Sonia Philipose

CRTH2 and D-type prostanoid receptor antagonists as novel therapeutic agents for inflammatory diseases.

Accumulation of type 2 T helper (Th2) lymphocytes and eosinophils is a hallmark of bronchial asthma and other allergic diseases, and it is believed that these cells play a crucial pathogenic role in allergic inflammation. Thus, Th2 cells and eosinophils are currently considered a major therapeutic target in allergic diseases and asthma. However, drugs that selectively target the accumulation and activation of Th2 cells and eosinophils in tissues are unavailable so far. Prostaglandin (PG)D2 is a key mediator in various inflammatory diseases including allergy and asthma. It is generated by activated mast cells after allergen exposure and subsequently orchestrates the recruitment of inflammatory cells to the tissue. PGD2 induces the chemotaxis of Th2 cells, basophils and eosinophils, stimulates cytokine release from these cells and prolongs their survival, and might hence indirectly promote IgE production. PGD2 mediates its biologic functions via 2 distinct G protein-coupled receptors, D-type prostanoid receptor (DP), and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). DP and CRTH2 receptors are currently being considered as highly promising therapeutic targets for combating allergic diseases and asthma. Here, we revisit the roles of PGD2 receptors in the regulation of eosinophil and Th2 cell function and the efforts towards developing candidate compounds for clinical evaluation.

The Prostaglandin E2 Receptor EP4 Is Expressed by Human Platelets and Potently Inhibits Platelet Aggregation and Thrombus Formation

Objective—Low concentrations of prostaglandin (PG) E2 enhance platelet aggregation, whereas high concentrations inhibit it. The effects of PGE2 are mediated through 4 G protein-coupled receptors, termed E-type prostaglindin (EP) receptor EP1, EP2, EP3, and EP4. The platelet-stimulating effect of PGE2 has been suggested to involve EP3 receptors. Here we analyzed the receptor usage relating to the inhibitory effect of PGE2. Methods and Results—Using flow cytometry, we found that human platelets expressed EP4 receptor protein. A selective EP4 agonist (ONO AE1-329) potently inhibited the platelet aggregation as induced by ADP or collagen. This effect could be completely reversed by an EP4 antagonist, but not by PGI2, PGD2, and thromboxane receptor antagonists or cyclooxygenase inhibition. Moreover, an EP4 antagonist enhanced the PGE2-induced stimulation of platelet aggregation, indicating a physiological antiaggregatory activity of EP4 receptors. The inhibitory effect of the EP4 agonist was accompanied by attenuated Ca2+ flux, inhibition of glycoprotein IIb/IIIa, and downregulation of P-selectin. Most importantly, adhesion of platelets to fibrinogen under flow and in vitro thrombus formation were effectively prevented by the EP4 agonist. In this respect, the EP4 agonist synergized with acetylsalicylic acid. Conclusion—These results are suggestive of EP4 receptor activation as a novel antithrombotic strategy.

EP4 receptor stimulation down-regulates human eosinophil function

Accumulation of eosinophils in tissue is a hallmark of allergic inflammation. Here we observed that a selective agonist of the PGE2 receptor EP4, ONO AE1-329, potently attenuated the chemotaxis of human peripheral blood eosinophils, upregulation of the adhesion molecule CD11b and the production of reactive oxygen species. These effects were accompanied by the inhibition of cytoskeletal rearrangement and Ca2+ mobilization. The involvement of the EP4 receptor was substantiated by a selective EP4 antagonist, which reversed the inhibitory effects of PGE2 and the EP4 agonist. Selective kinase inhibitors revealed that the inhibitory effect of EP4 stimulation on eosinophil migration depended upon activation of PI 3-kinase and PKC, but not cAMP. Finally, we found that EP4 receptors are expressed by human eosinophils, and are also present on infiltrating leukocytes in inflamed human nasal mucosa. These data indicate that EP4 agonists might be a novel therapeutic option in eosinophilic diseases.

Distinct composition of human fetal HDL attenuates its anti-oxidative capacity

In human high-density lipoprotein (HDL) represents the major cholesterol carrying lipoprotein class in cord blood, while cholesterol is mainly carried by low-density lipoprotein in maternal serum. Additionally, to carrying cholesterol, HDL also associates with a range of proteins as cargo. We tested the hypothesis that fetal HDL carries proteins qualitatively and quantitatively different from maternal HDL. These differences then contribute to distinct HDL functionality in both circulations. Shotgun proteomics and biochemical analyses were used to assess composition/function of fetal and maternal HDL isolated from uncomplicated human pregnancies at term of gestation. The pattern of analyzed proteins that were statistically elevated in fetal HDL (apoE, proteins involved in coagulation, transport processes) suggests a particle characteristic for the light HDL2 sub-fraction. In contrast, proteins that were enriched in maternal HDL (apoL, apoF, PON1, apoD, apoCs) have been described almost exclusively in the dense HDL3 fraction and relevant to its anti-oxidative function and role in innate immunity. Strikingly, PON1 mass and activity were 5-fold lower (p<0.01) in the fetus, which was accompanied by attenuation of anti-oxidant capacity of fetal HDL. Despite almost equal quantity of CETP in maternal and fetal HDL, its enzymatic activity was 55% lower (p<0.001) in the fetal circulation, whereas LCAT activity was not altered. These findings indicate that maternally derived HDL differs from fetal HDL with respect to its proteome, size and function. Absence of apoA-1, apoL and PON1 on fetal HDL is associated with decreased anti-oxidative properties together with deficiency in innate immunity collectively indicating distinct HDLs in fetuses.

Interaction of eosinophils with endothelial cells is modulated by prostaglandin EP4 receptors

Eosinophil extravasation across the endothelium is a key feature of allergic inflammation. Here, we investigated the role of PGE2 and its receptor, E‐type prostanoid receptor (EP)‐4, in the regulation of eosinophil interaction with human pulmonary microvascular endothelial cells. PGE2 and the EP4 receptor agonist ONO AE1‐329 significantly reduced eotaxin‐induced eosinophil adhesion to fibronectin, and formation of filamentous actin and gelsolin‐rich adhesive structures. These inhibitory effects were reversed by a selective EP4 receptor antagonist, ONO AE3‐208. PGE2 and the EP4 agonist prevented the activation and cell‐surface clustering of β2 integrins, and L‐selectin shedding of eosinophils. Under physiological flow conditions, eosinophils that were treated with the EP4 agonist showed reduced adhesion to endothelial monolayers upon stimulation with eotaxin, as well as after TNF‐α‐induced activation of the endothelial cells. Selective activation of EP1, EP2, and EP3 receptors did not alter eosinophil adhesion to endothelial cells, whereas the EP4 antagonist prevented PGE2 from decreasing eosinophil adhesion. Finally, eosinophil transmigration across thrombin‐ and TNF‐α‐activated endothelial cells was effectively reduced by the EP4 agonist. These data suggest that PGE2–EP4 signaling might be protective against allergic responses by inhibiting the interaction of eosinophils with the endothelium and might hence be a useful therapeutic option for controlling inappropriate eosinophil infiltration.

The EP3 Agonist Sulprostone Enhances Platelet Adhesion But Not Thrombus Formation Under Flow Conditions

Platelets express the EP2, EP3 and EP4 receptors. Prostaglandin (PG) E2 has a biphasic effect on platelets. Low concentrations of PGE2 enhance platelet aggregation through the activation of the EP3 receptors, while at high concentrations it attenuates aggregation via the EP4 receptor. Consequently, EP3 receptor inhibition was shown to inhibit artherothrombosis, but had no influence on bleeding time in vivo. In this study, we investigated the role of the EP3 receptor in adhesion and thrombus formation under flow conditions in vitro. The EP3 agonist sulprostone caused an increase in the adhesion of washed platelets to fibrinogen as well as to collagen under low shear stress, an effect that was blocked by the EP3 antagonist L-798106. In contrast, when whole blood was perfused over collagen-coated surfaces, sulprostone did not enhance binding and thrombus formation of platelets on collagen; at high concentrations it even attenuated this response. We conclude that in more physiological models of thrombus formation, the role for EP3 receptors is limited, indirectly suggesting that the primary action of PGE2 in haemostasis might be an inhibitory one.

Laropiprant attenuates EP3 and TP prostanoid receptor-mediated thrombus formation

The use of the lipid lowering agent niacin is hampered by a frequent flush response which is largely mediated by prostaglandin (PG) D2. Therefore, concomitant administration of the D-type prostanoid (DP) receptor antagonist laropiprant has been proposed to be a useful approach in preventing niacin-induced flush. However, antagonizing PGD2, which is a potent inhibitor of platelet aggregation, might pose the risk of atherothrombotic events in cardiovascular disease. In fact, we found that in vitro treatment of platelets with laropiprant prevented the inhibitory effects of PGD2 on platelet function, i.e. platelet aggregation, Ca flux, P-selectin expression, activation of glycoprotein IIb/IIIa and thrombus formation. In contrast, laropiprant did not prevent the inhibitory effects of acetylsalicylic acid or niacin on thrombus formation. At higher concentrations, laropiprant by itself attenuated platelet activation induced by thromboxane (TP) and E-type prostanoid (EP)3 receptor stimulation, as demonstrated in assays of platelet aggregation, Ca flux, P-selectin expression, and activation of glycoprotein IIb/IIIa. Inhibition of platelet function exerted by EP4 or I-type prostanoid (IP) receptors was not affected by laropiprant. These in vitro data suggest that niacin/laropiprant for the treatment of dyslipidemias might have a beneficial profile with respect to platelet function and thrombotic events in vascular disease. Citation: Philipose S, Konya V, Lazarevic M, Pasterk LM, Marsche G, et al. (2012) Laropiprant Attenuates EP3 and TP Prostanoid Receptor-Mediated Thrombus Formation. PLoS ONE 7(8): e40222. doi:10.1371/journal.pone.0040222 Editor: Christian Schulz, Heart Center Munich, Germany Received April 17, 2012; Accepted June 2, 2012; Published August 1, 2012 Copyright: 2012 Philipose et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Dr. Philipose was funded by the PhD Program in Molecular Medicine of the Medical University of Graz. This study was supported by the Jubilaumsfonds of the Austrian National Bank (OeNB, grants 13487 and 14263) and the Austrian Science Fund (FWF; grants P22521-B18, P19473-B05, P21004-B02 and P22976-B18. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: akos.heinemann@medunigraz.at

Prostaglandin E2 acts via the EP4 receptor to inhibit platelet aggregation.

Background Platelets play a central role in haemostasis. Blood vessel injury leads to platelet aggregation and also invokes an inflammatory response leading to the formation of prostanoids like prostaglandin E2 (PGE2) and prostacyclin (PGI2). It is known that low concentrations of PGE2 enhance and high concentrations inhibit platelet aggregation. PGE2 mediates its effect through four receptors: EP1 (Gαq signalling), EP3 (three isoforms present; signals via Gi, Gs or Gq based on cell type), EP2 and EP4 (Gs signalling). PGI2 is known to inhibit platelet aggregation through its IP receptor (Gs signalling). The role of EP3 in exacerbating platelet aggregation has been well described. However, the role of EP4 which acts via the same G protein coupling like IP has not been explored in detail. The aim of this study was to investigate the role of EP4 in platelet aggregation.

A biological target for antiplatelet therapy: the prostaglandin E2 receptor EP4.

Background Acute myocardial infarction is one of the leading causes of death in the world which is caused by coronary artery thrombosis. Platelets play a central role in cardiovascular thrombosis. Platelet aggregation caused due to a ruptured artherosclerotic plaque could eventually lead to vascular occlusion. Another important component of vascular diseases is inflammation. During inflammation, prostaglandins (PG) like PGI2, PGE2 and PGD2 are released which are also involved in thrombosis. Lower concentrations of PGE2 enhance platelet aggregation whereas higher concentrations inhibit aggregation. PGE2 acts via 4 receptors: EP1, EP2, EP3 and EP4 (Gs signalling). The role of the EP3 receptor in enhancing platelet activation and aggregation has been looked at in detail but the role of the EP4 receptor is largely unknown. We were interested in how this receptor modulates platelet aggregation and what are the signalling mechanisms involved in this process.