Cyclic guanosine monophosphate signalling pathway in pulmonary arterial hypertension

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Cyclic guanosine monophosphate signalling pathway in pulmonary arterial hypertension Chien-nien Chen, Geoffrey Watson, Lan Zhao ⁎ a r t i c l e

i n f o

Article history: Received 6 July 2012 Received in revised form 28 August 2012 Accepted 4 September 2012 Keywords: Pulmonary arterial hypertension cGMP

a b s t r a c t During the last decade, it emerged that cyclic guanosine monophosphate (cGMP) is a novel drug target for the treatment of pulmonary arterial hypertension (PAH). cGMP regulates many cellular functions, ranging from contractility to growth, of relevance to the disease. Generated from guanylyl cyclases in response to natriuretic peptides or nitric oxide (NO), cGMP transduces its effects through a number of cGMP effectors, including cGMP-regulated phosphodiesterases and protein kinases. Furthermore, the cGMP concentration is modulated by cGMP-degrading phosphodiesterases. Data to date demonstrate that increasing intracellular cGMP through stimulation of GCs, inhibition of PDEs, or both is a valid therapeutic strategy in drug development for PAH. New advances in understanding of cGMP are unravelled, as well as the pathobiology of PAH. cGMP remains an attractive future PAH drug target. This review makes a more detailed examination of cGMP signalling with particular reference to PAH. © 2012 Elsevier Inc. All rights reserved.

1. Introduction It has been almost a half century since the nucleotide second messengers, cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP), were first identified (Beavo and Brunton, 2002). The discovery of cAMP earned Earl W. Sutherland Jr. the Nobel Prize in Physiology or Medicine in 1971 and promoted widespread interest in cyclic nucleotide signalling. The discovery of upstream mediators, atrial natriuretic peptide (ANP) and nitric oxide (NO), in the 1980s focused attention on the biological importance of cGMP in vascular biology. Following the pioneering studies of Robert F. Furchgott, Louis J. Ignarro and Ferid Murad, who were duly awarded the Nobel Prize in 1998 (Murad, 2006), it is now established that the NO/cGMP signalling pathway has a crucial role in the maintenance of vascular tone. Perturbations to the cGMP signalling cascade, from synthesis and activation to degradation, have been implicated in a number of cardiovascular diseases, including systemic and pulmonary hypertension, atherosclerosis, cardiac hypertrophy and heart failure. In the past decade, cGMP signalling has emerged as an attractive therapeutic target in cardiovascular disease with several licensed products now on the market. Pulmonary arterial hypertension (PAH) has been a particular beneficiary of the efforts of the pharmaceutical industry in this domain. PAH, defined by an abnormal sustained elevation in pulmonary arterial pressure, is a progressive disease with a poor clinical outcome, even in the modern era. The vascular pathology of PAH is characterized by pulmonary vasoconstriction, vascular remodelling, thrombosis in ⁎ Corresponding author at: Experimental Medicine, Imperial College London, Hammersmith Hospital, London W12 0NN, UK. E-mail address: [email protected] (L. Zhao). 1537-1891/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.vph.2012.09.001

situ and inflammation (Schermuly et al., 2011a). The current treatment for PAH is based on prostanoid replacement, endothelin receptor antagonists (ERA) and phosphodiesterase type 5 (PDE5) inhibitors, all of which target directly or indirectly cGMP or cAMP signalling (O'Callaghan et al., 2011). Prostanoids stimulate the conversion of adenosine triphosphate (ATP) to the active form, cAMP, while PDE5 inhibitors restore NO–cGMP signalling. cGMP remains an attractive target for further drug development, and agents that stimulate or activate soluble guanylate cyclase are in clinical trials. This review makes a more detailed examination of cGMP signalling with particular reference to PAH. 2. cGMP signalling The major enzyme sources of cGMP in tissues are soluble guanylate cyclase (sGC) and particulate guanylate cyclase (pGC). NO stimulates sGC while the natriuretic peptides (atrial or A-type, brain or B-type and C-type, otherwise known as ANP, BNP and CNP) stimulate specific natriuretic peptide receptors (pGCs) to catalyse guanosine 5′-triphosphate (GTP) to cGMP. In turn, cGMP stimulates cGMPdependent protein kinases (PKG) and cyclic nucleotide-gated ion channels. The metabolism of cGMP is controlled by phosphodiesterases (PDEs), predominantly PDE5 (Fig. 1). 2.1. cGMP generators 2.1.1. Soluble guanylate cyclase Soluble guanylate cylclase (sGC) is an intracellularly located heterodimeric haemoprotein composed of two different subunits: alpha (α1, α2 and αi2) and haem-binding beta (β1, β2 and β3) (Kamisaki et al., 1986). The existence of two isoforms of sGC, α1β1

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Prostacyclin receptor agonists

Endothelial cells Prostacyclin synthase

eNOS BH4

Prostacyclin

NO donors

Prostanoids

ERAs

ET-1

Natriuretic peptides

NO

sGC stimulators activators

ETR

AC GTP

pGC

ATP

cAMP

sGC

cGMP

PDE3/4 AMP PDEs

GMP

Ion channels

cGKs AM

Smooth muscle cells

PDE5 inhibitors

VIP

Rho/ROCK

Fig. 1. Cyclic GMP (cGMP) is catalysed from GTP by endothelium–NO-mediated soluble guanylate cyclase (sGC) and natriuretic peptide-mediated particulate guanylate cyclase (pGC), and is hydrolysed and inactivated by phosphodiesterases (PDEs). sGC-dependent cGMP generation can be further augmented by increased NO bioactivity through tetrahydrobiopterin (BH4), the cofactor of endothelial NO synthase (eNOS), or direct stimulation and activation of sGC. cGMP activates cGMP-dependent protein kinases (cGKs) and results in the vasorelaxation and inhibits proliferation of vascular smooth muscle cells. These effects can be enhanced by reduction of cGMP breakdown via PDE inhibition. Prostacyclin and its analogues (prostanoids) promote relaxation and inhibit cell proliferation via activation of adenylate cyclase (AC) to increase cyclic AMP (cAMP). Endothelin-1 (ET-1), secreted from endothelial cells, leads to vasoconstriction, which is antagonised by endothelin receptor antagonists (ERAs). Rho/Rho kinase (Rho/ROCK), adrenomedullin (AM) and vasoactive intestinal polypeptide (VIP) also participate in the regulation of vascular tone and vascular remodeling through the cyclic nucleotide pathway.

and α2β1, has been described. In mammalian physiology, the α1β1 isoform is present in almost all tissues, whereas a higher level of α2β1 isoform is expressed exclusively in the brain. The presence of other combinations has only been identified through cloning and their biological relevance is largely unknown. On binding to NO with its N-terminal domain (haem-NO/oxygen binding domain, H-NOX domain), the sGC C-terminus is activated and catalyses the conversion of GTP to cGMP. The central PAS (Period clock protein, ARNT protein and Single minded protein) domain of sGC, followed by an extended coiled-coil region, has been implicated in heterodimer formation and a regulatory role in signal propagation from N-terminal to C-terminal domains (Ma et al., 2008). Distinctive from other haemoproteins, the H-NOX domain of sGC is highly selective for NO. NO binding results in at least a 200-fold increase in sGC activity compared with only a 4-fold increase stimulated by carbon monoxide (CO) (Ma et al., 2007). The absence of a specific tyrosine residue in the haem pocket of sGC, which is essential for O2 binding, may be responsible for the kinetic selection towards NO (Boon et al., 2005). In addition to NO, several endogenous regulators have emerged to manipulate sGC activity, such as calcium (Ca 2+), adenosine triphosphate (ATP), protein kinases and heat shock protein (Hsp). 2.1.2. Particulate guanylate cyclases Particulate guanylate cyclases (pGC) are plasma membraneintegrated natriuretic peptide receptors, which contain a N-terminal extracellular ligand-binding domain, a single transmembrane region, and an intracellular guanylate cyclase domain (Kuhn, 2003). pGC comprise a protein family of at least seven members, which are GC-A through GC-G. Among them, GC-A and GC-B are also known as natriuretic peptide receptors NPR-A and NPR-B. GC-A (NPR-A), activated by ANP and BNP, is the most widely expressed and is found in many tissues including the lung and immune system. ANP and BNP

are cardiac hormones secreted in response to myocardial stretch, leading to diuretic, vasorelaxant and cardioprotective effects. GC-B (NPR-B), localizing more specific to the lung, heart, kidney and brain, binds to CNP and, in addition to vascular effects, plays a role in cardiac function and bone growth. In addition to natriuretic peptides, adenine nucleotides such as ATP and its analogues are able to enhance pGC activity and generate synergistic effects with peptide ligands (Kurose et al., 1987). ATP-regulatory effects have been reported with GC-A, GC-B and GC-C (Goraczniak et al., 1992; Duda et al., 1993).

2.2. cGMP effectors 2.2.1. cGMP-dependent protein kinases The cGMP-dependent protein kinases (cGKs, PKG) are serine/ threonine kinases. Two families of cGKs, coded by separate genes (cGKI and cGKII) have been identified in mammals: the cytosolic cGK type I (cGKI), of which there are two isoforms, cGKIα and cGKIβ, and the membrane-bound cGK type II (Francis et al., 2005; Hofmann, 2005). Structurally, the two cGKs are homodimers, with each subunit containing a N-terminal domain mediating homodimerization, a regulatory domain binding to cGMP and a catalytic domain phosphorylating the serine/threonine side chains on substrate proteins (Surks, 2007). Generally, cGKI mediated NO/sGC-derived cGMP signalling transduction is involved in cardiovascular homeostasis, whereas cGKII plays a role in pGC-derived cGMP‐mediated electrolyte transport and bone formation (Feil et al., 2003). cGKI is highly expressed in smooth muscle cells (SMCs), platelets and fibroblasts, and to a lesser degree in vascular endothelium and cardiomyocytes. cGKII is more specific to renal cells, exocrine cells in distal airways, the gastrointestinal tract, salivary glands, chondrocytes and some brain nuclei, and relatively absent in the cardiovascular system.

cGKI contributes to smooth muscle relaxation and plays an essential role in the regulation of vascular tone. The cGKI knockout mice exhibit defective vasorelaxation, dysfunctional platelet adhesion and activation, and have a shorter life span (Pfeifer et al., 1998). These phenotypes are rescued by restoring cGKIα and cGKIβ smooth muscle expression (Weber et al., 2007). cGKI reduces cytosolic Ca 2+ levels, but also inhibits RhoA signalling and phosphorylation of myosinbinding protein (Sauzeau et al., 2000; Sawada et al., 2001). Changes in cGKI activity play a role in the pathological vascular remodelling process seen in PAH. However, the impact of cGMP/ cGKI on cell growth remains complicated and controversial, e.g. NO/ cGMP/cGKI transduction promotes growth in primary vascular SMCs but inhibits growth in subcultured cells (Sarkar and Webb, 1998; Koyama et al., 2001). cGMP/cGKI-stimulated cell proliferation has been shown to participate in the cross-talk between mitogenactivated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt kinase signaling (Komalavilas et al., 1999; Wolfsgruber et al., 2003). cGKI is also implicated in regulating of platelet adhesion and activation, and inotropic effects on the heart (Wegener et al., 2002). 2.2.2. Cyclic nucleotide-gated ion channels Two groups of ion channel proteins are regulated by cyclic nucleotides, the cyclic nucleotide-gated ion channels (CNGs) and the hyperpolarization-activated cyclic nucleotide-gated ion channels (HCNs) (Craven and Zagotta, 2006; Biel and Michalakis, 2007; Wahl-Schott and Biel, 2009). As members of the superfamily of voltage-gated ion channels, the activation of these two channels regulates the flow of ions across the plasma membrane (membrane hyperpolarization) in response to direct binding of cGMP and cAMP. The CNGs show higher affinity for cGMP while HCNs select cAMP over cGMP (Kaupp and Seifert, 2002). 2.2.3. Phosphodiesterases Phosphodiesterases (PDEs), the enzymes that catalyze hydrolytic cleavage of the 3′ phosphodiester bond of the cyclic nucleotides, regulate both intracellular cGMP and cAMP levels (Omori and Kotera, 2007). PDEs have a wide distribution in normal tissues and are subdivided into 11 distinctive families on the basis of substrate affinity and selectivity, sequence homology and regulatory mechanisms (Kass et al., 2007). Of these enzymes, PDE5, PDE6, and PDE9 are highly selective for cyclic GMP; PDE1, PDE2, and PDE11 bind cyclic GMP and cyclic AMP with equal affinity; and PDE3 and PDE10 are cyclic GMP-sensitive but cyclic AMP-selective. PDE5 was the first cGMPselective PDE to be discovered and its selectivity makes it an important target for drug development in the cardiovascular field (Zhang and Kass, 2011). In addition to breaking down cGMP, several PDEs are regulated by cGMP and its downstream mediators cGKs (Corbin et al., 2000). cGKs induce phosphorylation of PDE, which enhances binding affinity and catalytic activity, while cGMP binding to the GAF domain (found in cGMP-mediated PDEs, adenylate cyclases and FhlA) leads to phosphorylation of PDEs (Rybalkin et al., 2003; Zoraghi et al., 2005). Furthermore, elevated cGMP can alter cAMP levels via modulation of PDEs (Zaccolo and Movsesian, 2007). 3. cGMP in pulmonary arterial hypertension Impaired cGMP signalling, particularly via perturbation of NO synthesis, has a prominent role in PAH. 3.1. Nitric oxide NO is a potent vasodilator, synthesized by the three isoforms of nitric oxide synthase (NOS), endothelial (eNOS), neuronal (nNOS) and inducible (iNOS). It is also recognized as an inflammatory mediator, platelet inhibitor and regulator of SMC proliferation. In the

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pulmonary vasculature, eNOS-derived NO produced by endothelial cells diffuses freely across the cell membrane to the surrounding SMCs and activates intracellular sGC (Sim, 2010). Studies have shown that eNOS activity is related to various vasoactive factors and physiological stimuli, such as hypoxia, inflammation and oxidative stress (Moncada and Higgs, 1993). PAH patients have decreased eNOS expression in lung tissue and low NO levels in their exhaled breath (Giaid and Saleh, 1995; Girgis et al., 2005). In animal models, deficiency in eNOS increases susceptibility to hypoxia-induced pulmonary hypertension while pulmonary gene transfer of eNOS is protective (Champion et al., 2002). The availability of tetrahydrobiopterin (BH4), a cofactor in eNOS synthesis, to the endothelium has been shown to regulate the response of the pulmonary circulation to hypoxia (Khoo et al., 2005). The deficiency of cofactors such as BH4 leads to eNOS uncoupling and the production of reactive oxygen species rather than NO (Vasquez-Vivar et al., 1998; Xia et al., 1998). Decreased eNOS–NO–cGMP signalling contributes to pathological vascular constriction and increased vascular resistance in PAH. Inhaled NO has been approved for clinical use since 1999. NO selectively dilates pulmonary vessels at low levels (below 0.8 part per million) (Beghetti et al., 1995) and reduces elevated pulmonary vascular resistance with effects on the systemic circulation at 2–80 ppm (Roberts et al., 1993; Day et al., 1995). Inhalation of NO has been shown to be highly effective in the treatment of persistent pulmonary hypertension of the newborn and is associated with a significant increase in plasma cGMP (Turanlahti et al., 2001). However, NO therapy is limited by the need for continuous inhalation, NO reactions with oxygen to form nitrogen dioxide, and special delivery devices. Endogenous NO production is influenced by endothelin-1 (ET-1), a potent endothelium-derived vasoconstrictor and mitogen (Hassoun et al., 1992). ET-1 binding to ETA and ETB receptors on pulmonary arterial SMCs leads to increased intracellular calcium and subsequent vasoconstriction, whereas ET-1 binding to the ETB receptor on endothelial cells stimulates NO and prostacyclin release and favours vasodilation (Benigni and Remuzzi, 1999; Fagan et al., 2001). Increased circulating levels of ET-1 as well as increased local production in the pulmonary vasculature are well documented in patients with PAH (Giaid et al., 1993; Allen et al., 1993). The success of ERA in PAH testifies to the importance of ET in this condition but there is yet no conclusive evidence that selective blockade of the ETA receptors offers therapeutic advantage over dual ETA and ETB blockade. 3.2. Natriuretic peptides Natriuretic peptides stimulate cGMP synthesis through pGC. In PAH patients, circulating levels of ANP and BNP are elevated, and levels are used as a biomarker of RV dysfunction and clinical prognosis (Casserly and Klinger, 2009). Mice with genetic disruptions of ANP and its receptor demonstrated increased susceptibility to pulmonary hypertension when expose to chronic hypoxia (Klinger et al., 1999). NPR-A knockout results in augmented gene and protein expression of ANP and BNP in the ventricles which correlated with the degree of cardiac hypertrophy and fibrosis but disproportionate to the elevated blood pressure (Ellmers et al., 2002). Natriuretic peptides show specific anti-proliferative effects on vascular SMCs from different vascular beds (Arjona et al., 1997). For example, pulmonary artery-derived SMCs are more sensitive to ANP-dependent antihypertrophic effects than those derived from thoracic aorta. In addition to direct effects of vascular tone and natriuresis, natriuretic peptides inhibit ET-1 synthesis and ET-receptor expression (Emori et al., 1993). 3.3. Phosphodiesterases Among the 11 families of phosphodiesterases (PDEs), PDE5 and PDE1 have attracted the most interest as drug targets by virtue of

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their tissue distribution. PDE5 is ubiquitously expressed in an array of tissues but the levels and activity in the lung are up to 15-fold higher relative to the heart, matching those measured in penile corpus cavernosum. In the lung, PDE5 is the predominant cGMP PDE subtype, and it is most abundant in pulmonary SMCs (Corbin et al., 2005). PDE5 expression is significantly upregulated in the lungs of PAH patients (Wharton et al., 2005). Immunohistochemical studies have demonstrated co-localization of PDE5 to SMCs in the medial layer of the remodelled pulmonary vessels. PDE1 is expressed at low levels in healthy lung tissue (Schermuly et al., 2007), but it is significantly increased in proliferating pulmonary vascular SMCs (Phillips et al., 2005; Murray et al., 2007; Dony et al., 2008). PDE1C and PDE1A isoforms are increased in lung explants and pulmonary SMCs from patients with idiopathic PAH (Schermuly et al., 2007). Selective inhibition of PDE1 in vivo and in vitro has shown beneficial effects on vascular remodeling, indicating that PDE1, especially PDE1C, would be an interesting target for future treatments for PAH (Chan and Yan, 2011). 3.4. cAMP signalling pathway Endothelial prostacyclin production from prostaglandin H2 (PGH2) via prostacyclin synthase drives cAMP vasodilation and inhibits platelet aggregation. Prostacyclin synthase expression is reduced in small and medium-sized pulmonary arteries in PAH patients, while levels of thromboxane A2 (TXA2), its counterpart, are increased (Tuder et al., 1999). Consistent with this, urinary metabolites of prostacyclin are reduced in PAH patients but urinary TXA2 metabolites are increased (Christman et al., 1992). Prostacyclin analogues induce vasorelaxation and inhibit cell growth. Epoprostenol was the first agent to be used successfully as a targeted PAH therapy. Since then there have been a number of prostacyclin analogues developed and investigated as medical therapies for PAH (Rubin et al., 1990; Vachiery, 2011). Other endogenous peptides, such as vasoactive intersinal peptide (VIP) and adrenomedullin (AM), also act through cAMP. VIP is a 28 amino-acid peptide belonging to the glucagon-growth hormone‐ releasing factor secretion superfamily which acts through two receptors, VPAC-1 and VPAC-2. Stimulation of these receptors activates the cAMP and cGMP pathways which have been shown to mediate the actions of such substances as prostacyclin and NO. Circulating VIP levels in PAH patients are decreased (Petkov et al., 2003), while the VPAC-1 and VPAC-2 expression and binding affinity are upregulated in PAH patient lungs. VIP deficient mice develop moderate to severe PH (Said et al., 2007). Selective pulmonary vasodilatory effects with improved tissue oxygenation were suggested in a neonatal piglet model (Haydar et al., 2007). An initial pilot study of inhaled aviptadil, a VIP analogue, reported an improvement in pulmonary hemodynamics and right ventricular workload with transient but selective pulmonary vasodilation in patients with idiopathic PAH (Leuchte et al., 2008). However, a recent phase II study has failed to replicate these findings. AM is a 52 amino-acid peptide and a member of the calcitonin gene-related peptide superfamily. AM elicits vascular protective effects through stimulation of NO–cGMP and cAMP signalling, as well as via the PI3K/Akt pathway (Kato et al., 2005). Plasma AM levels are elevated in patients with PAH (Kakishita et al., 1999; Nishida et al., 2008). Intravenous AM attenuates the pulmonary hypertension phenotype in rodent models with marked amelioration in vascular remodelling (Zhao et al., 1996; Nagaya et al., 2000, 2003; Matsui et al., 2004). The obvious concern is the AM hypotensive effects in systemic circulation; this may be overcome by AM inhalation therapy for pulmonary selectivity (Nagaya et al., 2004). 3.5. Cross-talk between cAMP and cGMP signaling Several families of PDEs that are regulated by cGMP have direct impact on cAMP-hydrolyzing activity and thereby control intracellular

cAMP levels. Specifically, PDE3 binds with high affinity to both cAMP and cGMP but catalyses cAMP at a higher rate; cGMP therefore acts as a competitive inhibitor of cAMP hydrolysis (Zaccolo and Movsesian, 2007). PDE3 and PDE4 co-regulate the metabolism of cAMP in the lung and both are upregulated in experimental PAH models (MacLean et al., 1997). Inhibition of PDE3/4 by tolafentrine improves endothelial function via the asymmetrical dimethylarginine (ADMA)–dimethylarginine dimethylaminohydrolase (DDAH) axis and elevates NO/cGMP levels (Pullamsetti et al., 2011). PDE3 is also a crucial regulator of myocardial contractility through cAMP signaling. PDE3 inhibitors have inotropic and cardioprotective effects, especially when adrenergic stimulation is inadequate. Several PDE3 selective inhibitors are used to treat heart failure clinically (Rao and Xi, 2009). Combining cAMP-elevating agents, for example, iloprost or epoprostenol, with inhibition of cGMP degradation appears to bring synergistic effects and therapeutic efficacy (Ghofrani et al., 2002; Simonneau et al., 2008). 4. Therapeutic choices in PAH At present, three pathways are exploited for therapeutic gain in PAH: PDE5 inhibitors, endothelin receptor antagonists (ERA) and prostanoids (Galie et al., 2009b; Macchia et al., 2010; Humbert et al., 2010). 4.1. PDE5 inhibitors PDE5 inhibitors block the breakdown of cGMP, resulting in the accumulation of cGMP in the tissue and leading to vasodilation. PDE5 inhibitors were originally investigated for their potential use in angina, although the most widely known use is for erectile dysfunction. PDE5 inhibitors evolved as a treatment for PAH based on the abundance of PDE5 in the lung, particularly the pulmonary arterial wall. Three PDE5 inhibitors have been investigated in PAH: sildenafil, tadalafil and vardenafil. Structurally, sildenafil, and vardenafil share chemical similarities with cGMP and inhibit PDE5 by competitively binding to the catalytic site. Tadalafil has a longer half-life of 17.5 h compared to 4–5 h with sildenafil and vardenafil (Klinger, 2011). In initial experimental studies in animal models, chronic dosing with PDE5 inhibitors prevents and partially reverses elevated pulmonary artery pressure, right heart hypertrophy and pulmonary vascular remodelling, and improves survival in the monocrotaline-induced pulmonary hypertension rodent model (Zhao et al., 2001; Sebkhi et al., 2003; Schermuly et al., 2004). Studies in genetically manipulated mice revealed that both eNOS and natriuretic peptides contribute to the pharmacological effects of PDE5 inhibitors (Zhao et al., 2003). In PAH patients, sildenafil has profound vasodilatory effects, comparable to inhaled NO, in the pulmonary circulation (Klinger et al., 2006). Its effect is comparable to inhaled NO at clinically relevant doses and has shown benefit in patients with idiopathic PAH and other primary subtypes. Following a number of small-scale clinical studies, a pivotal regulatory study, SUPER-1, confirmed its haemodynamic and functional benefits, and sildenafil 20 mg three times daily gained approval for PAH in 2005 (Galie et al., 2005). Tadalafil was approved for PAH treatment in 2009 (Galie et al., 2009a). The longer plasma half-life makes tadalafil suitable for once daily use. PDE5 inhibition in combination with other treatments has found its way into clinical practice ahead of scientific evidence. Combinations of sildenafil with the ERA, bosentan, or the inhaled prostanoid, iloprost, have shown synergistic benefits in small studies (Ghofrani et al., 2002, 2003). A larger study has reported an improvement in exercise capacity (6 min walk distance) and a reduction in time to clinical worsening when sildenafil was added to epoprostenol (Simonneau et al., 2008). A large scale study investigating the benefits of combining

tadalafil and ambrisentan (a selective ETA antagonist) is underway (clinicaltrials.gov identifier: NCT01178073). 4.2. Endothelin receptor antagonists Activation of ETA on SMCs results in vasoconstriction and proliferation of vascular SMCs, whereas ETB simultaneously mediates pulmonary endothelin clearance and induces NO and prostacyclin production, which supports the rationale of using selective endothelin receptor antagonists (ERA) (Benigni and Remuzzi, 1999). Nonetheless, bosentan, a combined ETA and ETB antagonist, has proven effective at improving haemodynamics, exercise capacity and functional class in PAH (Rubin et al., 2002). Subsequent ERAs have targeted the ETA receptor (ambrisentan, sitaxsentan) but with little evidence to date that this confers any therapeutic advantage. Sitaxsentan was withdrawn in 2011 because of unacceptable liver toxicity. Ambrisentan is better tolerated in that respect, suggesting that the effect on the liver is not a property of ETA antagonism. But ambrisentan has to be used with care because of concern over fluid retention. 4.3. Prostanoids Prostacyclin exerts potent vasodilation as well as anti-thrombotic, anti-proliferative, anti-mitogenic, and immunomodulatory effects through enhancing cAMP levels. Given parenterally, it has a short half-life and its use associated with risk of infection. It is the only treatment that has been shown to improve survival in PAH (McLaughlin et al., 2002). Other more stable analogues have been developed—subcutaneous (treprostinil), oral (beraprost) or by inhalation (iloprost) (O'Callaghan et al., 2011). The challenge is to develop an orally active agent that avoids systemic side effects. 5. Emerging therapies A meta‐analysis of current treatments suggests that the targeted therapies improve survival (Galie et al., 2009b; Macchia et al., 2010) but it is clear from observational studies that they do not provide a cure (REVEAL Registry) (Badesch et al., 2010). cGMP signalling remains an attractive target for new drugs. 5.1. sGC stimulators and activators Despite the high efficacy of PDE5 inhibitors, it is estimated that up to 60% of patients with PAH are refractory to the therapy with sildenafil. The therapeutic effects of PDE5 inhibitors largely rely on the baseline levels of pulmonary cGMP, which are presumed to be inadequate in PAH patients (Leuchte et al., 2004; Chockalingam et al., 2005). Increasing sGC-dependent cGMP production offers an alternative approach to preserving endogenous cGMP production in disease. Two categories of compounds can act on sGC: sGC stimulators and sGC activators. Simulators sensitize sGC when there is a low bioavailability of NO, whereas activators act independent of ambient NO availability (Evgenov et al., 2006). Riociguat is a sGC stimulator currently in clinical development. First shown to be effective in hypoxic and monocrotaline-induced PH in rodent models (Dumitrascu et al., 2006; Schermuly et al., 2008), Riociguat has demonstrated efficacy in terms of improved haemodynamic parameters and exercise capacity is a Phase II study in patients with idiopathic PAH and other forms of PH such as chronic thromboembolic PH (Ghofrani et al., 2010). These initial clinical results have encouraged further randomized, double-blind, placebocontrolled phase III studies (CHEST and PATENT), involving multiple centres and enrolling more than 400 patients (Schermuly et al., 2011b). The results of these studies are expected soon.

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5.2. Tetrahydrobiopterin Tetrahydrobiopterin (BH4) is a critical cofactor to all three NOS isoforms and so NO synthesis (Vasquez-Vivar et al., 1998). Biosynthesized from GTP via a de novo pathway, BH4 can be retained through a recycling salvage pathway. Reduced availability of BH4 leads to uncoupled NOS, which generates superoxide instead of NO and may contribute to endothelial dysfunction in vascular disease (Thony et al., 2000). Early studies showed BH4 replacement augments NO-dependent vasodilation in several cardiovascular diseases. Mice deficient in BH4 biosynthesis (hph-1 mice) exhibit a pulmonary hypertension phenotype, including pulmonary vascular remodelling and right ventricular hypertrophy, and are more susceptible to hypoxia (Nandi et al., 2005; Khoo et al., 2005). Interestingly, the pulmonary vascular pathology in hph-1 mice is more severe than that in eNOS-deficient mice, which suggests additional factors to loss of endothelial derived NO in the vascular pathology. It is hypothesized that BH4 administration can restore eNOS coupling to increase NO bioavailability and eventually benefit PAH through cGMP-dependent cardiovascular protection. Chronic treatment with BH4 ameliorated hypoxia and monocrotaline-induced pulmonary hypertension in rodent models, associated with increased eNOS activity, elevated cGMP levels and reduced superoxide production (unpublished). The safety of BH4 in PAH patients has recently been tested and further studies to explore the therapeutic potential are favoured (Robbins et al., 2011). A recent clinical study of BH4 treatment in patients with coronary artery disease indicated elevated BH4 levels in blood, but its effect is significantly limited by systemic oxidation of exogenous BH4 to BH2 (Cunnington et al., 2012), suggesting an antioxidant coadministration. 5.3. Natriuretic peptides Another strategy for restoring cGMP levels in PAH patients is through the natriuretic peptide–pGC pathway. ANP has protective effects in hypoxia-induced pulmonary hypertension. Continuous infusion of ANP has been shown to attenuate the pulmonary hypertension phenotype in hypoxic animals, associated with increased plasma and pulmonary cGMP levels (Klinger et al., 1999). Furthermore, ANP-mediated vasodilation can be enhanced by PDE5 inhibition (Zhao et al., 2003; Baliga et al., 2008). However, a major challenge is that rapid degradation of natriuretic peptides makes long-term administration impractical. Inhibitors of neutral endopeptidase, which is responsible for the hydrolyzation of endogenous natriuretic peptides, may offer a mechanism for elevating endogenous natriuretic peptide levels and, in combination with PDE5 inhibitors, may provide a novel treatment combination for PAH (Baliga et al., 2008). 5.4. Rho kinase Vascular SMC contraction is controlled by a Ca 2+-independent pathway, Rho and its downstream effector Rho-kinase (ROCK), a G-protein-coupled system. The Rho/ROCK pathway regulates myosin light chain dynamics and therefore actin-dependent processes such as cell adhesion, motility and survival, relevant to the pathogenesis of PAH (Oka et al., 2008; Wojciak-Stothard, 2008). cAMP- and cGMP-dependent protein kinases (PKA and PKG) influence Rho/ ROCK activation and regulation (Sauzeau et al., 2000, 2003). Inhibition of Rho/ROCK activity can promote pulmonary vascular SMC Ca 2 +-sensitization and that maybe beneficial in PAH. The ROCK inhibitor, fassidil, ameliorates vasoconstriction in experimental models (Abe et al., 2004; Oka et al., 2007) and causes acute vasodilator effects in patients with PAH (Ishikura et al., 2006). However, ROCK inhibitors are not selective for the pulmonary circulation and systemic hypotension is a concern.

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5.5. Inorganic nitrite and nitrate Inorganic nitric (NO2−) and nitrate (NO3−) serve as a NO donor via biochemical reduction. This has been demonstrated in studies using inhaled nitrite as an alternative to NO gas inhalation. In sheep, mouse, and rat models with PAH, nitrite has been shown to act as a pulmonary vasodilator (Sparacino-Watkins et al., 2012). Nitrite inhalation decreases pulmonary arterial pressure induced by hypoxia and thromboxane (Hunter et al., 2004) and can prevent or reverse established right ventricle (RV) pressure increases, RV hypertrophy, and pulmonary vascular remodeling in hypoxia or monocrotaline‐induced PAH (Zuckerbraun et al., 2010). Sodium nitrite injection attenuates PAH induced by monocrotaline, hypoxia, and/or thromboxane (Lundberg et al., 2008; Casey et al., 2009). Recently, Baliga and colleagues have reported that oral nitrite and nitrate attenuate RV pressure, RV mass, and pulmonary vascular remodelling induced by hypoxia and bleomycin (Baliga et al., 2012). The pharmacological effects are associated with increased plasma and pulmonary cGMP levels and might be eNOS-dependent, providing a potential orally active therapy for PAH via the nitrate-nitrite-NO pathway. 5.6. Multidrug resistance protein inhibitors In addition to PDE-dependent degradation, intracellular homeostasis of cyclic nucleotides is also affected by a process involving active efflux out of cells. Multidrug resistance‐associated protein 4 (MRP4), a member of a large family of transmembrane proteins (ATP-binding cassette transporter family class C), has been shown to function as an energy-dependent transporter for cyclic nucleotides (van Aubel et al., 2002; Wielinga et al., 2003). MRP4 plays a role in vascular SMC proliferation (Sassi et al., 2008) and its expression is increased in IPAH patients and experimental PH models. Oral administration of the MRP4 inhibitor MK571 to mice or MRP4 knockout mice was protected from hypoxia‐induced PH (Hara et al., 2011). Inhibition of MRP4 in vitro was accompanied by increased intracellular cAMP and cGMP levels and PKA and PKG activities, implicating cyclic nucleotide-related signalling pathways in the mechanism underlying the protective effects of MRP4 inhibition. These data suggest that MRP4 may be a potential therapeutic target for PAH. 6. Conclusions During the last decade, enhancing cGMP activity in the pulmonary vasculature has emerged as a novel and effective treatment strategy for PAH. cGMP regulates many cellular functions, ranging from contractility to growth, of relevance to the disease. In validating cGMP as a signalling molecule to target, it provides a relatively low risk option for new drugs. Direct stimulation of sGC holds promise for PAH and other forms for pulmonary hypertension, such as chronic thromboembolic disease. The combination of PDE5 inhibitors with neutral endopeptidase inhibitors to increase natriuretic peptide activity is another avenue to follow. Perhaps the combination of BH4 and an antioxidant is worth further study. Another option lies with using endothelial progenitor cells transfected with eNOS to encourage vascular repair (clinicaltrials.gov identifier: NCT00469027). PAH is a formidable challenge for any drug treatment and the success enjoyed to date has marked a step change in the management of the disease. Whether we have seen the full benefit from manipulating cGMP in PAH may be a question that will soon be answered. References Abe, K., Shimokawa, H., Morikawa, K., Uwatoku, T., Oi, K., Matsumoto, Y., Hattori, T., Nakashima, Y., Kaibuchi, K., Sueishi, K., Takeshit, A., 2004. Long-term treatment with a Rho-kinase inhibitor improves monocrotaline-induced fatal pulmonary hypertension in rats. Circ. Res. 94, 385–393.

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