahrensburg, Germany) as previously described [23–25]. Aggregation of washed platelets was measured as the increase in light transmission for 6 min starting with the addition of the proaggregatory stimulus collagen (1.25–5 μg/mL). 17-pt-PGE2 (10–1000 nM) was added 5 min prior to platelet stimulation. In additional experiments the EP3 antagonist L-798,106 (75 nM) was added 10 min prior to agonist treatment. Data were expressed as percent of maximum light transmission, with non-stimulated washed platelets being 0% and Tyrode buffer 100%.

Fig. 1. 17-pt-PGE2 prevents LPS-induced neutrophil infiltration in BAL fluid via EP4 receptors. BAL fluid was collected 4 h after intranasal application of (A,B) LPS (20 μg) or vehicle (veh) in presence or absence of 17-pt-PGE2. Pretreatment of the mice with the (A) EP1 receptor antagonist SC51089 (SC; sc. 30 min before), or (B) with the EP4 antagonists ONO AE3-208 (ONO AE3; 10 mg/kg sc.) and GW627368X (GW; 10 mg/kg sc.). (C) Concomitant intranasal administration of the EP1 agonist ONO DI-004 (20 μg) and LPS (20 μg) or veh. Data were analyzed using one-way ANOVA and multiple comparisons were calculated with Tukey post test. *p b 0.05, ** p b 0.01, *** p b 0.001. Animals used per group: [A:veh = 5; LPS =5; LPS&17-ptPGE2 = 9; & SC 25 mg = 3] [B: veh = 9, LPS = 11, LPS&17-pt-PGE2 = 12, & ONO AE3 = 10: & GW = 5][C: veh = 5; LPS = 6; ONO DI-004 = 6].

Please cite this article as: A. Theiler, et al., The EP1/EP3 receptor agonist 17-pt-PGE2 acts as an EP4 receptor agonist on endothelial barrier function and in a model of LPS-induced pulmonary inflammation, Vascul. Pharmacol. (2016), http://dx.doi.org/10.1016/j.vph.2016.09.008

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Dunnett's or Tukey multiple comparison test; aggregation experiments were analyzed with one-way ANOVA followed by Dunnett's or Tukey multiple comparison test; the different treated groups of mice were compared with one-way ANOVA followed by Tukey post-hoc test. Data from EP4 receptor knock-down experiments were compared with paired t-test. Probability values of p b 0.05 were regarded as statistically significant.

3. Results 3.1. 17-pt-PGE2 inhibits LPS-induced neutrophil accumulation in the murine lung via EP4 receptors

Fig. 2. 17-pt-PGE2 inhibits LPS-induced pulmonary vascular leakage. Evans blue (EB; 60 mg/kg) extravasation in the lung. Intranasal application of veh or LPS (20 μg) in the presence or absence of 17-pt-PGE2 (20 or 60 μg) Data are shown as Evans Blue (EB) mg/g wet weight and were analyzed using one-way ANOVA and multiple comparisons were calculated with Tukey post test *p b 0.05, ***p b 0.001. Animals used per group: [veh n = 5; LPS n = 6; LPS & 17-pt-PGE2 20 μg n = 5, 60 μg n = 6].

2.11. Data handling and statistics All data are shown as mean + SEM for n observations. Statistical analyses were performed using GraphPad Prism software 6.0 (La Jolla, CA; USA) Experiments using endothelial cells were performed in duplicates; electrical resistance-measurements were analyzed by two-way ANOVA for repeated measurements. Groups were compared by

Acute pulmonary inflammation in mice was induced by the intranasal application of LPS (20 μg), which caused a pronounced accumulation of neutrophils in the BAL fluid after 4 h. Co-administration of 17-ptPGE2 (20 μg) with LPS resulted in an inhibition of LPS-induced neutrophilia in the airways (Fig. 1 A,B). In contrast, concomitant intranasal application of LPS and the EP1 receptor agonist ONO DI-004 (20 μg), did not affect the recruitment of neutrophils into the BAL fluid (Fig. 1 C). To further investigate whether the EP1 or EP4 receptors mediate this effect, mice were pretreated with the EP1 receptor antagonist SC51089 (25 mg/kg) or the EP4 receptor antagonists ONO AE3-208 and GW627368X (10 mg/kg, both). The EP1 and EP4 antagonists by themselves had no effect on the neutrophil infiltration (data not shown). While the EP1 antagonist did not influence the inhibitory effect of 17pt-PGE2 (Fig. 1 A), the EP4 antagonists reversed the effect (Fig. 1 B), suggesting that in the mouse model of LPS-induced acute pulmonary

Fig. 3. 17-pt-PGE2 increases endothelial barrier function of human lung microvascular endothelial cells. HMVEC-L were treated with vehicle, (A) 17-pt-PGE2, (B) the EP1 agonist ONO DI004 or (C) the EP3 receptor agonist sulprostone (concentrations as indicated). An arrow indicates start of treatment. Endothelial resistance is shown as mean + SEM of normalized resistance of 3–4 independent experiments performed in duplicates. Data were analyzed by two-way ANOVA for repeated measurements and multiple comparisons were calculated with Dunett's post test. ***p b 0.001, compared to vehicle.

Please cite this article as: A. Theiler, et al., The EP1/EP3 receptor agonist 17-pt-PGE2 acts as an EP4 receptor agonist on endothelial barrier function and in a model of LPS-induced pulmonary inflammation, Vascul. Pharmacol. (2016), http://dx.doi.org/10.1016/j.vph.2016.09.008

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inflammation 17-pt-PGE2 strengthens the endothelial barrier via activation of the EP4 receptor.

3.3. The endothelial barrier-promoting effect of 17-pt-PGE2 is mediated by EP4 receptors in vitro

3.2. 17-pt-PGE2 counteracts LPS-induced pulmonary vascular leakage

As we observed a beneficial effect of 17-pt-PGE2 regarding neutrophil infiltration and pulmonary vascular leakage in murine model of acute pulmonary inflammation, we next investigated its effect on the barrier function in vitro. 17-pt-PGE2 caused a significant, concentration-dependent increase in the resistance of HMVEC-L monolayer, which peaked at 40–60 min after application and showed a long lasting effect of N 3 h (Fig. 3A). In contrast, the selective EP1 agonist ONO DI-004 and the EP3 agonist sulprostone had no effect on the resistance up to concentrations of 1 μM (Fig. 3 B,C). To further investigate which of the EP receptors is involved in the barrier promotion by 17-pt-PGE2, we pretreated HMVEC-L with the specific EP1 receptor antagonists SC51322 (1 μM) and ONO-8711 (1 μM) and the EP3 antagonist L-161,982 (1 μM) 15 min before 17-pt-PGE2 (100 nM) application. Neither EP1 receptor antagonists significantly reduced the effect of 17-pt-PGE2 (Fig. 4 A,B), confirming the results we

In addition to neutrophil infiltration, acute pulmonary inflammation causes pulmonary vascular leakage. Intranasal administration of LPS (20 μg) caused significant pulmonary vascular leakage as determined by Evans Blue extravasation, which was inhibited by co-application with 17-pt-PGE2 (20 and 60 μg; Fig. 2). In a separate set of experiments, mice were pretreated with the EP4 receptor antagonist ONO AE3-208 (10 mg/kg). Here we found that the EP4 receptor antagonist restored the LPS-induced plasma extravasation (as statistically compared with vehicle-treated mice) that was abrogated by 17-pt-PGE2 (20 μg) (Supplementary Fig. S1). Therefore, EP4 receptor activation seems to be involved in the inhibition by 17-pt-PGE2 of the LPS-induced pulmonary vascular leakage, similar to its inhibitory effect on neutrophil accumulation in the lung (Fig. 1).

Fig. 4. The 17-pt-PGE2 induced endothelial barrier enhancement is mediated by EP4 receptors. HMVEC-L grown on gold microelectrodes were pre-treated with EP1 receptor antagonist (A) SC51322 (1 μM) or (B) ONO-8711 (1 μM) (C) the EP3 antagonist L-798,106 (1 μM) or the EP4 receptor antagonists (D) ONO AE3-208 (1 μM) or (E) L-161,982 (1 μM) for 15 min (as indicated by arrowheads) before treatment with 17-pt-PGE2 or vehicle (100 nM; indicated by arrow). Endothelial resistance is shown as mean + SEM of normalized resistance of 3 independent experiments performed in duplicates. Data were analyzed by two-way ANOVA for repeated measurements and multiple comparisons were calculated with Tukey post test. **p b 0.01, ***p b 0.001, as compared to vehicle, ##p b 0.01, ###p b 0.001, as compared to 17-pt-PGE2.

Please cite this article as: A. Theiler, et al., The EP1/EP3 receptor agonist 17-pt-PGE2 acts as an EP4 receptor agonist on endothelial barrier function and in a model of LPS-induced pulmonary inflammation, Vascul. Pharmacol. (2016), http://dx.doi.org/10.1016/j.vph.2016.09.008

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obtained with the EP1 agonist, suggesting that EP1 receptor activation is not involved in the barrier enhancing effect. Since 17-pt-PGE2 is also known to activate EP3 receptors, we tested whether the barrier promoting effect was mediated via EP3 receptors [26]. However, pre-treatment with the specific EP3 receptor antagonist did not counteract the effect of 17-pt-PGE2 (Fig. 4 C). Since we previously found that activation of the EP4 receptor augments the endothelial resistance on HMVEC-Ls [14], we pretreated cells with the EP4 antagonists ONO AE3–208 (1 μM) and L-161,982 (1 μM), which totally abolished the effect of 17-pt-PGE2 (Fig. 4 D,E). Also in HUVECs, 17-pt-PGE2 increased resistance via EP4 receptor activation, but this effect was less pronounced (Supplementary Fig. S2). We already demonstrated that HMVEC-L express EP1, EP3 and EP4 receptors, but lack EP2 receptors as determined by flow cytometry [14]. Here we investigated the mRNA expression with real-time RTPCR and found mRNA of the EP4 receptor, and very low levels of EP1 mRNA as indicated by the higher delta CT values, but we could not detect EP2 or EP3 receptor mRNA (supplementary Fig. S3A). EP2–4 receptors were previously described to be present on peripheral blood monocytes [27], hence we used monocytes as positive control for EP receptor expression (supplementary Fig. S3B). In order to confirm our findings of 17-pt-PGE2 acting via EP4 receptors we performed EP4 receptor knock-down in HMVEC-L using siRNA approach. We found that silencing the EP4 receptors completely abolished the barrier promoting effect of both 17-pt-PGE2 and the EP4 receptor agonist ONO AE1-329 (Fig. 5 A,B). Knock-down of EP4 receptor was confirmed using real time RT-PCR as shown in Fig. 5 C. Therefore, our results unequivocally suggest that 17-pt-PGE2 acts as an EP4 receptor agonist on HMVEC-Ls. Finally, we addressed the involvement of cAMP in HMVEC-L, but found no increase in cAMP production in response to 17-pt-PGE2, whereas the IP receptor agonist iloprost (300 nM) and the adenylyl cyclase activator forskolin (20 μM) induced cAMP (Supplementary Fig.

S4). This was even more pronounced when cells were pretreated with the phosphodiesterase inhibitor isobutylmethylxanthine (IMBX, 0.5 mM for 10 min; Supplementary Fig. S4). These results are in line with findings that the barrier-enhancing effect mediated via EP4 receptor activation does not depend on cAMP [14]. 3.4. 17-pt-PGE2 protects endothelial junctional integrity The integrity of the endothelial cell layer is maintained by endothelial adherens junctions connected to cytoskeletal filamentous-actin (F-actin) network. We assessed the regulatory role of 17-pt-PGE2 on VE-cadherin expression in the endothelial junctions and changes of F-actin polymerization by using immunofluorescence microscopy. Generally, vehicletreated cells showed intact adherens junction complexes between adjacent cells and peripheral F-actin polymerization. After 15 min treatment with 17-pt-PGE2 (100 nM) VE-cadherin expression in the adherens junctions appeared to be more intense and we observed a more pronounced F-actin network in the paracellular regions. Treatment with thrombin (0.5 U/mL) caused disruption of endothelial adherens junctions, remodeling of the F-actin network into stress fibers, and formation of paracellular gaps. This was prevented by the pretreatment with 17-pt-PGE2 (Fig. 6). 3.5. 17-pt-PGE2 potentiates collagen-induced platelet aggregation via the EP3 receptor Platelets have been implicated in inflammatory reactions [28–30] and we have described that EP4 receptors confer inhibition of platelet aggregation and activation [23]. Since we were able to show that 17pt-PGE2 acts as an EP4 receptor agonist in human pulmonary endothelial cells, we hypothesized that this may be true for other cell types as well. Washed platelets were treated with 17-pt-PGE2 (10, 100,

Fig. 5. Knock-down of EP4 receptors inhibits the barrier promoting effect of 17-pt-PGE2. Untransfected, control siRNA or EP4 siRNA transfected endothelial cells were grown on gold microelectrodes and were either treated with (A) 17-pt-PGE2 (100 nM) or the (B) EP4 receptor agonist ONO AE1-329 (100 nM; indicated by arrow). Endothelial resistance is shown as normalized mean + SEM of 3 independent experiments performed in duplicates. Data were analyzed by two-way ANOVA for repeated measurements and multiple comparisons were calculated with Tukey post test. **p b 0.01, ***p b 0.001, as compared to control siRNA. EP4 receptor knock-down was confirmed with real time RT-PCR (C). HMVEC-L were transfected with non-silencing control siRNA or EP4 siRNA (50 nM each) and incubated for 48 h. EP4 receptor expression is shown as percentage of control siRNA of 3 independent experiments. Data were analyzed by paired t-test. **p b 0.01, as compared to control siRNA.

Please cite this article as: A. Theiler, et al., The EP1/EP3 receptor agonist 17-pt-PGE2 acts as an EP4 receptor agonist on endothelial barrier function and in a model of LPS-induced pulmonary inflammation, Vascul. Pharmacol. (2016), http://dx.doi.org/10.1016/j.vph.2016.09.008

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Fig. 6. 17-pt-PGE2 prevents the thrombin-induced disruption of endothelial junctions. HMVEC-L was cultured in chamber slides until they reached confluence. Cells were pre-incubated with 17-pt-PGE2 (100 nM) for 10 min followed by exposure to vehicle or thrombin (0.5 U/mL, 15 min) and then stained with anti-VE-cadherin antibody (green) or phalloidin-Texas Red-X conjugate (red) and DAPI (blue). Pictures were taken with an Olympus IX70 fluorescence microscope and Olympus UPlanApo-60×/14.2 oil immersion objective. Images are representative of 3 independent experiments.

1000 nM) 5 min prior to stimulation with collagen. The concentration of collagen (1.25 to 10 μg/mL) was adjusted to cause submaximal aggregation as previously described [23–25]. We found that 17-pt-PGE2 increased the collagen-induced aggregation of washed platelets, which was significantly different from vehicle-treated platelets at 1 μM of the prostaglandin (p b 0.01) (Fig. 7 A). Pretreatment with the highly potent EP3 receptor antagonist L-798,106 (75 nM) 10 min prior to 17-pt-PGE2 (1 μM) counteracted the enhancement of collagen-induced aggregation, demonstrating the involvement of EP3 receptors (Fig. 7 B, C). 17-pt-PGE2 did not impact Ca2+ flux in human platelets alone and did not influence the Ca2+-inducing effect of ADP. The same was observed with the EP3 receptor agonist sulprostone (data not shown). To investigate whether a high concentration of 17-pt-PGE (10 μM) causes an inhibition of stimulus-induced aggregation, which may be due to activation of the EP4 receptor, collagen was used at a concentration that caused about 70–80% aggregation. However, under these conditions we could not detect an inhibitory effect on platelet aggregation (n = 3, data not shown). 4. Discussion In this study we demonstrate that the purported EP1/EP3 receptor agonist 17-pt-PGE2 concentration-dependently increases the endothelial barrier function of HMVEC-L as determined by an increase in electrical resistance, and protects against thrombin-induced disruption of

endothelial junctions and stress fiber formation. These effects were comparable to PGE2 [14]. Interestingly, the more specific EP1 receptor agonist, ONO DI-004 and the EP3 receptor agonist sulprostone failed to mimic this effect. The lack of involvement of EP1 and EP3 receptors was confirmed by EP1 and EP3 antagonists. In contrast, the barrierstrengthening effect of 17-pt-PGE2 was reversed by EP4 antagonists as well as by silencing of the EP4 receptor. Monolayers of endothelial cells are accomplished by junctional structures, where VE-cadherin plays a major role in adherens junction formation. We found a protective effect of 17-pt-PGE2 on thrombin-induced disruption of the endothelial junctions and stress fiber formation, which was recently demonstrated to be mediated via EP4 receptor activation [14]. Strengthening of vascular barrier function in vitro was shown for PGI2 and PGE2 via cAMP mediated activation of PKA-, Epac/Rap1- and Tiam1/Vav2-dependent pathways of Rac1 activation [6]. Recently, we demonstrated that EP4 receptor activation is responsible for the barrier-enhancing effect of PGE2, which was independent of cAMP/PKA, PKC, NO or Rac signaling but was mediated by cytoskeletal rearrangements [14]. This is in line with our findings, as 17-pt-PGE2 did not induce cAMP synthesis in HMVEC-L. In addition, wound healing was facilitated by EP4 receptor activation but was not influenced by EP2 and EP1/3 agonists [14]. Here we demonstrate that the barrier enhancing effect or 17pt-PGE2 is mediated solely by EP4 but not by EP1 or EP3 receptor activation.

Please cite this article as: A. Theiler, et al., The EP1/EP3 receptor agonist 17-pt-PGE2 acts as an EP4 receptor agonist on endothelial barrier function and in a model of LPS-induced pulmonary inflammation, Vascul. Pharmacol. (2016), http://dx.doi.org/10.1016/j.vph.2016.09.008

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Fig. 7. 17-pt-PGE2 augments collagen-induced aggregation in washed platelets via EP3 receptor activation. Platelet aggregation was induced by collagen, the concentrations (1.25–5 μg/mL) were adjusted to give a submaximal, 20–40% aggregation. (A) Freshly prepared washed platelets were pretreated with 17-pt-PGE2 (10–1000 nM) for 5 min, before stimulation with collagen. (B) Pre-treatment of platelets with the EP3 antagonist L-798,106 (75 nM) 10 min before addition of 17-pt-PGE2. (C) Original tracing showing that the proaggregatory effect of 17-pt-PGE2 (1000 nM) is inhibited by the EP3 receptor antagonist L-798,106. Results are shown as mean + SEM of n = 3–4 independent experiments and are normalized to the respective vehicle. Data were analyzed using one-way ANOVA and multiple comparisons were calculated with Tukey post test *P b 0.05, **P b 0.01, as compared to vehicle.

Inflammatory stimuli cause disruption of the endothelial barrier by dissociation of cell-cell junctions, leading to plasma extravasation and infiltration of inflammatory cells. Our second main finding was that in a murine model of LPS-induced acute lung inflammation, 17-pt-PGE2 reduced pulmonary plasma extravasation and ameliorated the recruitment of neutrophils into the BAL fluid, which account as characteristic clinical complications of acute lung injury [31]. This effect was mediated by EP4 receptor activation, which is in agreement with our in vitro findings on the barrier function. PGE2 is well known to reduce the accumulation of neutrophils within the BAL fluid in LPS-induced acute lung inflammation [32,33]. We showed recently that the effect of PGE2 on LPS-induced neutrophil infiltration and LPS- as well as oleic acid-induced plasma extravasation is mediated mainly via activation of the EP4 receptor [15]. In addition it was demonstrated in EP4-, but not in EP1-, EP2- and EP3-deficient mice that only the EP4 receptor seems to be responsible for the anti-inflammatory activity in mouse models of lung inflammation [12]. The action of 17-pt-PGE2 as an EP1 receptor agonist [34] was demonstrated in several experimental settings, including cancer, neurons, vascular system, kidney and was confirmed by application of EP1 receptor selective antagonists [35–41]. Receptor binding studies in human and rat EP4 receptor-expressing systems support our findings that 17pt-PGE2 also can act as an EP4 receptor agonist [42–44], whilst opposing findings are published for a murine EP4 receptor expressing system [26]. In addition it was shown that 17-pt-PGE2 activates human as well as murine and guinea pig EP3 receptors [26,34]. With regard to platelets, 17-pt-PGE2 has been described as aggregation-potentiating substance in human platelets [45]. Since it is known that EP3 receptor activation is responsible for enhancing stimulus-induced aggregation in human platelets [46–48] we investigated the

possible involvement of EP3 receptor. It is well established that prostaglandins have a biphasic effect on platelet aggregation, where activation of the EP3 receptor has pro-aggregatory and activation of the EP4 receptor anti-aggregatory effects [23,24]. Here we show that the proaggregatory effect of 17-pt-PGE2 is mediated via EP3 receptor activation. In this model, we did not find an involvement of the EP4 receptor, as even high concentration of 17-pt-PGE2 (10 μM) was not capable of inhibiting aggregation induced by collagen. EP1, EP3 and EP4 receptor expression on HMVEC-L was shown recently by flow cytometry [14]. However, only EP4 mRNA expression was previously described in these cells [49]. Our data obtained with RT-PCR showed EP4 mRNA and very low levels of EP1 mRNA, but no EP2 and EP3 mRNA expression was detectable in HMVEC-L. Although EP1 receptor mRNA was detected, the effects of the purported EP1/ EP3 receptor agonist 17-pt-PGE2 on human pulmonary microvascular endothelial cells were exclusively mediated via EP4 receptor activation. In contrast, 17-pt-PGE2 acted on human isolated platelets as an EP3 receptor agonist. The preferential activation of EP4 receptors rather than EP3 receptors can be explained by the lack of EP3 receptor expression and comparatively low amount of EP1 receptors on HMVEC-L. 5. Conclusion In conclusion, our results provide evidence that the purported EP1/ EP3 receptor agonist 17-pt-PGE2 possesses anti-inflammatory properties, promotes microvascular endothelial barrier function in vitro and further abates neutrophil recruitment and plasma extravasation in a murine model of pulmonary inflammation. Our data suggest that these effects are mediated by EP4 receptor activation and that its mode of action might be tissue and cell specific. We show here for the

Please cite this article as: A. Theiler, et al., The EP1/EP3 receptor agonist 17-pt-PGE2 acts as an EP4 receptor agonist on endothelial barrier function and in a model of LPS-induced pulmonary inflammation, Vascul. Pharmacol. (2016), http://dx.doi.org/10.1016/j.vph.2016.09.008

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Please cite this article as: A. Theiler, et al., The EP1/EP3 receptor agonist 17-pt-PGE2 acts as an EP4 receptor agonist on endothelial barrier function and in a model of LPS-induced pulmonary inflammation, Vascul. Pharmacol. (2016), http://dx.doi.org/10.1016/j.vph.2016.09.008