British journal of pharmacology 2015 volume 172 part 6

in patients with pulmonary hypertension caused by systolicleft ventricular dysfunction, an indication with no approved medication, shows that treatment with riociguat did not meet the pr

Themed Section: Pharmacology of the Gasotransmitters

Andreas Papapetropoulos1,2, Roberta Foresti3,4and Péter Ferdinandy5,6

1Faculty of Pharmacy, University of Athens, Athens, Greece,2‘George P Livanos and Marianthi

Simou Laboratories’, Evangelismos Hospital, 1 st Department of Critical Care and Pulmonary

Services, University of Athens, Greece,3Université Paris-Est, UMR_S955, UPEC, F-94000, Créteil,

France,4Inserm U955, Equipe 12, F-94000, Créteil, France,5Pharmahungary Group, Szeged,

Hungary and6Department of Pharmacology and Pharmacotherapy, Semmelweis University,

pharmahungary.com

LINKED ARTICLES

This article is part of a themed section on Pharmacology of the Gasotransmitters To view the other articles in this section visithttp://dx.doi.org/10.1111/bph.2015.172.issue-6

The current themed issue collates a number of reviews and

original papers on the pharmacology of NO, CO and H2S

These three molecules have been grouped together to form a

family of signaling mediators that has become known as

‘gasotransmitters’ Authors of the articles in this issue are

members of ENOG- the European Network On

Gasotransmit-ters (COST Action BM1005, www.gasotransmitGasotransmit-ters.eu) ENOG

currently numbers more than 200 researchers from 24

European Countries and is funded through the European

Science Foundation Work from ENOG researchers and

col-leagues around the world have contributed to the

under-standing of the role of these molecules in physiology and

disease initiation and progression In addition, substantial

progress has been made in recent years in the pharmacology

of CO and H2S with the development of several CO- and

H2S-donors

The NO field is more than 3 decades old, but readers can

find in this issue reviews on novel aspects of NO/cGMP

sig-naling and on the therapeutic usefulness of components of

this pathway in cardiovascular diseases (Papapetropoulos

et al., 2015) with or without co-morbidities, such as

meta-bolic diseases (Pechánová et al., 2015) Sexual dysfunction

(Yetik-Anacak et al., 2015) and male infertility (Buzadzic et al.,

2015) are additional fields where modulation of NO signaling

bears therapeutic potential S-nitrosation, a NO-induced

post-translation modification of proteins is discussed by Santos

et al (2015) in the context of neuronal plasticity.

The H2S field has recently experienced a booming interest

as evidenced by the exponentially increasing number ofpublished articles in the field Papers on the role of H2S in

ischaemic diseases (Bos et al., 2015), as well as blood pressure

regulation and hypertension (Snijder et al., 2015;

Brancaleone et al., 2015) can be found in this issue

Interac-tions of H2S with myeloperoxidase are reported in an original

paper by Pálinkás et al (2015); the inhibitory effect of H2S onmyeloperoxidase is expected to contribute to the actions of

H2S in the context of inflammation

CO is a unique gasotransmitter, as its specific moleculartargets are still not known and it is a more stable molecule ascompared to NO or H2S However, the strong affinity of CO formetal centers can guide us in the search for the putativecellular targets E.g mitochondria rich in haeme-iron proteinsare potential candidates for molecular targets for CO This

concept is discussed in the review of Queiroga et al (2015) in

the context of the role of endogenous and exogenous CO inpathologies of the central nervous system In addition, ionchannels have been recognized as possible effectors of COsignaling and it appears that modulation of the activity ofchannel proteins is part of the mechanism contributing to the

physiological and therapeutic actions of CO (Peers et al.,

2015) Comprehensive and conceptually challenging reviews

in this issue also summarize the anti-inflammatory and tissue

protective activity of CO in specific conditions, such as acute

gastrointestinal inflammation (Babu et al., 2015) and

preec-lampsia (Ahmed and Ramma, 2015), where both H2S and CO

exert anti-angiogenic properties

The interaction of NO, H2S, and CO at the cellular

level can be observed in several pathologies, such as

ischaemic heart disease and hypertension, allowing several

pharmacological approaches for modulation of these

gas-otransmitters in order to protect the ischaemic heart with or

without co-morbidities (Andreadou et al., 2015) and to

regu-late blood pressure (Wesseling et al., 2015) Cardiovascular

co-morbidities may alter cardioprotective signaling including

gasotransmitters, therefore, co-morbidities have to be taken

into account when developing cardioprotective therapies as

reviewed recently elsewhere (Ferdinandy et al., 2014).

The current issue also contains practical guides for

scien-tists just entering into the interesting field of gasotransmitter

research, including technical guidelines to measure NO in

biological samples (Csonka et al., 2015), basic guidelines

for H2S pharmacology (Papapetropoulos et al., 2015), and

the chemical characteristics and biological behaviors of

CO-releasing molecules (Schatzschneider, 2015)

The editors of this themed issue hope that the papers

gathered here will be useful for established researchers

already involved in gasotransmitter research, as well as for

young scientists just planning to enter the field, and for

teachers and students interested in the physiology,

pathol-ogy, and pharmacology of NO, H2S and CO

Acknowledgements

Authors acknowledge the support of the COST Action BM

1005 PF is a Szentágothai Fellow of the Hungarian National

Program of Excellence (TAMOP 4.2.4.A/2-11-1-2012-0001)

References

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A, Ferdinandy P (2015) The role of gasotransmitters NO, H2S and

CO in myocardial ischaemia/reperfusion injury and

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conditioning Br J Pharmacol 172: 1587–1606

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Papapetropoulos A, Hobbs AJ, Topouzis S (2015) Extending thetranslational potential of targeting NO/cGMP-regulated pathways inthe CVS Br J Pharmacol 172: 1397–1414

Papapetropoulos A, Whiteman M, Cirino G (2015) Pharmacologicaltools for hydrogen sulphide research: a brief, introductory guide forbeginners Br J Pharmacol 172: 1633–1637

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P (2015) Cardiac NO signalling in the metabolic syndrome Br JPharmacol 172: 1415–1433

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Hettiarachichi NT et al (2015) Diverse mechanisms underlying the

regulation of ion channels by carbon monoxide Br J Pharmacol172: 1546–1556

Queiroga CS, Vercelli A, Vieira HL (2015) Carbon monoxide andthe CNS: challenges and achievements Br J Pharmacol 172:1533–1545

Santos AI, Martínez-Ruiz A, Araújo IM (2015) S-nitrosation andneuronal plasticity Br J Pharmacol 172: 1468–1478

Schatzschneider U (2015) Novel lead structures and activationmechanisms for CO-releasing molecules (CORMs) Br J Pharmacol172: 1638–1650

Snijder PM, Frenay AS, de Boer RA, Pasch A, Hillebrands J,

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Wesseling S, Fledderus JO, Verhaar MC, Joles JA (2015) Beneficialeffects of diminished production of hydrogen sulfide or carbonmonoxide on hypertension and renal injury induced by NOwithdrawal Br J Pharmacol 172: 1607–1619

Yetik-Anacak G, Sorrentino R, Linder AE, Murat N (2015) Gaswhat: NO is not the only answer to sexual function Br J Pharmacol172: 1434–1454

Themed Section: Pharmacology of the Gasotransmitters

Andreas Papapetropoulos1, Adrian J Hobbs2and Stavros Topouzis3

1School of Health Sciences, Department of Pharmacy, University of Athens, Athens, Greece,

2William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary

University of London, London, UK, and3Laboratory of Molecular Pharmacology, Department of

Pharmacy, University of Patras, Patras, Greece

Correspondence

Stavros Topouzis, Laboratory ofMolecular Pharmacology,Department of Pharmacy,University of Patras, Patras

combinations or second-generation compounds, are also being assessed in additional experimental disease models and inpatients in a wide spectrum of novel indications, such as endotoxic shock, diabetic cardiomyopathy and Becker’s musculardystrophy There is well-founded optimism that the modulation of the NO-sGC-cGMP pathway will sustain the development

of an increasing number of successful clinical candidates for years to come

The recent progress in the generation of additional,

therapeu-tic molecules that target the NO transduction pathway is in

large part due to a more detailed understanding of the

bio-chemical and mechanistic complexities of the downstream

pathways this molecule triggers That is, the soluble GC

(sGC)–cGMP axis cGMP is a ubiquitous intracellular

signal-ling molecule that affects a wide spectrum of cellular, and

thus physiological, processes from cell growth and apoptosis

to ion channel gating Especially in the CVS in which it has

been best studied, cGMP regulates many vital homeostatic

mechanisms, including endothelial cell permeability,

vascu-lar smooth muscle contractility and cardiomyocyte

hypertro-phy (Francis et al., 2010) Of the two distinct GC systems that

generate cGMP, this review exclusively focuses on the

contri-bution of the NO-responsive arm to the detriment of the

cGMP pool generated by natriuretic peptide hormones acting

on membrane-bound, particulate forms of GC Whereas there

is considerable functional convergence of the two systems

downstream, there is overwhelming evidence of spatial

com-partmentalization that results from the specific cellular

co-localization of both the cGMP-generating systems as well

as the cGMP-degrading PDEs, exemplified by the ability of

PDE2 to selectively interfere with the natriuretic-stimulated

cGMP pool, whereas PDE5 targets mainly the cytosolic cGMP

pool, in cardiomyocytes (Castro et al., 2006; Piggott et al.,

2006; Nausch et al., 2008; Tsai and Kass, 2009; Zhang and

Kass, 2011)

This review will highlight the molecules and mechanisms

within this pathway whose further study has recently

gener-ated successful entries in the medical arsenal, including use in

some novel medical indications, thus showing great future

promise in contributing to the treatment and elimination ofhuman disease, especially disorders of the CVS

Basic biology of the NO-sGC-cGMP pathway

Enzymatic generation of NO

Three isoforms of NOS exist, each one with a different pattern

of expression (Alderton et al., 2001): neuronal NOS (nNOS or

NOS-1), inducible NOS (iNOS or NOS-2) and endothelial NOS(eNOS or NOS-3) nNOS and eNOS are expressed constitu-tively whereas iNOS is not found in healthy cells but proteinexpression is induced following tissue injury or infection(Nathan, 1997) NOSs are capable of associating with the cellmembrane, with cytosolic proteins or with the cytoskeleton,

thus exhibiting dynamic subcellular localization (Oess et al.,

2006) NOSs facilitate the five-electron oxidation of the minal guanidino moiety of the semi-essential amino acidL-arginine, utilizing NADPH and BH4 as electron sources, togenerate NO and L-citrulline in the presence of molecular

ter-oxygen (Alderton et al., 2001).

The regulation of NO bioavailability is complex and

con-trolled by numerous mechanisms impacting directly NO

levels, including NOS expression, substrate provision andchemical inactivation For example, production of reactive

oxygen species can inactivate NO (Münzel et al., 2005), and

endogenous asymmetric methylarginines appear to act asNOS inhibitors (Leiper and Nandi, 2011; Caplin and Leiper,2012) Arginase activity decreases the availability of the NOSsubstrate, L-arginine (Morris, 2009), uncouples NOS (result-ing in generation of cytotoxic superoxide) and is thought to

underlie nitrate tolerance (Khong et al., 2012) Modulation of

Tables of Links

TARGETS

β2-adrenoceptor Arginase

Endothelin receptors COX

Ligand-gated ion channelsb Endothelial NOS (NOS3)

NMDA receptor Inducible NOS (NOS2)

Nuclear hormone receptorsc Neuronal NOS (NOS1)

Glucocorticoid receptor PDE family

Isoprenaline TNF-αIsosorbide mononitrate YC-1

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://

www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are

permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (a,b,c,d Alexander et al., 2013a,b,c,d).

eNOS–caveolin interactions (Garcia-Cardena et al., 1996) acts

as an on/off switch for enzyme turnover and, more recently,

interactions of NO with somatic haemoglobin (Straub et al.,

2012) can reduce NO bioavailability Furthermore,

pharma-cological enhancement of NO signalling can also be achieved

indirectly For example, stimulation of theβ3-adrenoceptor in

the heart has been shown to be coupled to the NO–cGMP

pathway, to increase NO bioactivity and to prevent

experi-mental maladaptive myocardial remodelling caused by

iso-prenaline or angiotensin II, an effect that deserves to be

explored further clinically (Belge et al., 2014) Several

mol-ecules targeting the above mechanisms have been developed

and evaluated preclinically (e.g a NOS–caveolin disruptive

peptide; Bucci et al., 2000); fewer have advanced in clinical

trials The latter include the arginase inhibitor

N-hydroxy-nor-arginine, investigated in a phase I trial in coronary

disease (Shemyakin et al., 2012; NCT02009527) However, no

clinical approval of molecules targeting these mechanisms

has yet validated these approaches

cGMP biosynthesis in response to NO

The major biosensor of the generated NO is the enzyme sGC,

which is found as an obligate heterodimer ofα (α1 andα2)

andβ1 subunits; the α1β1 dimer seems to be the prevalentactive form in most tissues with the exception of the nervoussystems where equal amounts ofα1/β1andα2/β1are detected.Each sGC subunit consists of (i) an N-terminal regulatory,haem-NO/oxygen (H-NOX) domain; (ii) a central Per-Arnt-Sim domain; (iii) a coiled-coil domain; and (iv) a C-terminalcatalytic domain (Derbyshire and Marletta, 2012) There isone haem prosthetic group per heterodimer (Figure 1) thatserves as the NO sensor and that is stimulated by nM con-centrations of NO leading to an increase in enzymatic activity

up to 400-fold (Kamisaki et al., 1986; Tsai and Kass, 2009).

Theα and β subunits have been proposed to be organized in

a parallel fashion and the low basal activity of sGC is thought

to result from the inhibitory action exerted by the binding ofthe catalytic domain to the regulatory domain; this inhibi-tion is relieved upon NO binding The presence of a reduced(Fe2 +, ferrous) haem group is critical in NO sensing by sGC.For example, environmental cues, that increased the presence

of reactive species such as superoxide (.O2 −) and peroxynitrite(ONOO−) are translated into changes in the redox status ofthe haem group and therefore in the ability of sGC to respond

to low concentrations of NO (Weber et al., 2001; Stasch and

Hobbs, 2009; Figure 1) The implications of this in disease are

Figure 1

Schematic representation of the major targetable components of the NO pathway Disease-modifying NO can be generated from three main,well-studied sources: (i) cellular conversion from L-arginine; (ii) bacterial-based, enterosalivary bioconversion of food nitrates; and (iii) nitrate drugssuch as glyceryl trinitrate, either spontaneously or through cellular conversion The bioavailability of NO is regulated by its generation by thesynthetic NOS enzymes and by the tissue complexation and conversion of NO, for example, to nitrosyl-free radicals Initially, NO bioactivity is inmajor part determined by its best-described cellular ‘biosensor’: sGC coupled to reduced haem sGC ‘stimulators’ such as riociguat, which wasrecently approved for treatment of two forms of PH, can by themselves activate sGC or synergize with NO Chemical modification of sGC oroxidation of the haem prosthetic group and dissociation from sGC can occur in pathophysiological situations such as PH and heart ischaemia.Apo-sGC has an impaired ability to respond to NO, thus ‘uncoupling’ the NO pathway This form of sGC can be ‘resuscitated’ by sGC ‘activators’such as cinaciguat and ataciguat PDEs are themselves regulated by and participate in the catabolism of cGMP PDE5 inhibitors such as sildenafiland tadalafil are approved for erectile dysfunction and treatment of PH NO pathway modifying drugs are increasingly evaluated in clinical trials

in indications as varied as heart failure, traumatic cerebral oedema and forms of skeletal muscle dystrophies

crucial: it is thought that oxidative stress, a typical trigger for

cardiovascular disease, can produce an NO-unresponsive

(Fe3+, Ferric) sGC that is rapidly ubiquitinylated and degraded

(Evgenov et al., 2006; Stasch and Hobbs, 2009) Furthermore,

this sGC ‘uncoupling’ may result from S-nitrozation of

vicinal thiols in theβ1subunit in addition to oxidation of the

haem group (Stasch et al., 2006; Sayed et al., 2008) Such

impairment of sGC activity in cardiovascular disease, coupled

to concomitant decreases in NO bioavailability, has been the

bedrock on which novel NO and/or haem-independent sGC

stimulators and activators have been developed and which

will be examined below (Evgenov et al., 2006; Follmann et al.,

2013; Gheorghiade et al., 2013).

In addition to its upstream, direct effects on NO

availabil-ity and sGC function, cellular oxidative stress may also

interfere with the NO/cGMP pathway by inducing

post-translational activation of the downstream cGMP effector

PKG-Iα and thus affect adversely the progress of disease,

something that has been experimentally shown to occur in

sepsis (Rudyk et al., 2013) Due to this complex, and in some

cases antithetical, regulation of NO bioactivity, in such

pathological settings a dual-pronged therapeutic approach,

that combines upstream restoration of physiological cGMP

generation and pharmacological intervention (e.g

anti-oxidants) could be optimal to preserve the physiological

function of downstream effectors

It is important to note that the downstream biochemical

pathway of NO is far from limited to cGMP-mediated effects:

cGMP-independent changes are undeniably part of the NO

signalling repertoire, including NO-triggered protein

S-nitrozation (Lima et al., 2010) and effects on mitochondrial

respiration and oxygen utilization (Erusalimsky and

Moncada, 2007) One should keep in mind, however, that

genetic inactivation of sGCβ1 (Friebe et al., 2007) and

cGMP-dependent kinase I (PKG1) abolishes the hallmark

physiologi-cal effect of NO, that is, vasorelaxation (Pfeifer et al., 1998),

emphasizing the crucial involvement of cGMP in the effects

of NO It is also clear that the ‘canonical’ (cGMP dependent)

NO pathway has provided the major impetus for translational

progress and thus constitutes the central focus of the review

Direct downstream signalling of cGMP

Two main enzyme families are directly regulated and respond

to cGMP, to impact the pathophysiology of the CVS:

cGMP-dependent PKs (PKGs) and PDEs (Figure 1) In addition, ion

channel function is also directly or indirectly (e.g via

PKG-dependent pathways) regulated by cGMP levels, although

this phenomenon is largely restricted to sensory transduction

(Biel and Michalakis, 2007; Francis et al., 2010) To date,

suc-cessful translational efforts have, however, focused primarily

on the two upstream enzymatic targets: sGC and PDE The

ability of sGC to associate with the plasma membrane (Linder

et al., 2005) and the possible compartmentalization of cGMP

degrading PDEs (Castro et al., 2006; Nausch et al., 2008;

Zhang and Kass, 2011) may further complicate the

down-stream functions of spatially regulated cGMP levels and the

therapeutic targeting of enzymes that regulate its levels in

distinct diseases

The dominant PKG in the CVS is PKG type 1, which

consists of two isoforms: α and β (Hofmann et al., 2006;

Burley et al., 2007) The binding of cGMP to a regulatory

region of the kinase results in a conformational change that

‘unrepresses’ the catalytic activity of the kinase and permitsphosphorylation on Ser/Thr residues of client proteins Phar-macological targeting of PKG is attractive but has not beensuccessful up to now, because selective PKG activators andinhibitors are lacking In addition, PKG inhibition may result

in smooth muscle dysfunction, based on experimental dence provided by mice with genetic deletion of cGMP kinase

evi-I (Pfeifer et al., 1998) Conversely, use of PKG activators to

mimic the effects of sGC and pGC turnover is theoreticallydesirable in cardiovascular disease, but chronic use may beultimately undesirable, given that gain-of-function geneticmutations in PKG found in humans are causally associated

with aortic aneurysms and dissections (Guo et al., 2013).

The second cGMP-responsive system that has been studied comprises the PDE family of cyclic nucleotide-hydrolyzing enzymes, which have arguably been the mostsuccessful ‘cGMP-based’ therapeutic targets Of the 11 PDEfamilies (PDE1-11, each consisting of one to four isozymesand their multiple isoforms), PDEs-2, -3, -5, -6 and -11 areregulated by cGMP, of which PDE2, 3 and 5 are expressed inthe constituent cells of the CVS, with PDE11 being found inthe heart PDEs exist as dimers, each monomer comprises acharacteristic for the isotype N-terminal regulatory domainand a relatively high homology C-terminal catalytic domainthat can undergo post-translational prenylation or phospho-rylation (Conti and Beavo, 2007; Keravis and Lugnier, 2012).Whereas PDE2 and PDE5 are activated by cGMP binding totheir GAF regulatory domain, PDE3 is inhibited by competi-tive binding of cGMP to its catalytic site Of these three PDEs,PDE2 and PDE3 can hydrolyse both cGMP and cAMP, whilePDE5 is selective for cGMP (Bender and Beavo, 2006; Contiand Beavo, 2007) PDE5, which is highly expressed in thecorpus cavernosum and in the lung, is the target of small-molecule inhibitors that have been approved to treat erectiledysfunction and pulmonary arterial hypertension [PAH;World Health Organization (WHO) group I] (Rosen and

well-Kostis, 2003; Croom et al., 2008) Additional preclinical data

support a role for PDEs 1, 2, 3 and 10 in pulmonary tension, with proof-of-concept studies in cells and tissuesfrom patients with the disease, implying that pharmacologi-cal blockade of other PDE isoforms might be beneficial

hyper-(Phillips et al., 2005; Schermuly et al., 2007; Tian et al., 2011; Bubb et al., 2014) Further consideration of the therapeutic

potential of PDE inhibitors, particularly PDE5, is discussednext

New lead molecules targeting the NO-sGC-cGMP pathway

Innovation in targeting the NO-sGC-cGMP pathway derivesfrom either (i) development of new molecular entities; or (ii)extended clinical applications of already-approved therapeu-tic molecules Research that has been conducted in the past10–15 years has produced novel lead therapeutic moleculesthat have entered clinical evaluation and, on occasion, arenow approved medicines

Two main categories of novel chemical entities in theearly or late clinical arena that target the NO-sGC-cGMP axis

are briefly explored below First, there are established drugs

that have been coupled to an NO-donating group to alleviate

undesirable side effects of the ‘parent’ molecule However, far

more innovative is the second category, which includes sGC

‘stimulators’ and ‘activators’ and therefore this review will

draw attention to their preclinical pharmacology and mode

of action

NO-donating anti-inflammatory drugs

The most clinically advanced, major drug group that has

been used as NO-donating, ‘carrier’ scaffold has been the

steroidal and non-steroidal anti-inflammatory drugs

(NSAIDs), including aspirin These hybrid molecules are

being tested in a wide array of indications, from colon cancer

prophylaxis to reduction of vascular complications due to

hypercholesterolaemia, not all of which can be thoroughly

covered by this review

The molecules that are perhaps closest to approval are

NSAID conjugates whose therapeutic benefit relies (i) on the

presumed gastroprotection that released NO would provide to

the NSAID moiety, given the increased possibility of ulcer

development (del Soldato et al., 1999; Wolfe et al., 1999;

Bandarage and Janero, 2001); and (ii) on the

counterbalanc-ing of the modest, but significant, effect on blood pressure

that certain NSAIDs can cause in some patient populations

and that can limit the health benefit of the anti-inflammatory

drug (White et al., 2011) NSAIDs are among the most

pre-scribed drugs in the world; however, it is now well established

that their use carries the risk of upper gastrointestinal damage,

including life-threatening bleeding complications, as side

effects of their mode of action The risk varies with the NSAID

used and is especially frequent in certain populations prone to

bleeding (Chan et al., 2007) There are approved

pharmaco-logical strategies to prophylactically reduce the risk of

gastro-intestinal events due to NSAID intake, including, for example,

co-administration of proton pump inhibitors (Chan et al.,

2007; Graham and Chan, 2008)

There is now ample experimental evidence from

preclini-cal models that NO-releasing forms of approved steroidal and

NSAIDs, including COX inhibitors such as aspirin and

gluco-corticoids such as prednisolone and flunisolide, exhibit

similar or increased efficacy and a more favourable side effect

profile than the parent molecules in several preclinical

disease settings (Fiorucci et al., 2002; Paul-Clark et al., 2003;

Turesin et al., 2003; Wallace et al., 2004) Such

anti-inflammatory drug NO conjugates have been experimentally

shown to modulate ovarian (Bratasz et al., 2008) skin

(Chaudhary et al., 2013) or intestinal (Williams et al., 2004)

solid tumour growth, exert anti-inflammatory activity with

reduced symptoms of gastric damage properties (Wallace

et al., 2004; Fiorucci et al., 2007) and protect against or

accel-erate improvement of experimental colitis (Fiorucci et al.,

2002; Zwolinska-Wcislo et al., 2011) The increased

anti-inflammatory efficacy of at least one of them, the

predniso-lone derivative NCX-1015, may in part be attributed to

glucocorticoid receptor nitration resulting in more robust

signalling (Paul-Clark et al., 2003).

A number of NO-conjugated COX inhibitors have also

been evaluated in clinical trials For example, NCX4016 (an

aspirin-NO conjugate) has completed clinical testing in

pre-venting colorectal cancer in patients at high risk for

develop-ing this disease (ClinicalTrials.gov identifier: NCT00331786)and in improving walking distance in patients with periph-eral arterial occlusive disease (NCT01256775); however, nopublished report of trial outcomes is available at the writing

of this review Another 13 week clinical trial involves anaproxen–NO conjugate (naproxcinod) that is intended

to treat ‘hypertensive’ patients (mean arterial pressure

>125 mmHg) with osteoarthritis In these individuals, roxen induces a small rise (3–8 mmHg) in systolic BP, whichincreases significantly the risk of cardiac complications in thispopulation Naproxcinod exhibits a much lower tendency toincrease systolic BP than naproxen, sparing the need foranti-hypertensive drugs taken concomitantly by this popula-

nap-tion (White et al., 2011) However, the FDA has withheld

approval until longer term effects of the drug are presented

In sum, none of these molecules has yet progressed to scale clinical evaluation, while, for the moment, the clinicaluse of NO-donating NSAIDs awaits convincing clinical datathat for approval (Fiorucci and Distrutti, 2011)

large-sCG stimulatorsPharmacology and mode of action. Given that reduced NOproduction is a defining feature of many cardiovascular dis-eases, including PH, the use of PDE inhibitors is likely to belimited as the efficacy of such molecules is dependent onendogenous cGMP generation Thus, compounds that acti-vate sGC directly, or that synergize with NO in activating theenzyme, appear a perfect fit as drug candidates in such indi-cations The initial discovery, by Taiwanese researchers in the

mid-1990s, of the first ‘sGC stimulator’, YC-1 (Wu et al.,

1995), was paralleled by a wide search performed by a variety

of pharmaceutical companies for molecules that could act indual fashion: they synergize with NO in stimulating sGC anddirectly stimulate the enzyme in the absence of NO Bothactivities are, however, dependent on the presence of a

reduced, sGC-bound haem moiety (Hoenicka et al., 1999).

The mechanistic basis of sGC stimulation by these ecules has been extensively studied, but not conclusively

mol-elucidated (Follmann et al., 2013), mainly because there are

no X-ray data of the full-length crystallized enzyme Ramanspectroscopic studies with sGC stimulators and structuralmodelling studies (based on the somewhat tenuous similarity

to the AC catalytic domain) suggest that molecules such asYC-1 and BAY 41-2272 (i) induce a (indirect) change in theprosthetic haem group geometry that has bound NO, makingthe enzyme more active and stabilizing the nitrosyl–haemcomplex; and (ii) photoaffinity labelling of BAY-41-2272 andYC-1 analogues results in labelling of theα-subunit, follow-ing binding of the compound to a domain distinct fromthe catalytic site However, it is not absolutely clear that thebinding itself occurs on theα-subunit It is possible that thesite of binding is in the interface between the sGC subunitsand thus elicits an allosteric interaction that results in a moreactive conformational shift of the enzyme and in the label-ling of theα-subunit (reviewed in Derbyshire and Marletta,

2012; Follmann et al., 2013) Alternatively, sGC stimulators

have been suggested to relieve an autoinhibitory interactionbetween the H-NOX domain in the N-terminus, which har-bours the haem moiety and the C-terminus catalytic domain(Winger and Marletta, 2005) In a recently published study,

Purohit et al (2014) demonstrated that YC-1 binding to theβ1

sGC subunit overcomes the allosteric inhibition by the α1

subunit In all, the exact binding site of the sGC stimulators

has not been assigned with certainty yet, and more structural

studies have to be performed to finally understand how sGC

stimulators bind to the protein

Of the many molecules of the sGC stimulator class that

have been developed, riociguat (BAY 63-2521) is the one that

finished first in the translational race that led to its approval

in the past year in the United States, Canada and in the

European Union for the treatment of two forms of PH

(Conole and Scott, 2013) Many sGC stimulator molecules,

including YC-1, were abandoned because of lack of selectivity

(YC-1 also inhibited PDEs) and poor pharmacokinetic

char-acteristics (Stasch and Hobbs, 2009) One instructive reason

for riociguat’s success may be that very early, before full

preclinical evaluation, all fellow candidate molecules were

evaluated and discarded if they possessed a poor

pharmacoki-netic profile (Follmann et al., 2013), allowing research to

concentrate on candidates that were potent, selective and

possessed a favourable bioavailability/pharmacokinetic

profile At the outset, riociguat showed good bioavailability

and lack of interaction with the CYP metabolizing system,

thus presenting the considerable advantage of future

co-administration with other drugs (Follmann et al., 2013) In

vitro characterization of the drug showed strong synergy in

combination with NO, ability to induce sGC activity in the

absence of NO and dependence on a reduced haem prosthetic

group The preclinical evaluation of riociguat in key

experi-mental animal models in vivo displayed, crucially, a

long-preserved (several weeks) hypotensive effect in rats made

tolerant to organic nitrates, effective inhibition or reversal of

pulmonary vasoconstriction and remodelling

(musculariza-tion of small pulmonary arteries, hypertrophy of the right

ventricle) in the monocrotaline model of PH (Schermuly

et al., 2008; Stasch et al., 2011; Lang et al., 2012), and

reduc-tion of heart and kidney fibrosis in the Dahl hypertensive rat,

resulting in increased survival rates over time (Geschka et al.,

2011)

Clinical success of the sGC stimulator, riociguat. There are two

clinical areas where considerable progress has been made in

the last few years with the sGC stimulators: PH and heart

failure, with pulmonary hypertension being the most

suc-cessfully targeted clinical indication, based on riociguat’s

approval

PH is a progressive, debilitating, multifactorial disease and

exacts a high socio-economic toll Most of the approved

current treatments target one subgroup: PAH, a

life-threatening form of the disease that is characterized by

increased pulmonary vascular resistance, excessive

remodel-ling of small vessels and of the pulmonary artery that lead,

over time, to right heart failure and death (Baliga et al., 2011;

Galiè et al., 2011; Schermuly et al., 2011) Available

treat-ments for PAH include endothelin receptor antagonists, PDE

inhibitors, prostacyclin analogues and Ca2+channel blockers

(Baliga et al., 2011; Galiè et al., 2011) The necessity of

addi-tional supportive drug therapy to treat concurrent

patho-physiologies, which includes oral anticoagulants, digoxin for

arrhythmias and diuretics to regulate fluid accumulation and

blood pressure (reviewed by Galiè et al., 2011) increases the

risk of undesirable drug–drug interactions, especially with the

anticoagulants Approval of any new pharmacologicaloptions that are well-tolerated and display minimal drug–drug interactions would be a welcome addition to this thera-peutic arsenal

Among other PH forms, persistent PH of the neonate can

be effectively treated with administration of inhaled NO

(Roberts et al., 1992; Vosatka et al., 1994), but NO donors are

not clinically useful for chronic treatment of PH because ofpartial patient response, development of severe toleranceover time, short-lived duration of the pulmonary vasodila-tion and the danger of methaemoglobinaemia with high NO

doses (Ichinose et al., 2004; Galiè et al., 2011).

The exact molecular ‘defect’ in the NO-sGC-cGMP axisthat may contribute to the development of the various forms

of pulmonary hypertension in adults remains debatable andexperimental and clinical data seem often contradictory

(Giaid and Saleh, 1995; le Cras et al., 1996; Xu et al., 2004).

Pharmacological potentiation of the NO pathway (Rossaint

et al., 1993; Klinger, 2007; Vermeersch et al., 2007; Geschka

et al., 2011) has been the basis for the development of

small-molecule inhibitors of PDE5A such as sildenafil and tadalafil,which were introduced in this clinical area in the past decade

(Galiè et al., 2009; 2011; Stasch and Hobbs, 2009) The issue,

particularly in PAH, is reduced NO bioavailability: PAH isconsidered an NO-deficient state (Stasch and Evgenov, 2013).Because sGC expression is maintained or even up-regulated

in PH, targeting it with a sGC stimulator (which can synergizewith NO) seems a particularly beneficial approach

Clinical trials with the sGC stimulator, riociguat, in twoforms of PH were successfully concluded in 2013: the treat-ment met primary end points in patients diagnosed with PAHand with chronic thromboembolic pulmonary hypertension(CTEPH or WHO group IV) In the phase III trial (PATENT 1ClinicalTrials.gov) in PAH patients who received riociguatalone or in combination with approved endothelin receptorantagonists or prostanoids for 12 weeks, the 6 min walkdistance (6-MWD) increased by 36 m compared with placebo

In addition, there was significant improvement in pulmonaryvascular resistance, cardiac output, N-terminal pro-B-typenatriuretic peptide (NT-proBNP) plasma levels, time to clini-cal worsening, WHO functional class, Borg dyspnoea scoreand quality-of-life assessment In addition, the benefit was

also manifest at 24 weeks (Ghofrani et al., 2013b) The

second, 16 week phase III trial (CHEST-1) included patientsdiagnosed with CTEPH who were either inoperable or showedpersistent or recurrent PH despite having undergone pulmo-nary endarterectomy, a standard surgical option for thisgroup for which no pharmacological options exist Riociguatincreased the 6-MWD by 46 m compared with placebo andproduced significant improvement in pulmonary vascularresistance, cardiac output, N-terminal pro-B-type natriuretic

peptide level and WHO functional class (Ghofrani et al.,

2013a) In both trials, the safety profile of the sGC stimulatorwas reassuring, a major plus that warrants further evaluation

of the molecule in additional indications

In addition to the above indication, riociguat is also beingtested clinically, and has shown beneficial effects, in proof ofconcept, pilot or phase II studies in patients with PH second-ary to interstitial lung disease and chronic obstructive pul-

monary disease (Bonderman et al., 2013; Hoeper et al., 2013;

Stasch and Evgenov, 2013) The first report of a phase IIb trial

in patients with pulmonary hypertension caused by systolic

left ventricular dysfunction, an indication with no approved

medication, shows that treatment with riociguat did not

meet the primary end point, which was the decrease in mean

pulmonary artery pressure at 16 weeks (Bonderman et al.,

2013); however, it improved the secondary outcomes cardiac

index and systemic and pulmonary resistance Despite an

attempt to decipher possible effects in patient populations

after stratification, the study was not powered or designed to

answer some critical questions, for example, whether

riociguat elicited pulmonary vasodilation (inferred by the

calculated drop in pulmonary vascular resistance) or whether

variation of the drug dose and duration of treatment in

spe-cific patient subpopulations would successfully reach the

primary end point The mitigated results may leave the door

open for a more prolonged trial, where long-term ventricular

function is monitored and where, given riociguat’s safety

profile, higher doses are tested Riociguat is also in early

clinical stage evaluation for improvement of flow to the digits

in Raynaud’s syndrome patients (NCT01926847)

sGC activators

Preclinical pharmacology of sGC activators. Additional

screen-ing of a compound library followscreen-ing the discovery of sGC

stimulators at Bayer and further examination of hits revealed

that a second series of dicarboxylic acids could up-regulate

sGC activity in an NO-independent and haem-independent

manner, thus inaugurating a quite different molecular class,

termed sGC activators More companies also arrived at

similar-acting molecules (Schindler et al., 2006; Costell et al.,

2012; Follmann et al., 2013) Most of the second-generation

molecules contain only one monocarboxylic acid moiety An

example of an activator that lacks carboxylic acid moieties

also exists (HMR176) The mechanistic basis for the mode of

action of sGC activators is arguably better understood than

that of sGC stimulators Data from functional, mutational

and spectroscopic studies indicate that sGC activators bind in

the haem cavity within the H-NOX domain of theβ1subunit,

competing with the native ligand (Pellicena et al., 2004;

Martin et al., 2010; Follmann et al., 2013) The His105in theβ1

H-NOX domain, which serves as a fifth coordination for the

haem iron and is crucial for sGC activation, is displaced from

the ‘inactive’ form, causing the rotation of the helix that

harbours His105to a degree that depends on the sGC activator

used (Follmann et al., 2013) In this way, this class of

com-pounds activate sGC in the absence of a haem moiety

(Pellicena et al., 2004; Follmann et al., 2013) Of the sGC

activators, the molecular mechanism of action of BAY

58-2667 (cinaciguat) has been characterized in most detail

(Martin et al., 2010) The carboxylic groups of BAY 58-2667

displace the haem propionic acids and interact with Tyr135

and Arg139of theβ1subunit and sGC activation results from a

signal transmission triad composed of His105, Tyr135and Arg139

(Schmidt et al., 2004).

Cinaciguat, and possibly other sGC activators, can

prevent the degradation of sGC subunits that occurs

follow-ing haem oxidation, apo-sGC formation and subunit

ubiqui-tination in disease conditions The ability of cinaciguat to

closely mimic haem binding rescues sGC from proteasomal

degradation by stabilizing the apo-sGC structure and thus

possesses a dual mechanism of action (maintenance of sGC

levels and sGC activation) in diseases associated with

increased oxidative stress (Evgenov et al., 2006; Martin et al., 2010; Follmann et al., 2013).

A more conclusive assessment of the sGC haem redoxstate in whole cells and in tissues would help improve deci-sion making on which diseases might benefit from the

administration of sGC activators (Ahrens et al., 2011) There

are two, recently described, methods that may allow this indifferent contexts in the future, provided that they are vali-dated and confirmed by other laboratories Fluorescencedequenching can be measured after the attachment of thebiarsenical fluorophore FlAsH to the haem moiety (Hoffmann

et al., 2011) via energy transfer from this fluorophore to the

haem However, this technique for now is limited to live cells

in vitro and has yet to be extended to in vivo applications In

addition, a biochemical determination can be performed byassessing the degree of sGC-Hsp90 complexation: the binding

of Hsp90 is limited to the haem-lacking enzyme and Hsp90 isdissociated once sGC has incorporated a haem prostheticgroup (Ghosh and Stuehr, 2012) Similar methods, onceestablished, could be very useful in better directing the thera-peutic applicability of sGC activators

This class of NO- and haem-independent sGC activators,therefore, raised the possibility of therapeutic use in situa-tions where sGC is present in its haem-free form Increasedlevels of apo-sGC (leading to its ubiquitination and protea-somal degradation) occur during oxidative stress, exemplified

by either full-blown, acute inflammatory responses or

chronic, low-level inflammation (Stasch et al., 2002; 2006) In

these situations, the effect of PDE inhibitors or sGC

stimula-tors is inherently limited (Evgenov et al., 2006) due to a lack

of intact NO–sGC signalling Thus, sGC activators have beenextensively characterized in preclinical models of disease todetermine if they offer a greater therapeutic potential Forexample, drugs modifying the haem-oxidized or haem-freeenzyme would target diseased tissue This proved to be thecase with encouraging results observed in models of myocar-dial infarction, hypertension or congestive heart failure

(reviewed by Follmann et al., 2013) Cinaciguat, in a fast

ventricular pacing model of congestive heart failure in dogs

(Boerrigter et al., 2007), reduced mean arterial, right atrial,

pulmonary artery and pulmonary capillary wedge pressure;increased cardiac output and renal blood flow; and preservedglomerular filtration rate and sodium and water excretion,making it a prime therapeutic candidate for cardiovascularindications where sGC is impaired because of oxidative stress

In addition, cinaciguat was shown to antagonize crucial

pro-fibrotic mechanisms in vitro (Dunkern et al., 2007), thought

to operate in many pathological remodelling processes inchronic cardiovascular diseases GSK2181236A a sGC activa-tor developed by GlaxoSmithKline, was tested in spontane-ously hypertensive stroke-prone rats on a high salt/fat diet,demonstrating organ-protective effects and reducing left ven-

tricular hypertrophy (Costell et al., 2012) Yet another sGC activator, HMR 1766 (ataciguat), was shown to improve ex

vivo vascular function and reduce platelet activation (Schäfer

et al., 2010) Ataciguat also prevents and reverses pulmonary

vascular remodelling and right ventricular hypertrophy in a

mouse model of PH (Weissmann et al., 2009) Collectively,

these results warranted clinical evaluation in similarindications

Clinical testing of sGC activators. HMR1766 (ataciguat) has

been evaluated in two indications and trials have been

com-pleted: in the first, the primary end point was the reduction

of pain in patients with neuropathic pain (NCT00799656)

and in the second, the primary end point was improvement

of intermittent claudication in patients with Fontaine stage II

peripheral arterial disease (NCT00443287) The conclusions

from these trials are still being awaited

Cinaciguat has been tested in patients with acute

decom-pensated heart failure, an indication where it seemed to be

perfectly poised to succeed because of the strong evidence of

NO pathway impairment in this disease and because of the

experimentally based ability of the drug to limit fibrosis

(reviewed by Tamargo and López-Sendón, 2011; Gheorghiade

et al., 2013) Cinaciguat was delivered by i.v administration

at dose rates of 50–150μg·h−1 and patients were monitored

for up to 48 h The trial, however, was terminated

prema-turely because of an increased occurrence of hypotension

with all three doses (Gheorghiade et al., 2012; Erdmann et al.,

2013a), which is an unfavourable occurrence in this patient

population; in addition, there was no discernible effect of

this treatment on either dyspnoea or on cardiac index and

the small patient numbers did not allow stratification

(Gheorghiade et al., 2012).

Although some of these clinical results have been

disap-pointing, human genome-wide association studies have

iden-tified mutations in the genes encodingα1(GUCY1A3) andβ1

(GUCY1B3) subunits of sGC, and in the sGC-stabilizing

protein CCTη, which increase the risk of hypertension,

thrombosis and myocardial infarction (Ehret et al., 2011;

Erdmann et al., 2013b) Thus, there is strong evidence for a

direct involvement of sGC impairment in thromboembolic

human disease and in the regulation of blood pressure

Indi-viduals carrying such mutations may be prime candidates for

treatment with sGC stimulators or activators, as they are

likely to be disease modifying However, the ethnic

diver-gence in phenotype which is associated with GUCY SNPs

suggests that patient stratification to sGC modulating drugs

may be necessary

NOS cofactor supplementation

One particular approach aiming to augment NO production

is supplementation of the NOS cofactor tetrahydrobiopterin

(BH4) Its bioavailability is reduced in a variety of

cardiovas-cular pathologies, such as in atherosclerosis, at least in part as

a result of overproduction of oxygen radicals, and correlates

with NOS uncoupling (Förstermann and Li, 2011; Li and

Förstermann, 2013) Pharmacological augmentation of BH4,

therefore, aims to re-establish a healthy cofactor

stoichiom-etry (Alkaitis and Crabtree, 2012; Starr et al., 2013) and direct

eNOS catalytic activity towards producing NO rather than

O2 − To achieve just that, several clinical trials have been

conducted or are in progress in disease conditions that

include systolic or systemic hypertension and peripheral

artery disease; however, for the moment, results from these

trials either do not reveal statistically significant changes

or are still not reported (Alkaitis and Crabtree, 2012;

Cunnington et al., 2012) Characteristically, supplementation

of BH4 in patients with coronary artery disease, although it

produced increased levels of BH4 in saphenous vein (but not

in internal mammary artery), resulted in the presence of the

oxidation product BH2, which lacks NOS cofactor properties,and failed to either reduce superoxide levels or improve vas-

cular function (Cunnington et al., 2012) These results

dem-onstrate that, while supplementation of NOS cofactor(s) isbased on a sound therapeutic rationale, the establishment of

a favourable target BH4 : BH2 ratio is hard to achieve fore, a fundamentally different approach targeting BH4 may

There-be more useful, such as indirectly increasing its recycling andpreservation Indeed, in atherosclerotic patients, supplemen-tation with 5-methyl-tetrahydrofolate (which preventsperoxynitrite-driven oxidation of BH4) has been shown toreduce peroxynitrite-mediated BH4 oxidation, to amelioratethe BH4/total biopterin ratio and to increase NOS coupling,

thus preserving in vivo and ex vivo vascular endothelial tion (Antoniades et al., 2006).

func-Repositioning of existing medicines and combination approaches

New molecular entities and modes of action have tionably boosted excitement in the NO field, and haveadvanced understanding of the physiology and pathology ofsGC–cGMP signalling However, significant translational pro-gress has also been made with older, approved drugs Quite afew of these have been, or are currently being, evaluated inindications that are either poorly served by available medica-tions, or where an improvement of the currently obtainabletherapeutic effect is desired

unques-One such example is the small (six patient), pilot clinicaltrial with a combination of the tried-and-tested organicnitrate, isosorbide mononitrate (ISMN), and the PDE5 inhibi-tor, sildenafil, in achieving better regulation of the bloodpressure in patients afflicted with ‘resistant’ hypertension

(Oliver et al., 2010) Monotherapy with either drug alone

effectively reduced brachial systolic and diastolic blood sure, and central systolic and diastolic arterial pressure Com-bination of sildenafil and ISMN elicited significantly strongerreduction of brachial systolic blood pressure and central arte-rial systolic pressure, compared with either drug alone.Reduction of central arterial pressure with the combinationreached a maximum of 26/18 mmHg (systolic blood pressure/

pres-diastolic blood pressure) compared with placebo (Oliver et al.,

2010), thus opening the way for a study involving morepatients and evaluation of longer administration of this com-bination in this challenging patient population

Sildenafil also showed improvement in non-ischaemic,non-failing diabetic cardiomyopathy (i.e at a relatively earlystage) in a small, 3 month trial in 59 diabetic patients(NCT00692237), improving left ventricle contraction andpreventing cardiac remodelling through, presumably, directintramyocardial effects, independent of endothelial vasodila-

tation (Giannetta et al., 2012) Longer term results are

expected in the next 48 months

More impressively, in a 1 year prospective trial in 45patients with stable, systolic heart failure, sildenafil, at 6months and 1 year, improved left ventricle ejection fractionand elicited reverse remodelling of left atrial volume indexand left ventricle mass index These structural and functionalameliorations by sildenafil were coupled with improved exer-

cise performance, ventilation efficiency and quality of life,

thus making sildenafil the first PDE5 inhibitor that

demon-strably elicits structural and functional changes in the human

heart (Guazzi et al., 2011) A year later, the same group

(Guazzi et al., 2012) reported that sildenafil succeeded, in a

group of patients with heart failure that presented oscillatory

breathing during exercise (attributed to pulmonary

vasocon-striction), to almost eliminate (in∼90% of the patients at 6

and 12 months) oscillatory breathing, a sign of poor

progno-sis for the progress of the disease, as well as to improve

functional performance These results were accompanied by

reductions of pulmonary vascular resistance and pulmonary

arterial pressure Unfortunately, in the longer term follow-up

RELAX study (Effectiveness of Sildenafil at Improving Health

Outcomes and Exercise Ability in People With Diastolic Heart

Failure), treatment of HFpEF patients with sildenafil failed to

produce a significant change in exercise capacity, its primary

outcome measure (Redfield et al., 2013), despite the positive

outcome achieved in systolic heart failure patients (Guazzi

et al., 2011).

Sildenafil was also tested in a 12 week clinical trial

(NCT00517933) in patients with idiopathic pulmonary

fibro-sis (Zisman et al., 2010) Although the primary end point

(increase in the 6 min walk distance by more than 20%) was

not met, secondary symptomatic end points such as

oxygena-tion, dyspnoea and quality of life score were improved by

sildenafil (Zisman et al., 2010), raising the possibility of an

expanded clinical investigation in the future

Yet another approved PDE5 inhibitor, tadalafil, was the

second molecule of its class to be approved for PAH in 2009

(Rosenzweig, 2010) Furthermore, in 2012, in a small pilot

study, tadalafil proved effective in normalizing blood flow to

the muscles of patients with Becker’s muscular dystrophy

(BMD) This genetic disease is linked to mutations in the gene

encoding the skeletal muscle protein dystrophin, which

induces defective sarcolemmal targeting of proteins, among

which nNOSμ, and progressive muscle damage and wasting

(Bushby et al., 2010a,b) There is no pharmacological

treat-ment directed to this disease, which is associated with

car-diomyopathy and results in loss of ambulation The

investigators tested a small patient group (and a matched

cohort control, n= 10 each) for restoration of the

exercise-induced attenuation of reflex sympathetic vasoconstriction

This is a physiological reflex that optimizes perfusion to the

exercising muscles This reflex was absent in 9/10 men

carry-ing the disease and tellcarry-ingly correlated with misscarry-ing

sar-colemmal nNOSμ Tadalafil, given once, normalized this

adaptive blood flow in response to sympathetic

vasoconstric-tion in all participant patients (Martin et al., 2012) and can

therefore benefit people with BMD by preventing muscle

damage due to pathological vasoconstriction during exercise

In addition, in a promising preclinical study in a related

indication sildenafil reversed cardiac dysfunction in the mdx

mouse model of Duchenne muscular dystrophy (Adamo

et al., 2010).

It can safely be said that the expectation, broadly shared

by the research community, that PDE5 inhibitors would be

clinically useful in treating heart failure (Zhang and Kass,

2011) or other diseases with a critical cardiac and/or vascular

dysfunction (Kukreja et al., 2011) is slowly but steadily being

fulfilled, despite the occasional hiccup The clinical success

and failures of PDE5 inhibitors reveal both the potential andthe limitations of their therapeutic utility More diversifiedtrials may be expected to near completion in the next 2–3years, firmly positioning PDE5 inhibitors in the therapeuticarena for years to come

Thinking ‘outside the box’:

re-examination of existing work and targeting novel therapeutic areas

Innovative rethinking of the role of the NO pathway indisease can open new opportunities, described briefly in thesections below This is particularly true of the role of NO insepsis, which points towards ‘a window of opportunity’ forsGC activators In addition, dietary supplementation withinorganic nitrates offers an elegant example of how one canclinically improve cardiovascular disease by administering asimple, cheap and effective molecule The therapeutic advan-

tage of inhibition of the NO pathway has received relatively little attention, compared to efforts to increase NO activity;

however, there are situations where this could provide peutic benefit Lastly, the involvement of NO in energyexpenditure is a topic with immense translational potential

thera-in atherometabolic diseases

Time-sensitive apo-sGC stabilization

in sepsis?

After the recent withdrawal of recombinant activated protein

C from the market, there are no other specifically approvedmedications for sepsis, a largely (>50%) lethal indication

(Ranieri et al., 2012) The hypothesis that boosting NO

sig-nalling may be of therapeutic interest in this life-threateningdisorder is a novel concept, and directly opposite to theinitial notion that iNOS inhibitors, which reduce the exces-sive NO production associated with systemic expression ofthis NOS isozyme in sepsis, would be the better approach (athesis that failed to be substantiated in clinical evaluation;

López et al., 2004) The anti-inflammatory properties of NO

are well documented and augmenting NO signalling showspositive preliminary results in animal models of endotoxae-

mia (Da et al., 2007) and alleviates some symptoms in

humans presented with adult respiratory distress syndrome

(Taylor et al., 2004) Furthermore, nitrite generates NO tively in hypoxic conditions (Lundberg et al., 2008) and can

selec-rescue mice subjected to LPS- or TNF-α-elicited shock

(Cauwels et al., 2009), an effect mediated by cGMP produced

by sGCα1/β1(Buys et al., 2009) However, initial experimental

tests in endotoxin-exposed subjects that received inhaled NO

have not yielded positive results (Hållström et al., 2008) A recent study in mice (Vandendriessche et al., 2013), though,

has re-addressed this issue and has generated some very esting observations, namely that a beneficial effect may criti-cally depend on a combination of optimal timing and ofapo-sGC stabilization Mice that received an LPS injectionwere treated with sildenafil, the sGC stimulator BAY 41-2272

inter-or the sGC activatinter-or cinaciguat, 3 inter-or 8 h post-LPS challenge.Mortality was prevented only by cinaciguat, and only when itwas given at the late, 8 h, time point after LPS The effect

of late treatment with cinaciguat correlated with stabilized

body temperature and reduced cardiomyocyte apoptosis

(Vandendriessche et al., 2013) This preclinical work

demon-strates that ‘reactivation/preservation’ of apo-sGC is crucial

in endotoxaemic shock and that the response critically

depends on the time of treatment, when ‘rescued’ function of

haem-free sGC is optimally amenable to impact the course of

the disease It is therefore of particular importance in future

clinical trials in sepsis and systemic inflammatory response

syndrome to correctly estimate this target window of

apo-sGC responsiveness It should be stressed that in sepsis,

distinguishing between the effects of NO in the

macrocircu-lation and in the microcircumacrocircu-lation is important, and

genera-tion of NO selectively in the microcirculagenera-tion may provide

critical cytoprotective and tissue-protective effects Indeed,

treatment with nitrite, which is converted to NO selectively

in hypoxic/acidic conditions, characteristic of the septic

microvasculature, provides therapeutic benefit in preclinical

murine models based on challenge by LPS or TNF-α,

alleviat-ing telltale symptoms of sepsis, such as organ damage and

progressive hypothermia (Cauwels and Brouckaert, 2011)

Inorganic nitrite and nitrate

Although organic nitrates have been used for the treatment

of angina and heart failure for more than 150 years, the

physiological importance and pharmacodynamic properties

of inorganic nitrite (NO2 −) and nitrate (NO3 −) have only

recently been established (Lundberg et al., 2008; 2009)

Ini-tially considered to be simply inactive oxidation products of

NO, it is now clear that these molecules can be reduced,

preferentially under conditions of hypoxia and acidosis, to

bioactive NO This ‘non-canonical’ route of NO generation

(Figure 1) is dependent on reduction of nitrate to nitrite by

anaerobic bacteria that colonize the tongue, concentration of

nitrite in the saliva, followed by absorption through the gut

wall and entry into the systemic circulation (Lundberg et al.,

2008; Kapil et al., 2010b) Production of NO from nitrite is

then catalysed by ‘nitrite reductase’ enzymes, including

xan-thine oxidoreductase (Millar et al., 1998; Zhang et al., 1998;

Webb et al., 2008a) and globins (Doyle et al., 1981; Basu

et al., 2007; Tiso et al., 2011) In preclinical models,

augmen-tation of this ‘nitrate-nitrite-NO’ pathway lowers systemic

blood pressure, protects against ischaemia–reperfusion (I/R)

injury and ameliorates pulmonary hypertension (Hunter

et al., 2004; Webb et al., 2004; 2008b; Hendgen-Cotta et al.,

2008; Casey et al., 2009; Zuckerbraun et al., 2010; Baliga

et al., 2012) Such positive observations and the comparative

ease of pharmacological and/or dietary manipulation of

nitrite/nitrate levels has led to rapid translation of this

phe-nomenon to healthy volunteers and patients with

cardiovas-cular disease Inorganic nitrite and nitrate have both been

shown to lower systemic blood pressure in healthy

volun-teers (Cosby et al., 2003; Larsen et al., 2006; Webb et al.,

2008b; Kapil et al., 2010a) and dietary nitrate

supplementa-tion reduces blood pressure in hypertensive patients (Ghosh

et al., 2013) with a significantly increased potency,

suggest-ing the beneficial effects of modulatsuggest-ing nitrate-nitrite-NO

signalling for therapeutic benefit are enhanced in disease

Further clinical evaluation has been conducted in patients

presenting with acute myocardial infarction undergoing

per-cutaneous coronary intervention In a randomized,

placebo-controlled, double-blind phase II evaluation, a 5 min i.v.administration of sodium nitrite prior to angioplasty did notreduce infarct size (the primary end point), although a sub-group of patients with diabetes did show some improvement

(Siddiqi et al., 2013; NCT01388504 and ISRCTN57596739).

This lack of efficacy is disappointing, given the preclinicalobservations, but may be dose related as the 70μmol NaNO2

administrated was insufficient to significantly increase lating NO2 − concentrations, at least to levels shown to berequired for blood pressure effects in healthy volunteers andhypertensive patients Thus, further studies with higherdoses of nitrite (and/or nitrate) and using different routes ofadministration (e.g intracoronary) are warranted Severalfurther studies, primarily to assess the pharmacokinetic andsafety profile of inorganic nitrite or nitrate, are also under-way or nearing completion in patients with cardiovasculardisease (e.g cerebral vasospasm, sickle cell, peripheral arterialdisease) Nitrite, at least in part via bioconversion to NO, canalso provide tissue and organ protection following ischaemia

circu-(Rassaf et al., 2014), whether the experimental ischaemic

insult is established in heart, kidney, brain or liver In tion, nitrite also offers protection from experimental

addi-hypoxia-induced pulmonary hypertension (Rassaf et al.,

2014) Based on these data, the beneficial effects of inhalednitrite are currently being investigated in a phase I clinicaltrial, determining the changes in pulmonary vascular resist-ance in patients with pulmonary hypertension that undergoright heart catheterization (NCT01431313) In sum, raisingplasma nitrite levels by pharmacological or dietary meansrepresents a novel and inexpensive strategy to augment sGC–cGMP signalling for therapeutic gain

Therapeutic potential of NOS inhibitors

High NO concentrations can compromise the blood–brainbarrier and lead to brain oedema The expression of iNOS andthe levels of NO peak about 24–48 h after traumatic brain

injury in humans (Clark et al., 1996) The improved

pathol-ogy in mice subjected to cryogenic cerebral trauma that have

been subjected to genetic (Jones et al., 2004) or logical (Rinecker et al., 2003) ablation of iNOS indicates a

pharmaco-deleterious role for NO in the recovery in this disease setting.VAS203 (6R,S)-4-amino-5,6,7,8-tetrahydro-L-biopterin) is anallosteric NOS inhibitor which, in a preclinical mouse model

of intracranial oedema formation subsequent to cerebraltrauma, showed improvements in short-term (24 h) oedemaformation and in long-term functional preservation

(Terpolilli et al., 2009) VAS203 is being tested in the clinic

(NOSTRA: NO-Synthase inhibition in TRAumatic braininjury), in a European multicentre trial that is ongoing Pre-liminary phase IIa results (according to a communiqué of thecompany) seem promising; however, the end of the trial has

to be awaited to conclude on the efficacy of this molecule.Nonetheless, these findings are welcome because to date,iNOS inhibitors have failed to make the positive clinicalimpact predicted by animal models, particularly in thesetting of sepsis

NO production by NOS isoforms is regulated throughprotein–protein interactions In particular, nNOS has beenfound to exist in a ternary complex with the synaptic scaf-folding protein PSD95 and the NMDA receptor Activation ofthis complex by glutamate following stroke and excessive NO

production contributes to neuronal excitotoxicity and brain

damage, making nNOS–PSD95 uncoupling a therapeutic

approach to limit neurotoxicity (Cao et al., 2005)

Tat-NR2B9c is a chimeric peptide that consists of the HIV-1 Tat

protein transduction domain to facilitate cell penetration

fused to a sequence that binds to the PDZ domains of PSD95

disrupting downstream neurotoxic signalling pathways,

without blocking NMDA receptor activity It was

demon-strated that i.v administration of Tat-NR2B9c 1 h after

middle cerebral artery occlusion in non-human primates led

to a reduction in infarct volume by 70% after 30 days An

improved, dimeric version of this peptide (NA-1) was

gener-ated and first tested in mice with favourable results (Bach

et al., 2012) NA-1 was subsequently tested for its ability to

improve the outcome of iatrogenic strokes occurring during

aneurysm repair and assessed in a phase II trial (ENACT,

NCT00728182) Although no differences in infarct volumes

were observed between the saline and NA-1 groups, patients

who received NA-1 exhibited significantly fewer new brain

lesions than those receiving saline (Hill et al., 2012) This

landmark study provides a proof of concept that

neuropro-tection is feasible in humans; however, the efficacy of NA-1 in

community-onset stroke needs to be further established in

more extended studies

NO is also involved in nociceptive processing in the

brain and contributes to cerebral artery vasodilatation,

which is a symptomatic epiphenomenon of migraine

(Hoffmann and Goadsby, 2012) iNOS seems to play a role in

the pathogenesis of the disease, and for this reason iNOS

inhibitors have also been in various stages of preclinical and

clinical development to treat migraine, of which GW274150

is the most advanced This molecule has been clinically

tested both as a prophylactic treatment and as a treatment

in acute migraine (NCT00242866 and NCT00319137)

Results from both trials show that at doses that are predicted

to inhibit iNOS by 80–90%, GW724150 was ineffective in

reducing pain (Høye et al., 2009; Palmer et al., 2009; Høivik

et al., 2010) Taken together, therefore, these data suggest

that iNOS inhibition is unlikely to provide therapeutic relief

in this indication

iNOS inhibitors have also been, or are being, tested in

additional indications Elevated NO biosynthesis has been

linked with increased angiogenesis, bone resorption and

destruction of connective tissue in rheumatoid arthritis

(Farrell et al., 1992; Stefanovic-Racic et al., 1993; Sakurai et al.,

1995), manifestations that correlate with the pathogenesis

and progress of the disease GW274150 has been under

clini-cal evaluation for use in this indication (NCT00370435 and

NCT00379990) Final evaluation of 28 day treatment with

GW274150 in reducing synovial thickness and vascularity in

a rheumatoid arthritis in an early phase trial showed only a

non-statistically significant trend (Seymour et al., 2012) The

results of additional concurrent trials are being awaited Last,

GW274150 has also been tested in the treatment of mild

asthma (NCT00273013) The conclusions of the study were

negative, as GW274150 did not inhibit early or late asthmatic

challenges to allergen or to methacholine-induced responses

(Singh et al., 2007).

These mixed clinical results suggest that it may be

impor-tant in the future to focus testing selective NOS inhibitors in

a subset of carefully chosen clinical indications

Regulation of fat phenotype and energy expenditure by NO

Our understanding of the molecular mechanisms that mine adipose tissue phenotype and of the respective patho-physiological roles of white and brown fat has madeimpressive progress lately (Bartelt and Heeren, 2014) Hence,ways to pharmacologically control and modulate fat pheno-type can have a potentially enormous impact on variouspathologies, including atherometabolic diseases Pharmaco-

deter-logical inhibition of NO activity in vitro or eNOS genetic inactivation in vivo results in decreased mitochondrial bio-

genesis, which is ascribed to altered cGMP generation; theseinterventions also interfere with non-shivering thermogen-

esis by brown fat and with energy expenditure (Nisoli et al.,

2003) Conversely, eNOS transgenic mice (overexpressingeNOS under the pre-proendothelin promoter) on high fat dietdisplay increased systemic metabolic rate (not attributed tohyperthyroidism) and adipose cell hypertrophy, while theiradipose tissue shows signs of ‘browning’, with higher mito-chondrial activity and elevated PPAR-α and PPAR-γ expres-

sion (Sansbury et al., 2012) In addition to NO-dependent

pathways, natriuretic peptide signalling can also trigger abrown fat thermogenic programme in white adipocytes

(Bordicchia et al., 2012) Collectively, these data clearly show

anti-obesity effects of cGMP-mediated signalling and raise thepossibility that increased NO bioactivity may help controlsome crucial features of the metabolic syndrome Impor-

tantly, in the study by Sansbury et al., eNOS overexpression

did not affect blood glucose handling These exciting resultspoint to a novel biochemical pathway that can be effectivelytargeted, even with currently available medications, tocontrol clinical features of metabolic disorder associated withobesity

bio-a novel mechbio-anism of bio-action or tbio-arget moleculbio-ar components

of the system (Table 1) that had received poor attentionbefore (e.g riociguat and NA-1 respectively) It can be pre-dicted that an increasing number of new therapeutic candi-dates that target the NO-sGC-cGMP pathway will be seekingclinical assessment and approval in the next few years tobenefit the treatment of therapeutically challenging, or evenintractable, human pathologies (Figure 1)

A J H., A P and S T receive support from COST Action

BM1005: ENOG: European network on gasotransmitters

(http://www.gasotransmitters.eu) A P and S T are also

sup-ported by EU FP7 REGPOT CT-2011-285950 – ‘SEE-DRUG’:

‘ESTABLISHMENT OF A CENTRE OF EXCELLENCE FOR

STRUCTURE-BASED DRUG TARGET CHARACTERIZATION:

STRENGTHENING THE RESEARCH CAPACITY OF

SOUTH-EASTERN EUROPE’ (http://www.seedrug.upatras.gr)

Conflict of interest

A J H has acted as a consultant/advisory board member for

Bayer AG, Novartis, Merck and Palatin Technologies

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Themed Section: Pharmacology of the Gasotransmitters

1Institute of Normal and Pathological Physiology and Centre of Excellence for Regulatory Role of

Nitric Oxide in Civilization Diseases, Slovak Academy of Sciences, Bratislava, Slovak Republic,

2Faculty of Natural Sciences, Comenius University, Bratislava, Slovak Republic,3Cardiometabolic

Research Group, Department of Pharmacology and Pharmacotherapy, Semmelweis University,

Budapest, Hungary,4Laboratory of Physiological Studies, National Institutes of Health/NIAAA,

Bethesda, MD, USA, and5Pharmahungary Group, Szeged, Hungary

Correspondence

Olga Pechánová, Institute ofNormal and PathologicalPhysiology, Slovak Academy ofSciences, Bratislava 81371, SlovakRepublic E-mail:

It is well documented that metabolic syndrome (i.e a group of risk factors, such as abdominal obesity, elevated blood

pressure, elevated fasting plasma glucose, high serum triglycerides and low cholesterol level in high-density lipoprotein),which raises the risk for heart disease and diabetes, is associated with increased reactive oxygen and nitrogen species

(ROS/RNS) generation ROS/RNS can modulate cardiac NO signalling and trigger various adaptive changes in NOS andantioxidant enzyme expressions/activities While initially these changes may represent protective mechanisms in metabolicsyndrome, later with more prolonged oxidative, nitrosative and nitrative stress, these are often exhausted, eventually

favouring myocardial RNS generation and decreased NO bioavailability The increased oxidative and nitrative stress alsoimpairs the NO-soluble guanylate cyclase (sGC) signalling pathway, limiting the ability of NO to exert its fundamental

signalling roles in the heart Enhanced ROS/RNS generation in the presence of risk factors also facilitates activation of

mediators, and eventually the development of cardiac dysfunction and remodelling While the dysregulation of NO signallingmay interfere with the therapeutic efficacy of conventional drugs used in the management of metabolic syndrome, themodulation of NO signalling may also be responsible for the therapeutic benefits of already proven or recently developed

above-mentioned pathological processes may ultimately lead to more successful therapeutic approaches to overcome

metabolic syndrome and its pathological consequences in cardiac NO signalling

Although NO was discovered decades ago, scientific interest

in this gasotransmitter is continuously increasing Enzymic

and non-enzymic formation of NO and cGMP-dependent

and independent NO signalling has been reviewed in detail

in the current Themed Issue (Csonka et al., 2015) and

else-where (Ferdinandy and Schulz, 2003; Stasch et al., 2011; Tang

et al., 2013; Rassaf et al., 2014) Intercellular and intracellular

NO signalling is very complex, reflecting its many pathways

and interactions with other free radicals to form additional

signalling molecules Reactive oxygen species (ROS),

espe-cially the superoxide anion radical, can react with NO

non-enzymically with an extremely high-rate constant limited

only by diffusion These reactions produce peroxynitrite

(ONOO−) and other highly reactive oxygen and nitrogen

species (ROS/RNS), which in concert with NO act as

signal-ling molecules and also account for oxidative, nitrative and

nitrosative stress (Ferdinandy, 2006; Pacher et al., 2007;

Pechanova and Simko, 2009) Most techniques available for

the measurement of NO and its reactive metabolites have

numerous technical limitations (reviewed in this Themed

Issue by Csonka et al., 2015) which further complicates the

interpretation of results on the role of NO signalling in

physi-ology and pathphysi-ology

NO plays an important role in the regulation of

cardio-vascular functions in health and disease by, for example,

promoting vasodilation, inhibiting vascular smooth musclecell growth, platelet aggregation, and leukocyte adhesion,apart from by regulating myocardial function and providing

cardioprotection (see Pacher et al., 2007; Ferdinandy and

Schulz, 2003; and reviewed in this Themed Issue by

Andreadou et al., 2015) The metabolic syndrome,

compris-ing hypertension, hyperlipidaemia and insulin resistance/diabetes, is the major cardiovascular risk factor and thusaccounts for leading causes of morbidity and mortality inindustrialized societies Publications on the role ofNO-related pathways in these pathologies are continuouslygrowing In this review, we attempt to summarize the knowl-edge related to the role of NO signalling in the heart in thepresence of the major cardiovascular risk factors that areassociated with the metabolic syndrome Our review focuses

on the effect of the metabolic syndrome on NO signalling inthe non-ischaemic heart

The role of NO in myocardial ischaemia/reperfusioninjury and cardioprotection by ischaemic conditioning in thehealthy heart and in different co-morbidities is reviewed indetail elsewhere (Ferdinandy and Schulz, 2003; Andreadou

et al., 2015) In brief, NO itself protects the heart against

ischaemia/reperfusion injury However, accumulation ofexcess NO during prolonged ischaemia contributes to reper-fusion injury via an increased oxidative/nitrative stress Therole of endogenous NO in cardioprotection induced by

ischaemic preconditioning is still controversial (Csonka et al.,

DPP-4, dipeptidyl peptidase-4 β3-adrenoceptor

iNOS ATP-sensitive K+channels, Kir6.x

nNOS RyR, ryanodine receptors

PDE2 GLUT 4, glucose transporter

(SLC2A4)PDE5

Nuclear hormone receptorse

PKG

PPARαROCK, Rho kinase

PPARβ/δSERCA

Atorvastatin Niacin,

nicotinic acidAtrial natriuretic peptide

NOBH4, tetrahydrobiopterin

PioglitazoneCaptopril

PravastatincGMP

RosiglitazoneCinaciguat

RosuvastatinEnalapril

SildenafilFasudil

SimvastatinFluvastatin

TadalafilGIP

VardenafilGLP-1

Vitamin CGW7647

ZofenoprilInsulin

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://

www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are

permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (a,b,c,d,e Alexander et al., 2013a,b,c,d,e).

1999; Nakano et al., 2000; Post et al., 2000) Nevertheless, it

seems that mild oxidative/nitrative stress induced by

exog-enous or endogexog-enous NO is necessary to trigger both pre- and

post-conditioning (Nakano et al., 2000; Csonka et al., 2001;

Heusch, 2001; Kupai et al., 2009).

NO signalling in the heart

In the heart tissue, coronary and endocardial endothelial cells

and cardiac myocytes are major sources of NO However, NO

may also derive from intracardiac ganglia and some nerve

fibres located close to cardiac blood vessels Endothelial NOS

(eNOS) is expressed typically in the coronary and cardiac

endothelium, whereas neuronal NOS (nNOS) is mainly

located in the cardiac myocytes (see Pacher et al., 2007; Tirziu

and Simons, 2008) In coronary vascular endothelial cells, the

eNOS-caveolin-1 interaction in the caveolae is important for

normal eNOS activity (Feron and Balligand, 2006) The

physi-ological triggers for NO release from endothelial cells are the

flow-induced shear stress and mechanical deformations of

the endothelium during the cardiac cycle (Michel, 2010) In

cardiac myocytes, eNOS is co-localized with caveolin-3 in the

T tubules of plasmalemmal caveolae, nNOS is localized in the

sarcoplasmic reticulum (Shah and MacCarthy, 2000), and

the putative mitochondrial NOS (mtNOS) in cardiac

mito-chondria (Dedkova and Blatter, 2009) The normal

intracel-lular function of eNOS and nNOS in cardiomyocytes depends

on discrete coupling mechanisms in the local cytosolic

envi-ronments These mechanisms can be affected by altered

metabolism due to the metabolic syndrome (see Huang,

2009; Pechanova and Simko, 2010)

In fact, NO generated by inducible NOS (iNOS) may have

its origin in the myocytes or neutrophils that migrate in the

proximity of myocytes during inflammation and also in

acti-vated fibroblasts iNOS, when expressed in cardiac myocytes,

can regulate the response to β-adrenoceptor stimulation

However, as the neutrophils migrate to sites close to the

myocytes, iNOS becomes essential for the ability of

neutro-phils to damage myocytes (Poon et al., 2003) Indeed, an

increase in iNOS expression in the heart with substrate

limi-tation leads to uncoupled iNOS producing superoxide

anions and contributing to contractile dysfunction (Heusch

et al., 2010) These findings demonstrate that cellular source

and local cytosolic environment strongly modulate the

effects of different NOS isoforms, as reviewed elsewhere

(Tirziu and Simons, 2008; Huang, 2009; Pechanova and

Simko, 2010)

NO may affect myocytes in a number of different ways

NO signalling via cGMP-dependent or independent pathways

modulates the function of downstream proteins via specific

post-translational modifications, such as phosphorylation by

cGMP-dependent PK (PKG) or S-nitrosylation Interestingly,

an increase in intracellular cGMP induced by natriuretic

pep-tides or cGMP analogues was recently shown to modulate

both sarcolemmal and mitochondrial ATP-sensitive K+

channel opening in ventricular cardiomyocytes suggesting

further diverse actions of NO (Burley et al., 2014).

NO also affects mitochondrial function and

dyna-mics, thus regulating cardiac energy metabolism Under

pathological conditions, it may also contribute to the opment of myocardial dysfunction and heart failure

devel-(Davidson and Duchen, 2006; Azevedo et al., 2013; Dai

et al., 2013; Miller et al., 2013) Localization of NO

pro-duction within mitochondria seems to provide a distinctreciprocal regulation between mtNOS and intramitochon-drial Ca2+, pH, L-arginine and oxygen NO produced by theputative mtNOS may represent a mechanism of fine regu-lation of the respiratory complexes, enzymes of the citricacid cycle and energy metabolism as well (see Zaobornyj

and Ghafourifar, 2012; Csonka et al., 2015; Andreadou

et al., 2015) However, the existence of mitochondrial

mtNOS is still a controversial issue (Pacher et al., 2007),

and NO, which rapidly diffuses into mitochondria fromother cellular compartments or cells, is sufficient to effi-ciently regulate energy metabolism Despite these facts,very few studies investigated the role of NO signalling

in mitochondrial function in hearts with the metabolicsyndrome

The main physiological role of NO derived from eNOSand nNOS includes reduction of contractile frequency ofcardiomyocytes, attenuation of cardiac contractility, accelera-tion of relaxation and increasing distensibility of cardiomyo-cytes, and improvement of the efficiency of myocardialoxygen consumption In conditions of enhanced cardiacreserve and cardiac hypertrophy, NO derived from eNOSmodulates receptor-mediated signalling which ultimatelyleads to a moderate inhibition of cardiac contractility (Shahand MacCarthy, 2000; Yue and Yu, 2011) NO derived fromthe complex of nNOS-ryanodine receptor (RyR) stabilizes RyRcalcium release and increases the efficiency of Ca2+cycling insarcoplasmic reticulum by the inhibitory effects (Yue and Yu,2011) In swine, intracoronary infusion of an NO synthesisinhibitor, N-ω-nitro-L-arginine, markedly decreased left ven-tricle (LV) function, while peak LV pressure and mean coro-

nary arterial pressure were increased (Post et al., 2001).

Similarly, in healthy humans, inhibition of endogenous NOrelease also reduced, whereas replenishment with exogenous

NO increased left ventricular function, further emphasizingthat NO contributes to normal left ventricular function

(Rassaf et al., 2006) Thus, dysfunction of NOS induced by

altered expression, location, coupling and activity may tribute to the contractile dysfunction, adverse remodellingand myocardial hypertrophy – changes associated withvarious cardiac disease conditions, such as heart failure and

con-infarction (Tang et al., 2013).

Interestingly, eNOS expression was not affected by vascular risk factors like hypertension, obesity and insulin

cardio-resistance (Fulton et al., 2004; Bouvet et al., 2007), and

para-doxically was found to be increased in various pathological

states associated with oxidative stress (Li et al., 2002; Ding

et al., 2007; Zhen et al., 2008) This effect may be partly

medi-ated by limiting the availability of NO, thereby exerting anegative feedback on NOS expression through activation ofNF-κB (Zhen et al., 2008; Pechanova and Simko, 2009; 2010;

Vrankova et al., 2009) (Figure 1).

In conclusion, signalling functions of NO produced byspecific NOS isoforms seem to be compartmentalized in dis-tinct cellular microdomains and thus modulate cardiac func-tion differently Moreover, they may be further affected byrisk factors of the metabolic syndrome

NO signalling in the

hypertensive heart

Left ventricular remodelling and heart failure represent major

pathological consequences of chronic arterial hypertension

During the development of hypertension, differential signals

and metabolic abnormalities lead to the structural

remodel-ling of the cardiovascular system, as characterized by

myo-cardial hypertrophy and/or fibrosis, and coronary artery wall

hyperplasia which finally result in heart injury known as

cardiomyopathy (Kristek and Gerová, 1996; Babal et al., 1997;

Pechanova et al., 1997; Tribulova et al., 2000; Cebova and

Kristek, 2011) Pathological remodelling of the hypertensive

heart is due to an imbalance of stimulatory and inhibitory

signals of tissue proliferation Angiotensin II (Ang II),

aldos-terone or endothelin, with their vasoconstrictor and

pro-proliferative effects, stand on one side of the balance and NO,

prostacyclin, bradykinin or atrial natriuretic peptide, exerting

vasodilating and antiproliferative activities, provide the

counteracting factors (Swynghedauw, 1999; Cuspidi et al.,

2006; Pechanova and Simko, 2010) NO antagonizes the

effects of Ang II on vascular tone, cell growth and renal

sodium excretion, while it down-regulates the synthesis of

ACE and angiotensin AT1receptor On the other hand, Ang II

decreases NO bioavailability by promoting oxidative stress

(Zhou et al., 2004) Mice infused with Ang II displayed an

increase in blood pressure, cardiac hypertrophy and fibrosis

associated with enhanced collagen I content, TGF-β1 activity

and endoplasmic reticulum stress markers, which were,

however, blunted after endoplasmic reticulum stress

inhibi-tion (Kassan et al., 2012) Recently, however, Jin et al (2012)

demonstrated that myocardial nNOS is up-regulated by Ang

II which functions as an early adaptive mechanism to ate NADPH oxidase activity and facilitate myocardial relaxa-tion by promoting the cGMP/PKG pathway It was alsodocumented that activation of this pathway by novel solubleguanylate cyclase (sGC) stimulators, including riociguat (BAY63-2521), attenuates systemic hypertension and systolic dys-function, as well as fibrotic tissue remodelling in the myocar-dium in a rodent model of pressure and volume overload

attenu-(Geschka et al., 2011) This is in line with earlier data showing

impaired NO-sGC signalling pathways in hypertension andheart failure, and beneficial effects of sGC stimulators/activators in preclinical models of hypertension in attenuat-

ing myocardial hypertrophy and remodelling (Evgenov et al., 2006; Stasch et al., 2011) Validating this concept, recent

clinical trials with riociguat in pulmonary hypertension andchronic thromboembolic pulmonary hypertension showedencouraging results, which lead to the FDA approval of the

drug for these indications (Ghofrani et al., 2013a,b).

It is generally believed that increased production of ROSplays an important role in the pathology of hypertension, but

so far the limited number of clinical studies using specific antioxidants yielded mixed results Complicating thepicture, it should also be noted that temporarily increasedROS generation in hypertension is not necessarily harmful, as

non-it may stimulate the activnon-ity of the antioxidant defencesystem and improve the NO signalling pathway, resulting inthe establishment of a new equilibrium between increasedoxidative load and the stimulated NO pathways, thus main-taining sufficient NO availability (Dröge, 2002) However, inhypertension associated with obesity or diabetes, ROS mayfavour activation of pro-inflammatory NF-κB-dependent

Figure 1

NO signalling and metabolic syndrome-related pathways ROS generated by NADPH oxidases and other sources (e.g mitochondria, XO,uncoupled NOS, among others) leads to increased NF-κB activity followed by eNOS and iNOS up-regulation eNOS produces NO which preventsactivation of both NADPH oxidase and NF-κB The leptin/STAT3 pathway may also up-regulate the gene for iNOS whereas the leptin/JAK2/IRS-1pathway increases eNOS activity via Akt stimulation, as does insulin Increased circulating free fatty acids lead to ceramide elevation with increasingeffects on NADPH oxidase activity and diminishing effects on Akt activation NO produced by neuronal NOS (nNOS) and putative mtNOS mayaffect heart function in metabolic syndrome by different specific routes

pathways (Figure 1) In these conditions, activation of NF-κB

increases levels of cytokines such as IL-6 and TNF-α that may

affect the phosphorylation of tyrosine kinases and decrease

NOS activity with a final decrease in NO generation (see Belin

de Chantemele and Stepp, 2012)

In conclusion, increased ROS formation during

hyperten-sion may activate NF-κB and promote pro-inflammatory and

pro-oxidant changes (increased expression of TNF-α, COX2,

iNOS, NADPH oxidase, etc.) or compensatory adaptive

mechanisms (increased expression of eNOS and antioxidant

enzymes) Prolonged ROS/RNS formation may also lead to

uncoupling of eNOS/iNOS and impaired NO-sGC signalling

in hypertensive cardiovascular system

NO signalling in the

obese/hyperlipidaemic heart

In obesity, cardiac output increases to serve the larger body

mass of the obese individual (Kardassis et al., 2012) The

increase in cardiac output is due to a larger blood volume

resulting in elevated venous return and an increased

activa-tion of the sympathetic nervous system, both prevalent in

the obese population An increase in cardiac output elevates

cardiac oxygen consumption Consequently, the need for

perfusion is increased (Alvarez et al., 2002; Frohlich and Susic,

2008) In mice fed a high-fat diet, obesity suppressed left

ventricular ejection fraction, increased left ventricular

remodelling, and led to diminished circulating endothelial

progenitor cells level and impaired recovery of damaged

endothelium (Tsai et al., 2012).

The importance of two adipocyte-derived hormones –

leptin and angiotensinogen – in the pathological

conse-quence of obesity has been highlighted (Coatmellec-Taglioni

and Ribière, 2003) Leptin regulates energy balance and

metabolism by a variety of peripheral and central

mecha-nisms through specific cell surface receptors (Koh et al.,

2008) Leptin infusion was shown to reduce blood pressure

and heart rate, which may be reversed by an increased NO

synthesis (Frühbeck, 1999) In vitro studies demonstrated that

leptin elicited endothelium-dependent NO-mediated

vasore-laxation in rats (Lembo et al., 2000) In the mouse heart,

disruption of leptin signalling may contribute to

obesity-related cardiac disease, as leptin-deficient (ob/ob) mice display

cardiac hypertrophy, increased cardiac apoptosis and reduced

survival These changes were linked to decreased cardiac

expression of nNOS and NO production, with a concomitant

increase in xanthine oxidase (XO) activity and oxidative

stress, resulting in nitroso-redox imbalance (Saraiva et al.,

2007) Furthermore, cardiac β3-adrenoreceptor expression

and function were shown to be dependent on leptin as they

were severely diminished in the same model (ob/ob mice) It

was proposed that diminished β3-adrenoreceptor signalling

may be the critical element to explain the direct effects of

leptin on the myocardium and suggest an important role of

leptin in obesity-related cardiac hypertrophy and heart

failure (Larson et al., 2012) Leptin may up-regulate iNOS to

generate large amounts of NO that induce nitrosative and

nitrative stress and impair endothelial and myocyte functions

(Koh et al., 2008) In ventricular myocytes isolated from male

Sprague-Dawley rats, leptin-induced NO generation inhibitedmyocyte contraction which was prevented by the NOS

inhibitor L-NAME (Nickola et al., 2000) In addition,

hyper-leptinaemia may result in the overdrive of pituitary-adrenal axis (HPA axis) and the sympatheticnervous system, as well as in impaired insulin secretion andinsulin resistance HPA axis overdrive would account formetabolic abnormalities such as central adiposity, hypergly-caemia, dyslipidemia, hypertension and other cardiovasculardiseases which are well-known clinical aspects of the meta-

hypothalamus-bolic syndrome (Peters et al., 2002).

Cardiac lipotoxicity caused by the accumulation of lipidshas been well described in rodent models of obesity, hyper-

lipidaemia and diabetes (Zhou et al., 2000; Chiu et al., 2001; 2005; Young et al., 2002) Feeding mice a palmitate-rich diet

led to the accumulation of medium- and long-chain mides and sphingomyelins, which were incorporated intocellular membrane, thus changing the micro-domain struc-ture of the plasma membrane of cardiomyocytes Thepalmitate-rich diet also resulted in a decreased expression ofcaveolins, structural components of plasmalemmal rafts, the

cera-caveole (Knowles et al., 2011; 2013) In addition, ceramides

may activate NADPH oxidase leading to an increased

oxida-tive stress (Zhang et al., 2003) (Figure 1) In cardiomyocytes,

eNOS localizes to caveolae, which containsβ-adrenoceptors

and L-type calcium channels as well (Garcia-Cardena et al.,

1996; Feron and Balligand, 2006) The co-localization ofcaveolin-3 and eNOS may facilitate both eNOS activation bycell surface receptors as well as NO release at the cell surfacefor intercellular signalling (Feron and Balligand, 2006).Immunohistochemistry findings in human cardiac tissuesamples from obese humans showed a drastic reduction of

caveolin-3 expression in cardiomyocytes (Knowles et al.,

2013), further signifying the role of caveolin proteins inobesity

In conclusion, it seems that the dual effect of leptin in theobese heart depends on eNOS or iNOS activation by differentmechanisms Elevation of ceramide levels in obesity mayinhibit eNOS activity by decreasing caveolin proteins andpromoting oxidative stress

NO signalling in the hypercholesterolaemic heart

It is well documented that hypercholesterolaemia profoundlyaffects cardiac NO metabolism It has been previouslyreported that in cholesterol-fed rats, cardiac NO level

decreases (Ferdinandy et al., 1997; Giricz et al., 2003; Onody

et al., 2003) and that hypercholesterolaemia blunts activity of

downstream signalling elements of NO as indicated by a

lower PKG activity (Giricz et al., 2009) Reports on the effect

of hypercholesterolaemia on the phosphorylation of dial eNOS, which reflects its activity, however, are controver-sial In cholesterol-fed rats, Zhang showed a decreasedp-eNOS level in parallel with an elevated apoptosis (Zhang

myocar-et al., 2012); meanwhile, in hearts of hypercholesterolaemic

LDLr(−/−) mice, eNOS phosphorylation was unchanged

(Ou et al., 2011) Similarly, eNOS protein concentrations

were found to be unchanged in cholesterol-fed rabbits

(Rajamannan et al., 2005) and rats (Giricz et al., 2003) These

discrepancies might be attributed to the vast differences

between the animal models It has been also uncovered

that the decrease in NO content in hypercholesterolaemic

animals is supposedly not due to a decreased activity of NOS

isoenzymes, but instead a result of an increased clearance of

NO, as assessed by elevated markers of oxidative stress, such

as dityrosine, nitrotyrosine (Giricz et al., 2003) and

superox-ide anion formation due to at least, in part, XO activity

(Onody et al., 2003), and elevated expression and activity of

NADPH oxidase (Onody et al., 2003; Varga et al., 2013) These

reports were confirmed by Stokes et al (2009) who found

cardiac S-nitrosothiol (SNO) levels elevated and cardiac

nitrite levels decreased in hypercholesterolaemic mice In

genetic models of hypercholesterolaemia, similar findings

cholesterol-enriched diet increased cardiac superoxide anion

generation and NADPH oxidase expression in parallel with an

elevated cardiac nitrotyrosine level (Csont et al., 2007).

LDLr(−/−) mice also have a higher net production of ROS and

susceptibility to develop membrane permeability transition,

and increased ROS production in mitochondria can be

observed (Oliveira et al., 2005) These findings strongly

emphasize that cardiac NO production is diminished, while

its elimination is accelerated in diet-induced and genetic

models of hypercholesterolaemia as well Meanwhile, there is

an apparent dearth of reports on the successful

pharmaco-logical restoration of hindered NO-related mechanisms:

fasudil, a selective Rho-associated PK (ROCK) inhibitor

elevated activity of antioxidant enzymes and the expression

of eNOS as well as cardiac NO, and elsewhere atorvastatin

increased eNOS protein concentrations and serum nitrite

concentrations in cholesterol-fed rabbits (Rajamannan et al.,

2005) This scarcity of direct evidence is quite interesting,

especially in view of the high number of antioxidant and

anti-hyperlipidaemic treatments that have been under

devel-opment recently Therefore, it is likely that novel

pharmaco-logical targets will have to be explored aiming to restore

cardiac NO homeostasis in hypercholesterolaemia

One can speculate that disturbed NO metabolism might

affect cardiac function Indeed, it has been demonstrated in

guinea pigs fed with a cholesterol-enriched diet that

increased plasma XO activities were associated with a

pro-found myocardial and coronary endothelial dysfunction

(Schwemmer et al., 2000) Similarly, cholesterol feeding

resulted in the deterioration of cardiac function in rats

(Onody et al., 2003) This notion is further supported by

other studies where positive chronotropic effect of atropine

was selectively lost in genetically hypercholesterolaemic

apoE−/− mice, which was restored after a rosuvastatin

treat-ment (Pelat et al., 2003) This latter paper also reported that

cardiac expression of caveolin-1 was elevated in apoE−/−mice,

further evidencing a disturbed NO metabolism in

hypercho-lesterolaemia Similarly, LDLr(−/−) mice demonstrated a

decrease in left atrial contractility and eNOS expression

rela-tive to wild-type mice Interestingly, LDLr(−/−) mice fed with

an atherogenic diet for 15 days showed increased left

ven-tricular mass and enhanced expression of NOS isoforms,

which was reversed by the administration of

S-nitroso-N-acetylcysteine (Garcia et al., 2008) These results highlight

that, although it is well studied, the contribution of disturbed

NO signalling to the deteriorated cardiac function in cholesterolaemia is not completely understood

hyper-Isolated hypercholesterolaemia in humans is rarely seen;however, it is a major contributor to numerous pathologicalconditions, such as atherosclerosis and diabetes NO metabo-lism in the human heart has been studied in even rarer cases

In hypercholesterolaemic patients, tetrahydrobiopterin (BH4)attenuated acetylcholine (ACh)-induced decrease in coronarydiameter and restored ACh-induced increase in coronaryblood flow, which was not shown in normocholesterolaemic

patients (Fukuda et al., 2002) Asymmetric dimethylarginine

(ADMA) is an endogenous NOS inhibitor and an established

cardiovascular risk factor in adults (Wu, 2009; Wu et al.,

2009) Serum concentration of ADMA is elevated in holesterolaemic adults, which contributes to NO-dependent

hyperc-endothelial dysfunction (Böger et al., 1998; for review, see

Horowitz and Heresztyn, 2007), but not in children withhypercholesterolaemia type II, possibly due to an increase

in dimethylarginine dimethylaminohydrolase activity

(Chobanyan-Jürgens et al., 2012) However, whether ADMA

influences NO bioavailability in the heart, it has yet to beassessed

In addition to decreased NO bioavailability, the NO-sGCsignalling is also pathologically impaired in atherosclerosis,which can be successfully restored by novel sGC stimulators/activators in preclinical rodent models of atherosclerosis andrestenosis, where these drugs attenuate inflammation and

other pathological changes (Evgenov et al., 2006; Stasch et al.,

2011)

In conclusion, in animal models and humans, lesterolaemia hinders cardiac NO metabolism and, in theseconditions, increased oxidative stress plays a major role Fur-thermore, diminished NO availability and, most likely,impaired NO-sGC signalling in the heart tissue manifests indeteriorated cardiac function and would contribute to thedevelopment of other cardiovascular pathologies

hypercho-NO signalling in the diabetic heart

In diabetic patients, independent of vascular complications, aspecific form of cardiomyopathy develops known as diabeticcardiomyopathy Many factors may contribute to the evolu-tion of this pathology, including metabolic disturbances(glucotoxicity, lipotoxicity), inflammatory processes, mito-chondrial uncoupling, enhanced oxidative stress and deterio-

rated NO signalling (Pacher et al., 2005) Several publications

highlight the role of altered NO metabolism in diabetic diomyopathy, but surprisingly there is limited information

car-on the direct measurement of cardiac NO levels obtained by

strictly NO-specific methods (see Csonka et al., 2015) As

assessed by electron paramagnetic resonance spectrometry, agold standard NO-specific method, NO level was increased inthe hearts of streptozotocin-induced diabetic rats (Amour

et al., 2007) In line with this finding, an increase in cardiac

NO metabolites (nitrite, nitrate) has been reported in the

Goto-Kakizaki rat model of type 2 diabetes (Desrois et al.,

2010) Although these reports indicate that cardiac NOmetabolism is influenced by diabetes, to date no data havebeen published on cardiac levels or bioavailability of NOfrom diet-induced animal models, let alone diabetic patients

Diverse mechanisms have been proposed in

diabetes-induced dysfunction of NO signalling The pivotal role of

altered eNOS function as the rate-limiting step in NO

bio-availability is emphasized in the pathomechanism (Münzel

et al., 2005; Zhang et al., 2011) The mechanisms responsible

for eNOS dysfunction, however, remain elusive Availability

of cofactors for the eNOS complex, especially of BH4,

deter-mines the ratio of NO or superoxide anion produced by the

enzyme (Gielis et al., 2011) Furthermore, a decrease in the

dimer to monomer eNOS ratio within the myocardium of

diabetic animals has been reported (Zou et al., 2002; Jo et al.,

2011) Monomerization and subsequent uncoupling of NOS

results in increased oxidative stress and decreased NO

bio-availability that has been implicated in the pathophysiology

of many cardiovascular diseases

Of the three major NOS isoforms, two (iNOS and

eNOS) are known to be increased in the diabetic heart

(Stockklauser-Färber et al., 2000; Farhangkhoee et al., 2003;

Jesmin et al., 2006; Rajesh et al., 2012) The increase in NOS

expression in the diabetic heart is associated with an increase

in lipid peroxidation and nitrotyrosine formation, which

might be related to the uncoupled and monomer state of the

enzyme Indeed, inhibition of NOS activity in diabetes (by

L-NAME or L-NMMA) improves myocardial function,

sug-gesting that the increased production of superoxide anion

and peroxynitrite rather than NO is a major contributor of

suppressed contractile function (Smith et al., 1997; Esberg

and Ren, 2003) Moreover, it seems that

peroxynitrite-induced nitrative stress contributes to inactivation of

succinyl-CoA:3-oxoacid CoA transferase causing

deteriora-tion of energy metabolism of the diabetic heart (Turko et al.,

2001) In addition, restoration of iNOS coupling by BH4

administration improves ischaemic tolerance, reduces

iNOS-derived superoxide anion generation, and increases NO

bio-availability in the diabetic heart The authors also imply that

iNOS-derived NO-mediated cardioprotection occurs through

protein S-nitrosylation but not cGMP-dependent signalling

in the diabetic heart (Okazaki et al., 2011) The central role of

oxidative stress in impaired NO bioavailability and signalling

in diabetic hearts is further substantiated by Rajesh et al.

(2009), demonstrating that the XO inhibitor allopurinol not

only attenuated the myocardial oxidative stress, but also

attenuated the pathologically increased nitrosative/nitrative

stress, cell death, remodelling and cardiac dysfunction in

diabetic mice hearts (see Ansley and Wang, 2013)

Much less is known about the NO-related downstream

pathways (cGMP-PKG and NO-dependent post-translational

modifications) in the diabetic heart Recently, in patients

with heart failure with preserved ejection fraction (obese and

diabetic subjects), myocardial cGMP content as well as PKG

activity is decreased, which might be related to the increase

in oxidative/nitrosative stress (van Heerebeek et al., 2012).

However, it seems that natriuretic peptide-induced

cGMP-PKG signalling is not affected by diabetes, as shown by

Rosenkranz et al (2003) They reported that B-type

natriu-retic peptide is a suitable anti-hypertrophic strategy in the

diabetic myocardium, where NO-dependent (bradykinin –

ACE inhibitor) mechanisms fail to positively affect the

development of hypertrophy (Rosenkranz et al., 2003).

cGMP-independent effects of NO are mainly mediated by

S-nitrosylation, the covalent modification of a protein

cysteine thiol by an NO group to generate SNO Puthanveetil

et al reported recently that in the diabetic myocardium,

iNOS-dependent S-nitrosylation of GAPDH and caspase-3contributes to increased poly[ADP-ribose] polymerase-1(PARP-1) activity, and thereby initiates cell death activation

in hyperglycaemia (Puthanveetil et al., 2012) This is also in

line with data confirming the central role of PARP in diabetic

cardiac complications (Pacher et al., 2002; Pacher and Szabó,

2005)

In conclusion, diabetes markedly decreases NO ity in the heart that is related to increased superoxide (fromvarious sources including uncoupled NOS) and peroxynitriteformation As a consequence of increased oxidative/nitrosative stress, downstream signalling of NO (cGMP-PKGand protein S-nitrosylation) is also profoundly affected

availabil-Cardiac NO signalling as a pharmacological target

NO donors

NO donors are pharmacologically active substances thatspontaneously release NO, or are metabolized to NO or itsredox congeners and provide a wide scope for pharmaco-

therapy in cardiovascular medicine (Ignarro et al., 2002).

Several NO donors have been used in clinical settings fordecades, such as nitroglycerin and sodium nitroprusside.Nitrate tolerance, however, has become a limiting factor

for their clinical use (Kojda et al., 1995; 1998; Csont and

Ferdinandy, 2005) The underlying mechanisms responsiblefor nitrate tolerance may include neurohormonal counter-regulatory factors, intravascular volume or intrinsic abnor-malities such as desensitization of the target enzymeguanylate cyclase or a decrease in biotransformation of NO

donors (Munzel et al., 1995; Dikalov et al., 1997; 1998; 1999).

Molsidomine and pentaerythrityl tetranitrate (PETN) sent more effective tolerance-devoid NO donors with a phar-macodynamically beneficial effect Molsidomine is one of thesydnonimines and it is metabolized to the active linsidomine.PETN is the nitrate ester of pentaerythritol, structurally verysimilar to nitroglycerin It was found to be the most active

repre-drug in cGMP production (Hinz et al., 1998; Mollnau et al.,

2005) Despite these facts, neither molsidomine nor PETNwas able to improve pathological changes of the cardiovas-cular system in adult spontaneously hypertensive rats

(Kristek et al., 2003).

The compound LA-419 is an analogue of isosorbide onitrate containing a protected thiol group in its molecularstructure Preclinical studies have shown that this compoundhas anti-atherogenic and antioxidant properties that make itapplicable for the treatment of chronic cardiovascular disor-ders (Megson and Leslie, 2009) Ruiz-Hurtado and Delgado(2010) demonstrated that LA-419 prevents left ventricularremodelling in rats with aortic stenosis at doses not affectingarterial blood pressure In their experiment, LA-419 evenrestored cardiac eNOS expression and enhanced the interac-tion between eNOS and its positive regulator, heat shockprotein 90, and re-established the normal cardiac levels ofcGMP The thiol group of LA-419 improved also NO stability

mon-by converting NO into nitrosothiols and protecting the

formed NO from reaction with ROS (Ruiz-Hurtado et al.,

2007; Ruiz-Hurtado and Delgado, 2010)

In conclusion, there are very few data about the effects of

NO donors on heart and/or cardiomyocyte functions in the

metabolic syndrome Nevertheless, the beneficial effect of

compound LA-419 seems to be a promising therapeutic

approach against cardiac remodelling due to the metabolic

syndrome and the associated risk factors as well However,

the impaired NO-sGC signalling in the metabolic syndrome

by oxidative stress is likely to represent a major obstacle for

the success of this approach

ROS scavengers

ROS are involved in several physiological cellular signalling

mechanisms However, pathological increase in oxidative

stress contributes to different pathologies including

themeta-bolic syndrome and cardiovascular disorders Accordingly,

one of the most powerful antioxidants,

4-hydroxy-2,2,6,6,-tetramethylpiperidine-1-oxyl (tempol), prevents

cardiovascu-lar damage in different experimental hypertension and

diabetes models (Ebenezer et al., 2009; Hasdan et al., 2002;

Nagase et al., 2007), decreases hypertrophic responses to

atrial natriuretic peptide in neonatal rat cardiac myocytes

(Laskowski et al., 2006), and reduces apoptosis in cardiac cells

exposed to hyperglycaemia or in diabetic rats (Fiordaliso

et al., 2007) Tempol decreased apoptosis in response to

increased aldosterone signalling via a non-genomic pathway

in cardiomyocytes (Hayashi et al., 2008) and inhibited the

Ca2+ transient within cardiac myocytes stimulated by

pressure-flow stress (Belmonte and Morad, 2008) Tempol

improved insulin sensitivity and dyslipidemia, reduced

weight gain and diastolic dysfunction and heart failure in

diet-induced preclinical models of the metabolic syndrome

(see Wilcox, 2010) Moreover, infusion of tempol into

hyper-glycaemic dogs normalized their coronary endothelial

dys-function and coronary wall shear stress in type 1 and 2

diabetes models (Gross et al., 2003) Chronic treatment with

another antioxidant, N-acetylcysteine (NAC), partially

attenuated the increase in blood pressure in young, but not in

adult spontaneously hypertensive rats (SHR) The antioxidant

action of NAC on lipid peroxidation, inhibition of NF-κB

expression and eNOS activation was greater in young than in

adult SHR, indicating preventive rather than therapeutic

effect of NAC (Pechánová et al., 2006) Melatonin, an

indola-mine with antioxidant properties, has been shown do

decrease blood pressure even in the established form of the

spontaneous hypertension An in vitro study revealed that

melatonin lowered the tone of phenylephrine-precontracted

femoral artery via both NO-dependent and NO-independent

components since vasorelaxation was preserved even after

the blockade of sGC by oxadiazolo[4,3-a]quinoxalin-1-one

(Pechánová et al., 2007) Melatonin treatment also prevented

the development or induced a reversal of left ventricular

fibrosis in the model of L-NAME-induced hypertension or in

spontaneously hypertensive rats (see Simko and Pechanova,

2010) It has been documented that melatonin reduces blood

pressure in patients with hypertension or non-dipping blood

pressure (Reiter et al., 2009) Interestingly, melatonin, leptin

and insulin have been found to activate the same

intracellu-lar signalling pathways, particuintracellu-larly PI3K and STAT-3

(Carvalheira et al., 2001) As a consequence, melatonin may

attenuate or reverse insulin resistance in obesity by ing the actions of insulin and leptin signalling via crosstalk

mimick-between these pathways (see Nduhirabandi et al., 2012).

Several studies described positive effects of different phenolic compounds on the heart by restoring the balancebetween ROS and NO production, in hypertension as well as

poly-in other components of the metabolic syndrome (Pechánová

et al., 2004; Galleano et al., 2010) Sutra et al (2008) showed

the preventive effects of different polyphenolic molecules,like catechin, resveratrol, delphinidin and gallic acid, oncardiac fibrosis associated with the metabolic syndrome.Similarly, protection of ROS/NO balance was suggested to beinvolved in the beneficial effect of resveratrol The results of

Penumathsa et al (2008) suggested that the effect of

resvera-trol is non-insulin-dependent but triggers some of the cellular insulin signalling components such as eNOS and Aktthrough the AMPK pathway in the myocardium Further-more, resveratrol was shown to regulate the caveolin-1 andcaveolin-3 status that might play an essential role in GLUT-4translocation and glucose uptake in streptozotocin-induced

intra-type 1 diabetic myocardium (Penumathsa et al., 2008)

Simi-larly, olive leaf extract containing polyphenols, such as uropein and hydroxytyrosol, was shown to reverse chronicinflammation and oxidative stress in rat model of diet-

ole-induced obesity and diabetes (Poudyal et al., 2010)

Resvera-trol also protected against diabetic cardiac dysfunction byinhibiting oxidative/nitrative stress and improving NO avail-

ability (Zhang et al., 2010).

Despite the fact that antioxidants represent great promise

in the treatment of hypertension and other components ofthe metabolic syndrome, data from clinical studies and trialswith non-specific antioxidants are not conclusive Forexample, in the HOPE (Heart Outcomes Prevention Evalua-tion) study, involving patients with atherosclerotic complica-tions or diabetes mellitus, vitamin E in the dose of 400 IUdaily, was not able to reduce blood pressure and morbidity

and mortality from cardiovascular reasons (Yusuf et al., 2000;

Ward and Croft, 2006) In contrast, a more recent studyconfirmed that subjects with type 2 diabetes after a 3 monthlong supplementation of vitamins C and E or their combina-tion demonstrated significantly lower level of hypertension,decreased levels of blood glucose, and increased superoxidedismutase (SOD) and GSH enzyme activity that could prob-ably reduce insulin resistance by attenuating oxidative stress

(Rafighi et al., 2013) Vitamin C was also shown to increase

BH4 levels by preventing its oxidation, which reduced eNOS

uncoupling (Landmesser et al., 2003) Thus, preservation of

BH4 may also explain the effects of long-term ascorbate ment on blood pressure in patients with hypertension (Duffy

treat-et al., 1999).

Several studies suggest that imbalance between ROS duction and mitochondrial antioxidants also contributes tothe pathogenesis of hypertension and associated vascular

pro-pathologies Ito et al (1995) found that hypertension and

cardiac hypertrophy were associated with decreased sion of SOD1 and SOD2 in spontaneously hypertensive ratscompared with Wistar-Kyoto rats Indeed, overexpression ofmitochondrial SOD2 and thioredoxin 2 reduced the produc-tion of both mitochondrial and cytoplasmic ROS (Widder

expres-et al., 2009) SOD2 overexpression also attenuated H2O2induced apoptosis, decreased lipid peroxidation, reduced

-age-related decline in mitochondrial ATP levels and decreased

blood pressure (see Dikalov and Ungvari, 2013)

Recently, extracellular antioxidant enzymes like EC-SOD

or covalent bienzymes like SOD-CHS-CAT conjugate

(super-oxide dismutase-chondroitin sulphate-catalase) started to be

of particular interest, as they demonstrated protective

actions against development of hypertension, heart failure

and diabetes mellitus in vivo (see Maksimenko and Vavaev,

2012)

Taken together, based on numerous promising preclinical

studies with mitochondrial antioxidants, XO and/or NADPH

oxidase inhibitors in models of hypertension, diabetes and

atherosclerosis, it appears that, instead of using non-specific

antioxidants, selectively targeting the sources of ROS with

more specific drugs may represent a better approach to

over-come metabolic syndrome and its complications

PDE inhibitors

The cGMP-dependent NO signalling is largely influenced by

the family of PDEs that control cGMP levels and therefore

affect the downstream effects of NO including PKG

stimula-tion Several PDEs, including PDE1, PDE2 and PDE5, play a

role in the regulation of cGMP in both vascular smooth

muscle cells and cardiac myocytes PDEs are

compartmental-ized providing selective interactions of a certain source of

up-regulated in chronic disease conditions such as

atheroscle-rosis, cardiac pressure-load stress, and heart failure, as well as

in response to long-term exposure to nitrates In

pathophysi-ological states with reduced NO availability, such as, for

example, diabetes and hyperlipidaemia (see above), using

selective PDE inhibitors may be particularly helpful (see Kass

et al., 2007) Because PDE-5 is widely distributed in the body,

selective PDE-5 inhibitors have been extensively developed

The first PDE-5 inhibitor sildenafil on the market is used for

the indication of erectile dysfunction However, recent

studies revealed several beneficial pleiotropic cardiovascular

effects of PDE-5 inhibitors in patients with erectile

dysfunc-tion and multiple co-morbidities, including coronary artery

disease, heart failure, hypertension and diabetes mellitus (see

Chrysant and Chrysant, 2012) For example, tadalafil

attenu-ates oxidative stress and inflammation and induces

cardio-protection in type 2 diabetic mice models (Varma et al., 2012;

Koka et al., 2013) Moreover, vardenafil attenuated

diabetes-induced cardiac dysfunction in type 1 diabetic rats (Radovits

et al., 2009).

In conclusion, PDE inhibition is a promising tool to

restore the downstream signalling pathway of NO in the

metabolic syndrome

sGC stimulators and activators

Activation of sGC has traditionally been achieved with

nitrovasodilator drugs extensively used in ischaemic heart

disease However, these drugs are associated with the rapid

development of tolerance and potentially deleterious

cGMP-independent actions (see Csont and Ferdinandy, 2005)

Fur-thermore, the NO-sGC signalling pathway is impaired in

hypertension, heart failure and atherosclerosis by ROS/RNS,

limiting the ability of NO to activate its own signalling

machinery (Evgenov et al., 2006; Stasch et al., 2011)

There-fore, NO- and haem-independent sGC activators have beendeveloped, such as, for example, cinaciguat and ataciguat.These compounds selectively activate the oxidized/haem-freeenzyme via binding to the haem pocket of the enzyme,thereby causing strong vasodilatation Accordingly, activators

of sGC may be beneficial in the treatment of a variety ofpathologies including systemic and pulmonary hyperten-sion, heart failure, atherosclerosis and peripheral arterial

disease (Evgenov et al., 2006; Stasch et al., 2011) Indeed,

NO-insensitive sGC activators attenuated left ventricularhypertrophy, preserved cardiac function, and increased sur-vival in spontaneously hypertensive stroke-prone rats with

high-salt high-fat diet (Costell et al., 2012), in salt-sensitive Dahl rats (Geschka et al., 2011), as well as in chronic L-NAME- treated rats (Zanfolin et al., 2006) sGC activators have dem-

onstrated beneficial effects not only in hypertension andheart failure models but also in models of atherosclerosis

and restenosis (see Evgenov et al., 2006; Stasch et al., 2011).

Following successful recent clinical trials, riociguat receivedFDA approval for the treatment of pulmonary hypertensionand chronic thromboembolic pulmonary hypertension inhumans, and clinical trials with other similar drugs areongoing in heart failure

In conclusion, the pharmacological activation of sGCmay be the most promising tool to restore the downstreamsignalling pathway of NO in the metabolic syndrome, whichshould be validated in future clinical trials

Interaction of pharmacological treatment of metabolic syndrome with cardiac NO signalling

Interaction of antihypertensives with cardiac

NO signalling

Three approaches have been developed to correct the ance between increased oxidative stress and simultaneouslydecreased NO synthesis in the cardiovascular system: (1)reducing ROS bioavailability by administration of antioxi-dant compounds; (2) increasing NO levels via administration

imbal-of NO donors such as nitroglycerin or mono/dinitrates; and(3) reducing ROS production and stimulating NO production,for example, by treatment with statins, ACE inhibitors, angio-tensin AT1 receptor antagonists, or β-adrenoceptor antago-nists (β-blockers) with NO-dependent properties such as

nebivolol (see Münzel et al., 2010).

Among antihypertensives, the third-generation blockers with stimulating effect on NOS and/or β3-adrenoceptors have the best described effect on cardiac NOsignalling Nebivolol achieved a marked improvement oncardiac mass, coronary flow, mRNA expression levels of sar-coplasmic reticulum Ca2 + ATPase (SERCA2a), and atrialnatriuretic peptide and phospholamban (PLN)/SERCA2a andphospho-PLN/PLN ratio in rats treated with isoprenaline

β-(Ozakca et al., 2013) In Zucker diabetic fatty rats, nebivolol

and atenolol showed a comparable reduction in blood sure; however, nebivolol appeared to achieve a better lipidprofile, left ventricular function and less left ventricularhypertrophy, compared with atenolol Moreover, a reduction

pres-in platelet aggregation and an pres-increased

endothelium-dependent and endothelium-inendothelium-dependent relaxation were

observed in the nebivolol group versus the atenolol group

Together with an attenuation of oxidative stress parameters,

nebivolol also better preserved antioxidant defence markers

(Toblli et al., 2010) Concerning NO signalling, nebivolol has

been shown to stimulate endogenous production of NO by

inducing phosphorylation of eNOS (Maffei et al., 2006)

which determines its favourable effects on cardiac function in

patients with heart failure when compared with classical

β-blockers The action of nebivolol on iNOS was also

con-firmed by real-time PCR experiments, showing cardiac

overexpression of iNOS, but not nNOS or eNOS, in male

C57BL/6N mice (Maffei et al., 2007).

Among other promising antihypertensives with NO

increasing and ROS reducing effect are the ACE inhibitors

with a thiol group such as captopril and the newer zofenopril

In our earlier studies, both captopril and enalapril increased

NOS activity in the heart of spontaneously hypertensive

animals but did not increase the expression of eNOS Both

ACE inhibitors increased the level of cGMP However, cGMP

levels were significantly higher in the captopril group

Cap-topril, besides inhibition of ACE, prevented hypertension by

increasing NOS activity and by simultaneous decrease of

oxi-dative stress which resulted in increase of cGMP

concentra-tion (Pechánová, 2007) Most of the clinical studies revealed

that captopril, besides decreasing blood pressure, has also

vasodilator effects and attenuates left ventricular

hypertro-phy (Konstam et al., 2000) The SMILE (Survival of

Myocar-dial Infarction Long-term Evaluation) program indicates that

zofenopril may favourably affect the prognosis of patients

with a recent myocardial infarction (Lombardi et al., 2012)

and even of patients with the metabolic syndrome (Borghi

et al., 2008) Accordingly, a 12 week zofenopril treatment

significantly decreased lipid peroxidation, reduced cardiac

hypertrophy and improved NO pathway in patients with

essential hypertension (Napoli et al., 2004).

In conclusion, antihypertensive drugs, such asβ- blockers

with NO-dependent effects and ACE inhibitors with a thiol

group, may successfully restore NO signalling in the heart in

the metabolic syndrome

Interaction of antidiabetic drugs with cardiac

NO signalling

For the treatment of diabetes, several classes of drugs are

available with markedly different mechanisms of action

Besides various synthetic insulin analogues, several other

non-insulin-related drugs were developed and marketed in

the last years The mechanism of action involves the

stimu-lation of endogenous insulin secretion, the sensitization of

peripheral tissues to insulin or the increase in incretin levels

Although these mechanisms are directly not related to NO

signalling, all of these drugs have some degree of interaction

with NO-related pathways

Insulin itself is a strong regulator of cardiac NO level by

affecting eNOS phosphorylation Administration of insulin in

vivo to healthy rats activates Akt through a PI3K-dependent

mechanism Phosphorylation of the eNOS and the

concur-rent increase in NO production is a result of Akt activation

(Gao et al., 2002) However, this NO-related effect of insulin is

attenuated in the diabetic myocardium (Zakula et al., 2011).

Sulfonylurea drugs are potent stimulators of endogenousinsulin secretion by acting on ATP-sensitive K+ channels.Although these drugs do not interact directly with myocar-dial NO production, experimental and clinical data suggestconsiderable interaction with NO signalling Cardioprotec-tion mediated by NO is mainly related to the opening ofmitochondrial ATP-sensitive K+ channels (Han et al., 2002; Ljubkovic et al., 2007) The non-selective nature of K+

channel inhibition results in the attenuation of NO-mediatedcardioprotection by sulfonylureas, limiting their clinicalapplicability in diabetic patients with ischaemic heart dis-

eases (Garratt et al., 1999).

Insulin-sensitizing drugs include biguanides (metformin

is the most often used) and the thiazolidinedione class ofantidiabetic drugs (rosiglitazone and pioglitazone) Thesedrugs mainly act at peripheral tissues by sensitizing them tothe action of insulin Metformin facilitates the activation ofAMP-activated PK (AMPK) in the heart that has been shown

to be cardioprotective during heart failure induced positive effects were associated with increased AMPKand eNOS phosphorylation, and reductions in insulin, TGF-β1, basic fibroblast growth factor, and TNF-α levels in the

Metformin-circulation and/or in the myocardium (Gundewar et al., 2009; Wang et al., 2011) Withdrawal of rosiglitazone from

the market due to adverse cardiovascular effects (increasedmortality, accentuation if ischaemic heart diseases) high-lighted the controversial cardiovascular effects of thiazolidin-ediones In experimental studies, both rosiglitazone (Gonon

et al., 2007) and pioglitazone (Ye et al., 2008a) reduced infarct

size possibly via increased eNOS phosphorylation However,the mechanisms that resulted in adverse effects in humansare still not known

Dipeptidyl peptidase-4 (DPP-4) inhibitors are a relativelynew class of antidiabetics By the inhibition of DPP-4, theyincrease the level of incretins (GIP and GLP-1), inhibitingglucagon release, which in turn increases insulin secretion,decreases gastric emptying and decreases blood glucose level(Figure 2) DPP-4 inhibitors were proven to be atheroprotec-

tive (Matheeussen et al., 2013) and to affect positively

dias-tolic function in the insulin-resistant Zucker diabetic fatty ratmodel by increasing phosphorylation of eNOS (Ser1177) and

the expression of total eNOS (Aroor et al., 2013).

In conclusion, antidiabetics (except for sulfonylureas)may positively affect tissue NO availability and NO signal-ling, thereby providing a promising tool to treat cardiac com-plications of the metabolic syndrome

Interactions of anti-hyperlipidaemic treatments and the cardiac NO signalling

anti-hyperlipidaemic medications Apart from their HMG-CoAreductase inhibitory function, statins reduce cardiovascularrisks associated with hypercholesterolemia via a wide range ofwell-documented pleiotropic effects For instance, in theheart, atorvastatin increases phosphorylation of a host ofmediators associated with NO signalling, such as ERK, PDK-1,Akt and eNOS itself, plausibly via an adenosine receptor-

dependent mechanism (Merla et al., 2007; Ye et al., 2008b).

Direct modulation of NO signalling by statins downstream ofNOS was also suggested by another study, where rosuvastatinadministration reverted the elevation in mean arterial blood

pressure and cardiac remodelling caused by a treatment with

a NOS inhibitor, L-NAME (Baraka et al., 2009) Statins

modu-late cardiac NO metabolism under hyperlipidaemic

condi-tions as well In OLETF rats, both atorvastatin and pravastatin

up-regulated cardiac eNOS expression compared with their

genetic controls (Yu et al., 2004; Chen et al., 2007)

Interest-ingly, not all statins are equally effective in the modulation of

cardiac NO metabolism For example, pravastatin induced

eNOS more effectively than atorvastatin (Chen et al., 2007),

and we have previously shown that the first-generation

statin, lovastatin, does not affect NO production or NOS

activity in cholesterol-fed animals (Giricz et al., 2003)

Simi-larly, in spontaneously hypertensive rats, pravastatin

treat-ment failed to modulate the expression of nNOS, eNOS, sGC

or the NADPH oxidase subunits p40Phox and Gp91 in

myo-cardial tissue (Herring et al., 2011), which highlights that NO

modulation is not a general characteristic of the whole class

of statins and the effects are strongly model dependent

Newer statins have been also shown to alter cardiac NO

bioavailability in other pathologies unrelated to

hyperlipi-daemia For instance, in a hypertension model of rats

over-expressing renin, rosuvastatin decreased the accentuated

myocardial gp91(phox), p40(phox), p22(phox) expression

and reduced the myocardial lipid peroxidation, nitrotyrosine

formation and malondialdehyde content, suggesting that it

increased NO bioavailability by reducing ROS formation

(Habibi et al., 2007) Elsewhere, simvastatin reduced iNOS

expression in cytokine-treated H9C2 cardiac myoblasts,

which appeared to be related to the cholesterol

biosynthesis-modulating effect of statins, since mevalonate, and

gera-nylgeranyl pyrophosphate could reverse these effects

(Madonna et al., 2005) However, other statins exerted

seemingly opposing effects on the cardiac NO productionmodulated by pro-inflammatory signals: lipophilic statinsfluvastatin and lovastatin increased IL-1β-induced nitrite pro-duction by cardiac myocytes, whereas hydrophilic pravasta-tin did not Fluvastatin also increased iNOS expression (Ikeda

et al., 2001) These data demonstrate clearly that, before

ini-tiating a statin treatment, compounds must be evaluatedindividually in the view of the other coexisting pathologies.Statins might have positive effects on age-related disturbance

of cardiac NO metabolism as well In 20-month-old rats,atorvastatin administration for 4 months reversed the age-related increase in cardiac malondialdehyde and decrease of

SOD, catalase and NOS activity (Han et al., 2012) Direct

effects of statins on cardiac NO signalling have been studied

in humans in a few publications Perioperative simvastatintherapy of patients undergoing non-coronary cardiac surgeryincreased nitrite and nitrate levels, expression and phospho-rylation of eNOS at Ser1177, phosphorylation of Akt, HSP90,and its association with eNOS in right atrial appendage

(Almansob et al., 2012) Furthermore, atorvastatin induced a

mevalonate-reversible inhibition of NOX2-NADPH oxidaseactivity in right atrial samples from patients who developedpost-operative atrial fibrillation (AF); however, it did notaffect ROS, or NOS uncoupling in patients with permanent

AF (Reilly et al., 2011) Although the general notion is that

statins improve cardiac NO metabolism, these data alsosuggest that differences in the biochemical background ofdiverse pathologies might profoundly influence the benefi-cial pleiotropic effects of several of the statins

The PPAR family of nuclear receptors has been a target fornumerous antidiabetic and anti-hyperlipidaemic agents,many of which are shown to modulate NO metabolism

Figure 2

Effect of drugs used in the metabolic syndrome on cardiac NO signalling Antihypertensives activate primarily eNOS or potentiate the release of

NO from SNOs Antidiabetics activate predominantly the kinases AMPK and Akt upstream of eNOS to induce its phosphorylation Sulfonylureasmay, however, interfere with NO-related downstream effectors (i.e mitochondrial KATPchannels) Anti-hyperlipidaemic drugs have pleiotropiceffects on NO signalling, serving as antioxidants and inducers of eNOS phosphorylation

GW7647, a potent PPARα inducer, enhanced cardiac eNOS

activation in isolated papillary muscles of rat hearts (Xiao

et al., 2010) WY-14643, another PPARα agonist, has also been

shown to increase the expression of eNOS and iNOS, as well

as nitrite/nitrate levels in the ischaemic myocardium of

Goto-Kakizaki and Wistar rats (Bulhak et al., 2009) This

publica-tion also demonstrated that PPARα activapublica-tion leads to the

induction of the downstream mediators of NO, as shown by

an elevated cardiac phosphorylation of Akt at Ser473 and

Thr308 However, elsewhere, fenofibrate, also a PPARα inducer,

did not alter cardiac NO or its metabolites in LPS-treated

Wistar rats (Jozefowicz et al., 2007) Similarly, PPARβ/δ

agonist GW0742 reduced the ischaemia/reperfusion-induced

increase in the expression of iNOS and normalized the

phos-phorylation of Akt and glycogen synthase kinase-3β in a rat

model of regional myocardial I/R in vivo (Kapoor et al., 2010),

demonstrating the involvement of these PPAR isoforms in

NO metabolism More information is available on the effects

of PPARγ induction on cardiac NO balance The endogenous

PPARγ ligand, 15-deoxy-Δ12,14-PGJ2(15D-PGJ2), attenuated the

cardiac ischaemia/reperfusion-induced increase in iNOS

mRNA expression in rats (Wayman et al., 2002) The

inhibi-tion of iNOS expression by 15D-PGJ2, but not by

rosiglita-zone, a synthetic PPARγ agonist, was confirmed in neonatal

cardiomyocytes pretreated with LPS (Hovsepian et al., 2010)

or IL-1β (Mendez and LaPointe, 2003) Negative correlation

between the activity of PPARγ and NOS enzymes was

con-firmed in another study, where pioglitazone down-regulated

iNOS expression in a murine cardiac allotransplantation

model (Hasegawa et al., 2011) However, pioglitazone seems

to have opposing effects on eNOS In diabetic OLETF rats,

cardiac expression of eNOS and phosphorylation of Akt was

reduced compared with non-diabetic controls, which was

reversed by the induction of PPARγ by pioglitazone (Makino

et al., 2009) The notion that disturbed eNOS signalling is

restored by PPARγ activation seems to be strengthened by

other experiments Phosphorylated eNOS was increased in

mice receiving rosiglitazone before ischaemia/reperfusion

(Gonon et al., 2007), and in diabetic db/db mice, the reduced

dilations of coronary arterioles in response to ACh and the

NO donor NONOate were augmented by rosiglitazone (Bagi

et al., 2004).

There is only a limited amount of data on the effect of

other less frequently prescribed anti-hyperlipidaemic agents

on cardiac NO signalling Although dietary supplementation

of niacin is often recommended for obese patients, its effect

on the NO-cGMP-PKG system has been revealed indirectly in

a single publication Niacin-bound chromium induced

myocardial phosphorylation of Akt, AMPK and eNOS

in streptozotocin-induced diabetic rats after

ischaemia-reperfusion injury, suggesting that beneficial effects of niacin

and chromium are mediated not only through the

modula-tion of metabolic pathways, but via the activamodula-tion of the NO

pathway as well (Penumathsa et al., 2009) Inhibition of

cho-lesterol absorption by ezetimibe, an inhibitor of the intestinal

Niemann-Pick C1-like 1 protein, has been shown to decrease

cardiac NADPH oxidase-mediated oxidative stress in

hyper-lipidaemic db/db mice (Fukuda et al., 2010), therefore,

plausi-bly increase NO bioavailability, known to be depressed in

hyperlipidaemia (Ferdinandy et al., 1997; Giricz et al., 2003;

Conclusions and perspectives

Published data show that NO availability and its signalling inthe heart is impaired in the presence of risk factors associatedwith the metabolic syndrome The decreased tissue availabil-ity of NO is a consequence of increased oxidative andnitrosative/nitrative stress rather than a decreased cardiac NOsynthesis The impaired NO signalling in the heart due to themetabolic syndrome leads to different pathophysiologicalprocesses including myocardial hypertrophy, fibrosis andeventually heart failure Therefore, in addition to treating theindividual risk factors related to the metabolic syndrome,restoration of NO signalling in the heart by pharmacologicaltools may be a promising therapeutic avenue to alleviatecardiac pathologies related to the metabolic syndrome

Acknowledgements

This study was elaborated within the projects APVV-0742-10,VEGA 2/0183/12 and 2/0144/14, the New Horizons Grant ofthe European Foundation for the Study of Diabetes, Hungar-ian Scientific Research Fund (OTKA K109737) and COSTAction BM1005, and the Intramural Program of NIH/NIAA.All authors contributed equally on literature search and meta-bolic syndrome models preparation

Conflicts of interest

The authors declare no conflict of interest

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Themed Section: Pharmacology of the Gasotransmitters

REVIEW

Gas what: NO is not the

only answer to sexual

function

G Yetik-Anacak1, R Sorrentino2, A E Linder3and N Murat4

1Department of Pharmacology, Faculty of Pharmacy, Ege University, I˙zmir, Turkey,2Department

of Pharmacy, University of Naples Federico II,, Naples, Italy,3Department of Pharmacology,

Universidade Federal de Santa Catarina, University Campus, Trindade, Biological Sciences

Centre, Santa Catarina, Brazil, and4Department of Pharmacology, Medical School, Dokuz Eylül

University, Izmir, Turkey

Correspondence

Gunay Yetik-Anacak, Department

of Pharmacology, Ege University,Faculty of Pharmacy, 35100,Izmir, Turkey E-mail:

gunayyetik@gmail.com

-Standard abbreviations conform

to BJP’s Concise Guide toPHARMACOLOGY (Alexander

et al.,2013a,b) and to the IUPHAR

The ability to get and keep an erection is important to men for several reasons and the inability is known as erectile

dysfunction (ED) ED has started to be accepted as an early indicator of systemic endothelial dysfunction and subsequently ofcardiovascular diseases The role of NO in endothelial relaxation and erectile function is well accepted The discovery of NO as

a small signalling gasotransmitter led to the investigation of the role of other endogenously derived gases, carbon monoxide

has also been confirmed In this review, we focus on the role of these three sister gasotransmitters in the physiology,

pharmacology and pathophysiology of sexual function in man, specifically erectile function We have also reviewed the role ofsoluble guanylyl cyclase/cGMP pathway as a common target of these gasotransmitters Several studies have proposed

alternative therapies targeting different mechanisms in addition to PDE-5 inhibition for ED treatment, since some patients donot respond to these drugs This review highlights complementary and possible coordinated roles for these mediators andtreatments targeting these gasotransmitters in erectile function/ED

Erectile physiology is the interplay of vascular, neurological

and endocrine factors, which leads to an increase in or

facili-tates the vasodilatation (tumescence) and/or reduces the

con-traction (detumescence) of the corpus cavernosum smooth

muscle (CCSM) cells Erection is the final outcome of acomplex integration of signals It is essentially a spinal reflexthat can be initiated by recruitment of penile afferents, butalso by visual, olfactory and imaginary stimuli and all thestimuli contribute to the increase in vasodilatation of penile

tissues (for details, see review by Cirino et al., 2006) Neuronal

and endothelial NO are considered as the most importantfactors for relaxation of penile vessels and CCSM cells