Nitric oxide and pulmonary arterial hypertension

The pathogenesis of pulmonary arterial hypertension remains undefined. Changes in the expression and effects mediated by a number of vasoactive factors have been implicated to play a role in the onset and progression of the disease. The source of many of these mediators, such as nitric oxide (NO), prostacyclin and endothelin-1 (ET-1), is the pulmonary endothelium. This article focus in the role of nitric oxide in PAH, reviewing the evidence for its involvement in regulation of pulmonary a vascular tone under physiological conditions, the mechanisms by which it can contribute to the pathological changes seen in PAH and strategies for the use of NO as a therapy for treatment of the disease.

. Plexiform lesions are composed of proliferating endothelial cells, matrix proteins and fibroblasts that obliterate the vascular lumen 17 . The reasons for their formation are poorly understood, however hypoxia, inflammation, shear stress, drugs, viral infections and genetic susceptibility have all been implicated 18 . A number of factors and agents responsible for initiating and progressing the increases in pulmonary artery pressure have been suggested. Given the variety of different forms of the disease, it's not surprising that so many different mediators and mechanisms are believed to be responsible ( Table 2), many of which have been reviewed elsewhere [1][2][3][4][5]19 . At the cellular level dysfunction of the pulmonary endothelium seems to underpin many of the changes seen in PAH. Endothelial cells regulate vascular tone, vascular remodelling and inflammation via the release a range of vasoactive molecules that interact with blood elements and the underlying vascular smooth muscle. These mediators include nitric oxide (NO), prostacyclin and endothelin-1 (ET-1). The role of both ET-1 and prostacyclin has recently been reviewed in this journal 2,3 . The focus of the present article is on the role of NO in the onset and progression of PAH as well as the use of NO therapies for the alleviation of the clinical symptoms and improving the quality of life of patients with PAH.

NITRIC OXIDE IN THE PHYSIOLOGY OF THE PULMONARY CIRCULATION
As with all other vascular beds, the production of NO by the pulmonary endothelium helps to regulate vascular tone. While a diverse range of endogenous chemical mediators have been identified to stimulate the release of NO from endothelial cells, the frictional force of the flow of blood over the surface of the endothelial cells (shear stress) is the principal physiological stimulus for NO release 20 . The role of endothelial cells in regulating vascular tone was first reported as the ''obligatory role'' of the vascular endothelium in mediating relaxation of the underlying smooth muscle in 1980 with the discovery of endothelium-derived relaxing factor (EDRF) 21 . It was not until 7 years later that the chemical nature of EDRF as NO was identified and the metabolic pathway leading to its synthesis elucidated. Nitric oxide is formed by the enzymatic cleavage of the terminal amino group from the amino acid L-arginine 22,23 . The enzyme, termed nitric oxide synthase (NOS) was specific for the L isomer, but had no activity against the D isomer of arginine (Figure 2). At approximately the same time, NO was also shown to account for the biological actions of nitrovasodilators, which are capable of generating NO from their chemical structure (so called NO donors) 24 .
The characterisation of the first of a number of inhibitors of NOS, such as L-N Gmonomethyl Arginine (L-NMMA), L-N G -Nitroarginine methyl ester (L-NAME) and N G -Methyl-L-arginine (L-NMA), allowed researchers to probe the role of NO in the vascular system, leading to the first demonstration that inhibition of NO increases blood pressure in animals and induces vasoconstriction in human blood vessels [25][26][27] . In addition, it was shown that the effects of NO are not confined to the vascular system alone. Besides the endothelial NOS two additional isoforms of the NOS enzyme, a neuronal isoform and an  inducible isoform, have been identified. These three isoforms release NO for a variety of different biological functions 28 . All NOS enzymes bind calmodulin and contain haem and utilise L-arginine and molecular oxygen as substrates and requires the cofactors reduced nicotinamide-adenine-dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and (6R-) 5,6,7,8-tetrahydrobiopterin (BH4). They differ in their distribution, dependence on calcium and the amount of NO that they produce (Table 3) 29 .
Most of the biological actions of NO depend on its ability to stimulate the enzyme soluble guanylate cyclase with the subsequent formation of cyclic 3 , 5 guanosine monophosphate (cGMP) 30 . This leads to the activation of cGMP-dependent kinases (cGKs), which leads to the activation myosin phosphate and a subsequent release of calcium from intracellular stores allowing smooth muscle cells to relax. cGKs may also activate transcription factors that may change gene expression in the cell and mediate other responses of the cell to NO or alter its response to other stimuli 31,32 . The bioavailability of NO is influenced by its inactivation by excessive amounts of oxygenderived species such as superoxide (O 2 − ) or changes in the protective effects of endogenous antioxidants.

Sources of O 2
− include NADPH oxidases, xanthine oxidase, cytochrome P450 enzymes and from complexes I and III of the electron transport chain in mitochondria. An additional source of O 2 − may come from uncoupling of NOS, which has been suggested to occur in several pathological conditions [33][34][35] . Unless inactivated by oxygen free radicals, the effects of NO are terminated by the breakdown cGMP by phosphodiesterase (PDE) enzymes. There are a number of different isoforms of PDEs that have specific distributions in different organs and vascular beds 36 .

Interactions between nitric oxide and other endothelium-derived vasoactive mediators
The endothelium was initially regarded simply as monolayer of cells that line the vasculature serving as a barrier between the blood and the vessel wall 37 , however it is now considered more as an organ in its own right that regulates vascular homeostasis. In addition to NO, the endothelium also releases prostacyclin (PGI 2 ) and ET-1, which also both have a direct effect on the vessel wall [38][39][40][41] . ET-1 is a powerful vasoconstrictor peptide that can be considered as a functional antagonist to the vasodilator effects of NO. ET-1 has also been implicated in the pathogenesis of PAH and inhibition of its effects is a pharmacological target for the treatment of the disease 42,43 . In contrast, PGI2 is a vasodilator and anti-aggregating substance. The effect of prostacyclin on platelet aggregation are enhanced by the presence of NO 44 . The effects of PGI2 are mediated via its action on cell surface receptors that in turn stimulate the accumulation of cAMP. Administration of PGI2, has been the most widely used, and to date most successful, therapeutic strategy for the treatment of patients with PAH. The labile nature of PGI2 has meant that such strategies have relied upon the development of stable analogues of PGI2 such as Iloprost, which unfortunately is not orally active and has to be inhaled or given as an I.V. infusion 45 . There have been some clinical trials that have combined PGI2 analogues or ET-1 receptor antagonists with compounds that promote the effect of NO 43 . The role of PGI2 in PAH, the therapeutic potential of PGI2 analogues and receptor agonists in the treatment of the disease and potential novel strategies to maximise drug delivery and effect have recently been reviewed in this Journal 3 .
In addition to its direct effects on soluble guanylate cyclase, NO has been implicated to play a role in the regulation of mitochondrial function via its ability to interact with components of the electron transport chain 46 . NO has also been shown to activate mitochondrial biogenesis via a cGMP-dependent mechanism 47 . In conditions associated with over-production of NO, such as septic shock, its been shown that low mitochondrial complex I activity is associated with raised nitrite/nitrate concentrations 48 . The irreversible nature of the inhibition of complex I suggest that it is due to the action of reactive nitrogen species, rather than an inhibitory effect of NO. The direct relevance to how NO regulation of mitochondrial function affects the pulmonary vasculature is currently been investigated. In the fetal lung, it has been shown that interactions between endothelial NOS and mitochondria reduce the levels of oxidative stress and facilitates vasodilation at birth 49 . The recent development of fluorescent molecular probes specific for the mitochondria and cytosol, that specifically and directly respond to NO have provided the opportunity to obtain a quantifiable and real-time readout of NO dynamics in different cellular compartments (Figure 3) 50 . This approach may be of use to track the intracellular actions of NO is pulmonary cells.

ROLE OF NITRIC OXIDE IN THE PATHOPHYSIOLOGY OF PAH
Given the vasodilator effect of NO, it would be easy to speculate that reduced expression or release of NO would be identified in patients with the disease. Indeed there is evidence that pulmonary arteries from patients with PAH due to a variety of reasons have reduced endothelial expression of eNOS 51 . However there are also studies that have shown no change or even increased expression of eNOS in vessels taken from hypertensive lungs 52 . Increased expression of endothelial NOS has been specifically localised to plexiform lesions 53 (Figure 4). While these studies have been able to show changes in expression of the enzyme responsible for NO synthesis by the endothelium, it remains unclear as to whether this translates to changes in the activity of the enzyme or sensitivity of the vessel wall to the action of NO. Indeed, patients can be classified as responders or non-responders in the ability of inhaled NO to reduce pulmonary vascular resistance (PVR) and mean pulmonary artery pressure (mPAP). Those with a reduction of >30% in PVR or >12% in mPAP were shown to have reduced mortality compared to patients who had responses below these thresholds [54][55][56] .
Reductions in endothelial NOS have been shown to contribute to impaired mitochondrial biogenesis an ovine model of PAH 57 . Changes in mitochondrial activity in the pulmonary arteries during PAH results in alterations to redox signalling and impaired oxygen sensing. These changes cause activation of transcription factors usually associated with hypoxia, such as hypoxia-induced factor 1a (HIF-1a), which persist even under normoxic conditions 58,59 . HIF-1a can regulate mitochondrial fission, fusion and metabolism of pulmonary artery smooth muscle cells, which contribute to proliferation of smooth muscle cells and remodelling of the vessel wall 60,61 . The regulatory role of NO on mitochondria and the activation of these hypoxia-related pathways remain to be determined.

Endogenous inhibitors of NOS enzymes
Another potential mechanism for impairment of eNOS is the action of circulating inhibitors of NOS enzymes. The first of these inhibitors to be characterised was asymmetric dimethylarginine (ADMA) 56 . ADMA is a methyl derivate of arginine, and is produced by the physiological degradation of methylated proteins ( Figure 5). Metabolism of ADMA is mediated by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), forming citrulline and dimethylamine 62 . Loss of DDAH activity leads to accumulation of ADMA and reductions in NO signalling 63 . Functional polymorphisms in the gene for DDAH2 have associated with increased with increased levels of circulating ADMA, suggesting that there may be an association between genetic variations in the expression of DDAH and cardiovascular risk 64,65 . ADMA is made in cells by arginine methylation and is released into the cytosol upon protein degradation 66 . The concentration of ADMA within endothelial cells may be 10-20 fold higher than that seen in the plasma 67 . Plasma levels of ADMA predict cardiovascular events and mortality and have been associated with a wide range of conditions including hyperlipidemia, hypertension, peripheral arterial disease, chronic renal failure, chronic heart failure, diabetes mellitus type II, preeclampsia and pulmonary hypertension [68][69][70][71] .

Inhaled NO gas
The vasodilator and anti-proliferative actions of NO make it an attractive tool for pharmacological treatment of PAH. Administration of NO gas by inhalation has been shown to be beneficial to patients with PAH, particularly in paediatric cases [72][73][74] . However, the usefulness of inhaled NO as a treatment is limited due cost, technical difficulties and the fact that not all patients respond to the therapy. The ability of NO to oxidise haemoglobin to form methaemoglobin, which has been shown to be related the cumulative exposure to the gas, may also limit its effectiveness 75 . Rapid withdrawal of inhaled NO therapy can also have deleterious effects with levels of oxygenation and pulmonary hypertension returning to levels worse than those seen prior to the commencement of therapy 76,77 . However, developments in delivery technology have allowed optimisation of inhaled NO therapy by administering pulsed dosing of the gas ( Figure 6A) [78][79][80] . These studies have shown a reduction in adverse events, no changes in methaemoglobin levels and no reports of syncope 79,81 . The pulsed delivery of NO was shown to be as effective as continuous therapy in reducing mean pulmonary artery pressure and pulmonary vascular resistance ( Figure 6B). The use of inhaled NO therapy has recently been shown to be cost effective for the treatment of infants in a trial of NO therapy for chronic lung disease 82 . Despite the cost of the therapy, those patients who received inhaled NO had a shorted hospital stay and ventilation period.

Phosphodiesterases inhibitors
PDEs are responsible for the degradation of cGMP and thus terminating the effects mediated by NO. There currently 12 different isoforms identifed of these enzymes that differ in their 3-dimensional structures and tissue distribution 83,84 . PDE5 is present in the cardiovascular system; with abundance in the pulmonary vascular smooth muscle and endothelial cells 85,86 . Inhibition of PDE5 has been shown to mediate anti-proliferative effects on pulmonary artery smooth muscle cells 87 . The development of PDE5 inhibitors, such as sildenafil, are effective pharmacological agents for the treatment of patients with PAH due to either ability to bind to the PDE5 enzyme, inhibit the breakdown of cGMP and thereby prolong the effect of endogenous NO (Figure 7) 88 .
The SUPER-1 (Sildenafil Use in Pulmonary Arterial Hypertension) trial showed that sildenafil improves the functional capacity and pulmonary haemodynamics in patients with PAH, with only mild adverse effects 89 . These effects are sustained over a period of 3 years in a follow-up study (SUPER-2) 90 . In addition to sildenafil, tadalafil and vardenafil, two newer PDE5 inhibitors, are currently being assessed for use in patients with PAH. Tadalafil, which is a once a day oral preparation has been shown to improve exercise capacity, pulmonary haemodynamics and time to clinical worsening, effects that last for up to 1 year in the PHIRST-1 trial (Pulmonary Arterial Hypertension and Response to Tadalafil) 91 . Similar results have been reported for vardenafil in the EVALUATION (Efficacy and Safety of Vardenafil in the Treatment of Pulmonary Arterial Hypertension) trial, albeit in a much smaller cohort of patients 92 .
PDE5 is also expressed in the heart and some small studies have suggested that sildenafil is capable of preventing the remodeling of the myocardium seen in heart failure 93,94 . However, in larger clinical trials this effect of sildenafil was not seen 95 . It has recently been suggested that this may be due to the expression of PDE9 in myocytes. While PDE5 is found in contractile filaments, where is degrades cGMP produced by the action of NO on the soluble guanylyl cyclase (sGC) receptor, PDE9 is located near the T-tubular' invaginations of the plasma membrane 96 . PDE9 in believed to be responsible for the degradation of cGMP produced by atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) hormones acting on the guanylyl cyclase type A (GC-A) receptor that is located the cardiomyocyte plasma membrane.

cGMP activators
As an alternative to inhibition of the breakdown of cGMP, a new class of drugs are now being developed that directly activates soluble guanylate cyclase, thereby mimicking the effects of NO by increasing levels of cGMP. One such drug is riociguat, which has been assessed for efficacy in the treatment of PAH 97 . Results of the PATENT-1 (The Pulmonary Arterial Hypertension Soluble Guanylate Cyclase-Stimulator Trial) trial showed an improvement in exercise capacity, pulmonary haemodynamics, N-terminal pro-brain natriuretic peptide levels and time to clinical worsening. While adverse effects were similar to PDE5 inhibitors there was a greater incidence of systemic hypotension and haemoptysis.

Combination therapy
The mainstay of treatment for PAH is still the use of prostacyclin mimetic compounds and more recently endothelin-receptor antagonists and PDE-5 inhibitors. There are potential advantages in using combinations of these drugs to enhance the clinical benefits that can be obtained when the drugs are used in isolation. The use of strategies that combine treatment with PDE-5 inhibitors with PGI2 analogues and/or ET receptor antagonists have recently been comprehensively reviewed 43 . The results of some of the trials of combination therapy do show some promise, however some results are contradictory. For example the COMPASS-2 (Combination of Bosentan and Sildenafil versus Sildenafil Monotherapy on Pulmonary Arterial Hypertension) trial was able to show an improvement in the 6-minute walk test after 16 weeks of treatment but failed to delay the time to the first morbidity/mortality event 98 . However, meta-analysis of data from studies that involve combination therapy do indicate that this approach is effective in the treatment of PAH 9 .

Future therapies
The current pharmacological strategies to augment the supply or duration of the action of NO have a number of limitations mainly relayed to the routes of administration, duration of action and systemic effects. Novel nanoparticles are being developed for the treatment of a number of different conditions (Figure 8) 99,100 . Of particular interest are fibres that are capable of releasing NO and that are suitable for delivery via inhalation to deliver NO directly to the pulmonary vasculature 101,102 . These nanofibres have recently been shown to be able to continuously release NO over an 8-hour period and relax pulmonary vessel of rats with pulmonary hypertension in vitro 103 . These initial observations suggest that nanofibres capable of releasing NO may be of use in the future treatment of patients with PAH.

CONCLUSIONS
NO plays a significant role in the pulmonary circulation. Changes to its synthesis, release or signalling contribute to the pathogenesis of PAH. While targeting NO in has resulted in advances the treatment of the disease, the quality of life and the prognosis for patients with PAH remains poor. There remains a substantial amount of work to be done in order to optimise strategies that target to NO system or utilise the delivery of NO as a pharmacological agent.