The Coxib case: Are EP receptors really guilty?

Atherosclerosis 249 (2016) 164e173

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Review article

The Coxib case: Are EP receptors really guilty? Francesca Santilli, Andrea Boccatonda, Giovanni Davì, Francesco Cipollone* Department of Internal Medicine, “G. d’Annunzio” University, Chieti, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 January 2016 Received in revised form 21 March 2016 Accepted 5 April 2016 Available online 6 April 2016

The effects of nonsteroidal anti-inflammatory drugs (NSAIDs) originate from the inhibition of cyclooxygenase (COX), which converts arachidonic acid (AA) to prostaglandin H2 (PGH2). COX consists of two isoforms, called COX-1 and COX-2. Increasing drug selectivity for COX-2 is associated with higher CV risk. Indeed, Coxibs are shown to favour a prothrombotic state, predisposing patients to myocardial infarction (MI) or thrombotic stroke, and to counter the effects of antihypertensive drugs. Indeed, Coxibs affect kidneys, by dysregulating glomerular filtration and salt/water homeostasis. Eventually, recent data associate Coxibs to “amazing” side effects such as acute hepatitis, hyperkalemia and atrial fibrillation or flutter. The circulating concentrations reached in vivo regulate the selectivity towards one of the two COX isozymes. Thus, both tNSAIDs and Coxibs seem to be able to interfere with COX-2 activity, but the interaction depends on the concentration at which each drug may inhibit PGs synthetase in different tissues. PG synthesis inhibition leads to a multiplicity of effects which can be due to the activation of four E-type prostanoid (EP) receptors, which show differential patterns of tissue distribution. Moreover, nitric oxide (NO) bioavailability and its relation with the endogenous nitric oxide synthase (NOS) inhibitors asymmetric dimethylarginine (ADMA) and l-NG-monomethylarginine (l-NMMA), renin release by juxtaglomerular cells and aldosterone pathway, seem to determine NSAID safety also. These changes may powerfully synergize with NSAID-induced prostaglandin (PG) modifications, thus regulating vascular side effects. © 2016 Elsevier Ireland Ltd. All rights reserved.

Keywords: Coxib NSAIDs Prostaglandin EP receptor Platelet

1. The investigated: NSAIDs, Coxibs and current clinical indications Nonsteroidal anti-inflammatory drug (NSAIDs) are among the most commonly employed drugs for symptom alleviation in patients with arthritis and other inflammatory conditions [1]. NSAIDs comprise traditional NSAIDs (tNSAIDs) and NSAIDs selective for cyclooxygenase-2 (Coxibs). The inhibition of cyclooxygenase (COX) is the main pharmacological mechanism of NSAIDs, by converting arachidonic acid (AA) to prostaglandin H2 (PGH2). PGH2 is in turn metabolized into several prostanoids, such as thromboxane A2 (TxA2), prostaglandin D2 (PGD2), prostacyclin (PGI2), prostaglandin E2 (PGE2) and prostaglandin F2 (PGF2) [2]. COX consists of different isoforms, that are differently regulated. Indeed, COX-1 is constitutively expressed, and catalyzes the production of prostaglandins (PGs) involved in several physiological

* Corresponding author. Department of Medicine and Aging, European Center of Excellence on Atherosclerosis, Hypertension and Dyslipidemia, “G. d’Annuzio” University, 66100 Chieti, Italy. E-mail address: [email protected] (F. Cipollone). http://dx.doi.org/10.1016/j.atherosclerosis.2016.04.004 0021-9150/© 2016 Elsevier Ireland Ltd. All rights reserved.

functions, such as renal function, gastrointestinal tract mucosal protection and TxA2 biosynthesis in platelets [3]. On the other hand, COX-2 activation can be induced by cytokines and other inflammatory mediators in several tissues, thereby regulating inflammation, fever and pain [3]. NSAIDs are grouped depending on their COX-2 selectivity, which is defined by the drug concentration that inhibits by 50% COX activity or COX-1 and COX-2 in vitro, called as ratio of IC50 [4]. Interestingly, several NSAIDs inhibit COX-2 to a greater extent than COX-1 in vitro, some of them displaying comparable COX-2 selectivity to some Coxibs, thus proving that COX-2 selectivity is a continuous variable [5]. Drug dose and the concentrations reached in vivo limit the degree of inhibition and the selectivity towards one of the two COX isozymes [5]. 2. The crime scene: Coxibs and their detrimental effects 2.1. NSAIDs and cardiovascular events Warnings about cardiovascular (CV) safety of NSAID led to withdrawal of two drugs (rofecoxib and valdecoxib) and in an

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overall reduction in their use [3]. The Food and Drug Administration (FDA) stated that CV risk is associated with all NSAIDs, with the exception of aspirin [6]. The American Heart Association (AHA), in its recommendations for the management of pain, suggested acetaminophen, tramadol, aspirin, and opioids as first line agents, followed by NSAIDs, with COXIBs considered lastly [7]. Moreover, the European Medicines Agency's (EMA) Committee for Medicinal Products for Human Use recommended that “COX-2 inhibitors must not be used in patients with established ischemic heart disease and/or cerebrovascular disease” [8]. The VIGOR study, comparing side effects of rofecoxib and naproxen in rheumatoid arthritis (RA) patients, reported an increased incidence of thrombotic accidents in patients receiving rofecoxib [9]. These observations were confirmed in the APPROVE study [10], with a two-fold increase in MI risk reported in patients treated with rofecoxib versus placebo, thus resulting in the removal of the drug from the market [10]. Similarly, in the Adenoma Prevention with Celecoxib trial, subjects receiving celecoxib were characterized by higher risk of vascular events when compared to placebo [11]. The Alzheimer's Disease Anti-inflammatory Prevention Trial [12] compared for the first time a tNSAIDs (naproxen) and a COX-2 selective agent (celecoxib) in a prospective outcome trial. Importantly, naproxen was not more harmless than celecoxib, showing a trend towards a higher incidence of CV events [12]. The Multinational Etoricoxib and Diclofenac Arthritis Long-term (MEDAL) trial showed that the rate of thrombotic accidents with etoricoxib was similar to that on diclofenac in osteoarthritis and RA patients [13]; Subsequent observational studies have also confirmed that no difference exists in CV outcomes comparing the two drugs [14]. The overall CV risk seems to be increased for both the drugs, and there are no clear data about major differences between NSAIDs and Coxibs (with the exception of naproxen) [8,15]. Indeed, naproxen showed the higher CV safety profile, possibly due to its unique pharmacodynamics of a twice-daily high-dose regimen, thus reflecting an aspirin-like phenotype [16]. High-dose diclofenac and ibuprofen are similar to Coxibs regarding cardiovascular risk, whereas high-dose naproxen is related to lower risk than other NSAIDs [15]. A direct comparison of celecoxib with naproxen did not demonstrate different CV risk, whereas both rofecoxib and etoricoxib were characterized by a higher risk [15]. Furthermore, a recent cohort study has confirmed NSAID correlation with acute coronary syndrome, showing higher CV risk for alkanones, followed by propionoicos as ibuprofen, thirdly arylacetic and finally for the Coxibs [17]. Differential CV risks seem to be related to baseline CV risk, drug dose and regimen [18,19]. No significant differences exist in the increased risk of major CV events associated with celecoxib and rofecoxib, despite a 20-fold diversity in COX-2 selectivity between the two, consistent with the COX-2 dependence of PGI2-mediated thromboresistance of the vessel wall [16]. Since the inhibition of platelet COX-1 activity and the inhibition of platelet activation in vivo are characterized by a nonlinear relationship, neither Coxibs nor some tNSAIDs are able to sufficiently inhibit platelet COX-1 activity, regardless their COX-2 selectivity [16], to attain an adequate inhibition of platelet activation. In particular, while aspirin and tNSAIDs inhibit both TxA2 and PGI2, Coxibs do not affect TxA2 synthesis, due to the lack of COX-2 in platelets, thus favouring a prothrombotic state [20]. Indeed, Coxibs may expose patients to myocardial infarction (MI) or thrombotic stroke risk, by inhibiting COX-2edependent formation of PGI2 [20]. NSAIDs may increase MI risk particularly at high doses and in patients with a history of a previous attack. Rofecoxib is the most toxic of all tested NSAIDs, while naproxen and etodolac may be the safest [21e23]. The APPROVE trial has shown that the CV risk is greater with NSAIDs long term use (18 months) [24]. In contrast, a

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European cohort study has reported that a short term use of NSAIDs may also increase the incidence of MI and mortality, especially in subjects with a previous MI [25]. A dose-related death due to reinfarction has been reported in the Danish population who used NSAIDs [26]. All doses of celecoxib and rofecoxib, but only high doses of tNSAIDs have been related to increased risk of death in subjects with previous MI [26,27]. Furthermore, although Coxibs have been proposed for stroke treatment, recent findings have reported a paradoxical increased risk of cerebrovascular disease following NSAID long-term use [28,29]. Some tNSAIDs such as diclofenac and aceclofenac have been shown to determine an increased risk of ischemic stroke, while both naproxen and ibuprofen have not been correlated [28]. Several factors such as patient baseline CV risk, treatment dose and duration, but not aspirin use, seem to regulate this risk [28]. Furthermore, previous administration of COX-2 inhibitors has been related to higher 30-day mortality after ischemic stroke, but not with hemorrhagic stroke [29]. Long-term treatment with rofecoxib has been shown to increase circulating levels of 20hydroxyeicosatetraenoic acid (20-HETE) [30], a molecule exerting detrimental effects in the brain, as proved by the fact that its blockade may be cerebroprotective against ischemic stroke and subarachnoid hemorrhage [31]. As the literature is highly controversial, no clear conclusion can be made regarding the possibility of an increased risk of stroke in NSAIDs users [32,33]. 2.2. Association with aspirin (ASA) or oral anticoagulants An increased risk of bleeding and, almost paradoxically, of thrombotic events have been correlated with NSAIDs administration in patients on chronic treatment with antithrombotic drugs. Concomitant administration of NSAIDs has been related to higher risk of bleeding in patients with a previous MI [34]. This association is substantial for every antithrombotic drug, and for both COX-2 inhibitors (rofecoxib and celecoxib) or tNSAIDs (ibuprofen and diclofenac) use [34]. A short term treatment has been also related to higher risk of bleeding in comparison to no NSAID use [34]. The Coxibs and traditional NSAID Trialists (CNT) analyses did not provide any evidence that the effect of Coxib therapy on the risk of major vascular events is attenuated by concomitant aspirin use at baseline [15]. However, a protective role of low-dose aspirin may be possible, as proved by the fact that COX-1 knockdown in animal model attenuates the prothrombotic effect due to COX-2 inhibition [35]. NSAIDs may counteract the antiplatelet effect of low-dose aspirin, by acting as a competing inhibitor with acetylsalicylic acid for a common docking site (arginine 120) in the COX-1 channel, thereby increasing thrombotic risk [16]. Consistently, concomitant antithrombotic and NSAID therapy in patients with a previous MI has been correlated with an increased CV risk, independently from ongoing antithrombotic treatment, type and duration of NSAID therapy, probably due to a reduction of the thrombotic protection provided by antiplatelet agents and oral anticoagulants [34]. Both dual antiplatelet (aspirin and clopidogrel) and aspirin alone have been related to increased CV risk in patients taking NSAIDs [34]. In contrast, a recent consensus paper exonerates Coxibs from the “accusation”of increasing rate of thrombotic events [8]. The proofs to support this hypothesis include the evidence that Coxibs have been shown to not affect platelet COX-1 activity and platelet aggregation or bleeding time [36,37]. Indeed, Coxibs may interfere with aspirin, depending on their activity against COX-1 [38]. In in vitro studies, Coxibs characterized by a low affinity for COX-1 and a high COX-2 selectivity have been shown to poorly counteract aspirin platelet COX-1 inhibition [38]. Thus, highly selective Coxibs,

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being weak COX-1 inhibitors, display a low ability to counteract the antiplatelet effect of low-dose aspirin both in healthy subjects and CV patients [8,38]. 2.3. NSAIDs and hypertension Coxibs have also been shown to increase blood pressure (BP), by several mechanisms including retention of salt and fluid, and to counteract the effects of antihypertensive medications [32]. NSAIDmediated elevation in blood pressure has been shown to significantly rise the incidence of stroke and coronary heart disease [39]. The mechanism for the increase in BP is most likely due to the impact of NSAIDs on vasoactive endothelium-derived factors, particularly via the inhibition of PG synthesis, important for the regulation of vascular tone and sodium excretion [40]. 2.4. Renal impairment Recent studies have highlighted the role of renal impairment as one of the major determinants of Coxib-related hypertension. In fact, Coxibs exert well-documented effects on the kidney, by dysregulating glomerular filtration and salt/water homeostasis [41]. Sodium retention seems to be mediated by the inhibition of COX-2, whereas GFR depression is related to COX-1 inhibition [32]. NSAIDs are associated with GFR reductions and thus acute renal failure [42], by inhibiting PG production and consequently reducing the blood flow to the kidneys and/or by induction of interstitial nephritis [32]. Patients with a history of hypertension, heart failure, or diabetes have higher chances to develop acute renal failure [42] while taking NSAIDs [32]. Moreover, NSAIDs cause chronic renal failure (CRF) secondary to interstitial nephritis or renal papillary necrosis [32]. Concomitant administration of aminoglycosides, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers and diuretics has been related to the risk of worsening renal failure, especially in elderly patients [32]. While regular doses of NSAIDs did not significantly elevate the risk of CRF, high doses significantly increased CRF risk [43]. NSAIDs with long half-life demonstrate a greater CRF risk, due to their sustained PG inhibition [44]. Finally, recent studies have related Coxibs to “amazing” side effects and negative outcomes such as acute hepatitis [45], hyperkalemia [46] and atrial fibrillation or flutter [47]. More and more studies are relating many favorable or detrimental Coxib effects to specific PGs and E-type prostanoid (EP) receptor activation pathways [31,48e51]. PGE2 (as well as the other PGs) is an autacoid that activates membrane receptors close to its site of synthesis, and its complex final effect depends on the activation of 4 different EP receptors [48]. EP-related cellular pathways have been investigated in animal studies based on EP subtypedeficient mice or using several chemical molecules highly selective to each EP subtype [50] (Table 1). Thus, EP receptors may finally solve the “Coxib case”, but are they really guilty? 3. The NSAID case: proofs from EP receptor studies 3.1. Role of EP receptors as determinants of NSAIDs CV outcomes One potential explanation for NSAIDs increased CV outcomes may be the presence of off-target effects of COX-2 inhibition [48]. In particular, all the proofs converge on COX-2-dependent PGs production, with the complicity of EP receptors [40,52]. The so-called “COX-2 hypothesis” suggested that the detrimental cardiovascular effects of COX-2 inhibitors are due to their COX-2 selectivity, thus leading to a greater prostacyclin/thromboxane imbalance [40]. Elevated CV risks seem to be strictly related to the degree of direct

pharmacologic COX-2 inhibition of any NSAIDdboth selective and traditional [53,54]. PGs, in particular PGE2, are responsible for a variety of biological effects which can be mediated by different EP receptors, characterized by a differential tissue distribution (Figs. 1 and 2) [52]. EP1 mRNA seems to be ubiquitous in tissues, while EP3 receptors are highly expressed in pancreatic and adipose tissues, kidney and vena cava [52]. Gastrointestinal mucosa, uterus, hematopoietic tissues and skin are characterized by the highest level of EP4 mRNA, whereas EP2 receptor are preferably expressed in the airways, ovary, bone marrow and olfactory epithelium [52]. EP1 receptor favours the release of intracellular Ca2þ and phosphatidylinositolbisphosphate hydrolysis, whereas the EP2 and EP4 receptors are bound to Gs protein and subsequential intracellular cAMP synthesis [55]. The EP3 receptor consists of several splice isoforms, differently linked to distinct signaling molecules, such as Gi and subsequent inhibition of cAMP formation, Gs and stimulation of cAMP formation, and Gq with an increased release of intracellular Ca2þ [55]. 3.2. The double-edged sword of NSAID effects on platelets NSAIDs have been related to increased bleeding risk and, almost paradoxically, high incidence of thrombotic accidents in patients receiving antithrombotic therapy [34]. Indeed, PGE2 can both inhibit and potentiate platelet function, the resultant effect depending on the concentration of the PGs and the balance between its effects on receptors [56]. PGE2, through its action at EP3 receptor, is able to overcome the inhibitory effects of antiplatelet agents which operate via Gs [56]. Indeed, PGE2 has been implicated in the initiation of arterial thrombosis through its action at EP3 receptor, and antagonism of the EP3 receptor has been proposed as a novel approach to antiplatelet therapy [56]. Moreover, the activation of EP4 receptor pathway on platelets has been shown to block platelet aggregation and in vitro thrombus formation [52]. EP4 receptor activation leads to reduced intracellular Ca2þ mobilization and phosphorylation of vasodilatorstimulated phosphoprotein, inhibition of glycoprotein IIb/IIIa activation, and downregulation of P-selectin, thereby exerting an antiaggregatory effect in stimulated platelets [52]. These findings may explain the dual controversial effect of PGE2 on platelet aggregation, that is potentiation of platelet aggregation at lower concentrations, while inhibition at higher concentrations [52]. Thus, the pro-aggregatory effect of PGE2 in platelets seems to be mediated by EP3 receptors, while EP4 activation may be related to its antiaggregatory properties (Fig. 3) [52]. 3.3. The double-edged sword of NSAID effects on the myocardium and vascular wall In a recent work of Nanhwan et al., ticagrelor has been shown to reduce MI size, but this effect is abrogated by COX2 inhibition with either a specific inhibitor or high dose aspirin [57]. Ticagrelor protective effect is dependent on adenosine-receptor activation with downstream upregulation of eNOS, cPLA2, and COX2 enzyme activation [57]. Ticagrelor upregulates COX2 mRNA levels and COX2 activity, resulting in increased levels of prostacyclin, thus explaining the association between higher doses of aspirin and decrease in the relative efficacy of ticagrelor [57]. Moreover, ticagrelor increases cPLA2 activity, the major enzyme that supplies AA to the COX2 for the production of protective PGs, and this effect is blocked by adenosine-receptor inhibitors and aspirin [57]. Furthermore, the safety of a Coxib, such as celecoxib, in angina patients taking dual anti-platelet therapy has been investigated in two trials (COREA-TAXUS trial and mini-COREA) [58,59]. The use of celecoxib for 3 months in these patients has been shown to be

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Table 1 Studies evaluating the different EP receptors pathways.

EP1 Kassem et al., 2014 [62] Harding et al., 2011 [74] EP2 Kassem et al., 2014 [62] Yang et al., 2012 [55] Yang et al., 2012 [55] Yang et al., 2012 [55] Kawada et al., 2012 [2] EP3 Glenn et al., 2012 [56]

Clinical implication

Study setting

Findings

Myocardial infarction Myocardial infarction

Cultures of neonatal rat ventricular fibroblasts PGE2 increases PAI-1 expression by dose dependent mechanisms, most likely related to EP1 receptor activation. Cultures of neonatal rat ventricular fibroblasts PGE2 treatment induced cell proliferation through EP1 receptors, by stimulating cyclin D together with both p42/44 MAP kinase pathway and the PI3K pathway activation.

Myocardial Cultures of neonatal rat ventricular fibroblasts EP2 agonist can reduce fibroblast accumulation, thus suggesting an inhibitory effect on infarction migration exerted by PGE2 through EP2 pathway. Blood Pressure EP2/ mice EP2 receptor can regulate vascular tone, by stimulating a Gs coupled pathway and increasing intracellular cAMP, thus leading to vasorelaxation. Blood Pressure EP2/ mice EP2 receptor may control PGE2-mediated natriuretic effect on kidneys. Aldosterone Cultures of cells from bovine adrenal glands Metabolism Blood Pressure EP2/ mice

PGE2-mediated aldosterone release seems to be related to cAMP-mobilization through EP2 receptor activation. PGE2 and PGI2 may stimulate EP2 receptors in JG cells, thus favouring renin granules excretion through PKA-dependent pathway.

Platelet activation

EP receptor antagonists on platelet-rich plasma PGE2, through its action at EP3 receptor, can overcome the inhibitory effects of obtained from healthy volunteers antiplatelet agents which operate via Gs. Indeed, PGE2 seems to be implicated in the initiation of arterial thrombosis through its action at EP3 receptor. Zhang et al., Atherosclerotic Vascular remodeling induced by wire injury in The blockage of EP3 receptor may result in loss of vascular smooth muscle cells 2013 [66] plaque femoral arteries of mice polarization and migration. EP3 knockout mice display a reduced rate of restenosis after wire injury. Kassem et al., Myocardial Cultures of neonatal rat ventricular fibroblasts PGE2 increases PAI-1 expression by dose dependent mechanisms, most likely related to 2014 [62] infarction EP3 receptor activation. Harding et al., Myocardial Cultures of neonatal rat ventricular fibroblasts PGE2 treatment induced cell proliferation through EP3 receptors, by stimulating cyclin D 2011 [74] infarction together with both p42/44 MAP kinase pathway and the PI3K pathway activation. Yang et al., Blood Pressure EP3 / mice EP3 receptor activation reduces plasma vasopressin-mediated cAMP synthesis. 2012 [55] Kawada et al., Blood Pressure EP3 / mice EP3 receptor seems to be necessary for a complete inhibition of vasopressin V2 receptor 2012 [2] signaling. Blood Pressure immature rabbit kidney PGE2 is able to counteract vasopressin effects through EP3 receptor, by blocking PKA Bonilla-Felix et al., 2004 activation. [78] EP4 Konya et al., Platelet EP4/ mice The activation of EP4 receptor pathway on platelets blocks platelet aggregation and 2013 [52] activation thrombus formation. EP4 receptor decreases the synthesis of inflammatory cytokines, thus reducing macrophages infiltration at sites of myocardial ischemia/reperfusion. Xiao et al., Myocardial EP4/ mice EP4 deficient mice are characterized by increased myocardial infarction size in 2004 [60] infarction comparison to wild-type mice. Kassem et al., Myocardial Cultures of neonatal rat ventricular fibroblasts PGE2 may decrease MMP-14 tissue levels by the activation of EP4 pathway. EP4 agonists 2014 [62] infarction can reduce fibroblast accumulation. Konya et al., Stroke Mouse model of cerebral ischemia EP4 activation decreases infarct size and prevents long-term motor deficits, thus 2013 [52] supporting a beneficial role for COX metabolites. Atherosclerotic Plaques from symptomatic and asymptomatic EP4 receptors are more abundant in MMP-rich symptomatic lesions and are associated Cipollone patients undergoing carotid endarterectomy with enhanced inflammatory reaction and thus plaque destabilization. et al., 2005 plaque [70] Yang et al., Blood Pressure EP4 / mice EP4 receptors can regulate vascular tone, by stimulating a Gs coupled pathway and 2012 [55] increasing intracellular cAMP, thus leading to vasorelaxation. Blood Pressure Aortic rings from mice EP4 receptors have been shown to modulate PGE2-induced vasodilation in isolated aortic Hristovska et al., 2007 rings in vitro, depending on eNOS activation as a result of dephosphorylation at Thr495. [76] Kawada et al., Blood Pressure EP4 / mice PGE2 and PGI2 may stimulate EP4 receptors in JG cells, thus favouring renin granules excretion through PKA-dependent pathway. 2012 [2] Li et al., 2009 Kidney function V2 receptor knock-out mice EP4 receptor stimulation regulates polyuria degree, by enhancing renal aquaporin 2 [79] protein expression through cAMP/PKA-mediated pathway. Yang et al., Aldosterone Cultures of cells from bovine adrenal glands The main pathway involved in PGE2-mediated aldosterone release seems to be related to 2012 [55] metabolism cAMP-mobilization through EP4 receptor activation. Abbreviations: PGE2, prostaglandin E2; PAI-1, plasminogen activator inhibitor-1; EP, prostaglandin E receptor; PI3K, phosphoinositide 3-kinase; Camp, cyclic adenosine monophosphate; PKA, protein kinase A; JG, juxtaglomerular; MMP, matrix metalloproteinase; COX, cyclooxygenase; eNOS, endothelial nitric oxide synthase.

useful to reduce in-stent late loss of drug-eluting stent (DES) and probably target-lesion revascularization, however it has not been associated with reduced major vascular events, due to an increase in thrombotic hard endpoints [58]. These findings have raised a warning about the concomitant use of Celecoxib in patients taking dual anti-platelet therapy, particularly due to the elevated thrombotic risk, and no recommendation has been derived about the use of Coxibs in patients undergoing PCI with DES to reduce re-stenosis and negative cardiac events [58].

PGE2 has been demonstrated to be protective in cardiac ischemia/reperfusion animal model, by activating EP4 pathway [60]. EP4 deficient mice are characterized by increased MI size in comparison to wild-type mice [60]. Mechanisms through which EP4 exerts cardioprotective effects have not been completely understood, but they may be related to EP4 receptor-mediated elevation of intracellular cAMP levels, thereby eliciting vasodilator and/or platelet inhibitory responses [61]. In addition, EP4 receptor seems to decrease the synthesis of inflammatory

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Fig. 1. EP1 receptor is expressed in all tissues, while adipose tissues, pancreas, kidney and vena cava display the higher levels of EP3 receptor. Gastrointestinal tract, uterus, hematopoietic tissues and skin are characterized by EP4 expression, whereas EP2 receptor mRNA has been found in airways, ovary, bone marrow and olfactory epithelium.

Fig. 2. NSAIDs can exert a variety of effects due to different tissue distribution of EP receptor subtypes. NSAIDs are shown to affect platelet activation, vascular tone, natriuresis, and to increase cardiac and cerebral infarct size after acute coronary syndrome and ischemic stroke.

cytokines, such as TNF-a, IL-1b, IL-6, and MCP-1, thus reducing macrophages infiltration at sites of myocardial ischemia/reperfusion [52]. PGE2 can also affect membrane metalloproteinase (MMP) synthesis and activity, through the activation of EP receptors, thus impacting on cardiac fibroblast migration [62]. PGE2 may decrease MMP-14 tissue levels by the activation of EP4 pathway [62]. MMP14 operates as activator of pro MMP-2, and lower levels of MMP-14 after PGE2 administration are related to decreased MMP-2 activity [62]. Moreover, increased expression of MMP-14 is responsible for negative myocardial remodeling after MI, increasing overall collagen content through the activation of TGFb and Smad2 signaling pathways [62]. In addition, the plasminogen-plasmin

system may increase the expression of MMPs, whereas plasminogen activator inhibitor-1 (PAI-1) may inhibit this mechanism [62]. COX-2 inhibitors are also correlated with an increased incidence of cerebrovascular events [33,63]. Rofecoxib and valdecoxib have been shown to be responsible for an increased risk of stroke, a finding that has contributed to their withdrawal from the market [64]. PG role in neurological disorders is still controversial [28,52]. In animal model of cerebral ischemia, the activation of EP4 receptor through a specific agonist decreased infarct size and prevented long-term motor deficits, thus supporting a beneficial role for COX metabolites [52]. In the brain EP4 receptors are present on neuronal cells and their expression may be increased in endothelial cells after ischemia, thus suggesting a EP4-mediated cerebroprotective

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Fig. 3. NSAIDs are shown to exert dual effect on platelet aggregation depending on PGE2 concentrations. Decreased PGE2 levels lead to platelet activation reducing cAMP concentrations through Gi-coupled EP3 receptor, while higher PGE2 levels are shown to increase cAMP concentrations through Gs-coupled EP4 receptor, thus exerting antiaggregatory properties. Abbreviations: NSAIDs, nonsteroidal anti-inflammatory drugs; AA, arachidonic acid; PGG2, prostaglandin G2; PGH2, prostaglandin H2; PGE2, prostaglandin E2; EP, prostaglandin E receptor; AC, adenylate cyclase; cAMP, cyclic adenosine monophosphate; AMPK, AMP-activated protein kinase; eNOS, endothelial nitric oxide synthase; PKA, protein kinase A; CREB, cAMP responsive element-binding protein; Epac, exchange protein directly activated by cAMP; ICER, inducible cAMP early repressor.

effect on these cells [52]. Furthermore, neurological deficits and decreased cerebrovascular reperfusion have been demonstrated in model of neuronal or endothelial EP4 deletion [52]. Otherwise, human atherosclerotic plaques are characterized by an increased COX-2 expression [48,65]. In the work of Zhang et al., genetically silenced COX-2 expression explained the beneficial effects on neointima formation, as reflected by lower luminal narrowing and reduction in intima-to-media ratio after vascular injury in COX-2 knockout mice [48,66]. Thus, Coxibs may be a new treatment for atherosclerotic lesion stabilization, and in turn for acute ischemic disease prevention. Endothelial cells may release PGI2, thus inhibiting platelet aggregation and cholesterol accumulation [67], whereas a variety of atherogenic eicosanoids, such as PGE2, is produced by macrophages [68]. Macrophage-mediated activation of the inducible COX/PGES can increase PGE2 synthesis, thus enhancing MMP production in atherosclerotic plaques [65]. Increased MMP-2 and MMP-9 activity has been shown in vulnerable regions of human carotid plaques, together with macrophage accumulation [69]. EP4 receptors are more abundant in MMP-rich symptomatic lesions and are associated with enhanced inflammatory reaction and thus plaque destabilization [70]. These findings are consistent with pharmacological studies, in which MMP induction by PGE2 in vitro is inhibited by the EP4 antagonist, but not by EP2 antagonist [70]. On the other hand, atherosclerotic plaques show a low presence of EP2, without expressing EP1 and EP3 receptors [70]. The blockage of EP3 receptor, in particular EP3a and EP3b subtypes, may result in loss of vascular smooth muscle cells (VSMCs) polarization and migration [66]. In vivo studies show that EP3 knockout mice have a reduced rate of restenosis after wire injury, a condition that was reverted by lentiviral re-expression of EP3a and EP3b [66]. Oxidized low-density lipoproteins (OxLDL) have been shown to decrease both the mRNA and protein levels of EP3 receptor, depending on dose reached in vivo [71]. Moreover, oxLDL can decrease the nuclear factor-kB (NF-kB)-dependent transcription of

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the EP3 gene by activating peroxisome proliferator-activated receptor-g (PPAR-g) [71]. PGE2 and IL-1b may synergize to enhance a phenotypical transition of smooth muscle cells, by cAMP-protein kinase A [72]. This mechanism of PLA2 activation is related to a different regulation exerted by the EP3 Gi-coupled PGE2 receptors toward adenylyl cyclase activated by the EP4 Gs-linked PGE2 receptor [72]. Thus, oxLDL may down-regulate EP3 expression, thereby impairing EP3-mediated anti-inflammatory properties. PGE2-mediated activation of either EP3 or EP4 may either stimulate or inhibit platelets in atherosclerotic plaque, respectively [73]. However, EP3-antagonists neither reduced platelet aggregation, GPIIb/IIIa expression, dense and alpha granule secretion in blood nor decreased thrombus formation under arterial flow in atherosclerotic plaques [73]. Therefore, EP4-antagonist pretreatment did not increased plaque-induced platelet activation, thereby suggesting that PGE2 does not stimulate the EP4-receptor in the atherosclerotic plaque [73]. Further studies are needed to disclose PGE2 role in human atherosclerotic plaques, and how it may modulate atherothrombosis following plaque rupture. Finally, Coxibs may also appear beneficial on myocardial fibrosis after MI, when considering EP1/EP3 receptor-mediated pathogenesis. PGE2 increases PAI-1 expression by dose dependent mechanisms, most likely related to EP1/EP3 receptor activation [62]. Findings from an in vitro study showed that cardiac fibroblasts expressed all EP receptors, and that PGE2 treatment induced cell proliferation through EP1/EP3 receptors, by stimulating cyclin D together with both p42/44 MAP kinase pathway and the PI3 kinase pathway activation [74]. Moreover, rofecoxib has been related to reduced fibroblast proliferation in a rat model of MI [75]. Indeed, both EP2 agonist and EP4 agonist can reduce fibroblast accumulation, thus suggesting an inhibitory effect on migration exerted by PGE2 through EP2 and EP4 pathways [62]. 3.4. EP receptors, NSAIDs and high blood pressure Several studies have shown NSAIDs to affect fluid balance and blood pressure, through the modulation of resistant vessel tone, renin release, vasopressin signaling, and tubular sodium reabsorption and filtration in kidneys [2]. TxA2 can induce vasoconstriction by activating thromboxane prostanoid receptors on vascular smooth muscle cells of resistant vessels, whereas PGI2 produced by endothelial cells may counteract thromboxane-mediated constriction [2]. EP1 receptor has been shown to induce smooth muscle cell constriction, with a pro-hypertensive role [55]. In some vascular beds, concomitant activation of EP1 and EP3 receptors leads to PGE2-mediated constriction through phosphatidylinositol pathway [55]. On the contrary, a direct activation of EP1 receptor on tubular cells may induce a natriuretic effect, thereby displaying antihypertensive property [55]. Moreover, as shown in animal studies, EP3 receptor activation seems to reduce plasma vasopressinmediated cAMP synthesis, although EP3 receptor deficit in animal model did not influence renal function and/or blood pressure [55]. Conversely, EP2/EP4 receptors can regulate vascular tone, by stimulating a Gs coupled pathway and increasing intracellular cAMP, thus leading to vasorelaxation [55]. EP2 receptor may also control PGE2-mediated natriuretic effect on kidneys [55]. EP4 receptors have been shown to modulate PGE2-induced vasodilation in isolated aortic rings in vitro, depending on eNOS activation as a result of dephosphorylation at Thr495 [76]. NO is responsible for an increased vasorelaxation in aortic rings, due to cGMP accumulation, in a guanylyl cyclase-dependent manner [55,76]. In a recent study, Kawada et al. have hypothesized that EP2 receptor natriuretic effect may be due to tubular sodium transport [2]. The amiloridesensitive epithelial sodium channel [77] and sodium-potassium

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adenosine triphosphatase are activated by the cAMP/protein kinase A (PKA) pathway, thereby favouring the mobilization of these proteins to cell membrane in collecting duct [2]. Thus, NSAIDS may counteract the natriuretic effect of EP2 receptor, by blocking ENaC activity. 3.5. EP receptors as determinants of blood flow and glomerular filtration rate in patient receiving NSAIDs PGE2 has been linked to renin-mediated hypertension. Coxibs can decrease renin excretion following low-salt diet, angiotensinconverting enzyme inhibition, angiotensin AT1 receptor antagonism, and reduced renal perfusion pressure [55]. COX-2 pathway may increase renin release from juxtaglomerular (JG) granular cells in macula densa [2]. Prostanoids generated by COX-2 metabolism, such as PGE2 and PGI2, may stimulate EP2 and EP4 receptors in JG cells, thus favouring renin granules excretion through PKAdependent pathway [2]. In addition, NSAIDs may increase vasopressin renal effects. Since PGE2 is able to counteract vasopressin effects through EP3 receptor, by blocking PKA activation [78]. Conversely, EP4 receptor stimulation in V2 receptor knock-out mice has been shown to regulate polyuria degree, by enhancing renal aquaporin 2 protein expression through cAMP/PKA-mediated pathway [79]. Thus, EP3 receptor seems to be necessary for a complete inhibition of vasopressin V2 receptor signaling, whereas EP4 receptor may increase free-water reabsorption, irrespective of vasopressin and V2 receptor activity [2]. 3.6. ADMA, NO bioavailability, aldosterone metabolism: other explanations for adverse NSAID effects? Reactive oxygen species derived from COX metabolism may affect the biologic activities of endothelium-derived NO, by favouring lipid peroxidation, that in turn can down-regulate NO synthesis and bioavailability [80]. In vivo studies have shown that traditional COX inhibitors, such as indomethacin and aspirin, may enhance endothelium-dependent dilation [81,82]. A recent study revealed alterations in the kidney of genes regulating the synthesis and metabolism of the endogenous NOS inhibitors asymmetrical dimethylarginine (ADMA) and monomethyl-l-arginine (l-NMMA), in COX-2edeficient mice [41]. Given the protective role of NO released from endothelial cells, inhibition of COX-2 in the kidney can regulate vascular side effects throughout the vasculature [41]. Protein arginine methyltransferases (Prmt) 1 enzyme, which methylates arginine residues in proteins leading to the release of mature ADMA and l-NMMA after proteolysis, was increased by COX-2 deletion, whereas both Agxt237 and Ddah131, enzymes that metabolize methylarginines, were decreased in COX-2/ renal medulla [41]. Plasma increases of ADMA and l-NMMA shown in COX-2/ mice, were associated with vascular dysfunction, characterized by reduced acetylcholineinduced vasodilator responses in endothelium segments of aorta [83]. These results are in agreement with a clinical study, in which ADMA levels increased in healthy male volunteers treated with standard doses of celecoxib or naproxen [41]. Finally, increased plasma aldosterone concentrations also seem to play a role in determining NSAID-related CV risk. In vitro studies have shown that PGE2 induce aldosterone release from adrenal zona glomerulosa cells [55]. The main pathway involved in PGE2mediated aldosterone release seems to be related to cAMPmobilization through EP2 or EP4 receptor activation [55]. Several tNSAIDs exert significant inhibitory effects on aldosterone metabolism, with a consequent potential increase in serum aldosterone concentrations [84]. tNSAIDs have been shown in vitro to inhibit aldosterone 18 beta-glucuronidation, especially diclofenac and

naproxen [84]. Therefore, in a recent research of Crilly et al., chronic celecoxib use, compared with diclofenac, was associated with a greater degree of aldosterone 18 beta-glucuronidation inhibition and with a higher augmentation index (AIX%) and lower reflected wave transit time (RWTT), both indices of arterial dysfunction. Thus, chronic celecoxib use seems to be correlated with a higher incidence of cardiovascular events and endothelial dysfunction degree [84]. 4. The judgement: are NSAIDS guilty or innocent? tNSAIDs and Coxibs seem to be both guilty with regard to the increase in CV risk. A quite consistent epidemiological evidence is supported by the clinical pharmacology, since studies about COX isoenzyme selectivity have established COX-2 selectivity as a continuous variable [14e16,85]. Thus, a dichotomous definition of selective and nonselective inhibitors does not appear justified, given an appreciable overlap in COX-2 selectivity [16]. Indeed, both tNSAIDs and Coxibs seem to be able to interfere with COX-2 activity, but the interaction depends on the concentration at which each drug may inhibit PGs synthetase in different tissues [16]. The standardization of COX-2 inhibition biomarkers can stimulate dose-findings studies leading to a better definition of the optimal dose for any single drug, and thus fostering the risk stratification of patients to whom tNSAIDs and/or Coxibs should be prescribed [5]. Indeed, the analysis of PGE2 levels in lipopolysaccharide-stimulated whole blood may be a useful tool to predict drug efficacy and safety, by reflecting COX-2 inhibition degree [86]. Different efficacy and tolerability of NSAIDs have been observed between individuals, resulting from genetic factors that affect the pharmacokinetics and pharmacodynamics of NSAIDs [87]. Indeed, a number of single-nucleotide polymorphisms in the genetic sequence of CYP2C9 were shown to alter the metabolism of some NSAIDs [88]. Thus, ongoing studies designed to characterized genetic biomarkers may identify individuals at accelerated risk of developing CVD [89]. New interesting findings on EP receptor pathways reinforce the idea that PG synthesis inhibition does not lead toward a unique adverse or positive result, but it will be important to understand which type of EP receptor and which agonist concentration may exert the beneficial or detrimental effects of COX inhibition on different tissues [48,52,55,60,61]. Further understanding of EP receptor functions may be helpful to develop novel strategies to reduce NSAID adverse effects and interactions with other drugs [2]. Unmet needs include the assessment of NSAID safety for patients in monotherapy with different drugs, for patients with dual therapy (e.g. dual antiplatelet therapy after drug-eluting stents) and on NOACs [38]. In this regard, it should be taken into account that NSAID safety seems to be not determined by prostanoids alone, but by a variety of other factors, such as NO bioavailability and its relation with the endogenous NOS inhibitors ADMA and l-NMMA, renin release by juxtaglomerular cells and aldosterone pathway [2,41,84]. These changes may powerfully synergize with NSAID-induced PG modifications, thus regulating vascular side effects throughout the vasculature. In conclusion, patient clinical background, particularly gastrointestinal and cardiovascular diseases, should be considered in every single patient, as important predictors of the risk of adverse reactions due to NSAIDs [8,90]. The “conclusive” data to close our case will arise from the Prospective Randomized Evaluation of Celecoxib Integrated Safety versus Ibuprofen or Naproxen (PRECISION) study, the first specifically designed to evaluate anti-inflammatory drug CV safety, that

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171

Table 2 Ongoing trials on NSAIDs and Cardiovascular Outcomes. Study

N

Drugs

Duration Patients

Outcomes

5 yrs

Patients with SpA/AS

Celecoxib vs Naproxen vs Placebo

3 yrs

Healthy adults

Parecoxib and Celecoxib vs Placebo

3 yrs

OA patients undergoing total knee arthroplasty

Describe the use of etoricoxib and other Coxibs/tNSAIDs in Swedish patients with SpA/AS, and estimate and compare the rates of clinical outcomes of special interest (gastrointestinal, renovascular, cardiovascular and cerebrovascular). COX-1 and COX-2 activity measured ex vivo using a whole blood assay and in vivo by quantifying concentrations of prostaglandin metabolites in urine. Evaluate the cumulative morphine consumption over postsurgical period and adverse events.

Parecoxib vs Placebo

4 yrs

Celecoxib 100e200 mg twice daily, vs Ibuprofen 600 mg e800 mg three times daily, vs Naproxen 375 mg to 500 mg twice daily.

10 yrs

Patients undergoing pulmonary resection by Compare the amount of morphine open thoracotomy consumption and adverse effect related to parecoxib. The first occurrence of CV death (including Patients with OA or rheumatoid arthritis with or at risk of developing CV disease and hemorrhagic death), non-fatal myocardial infarction, or non-fatal stroke (APTC requiring chronic, daily therapy with an composite endpoint). NSAID to control arthritis sign and symptoms.

16,671 Etoricoxib vs other Coxibs, vs Safety of Etoricoxib in tNSAIDs patients With SpA/AS in Sweden

Variability in response to non-steroidal antiinflammatory drugs

288

Efficacy and safety of 246 postoperative intravenous Parecoxib sodium followed by oral Celecoxib in OA patients Can parecoxib reduce post- 160 operative Ipsilateral shoulder pain? 24,222 Prospective randomized evaluation of Celecoxib integrated safety Vs Ibuprofen or Naproxen (PRECISION)

Abbreviations: Coxibs, COX-2 inhibitors; tNSAIDs, traditional nonsteroidal anti-inflammatory drugs; SpA, spondyloarthropathy; AS, ankylosing Spondylitis; OA, osteoarthritis; CV, Cardiovascular.

will provide decisive “proofs” from a direct comparison of celecoxib, ibuprofen, and naproxen [91] (Table 2). Author contributions Santilli F. contributed to the design of the review, analysis of the more relevant literature, drafting of the manuscript, critical revision for important intellectual content. Boccatonda A. made a comprehensive review of the available literature, contributed to the design and drafting of the manuscript and figures. Davi G contributed to the analysis of most relevant literature and made a critical revision for important intellectual content. Cipollone F contributed to the design of the review, drafting of the manuscript, critical revision for important intellectual content.

[4]

[5]

[6]

[7]

[8]

[9]

Conflict of interest The authors confirm that there are no conflicts of interest. Search strategy This document is based on a comprehensive review of the available literature. PubMed database was searched through February 2016 for articles in English reporting Coxibs and adverse cardiovascular risk and outcomes. The following search terms were used: ‘Coxibs’, ‘NSAIDs’, ‘prostaglandin’, ‘EP receptor’, ‘platelet’, ‘cardiovascular diseases’, ‘atherosclerosis’. Identified references were hand-searched to locate other potentially useful references. Both clinical and experimental studies were included; abstracts were excluded.

[10]

[11]

[12]

[13]

[14]

[15]

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