Prostaglandin E2 receptor subtypes in human blood and vascular cells

European Journal of Pharmacology 695 (2012) 1–6

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Perspective

Prostaglandin E2 receptor subtypes in human blood and vascular cells Nabil Foudi a,n, Ingrid Gomez b,c, Chabha Benyahia b,c, Dan Longrois b,d, Xavier Norel b,c a

Department of Pharmacy, Faculty of Medicine, Ferhat Abbas University, 19000 Setif, Algeria INSERM U698, CHU X. Bichat, 46 rue Henri Huchard, 75018 Paris, France University of Paris Nord, UMR-S698, Paris F-75018, France d CHU X. Bichat, Department of Anesthesia and Intensive Care, University of Paris Diderot, Sorbonne Paris Cite´, UMR-S698, 75018 Paris, France b c

a r t i c l e i n f o

abstract

Article history: Received 6 April 2012 Received in revised form 21 August 2012 Accepted 27 August 2012 Available online 3 September 2012

Prostaglandin E2 is produced in inflammatory responses via the cyclooxygenase pathway and regulates a variety of physiological and pathological reactions through four different receptor subtypes; EP1, EP2, EP3 and EP4. The role of the classical prostanoid receptors stimulated by prostaglandin I2 and thromboxane A2 in the blood circulation has been largely studied, whereas the other receptors such as EP activated by prostaglandin E2, have been recently shown to be also implicated. There is now increasing evidence suggesting an important role of EP3 and EP4 receptor subtypes in the control of the human vascular tone and remodeling of the vascular wall as well in platelet aggregation and thrombosis. These receptors are implicated in vascular homeostasis and in the development of some pathological situations, such as atherosclerosis, aneurysms and hypertension. The use of specific EP agonists/antagonists would provide a novel cardiovascular therapeutic approach. In this review, we discuss the role of prostaglandin E2 receptors in the control of human blood and vascular cells. & 2012 Elsevier B.V. All rights reserved.

Keywords: Prostaglandin Blood cell Endothelium Smooth muscle cell Atherosclerosis Vascular tone

1. Introduction Blood cells (erythrocytes, leukocytes and platelets) are in permanent interaction with vascular wall, especially with endothelial and smooth muscle cells (SMC), leading to numerous physiological and pathological processes (Norel, 2007; Ricciotti and FitzGerald, 2011). One major signal of the interactions among these cells are the prostanoids, these bioactive lipids derive from arachidonic acid metabolism. Prostanoids [prostanglandins (PG) and thromboxanes (Tx)] are ubiquitous and implicated in blood disorders, cardiovascular diseases and vascular homeostasis (Norel, 2007; Woodward et al., 2011). The recent discovery of specific agonists and antagonists has to a large extent characterized the role of each receptor activated by different prostanoids. This review concerns especially the role of PGE2, one of the most important inflammatory mediators, and their receptors expressed in human blood and vascular wall cells.

2. PGE2 synthesis PGE2 is derived from arachidonic acid liberated from phospholipids by the action of phospholipase A2 enzymes in the cell membrane (Norel, 2007; Ricciotti and FitzGerald, 2011). The cyclooxygenases (COX-1 and COX-2) use arachidonic acid as n

Corresponding author. Tel.: þ213 550854804; fax: þ 213 36620035. E-mail address: [email protected] (N. Foudi).

0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.08.009

substrate, leading to the formation of PGH2. This intermediate metabolite is used by PGE synthases (PGES) to produce PGE2 (Norel, 2007). Three PGES isoforms are described; cytosolic PGES (cPGES) and mircosomal PGES (mPGES-1 and mPGES-2). The expression of cPGES and mPGES-2 is constitutive. However, the expression of mPGES-1, as well as COX-2, is induced under inflammatory conditions (Jakobsson et al., 1999; Ricciotti and FitzGerald, 2011).

3. EP receptor subtypes expressed by vascular and blood cells The use of molecular biology, pharmacological tools and homology screening of cDNA libraries resulted in the identification of four PGE2 receptors: EP1, EP2, EP3 and EP4 with 8 isoforms described for the EP3 receptor in humans (Norel, 2007; Woodward et al., 2011). The EP receptor subtypes present on SMC and involved in regulation of vascular tone can be separated into two groups, EP1/3 and EP2/4 receptors (Table 1). In the first group, stimulation of EP3 receptor induces vasoconstriction by reducing intracellular cAMP content while stimulation of EP1 involves the same effect through inositol phosphate-3 pathway and Ca2þ release (Lang et al., 2006). Furthermore, the EP3 receptor is more frequently described than EP1 as being involved in vasoconstriction in human vasculature (Foudi et al., 2011; Longrois et al., 2012; Qian et al., 1994; Walch et al., 2001). On the other hand, activation of EP2/4 receptors is responsible for vasodilation through increased intracellular cAMP content (Besse et al., 1999). In human vessels, studies using selective agonist and

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Table 1 Effects mediated by EP receptor subtypes in human vascular cells. ( þ): activation; ( ): inhibition; IL: interleukin; VEGF; vascular endothelial growth factor; Ig: immunoglobulin; MMP: matrix metalloproteases; PG: prostaglandin. Cells

Endothelial

Smooth muscle

Receptors

Induced effects (þ activation,  inhibition)

References

EP2 EP3 EP4 EP4 EP2, EP4 EP1 EP3

þ þ þ þ þ þ þ

Nuclear Factor-kB activation and expression of the chemokine (C-C motif) ligand 2 MMP-9 expression Angiogenesis Vascular endothelial growth factor (VEGF)-induced cell proliferation and IL-8 production Proliferation, cytosolic cAMP levels Vasoconstriction in pulmonary vein Vasoconstriction in mammary, intercostal and pulmonary arteries

EP4

þ

Vasodilation in pulmonary and saphenous veins, in uterine and cerebral arteries

(Aoki et al., 2011) (Amano et al., 2009) (Zhang and Daaka, 2011) (Aso et al., 2012; Yanni et al., 2009) (Mori et al., 2011) (Walch et al., 2001) (Foudi et al., 2011; Longrois et al., 2012; Qian et al., 1994) (Baxter et al., 1995; Davis et al., 2004; Foudi et al., 2011; Foudi et al., 2008)

Table 2 Effects mediated by EP receptor subtypes in human blood cells. ( þ ): activation; ( ): inhibition; IL: interleukin; VEGF; vascular endothelial growth factor; Ig: immunoglobulin; MMP: matrix metalloproteases; TNF: Tumor Nucrosis factor. Cells

Platelets

Receptors

Induced effects (þ activation,  inhibition)

References

EP4

 Aggregation and thrombotic effect

EP2 EP3

 Aggregation þ Aggregation

(Iyu et al., 2010; Kuriyama et al., 2010; Philipose et al., 2010; Schober et al., 2011) (Petrucci et al., 2011; Smith et al., 2010) (Armstrong, 1996; Heptinstall et al., 2008; Jones et al., 2006; Petrucci et al., 2011; Schober et al., 2011; Smith et al., 2010)

Erythrocyte

Any study demonstrates EP receptor expression in Erythrocyte  Activation, migration EP2 EP4  Production de Leukotriene C4 Eosinophil EP4  Interaction with the endothelium, Chemotaxis, production of adhesion molecule CD11b and reactive oxygen species.  Leukotriene B4 and superoxide anion release EP2 Neutrophil EP4  Expression of Beta1-integrin Basophil Any study demonstrates EP receptor expression in Basophil Mastocyte EP1 and/ þ IgE-mediated histamine release or EP3 þ VEGF production EP2 EP2  Degranulation EP2, EP4  Intercellular adhesion molecule-1 expression EP3 þ Migration EP2, EP4 þ Expression, activity of chemokine receptor CCR7 and migration EP(1 or 2) þ Adhesion to endothelial cells EP2, EP4  Tumor necrosis factor-alpha (TNF-a) production and T-cell Monocyte proliferation  IL-5 and interferon-gamma production EP2 EP4  IL-12 production EP2, EP4 þ Maturation in monocyte derived dentritic cell EP(2 or 4) þ Cyclooxygenase-2 expression EP(2 or 4) þ Migration  Chemokine production and Beta1-integrin expression Macrophage EP4 EP4 þ IL-6 production EP4  Proliferation B þ Apoptosis Lymphocyte EP4 EP2  Apoptosis T EP3 þ MMP-9 production Lymphocyte EP4  IL-2 and IL-6 production

antagonists demonstrated that vasodilation is mainly due to EP4- and not EP2- receptor activation (Baxter et al., 1995; Davis et al., 2004; Foudi et al., 2011; Foudi et al., 2008). In these previous studies on human vascular tone, the effects of EP receptor subtypes were mainly described using isolated vascular preparations with endothelium (Table 1). Few studies have investigated an involvement of prostanoid receptors expressed on the endothelium. For example, the relaxation of human hand veins produced after PGE2 stimulation was reduced in the absence of endothelium (Arner et al., 1994). These results suggest the presence of endothelial prostanoid receptors and the involvement of an endothelial relaxing factor in these veins. The EP receptors expressed in blood cells are coupled to the same intracellular signal transduction mechanisms as those

(Sturm et al., 2008) (Mita et al., 2002) (Konya et al., 2011; Luschnig-Schratl et al., 2011) (Wheeldon and Vardey, 1993) (Profita et al., 2010) (Wang and Lau, 2006) (Abdel-Majid and Marshall, 2004) (Duffy et al., 2008) (Morichika et al., 2003) (Zeng et al., 1995) (Cote et al., 2009) (Passacquale et al., 2011) (Takahashi et al., 2005) (Okano et al., 2006) (Iwasaki et al., 2003) (Kubo et al., 2004) (Hinz et al., 2000) (Luft et al., 2002) (Hasegawa et al., 2010; Takayama et al., 2002) (Bayston et al., 2003) (Murn et al., 2008) (Prijatelj et al., 2011) (Goetzl et al., 1995) (Zeng et al., 1996) (Cosme et al., 2000; Zeng et al., 1998)

described above in the vascular wall. Their function varies according to cell type as indicated in Table 2. Among the EP receptor subtypes, on blood cells, the functions mediated by EP4and EP2- subtypes are the most frequently described and they are involved in the control of cytokines, chemokines and cell adhesion proteins expression (Table 2).

4. EP receptor subtypes and platelet aggregation The role of prostanoids in blood homeostasis through regulation of platelet aggregation is known since the 1970s (Moncada et al., 1976; Needleman et al., 1976; Shio et al., 1972). The first

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studies indicated that TxA2, produced by platelets, is a potent stimulator of aggregation, while PGI2 is known to inhibit this effect. In addition, PGE2 has been shown to play a biphasic concentrationdependent effect on platelet aggregation (Shio et al., 1972). In fact, high concentrations of PGE2 inhibit platelet aggregation while lower concentrations enhance it (Petrucci et al., 2011). However these effects of PGE2 are variable and depend on the aggregation stimuli (adenosine diphosphate, collagen) and on their concentrations. Enhancement of platelet function is known to be mediated via EP3  receptors leading to increased platelet aggregation (Heptinstall et al., 2008; Jones et al., 2006). Furthermore, EP3 receptor antagonists, such as DG-041, specifically and completely inhibited the synergistic effect of the EP3 receptor agonist sulprostone on platelet aggregation in blood (Smith et al., 2010). In contrast, the characterization of the EP receptors in human platelets, which are involved in the inhibition of aggregation, is somewhat controversial. Two studies demonstrated the antiaggregant effect of butaprost an EP2 receptor agonist (Petrucci et al., 2011; Smith et al., 2010). However, butaprost is not a full EP2 receptor agonist; it is provided either as a methyl ester or a free acid formulation. These two compounds have different affinity profiles for the prostanoid receptors (Abramovitz et al., 2000). In one study (Smith et al., 2010), the free acid formulation of butaprost, a more EP2 selective agonist was used and the authors showed an inhibitory effect of the EP4 receptor on platelet aggregation. In contrast, the EP4 receptor-mediated effect on platelet aggregation seems to be more clear since it was described many times using selective EP4 agonists/antagonists (ONO-AE1329; ONO-AE3-208; GW627368X) while the EP2 ligands were less or not effective (Iyu et al., 2010; Kuriyama et al., 2010; Philipose et al., 2010; Schober et al., 2011). These observation can therefore conclude that PGE2 exerts it antiaggregant effect through the EP4. EP receptor subtypes expressed on platelets and/or those present on monocytes could be also involved in the formation of human monocytes–platelets aggregates with an increase of their adhesion to endothelium. In fact, the formation of human monocytes–platelets aggregates is inhibited by the use of the DP/ EP1/EP2 antagonist (AH6809) (Passacquale et al., 2011).

5. EP receptor subtypes and hematopoiesis Numerous studies have defined hematopoietic regulatory mechanisms mediated by PGE2. However, there are few studies concerning the role and the implication of each EP receptor subtypes during hematopoiesis. Short-term exposure of hematopoietic stem cell (HSC) to PGE2 increases their migration (Frisch et al., 2009; Hoggatt and Pelus, 2010). The activation of EP2/4 receptors by PGE2 decreases macrophage colony stimulating factor (M-CSF) synthesis by bone marrow stromal cells (Besse et al., 1999). This observation suggests that PGE2 may down regulate hematopoiesis processes such as proliferation and differentiation of mononuclear phagocytes. In contrast, more recent studies demonstrated that PGE2 stimulates erythropoesis in cultured erythroblasts developed from human CD34 (þ) hematopoietic progenitors and in peripheral erythrocytes isolated from healthy donors (Rocca et al., 2004). These findings shed a new light on the role mediated by PGE2 to facilitate hematopoietic transplantation. This idea is currently being tested in clinical trials as a potential therapy to enhance HSC engraftment following a transplantation procedure.

6. Prostanoid EP receptor subtypes and eryptosis The term of eryptosis is used to distinguish the death of erythrocytes from apoptosis of nucleated cells. This process can

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be accelerated during anemia leading to the release of hemoglobin in the plasma. PGE2 is known to cause eryptosis (Lang et al., 2006). This effect is abolished by the COX inhibitors such as acetylsalicylic acid and diclophenac which supports a role of PGE2 in eryptosis (Shumilina et al., 2006). Furthermore several EP receptor subtypes seem to be involved in this process since high-doses of misoprostol (EP2,3,4 agonist) used for medical abortion, severely affect erythrocytes and cause an acquired acute hemolytic anemia (Filippini et al., 2007). In contrast, the preoperative use of misoprostol in patients with uterine fibroids reduces intraoperative blood loss in a randomized clinical trial (Kalogiannidis et al., 2011). This discrepancy could be related to the doses used.

7. Involvement of PGE2 receptors in angiogenesis Prostaglandin E2 is also known to inhibit proliferation of vascular SMC. In fact, incubation of SMC from human aorta with PGE2 or PGE1 decreased proliferation and DNA synthesis (Proudfoot et al., 1999; Walton et al., 1999). In addition, migration of SMC derived from pulmonary artery induced by platelet-derived growth factor is inhibited by PGE2 (Goncharova et al., 2003). In these studies the prostanoid receptors involved have not been characterized. However, an opposite effect of PGE2 has been described on endothelial cells proliferation. Recently, the use of EP subtype-selective agonists and antagonists suggested that EP4 receptor mediates the PGE2-induced tube formation of human microvascular endothelial cells through the inhibition of protein kinase A activity (Zhang and Daaka, 2011). This finding supports the use of selective EP4 receptor antagonists as a probable strategy to control vascular cells proliferation and to limit pathologic angiogenesis processes, in particular during tumor development.

8. EP receptor subtypes implicated in the cardiovascular diseases Prostaglandin E2 via EP receptor subtypes has important actions in the cardiovascular system, by controlling vascular tone, cell adhesion and participating in the pathogenesis of vascular diseases such as atherosclerosis, aneurysm and hypertension (Fig. 1). In pathophysiological conditions, these effects are frequently associated with infiltration of leukocytes such as monocytes/macrophages or eosinophils expressing the EP4 receptor which could have pro- or anti- inflammatory roles (Tang et al., 2012; Woodward et al., 2011). In atherosclerosis, the interaction of macrophages with SMC is mediated by PGE2 through the activation of macrophage EP4 receptor (Takayama et al., 2002). This finding is supported by the use of a selective EP4 antagonist (L-161,982) which completely reversed PGE2-mediated suppression of chemokine production in human macrophages co-cultured with SMC. Another recent study has also shown that in hyperlipidemic mice (ldlr  / and EP4 / ) displayed enhanced inflammation in their atherosclerotic plaques as compared with (ldlr  / and EP4þ / þ ) mice (Tang et al., 2011). These results suggest an anti-inflammatory role for EP4 receptor in atherosclerosis. However, EP4 receptors are co-localized with macrophages and associated with enhanced inflammatory markers in atherosclerotic plaques from symptomatic patients in comparison with asymptomatic one (Cipollone et al., 2005). In this later study, EP4 receptors were more abundant in matrix metalloproteinase (MMP)-rich lesions from symptomatic patients, whereas EP2 receptor density was not different. In addition, PGE2 via EP2/EP4 receptors is known to induce MMP expression in human monocyte and endometriotic epithelial and stromal cells (Lee et al., 2011; Shankavaram et al., 2001) This effect could be responsible for plaque instability. Thus, the use of EP4

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Fig. 1. Effect of PGE2 and its receptors on cardiovascular system and atherosclerosis. PG: prostaglandin; IL: interleukin; MMP: matrix metalloprotease; SMC: smooth muscle cells; k: decrease; m: increase.

ligands in treatment of atherosclerosis should consider their global effects in the atheroma plaque, since opposite effects on inflammation and MMP expression have been described. While anti-inflammatory roles mediated by EP4 receptor and PGE2 stimulation have been described in cardiac pathologies and atherosclerosis, it is probably not the case in human arterial aneurysm by Bayston and colleagues (2003) have shown that PGE2 stimulates production of interleukin (IL)-6 and apoptosis of SMC using explants from human abdominal aortic aneurysm (Bayston et al., 2003). This pro-inflammatory effect is mediated through the activation of EP4 receptors present on macrophages. Furthermore, the EP2 receptor expression was detected by immunohistochemistry in endothelial cells of cerebral aneurysm walls of human patients and not in control artery from post-mortem subjects (Aoki et al., 2011). In this study, EP2-induced expression of the chemokine (C–C motif) ligand 2, essential for cerebral aneurysm formation, was observed in primary cultures of human endothelial cells. In addition, recently, a protective effect against the risk of intracranial aneurysm rupture has been observed in patients treated with aspirin (Hasan et al., 2011). These findings support the use of EP2/4 antagonists to prevent patients from cerebral aneurysms progression. The EP4 receptor on eosinophils could control the extravasation of these cells across the endothelium in the pulmonary vasculature. In 2011, Konyia and colleagues have shown that PGE2 and a selective EP4 receptor agonist (ONO-AE1-329) significantly reduced eotaxin-induced eosinophil adhesion in human pulmonary microvascular endothelial cell culture. Under physiological flow conditions, eosinophils treated with the EP4 agonist showed reduced adhesion to endothelial cells (Konya et al., 2011). In this later study, this effect was not observed using selective EP1 , EP2  , and EP3  receptors agonists. These data suggest that EP4 activation could be protective against allergic responses by inhibiting the interaction of eosinophils with the endothelium. Finally, EP receptor subtypes appear to be involved in hypertension. A single nucleotide polymorphism in the gene encoding the PGE2 receptor subtype EP2 is associated to essential hypertension after a genetic association study comparing normotensive and hypertensive men (Sato et al., 2007).

9. Cardiovascular drugs and EP receptor subtypes The pharmacodynamics of several cardiovascular drugs, such as statins and angiotensin II receptor antagonists may pass through the activation of EP receptor signaling pathways (Alvarez et al., 2009; Mezzetti, 2005). In fact, these classes of drugs can stabilize

atherosclerotic plaques through modulation of COX-2/mPGES-1dependent biosynthesis. A study demonstrated that the stabilizing effect of atherosclerotic plaques by simvastatin or irbesartan, is caused by the reduction of inflammation with a decrease of PGE2dependent release (Mezzetti, 2005). In addition, a clinical trial has shown that statins and AT1 receptor blockers significantly reduce the incidence of cardiovascular events in humans through the COX-2 pathway (Naito et al., 2009). For example, angiotensin II can upregulate extracellular MMP-induced expression in macrophages via the AT1/COX-2/PGE2 signal transduction pathway. This effect is inhibited by losartan, an angiotensin II blocker and NS-398, a selective COX-2 inhibitor (Yang et al., 2010). In another study, the selective EP3 antagonist, DG-041, inhibits PGE2-induced platelet aggregation in vitro and ex vivo when the P2Y12 receptor has been blocked by clopidogrel (Singh et al., 2009). This effect indicates that EP3 antagonists potentially have a superior safety profile compared to P2Y12 antagonists and represent a novel class of antiplatelet agents suggesting a cross-talk between P2Y12 and EP3 receptors. Finally, the administration of atorvastatin 80 mg per day prevented the increase of PGE2 levels and MMP-9 activity measured in plasma during acute coronary syndromes (Gomez-Hernandez et al., 2008). Another study performed by the same laboratory showed that treatment with atorvastatin (80 mg per day, for one month) reduced EP1–4 receptor expression in atherosclerotic plaques and EP3 and EP4 expression in peripheral blood mononuclear cells in comparison to non-treated patients (Gomez-Hernandez et al., 2006). Thus, these results could explain, at least in part, some of mechanisms by which statins could modulate the COX-2/mPGES-1 proinflammatory pathway and induce plaque stabilization in humans. The PGE2 synthesis and its receptors emerged as therapeutic targets whose inhibition could reduce incidence of cardiovascular events. 10. Conclusion EP receptors seem to be important for vascular homeostasis and in several physiological and pathological situations. We can conclude that studies on PGE2 in blood and vascular wall have to receive more attention. The EP3 and EP4 receptors appear to be most implicated in comparison to other EP receptors subtypes in the human cardiovascular system. The selectivity of the EP synthetic ligands for the 4 different subtypes has been largely improved in the past 10–20 years, for this reason a few clinical trials are just arising. An EP4 antagonist (BGC20-1531) has been tested unsuccessfully in a PGE2 human model of headache (Antonova et al., 2011) while an EP3 antagonist (DG-041) is a potential inhibitor of human platelet aggregation. This last compound is currently in human clinical trials for the treatment of atherothrombosis (Singh et al., 2010) while a more recent study showed that the EP3 antagonism did not affect atherogenesis (Schober et al., 2011). These receptors are a possible new target for anti-atherosclerotic therapy. However, the specific role of these receptors in atherosclerotic plaque requires further studies. On one hand, the EP4 antagonism could reduce the production of MMP and on the other hand activation of this receptor could inhibit macrophage activation, inhibit the proliferation and the activation of T cells, suppress the release of cytokines and chemokines from macrophages and T cells (Cipollone et al., 2005; Magnone et al., 2009; Takayama et al., 2006). Clinical studies using EP agonists/antagonists as cardiovascular anti-inflammatory drugs, will be probably in large expansion in the near future and will determine the implicated EP receptor(s). References Abdel-Majid, R.M., Marshall, J.S., 2004. Prostaglandin E2 induces degranulationindependent production of vascular endothelial growth factor by human mast cells. J. Immunol. 172, 1227–1236.

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