C-type natriuretic peptide in vascular physiology and disease

Associate editor:R.M.WadsworthC-type natriuretic peptide in vascular physiology and diseaseRamona S.Scotland a ,Amrita Ahluwalia b ,Adrian J.Hobbs a ,*a Wolfson Institute for Biomedical Research,University College London,Cruciform Building Gower Street,London WC1E 6AE,UK bClinical Pharmacology,William Harvey Research Institute,Barts and The London,Charterhouse Square,London EC1M 6BQ,UKAbstractNatriuretic peptides play a critical role in coordination of fluid/electrolyte balance and vascular tone.The renal effects of circulating atrial natriuretic peptide (ANP)and brain natriuretic peptide (BNP)are distinct from the paracrine effects of vascular C-type natriuretic peptide (CNP).CNP is widely expressed throughout the vasculature and is found in particularly high concentrations in the endothelium.Recent studies demonstrate that CNP is a novel endothelium-derived hyperpolarising factor (EDHF)that complements the actions of other endothelial vasorelaxant mediators such as nitric oxide (NO)and prostacyclin.Since several cardiovascular disorders are associated with dysfunction of natriuretic peptide activity,selective modulation of the natriuretic peptide pathways represents an important therapeutic target;whilst this has been exploited to some degree in terms of ANP/BNP,the therapeutic potential of CNP has yet to be tapped.This review focuses on recent findings on the actions and mechanism of locally produced endothelial-derived CNP in the cardiovascular system and highlights many potential avenues for therapeutic intervention,via modulation of CNP-signalling,in cardiovascular disease.D 2004Elsevier Inc.All rights reserved.Keywords:Natriuretic peptide;Endothelium;Endothelial dysfunctionAbbreviations:ANP,atrial natriuretic peptide;BNP,B-type natriuretic peptide;BP,blood pressure;CNP,C-type natriuretic peptide;DNP,Dendroaspisnatriuretic peptide;EDHF,endothelium-derived hyperpolarising factor;ET-1,endothelin-1;GC,guanylate cyclase;I/R,ischaemia-reperfusion;NP,natriuretic peptide;NPR,natriuretic peptide receptor;RAAS,renin-angiotensin-aldosterone system.Contents 1.Introduction.........................................862.Structure and synthesis of C-type natriuretic peptide ..................863.Natriuretic peptide receptors ...............................874.Regulation of vascular tone ...............................874.1.C-type natriuretic peptide as an endothelium-derived hyperpolarising factor...884.2.C-type natriuretic peptide as a venodilator ....................884.3.Interactions with other endothelium-derived factors ...............885.Regulation of blood pressure...............................896.C-type natriuretic peptide and ischaemia/reprefusion injury ...............897.C-type natriuretic peptide and vascular inflammation ..................908.Future direction .....................................91Acknowledgments .......................................91References...........................................910163-7258/$-see front matter D 2004Elsevier Inc.All rights reserved.doi:10.1016/j.pharmthera.2004.08.011*Corresponding author.Tel.:+442076796611;fax:+442076913104.E-mail address:a.hobbs@ (A.J.Hobbs).Pharmacology &Therapeutics 105(2005)85–93/locate/pharmthera1.IntroductionThe natriuretic peptides (NP)play an important role in the regulation of vascular homeostasis,not only as regulators of blood volume but also by directly altering vascular reactivity.As such these peptides are obvious candidates as targets for therapeutic intervention in cardio-vascular disease.NP comprise a family of peptides including C-type natriuretic peptide (CNP),atrial (or A-type)natriuretic peptide (ANP),brain (or B-type)natriuretic peptide (BNP),urodilatin (derived from alternate splicing of pro-ANP and sometimes termed ANP 95–126),and the snake venom-derived Dendroaspis natriuretic peptide (DNP)(Schweitz et al.,1992)that may also be present in mammals (Schirger et al.,1999).These peptides oppose the renin-angiotensin-aldosterone system (RAAS)and thereby regu-late diuresis/natriuresis and smooth muscle tone and proliferation.Hypervolaemia-induced stretch of the atria and ventricles releases ANP and BNP into the circulation where these peptides act in an endocrine fashion to modulate blood volume and hence blood pressure.In contrast,CNP and urodilatin may be considered as the paracrine NP that act locally in the vasculature and kidney,respectively.Whilst ANP and BNP have been well characterised,less is known about the biological functions of CNP and the signal transduction mechanisms this peptide initiates.2.Structure and synthesis of C-type natriuretic peptide The NP consist of a common 17-aa ring structure formed by a disulphide bridge (Fig.1).This ring structure,important for receptor binding,is highly conserved among members of the NP family and also amongst species.Of the NP,CNP is the most highly conserved (including man,rodent,reptile,fish)and has been identified in primitive species (for review,see Takei,2001).Indeed,there is evidence that CNP may be theancestral precursor from which ANP and BNP evolved (Inoue et al.,2003).The NP genes each encode prepropeptides and in man,the CNP gene is located on chromosome 2,and contains two exons and one intron (ANP and BNP are located closely together on chromo-some 1and each comprises three exons separated by two introns).Following removal of the signal peptide by signal peptidase,126aa human prepro-CNP is converted to the propeptide (the form in which it is stored)(Fig.2).Pro-CNP is subsequently cleaved by furin (Wu et al.,2003),a ubiquitous proprotein convertase resident in the trans-Golgi network (Thomas,2002),to yield CNP-53.This peptide can be secreted and/or further processed,by an unidentified mechanism,to yield the active 22-aa CNP (although the extended 53-aa peptide is also found in brain tissue from human,pigs,and sheep as well as human endothelial cells (Stingo et al.,1992),and may also have biological functions).This pathway of CNP synthesis is similar to that of endothelin-1(ET-1)from pro-ET-1in human endothelial cells,which also requires furin (Blais et al.,2002).In contrast,pro-ANP and pro-BNP are not cleaved by furin but are processed in a similar manner by corin,a transmembrane serine protease (Yan et al.,2000).CNP was first identified as a 22-aa peptide isolated from porcine brain extract that caused potent relaxation of chick rectum,and had a similar structure to the previously identified ANP and BNP (Sudoh et al.,1990).CNP is expressed abundantly in the brain,but is also present in high concentrations in peripheral tissues,particularly vascular endothelial cells (Stingo et al.,1992).Whilst ANP and BNP are known as b the cardiac natriuretic peptides Q due to their predominant expression in the atria and ventricles,respec-tively,CNP is expressed in low amounts in the heart (V ollmar et al.,1993).Typically,in the absence of disease,plasma CNP levels in humans are low (Igaki et al.,1996;Barletta et al.,1998;Kalra et al.,2003)which,coupled with the high concentration of this peptide in endothelial cells,has given rise to the thesis that the primary actions ofCNPFig.1.Primary structure of the natriuretic peptide family.Each peptide consists of a highly conserved 17-aa ring created by a disulphide bond,which is required for receptor binding.Residues in white circles denote those that are conserved between the peptides.R.S.Scotland et al./Pharmacology &Therapeutics 105(2005)85–9386are paracrine/autocrine and that it is unlikely to act in an endocrine fashion.3.Natriuretic peptide receptorsThe biological effects of NP are transduced by specific cell surface receptors.ANP and BNP represent selective ligands for NP receptor (NPR)-A and CNP is a selective agonist at NPR-B.Both these NPR are particulate guanylate cyclase (pGC)-linked receptors (also termed GC-A and GC-B,respectively)that catalyse the conversion of GTP to cyclic guanosine monophosphate (cGMP).The different tissue distribution of NPR underlies the differ-ential effects of NP.NPR-A is predominantly expressed in the vasculature (particularly large conduit blood vessels)and NPR-B is predominantly expressed in the brain (Levin et al.,1998),intimating that CNP signalling is important in the CNS.A third NPR,NPR-C (the b clearance receptor Q )is a single transmembrane receptor with a short 37-aa C-terminal tail that lacks GC activity but contains a pertussis toxin-sensitive G i -binding domain (Anand-Srivastava et al.,1996).NPR-C is the most abundantly expressed NPR,comprising ~95%of the total NPR population,and has similar affinity for all NP (Maack,1992).The primary role of NPR-C is considered to be to modulate the availability of NP by binding and removing them from the circulation (Maack et al.,1987),as highlighted by mice deficient in NPR-C where the half-life of ANP is significantly elevated (Matsukawa et al.,1999).Moreover,administration of CNP in anaesthetised dogs concomitantly increases plasma NP and vice versa,suggesting that NP compete for NPR-Cand thereby influence plasma NP levels (Clavell et al.,1993).In addition to clearance by NPR-C,NP availability is also modulated by enzymatic degradation by neutral endopeptidase (NEP)24.11,a zinc metallopeptidase widely distributed throughout the vasculature (including heart,lung,kidney,and endothelium)that also degrades several other peptides including ET-1.NEP exhibits a higher rate of hydrolysis of CNP compared to other NP,suggesting that this enzyme may be more important in regulation of CNP bioavailability than sequestration by NPR-C (Kenny et al.,1993).4.Regulation of vascular toneThe relatively high expression of CNP in vascular endothelial cells and the presence of its receptors on the underlying smooth muscle intimate that this peptide has the capacity to affect vascular tone.Indeed,local infusion of CNP increases forearm blood flow,an effect that is independent of nitric oxide (NO)(Honing et al.,2001).It is well established that exogenous CNP is a potent vaso-and venodilator of isolated human (Wiley &Davenport,2001),rat (Drewett et al.,1995;Brunner &Wolkart,2001;Chauhan et al.,2003),canine (Wennberg et al.,1999),murine (Madhani et al.,2003;Steinmetz et al.,2004),and porcine (Barber et al.,1998)blood vessels.Whilst CNP is a dilator of many vascular beds in different species the mechanism and potency of these responses appear to vary according to the size of the vessel,a fact that most likely reflects the heterogeneity of NPR subtype expression.NPR-B is a b CNP-selective Q receptor and thus many oftheFig.2.Schematic diagram of the processing of human prepro-C-type natriuretic peptide (CNP)to the biologically active CNP-22.Removal of the N-terminal signalling peptide is followed by processing by the serine protease Furin to yield CNP-53.This peptide may possess biological activity per se,but is cleaved to the principal physiologically relevant form (CNP-22)by an as yet unidentified process.R.S.Scotland et al./Pharmacology &Therapeutics 105(2005)85–9387biological effects of CNP are attributed to activation of this receptor,such as in conduit arteries where smooth muscle relaxation to CNP is inhibited by the selective NPR-A/B antagonist HS-142-1(Drewett et al.,1995;Madhani et al., 2003).However,evidence is emerging that the clearance receptor NPR-C,when stimulated by CNP or the synthetic selective ligand cANF4–23(ring-deleted ANP;(Maack et al., 1987),can also elicit effects in a G i-dependent manner. 4.1.C-type natriuretic peptide as anendothelium-derived hyperpolarising factorThe involvement of NPR-C in regulation of vascular tone appears to be particularly prevalent in small resistance arteries(Chauhan et al.,2003;Steinmetz et al.,2004).This profile of reactivity to CNP in conduit and resistance arteries mirrors the pattern of activity observed with endothelium-derived hyperpolarising factor(EDHF),an as yet unidenti-fied endothelial mediator that elicits vasorelaxation via a characteristic smooth muscle hyperpolarisation.EDHF-mediated,endothelium-dependent relaxation,in response to hormones or physical stimuli,is more prominent in resistance than conduit arteries(Shimokawa et al.,1996). The fact that CNP is stored in endothelial cells and capable of causing vasorelaxation by hyperpolarisation(Barton et al.,1998)has fuelled speculation that CNP may represent (an)EDHF.Studies in isolated small arteries suggest that classical endothelium-dependent dilators acetylcholine (ACh)or bradykinin cause relaxation partly by the release of CNP(Wennberg et al.,1999).More recently,ACh has been shown to directly stimulate the release of CNP from vascular endothelium and cause relaxation of rat resistance arteries that is associated with membrane hyperpolarisation and mediated by NPR-C(Chauhan et al.,2003).This study also showed that relaxation to either CNP or ACh is abolished in the presence of pharmacological inhibitors known to block EDHF activity(for review,see Busse et al., 2002).In these arteries,dilatation to CNP/EDHF through NPR-C is mediated by direct coupling of G i with inwardly rectifying K+channels(GIRK)present in smooth muscle. These exciting new findings indicate that CNP/NPR-C signal transduction system is an important novel pathway in regulation of peripheral blood flow and that CNP acts as an EDHF.This hypothesis is supported by the recent finding that an identical CNP/NPR-C pathway exists in the rat coronary vasculature.As in the mesenteric circulation,CNP or cANF4–23reduce perfusion pressure in isolated rat heart, an effect that is blocked by inhibition of either EDHF or NPR-C pathways(Hobbs et al.,2004).These findings fit with previous data showing that CNP can cause relaxation of isolated porcine coronary arteries by hyperpolarisation (Barber et al.,1998;Barton et al.,1998)and increase coronary blood flow in vivo in dogs.Even though CNP is not considered to be a cardiac NP,CNP is measurable in the myocardium in man(Kalra et al.,2003)and infusion of ACh into isolated rat hearts stimulates the release of considerable amounts of CNP into the coronary circulation (Hobbs et al.,2004).Thus,there is compelling evidence that CNP is also an important mediator in the coronary circulation and that its effects are predominantly mediated by NPR-C.A similar mechanism may also account for the inhibition of myocardial L-type Ca2+currents by CNP(Rose et al.,2004).The existence of the CNP/NPR-C dilator pathway in different vascular beds suggests that similar mechanisms of regulation of vascular tone/blood flow are likely to exist in other organs and species.Importantly,the finding that these pathways predominate in resistance vessels provides an opportunity to selectively target these blood vessels in diseases characterised by excessive tone, such as hypertension.Although ANP and BNP also have the capacity to bind to NPR-C,unlike CNP,they do not appear to reduce tone of small resistance arteries through activation of this receptor.Indeed in arteries known to have functional NPR-C receptors(such as mouse and rat mesenteric resistance artery)ANP does not elicit significant dilatation (Osol et al.,1986;Madhani et al.,2003).Thus,dilatation of certain small human arteries by ANP(Hughes et al.,1989; Kublickiene et al.,1995;Wiley&Davenport,2001)appears to be mediated exclusively through activation of the NPR-A/cGMP pathway.The reason for this discrepancy in NPR-C activation is not known but may indicate that the interaction of other NP with NPR-C is different from CNP such that they initiate receptor internalisation but can not stimulate signal transduction.It is also possible that there is more than one subtype of NPR-C receptor(Anand-Srivastava,1997)that accounts for the divergent effects of CNP and other NP.4.2.C-type natriuretic peptide as a venodilatorCNP is a potent dilator of large canine and porcine veins (i.e.,saphenous and femoral veins)and this vasorelaxant activity,as with resistance arteries,involves smooth muscle hyperpolarisation(Wei et al.,1993;Banks et al.,1996). However,CNP has only modest dilator effects on human isolated saphenous veins(Protter et al.,1996).Interestingly, CNP levels also appear to be higher in veins than in arteries suggesting that physiologically it has considerable impact on venous pressure.The receptor subtype involved in venodilatation has not been extensively characterised but appears to be GC-linked NPR-B rather than NPR-C(Banks et al.,1996).The differential action of CNP in the venous and arterial circulation implies that the regulation of pre-load(venous)and peripheral resistance(arterial)by CNP are also distinct.4.3.Interactions with other endothelium-derived factorsThe effects of(exogenous)CNP on vascular tone are not dependent on the endothelium,consistent with its identi-fication as an EDHF.In keeping with this,removal of the endothelium does not abolish the vasorelaxant effects ofR.S.Scotland et al./Pharmacology&Therapeutics105(2005)85–93 88CNP(Wennberg et al.,1999).Paradoxically,the potency of CNP is increased in the absence of the endothelium(Wright et al.,1996)suggesting an interaction between CNP and other endothelium-derived factors.NO is an important endothelium-derived dilator that regulates vascular tone and whose effects are predominantly mediated by the stimulation of vascular smooth muscle soluble guanylate cyclase(sGC)and subsequent production of cGMP(Hobbs, 1997).Akin to NO,CNP can elevate smooth muscle cGMP through stimulation of NPR-B.Hence,it is reasonable to assume that there is considerable cross-talk in the biological effects of CNP and NO.Recent studies demonstrate that the NO/sGC/cGMP pathway complements NP/pGC/cGMP such that suppression of one pathway decreases the sensitivity of the other(Hussain et al.,2001;Madhani et al.,2003).Thereby,circulating ANP/BNP or locally produced CNP are functionally linked to the activity of endothelial-derived NO.This reciprocal relationship may have important consequences in disease states where NO bioavailability is compromised(e.g.,by excess superoxide production)since NP/pGC/cGMP can supplement dysfunc-tional NO/sGC/cGMP to regulate vascular tone.Similarly, reduced NP/pGC/cGMP activity may explain some of the side effects seen with nitrate tolerance,such as sodium retention.Interactions between CNP and ET-1also appear to be important physiologically;indeed,the vascular effects of CNP are directly opposite to those of ET-1(for review,see Han&Hasin,2003).For example,ET-1-induced vaso-constriction and cardiomyocyte hypertrophy is inhibited by CNP.Whilst CNP has little natriuretic and diuretic action compared to ANP or BNP,it is capable of modulating the vascular effects of local RAAS by opposing potent vaso-constriction to angiotensin II(ANGII).Not only does CNP functionally antagonise ET-1and ANGII but it can also directly modulate ET-1(Kohno et al.,1992)and ANGII (Davidson et al.,1996)synthesis.Therefore,the parallel production and activity of vasodilator CNP and vaso-constrictors such as ET-1and ANGII allows for tight local regulation of these vasoactive peptides and thus blood flow.5.Regulation of blood pressureThe potent vasodilator activity of CNP,particularly in resistance vessels,suggests that CNP has the capacity to affect blood pressure(BP).Interestingly,a study of N2000 Japanese subjects demonstrated that a polymorphism in the human CNP gene(G2628A)is a predictor of hypertension, particularly in the younger subpopulation of the study(Ono et al.,2002).However,hypertension does not appear to be associated with elevated plasma CNP per se(it is correlated with increased BNP;Cheung&Brown,1994).The inability to detect significant changes in plasma CNP in hypertension is perhaps not surprising given the rapid inactivation of CNP and the paracrine/autocrine nature of its action.Thus,local changes in CNP production or activity are not necessarily reflected in circulating plasma.The importance of this polymorphism and the link between CNP and hypertension are not known but it is clear that i.v.administration of CNP in healthy volunteers causes a significant decrease in BP (Igaki et al.,1996)and therefore exogenous CNP can modulate BP,at least acutely.Similarly,CNP administration lowers BP in primates(Seymour et al.,1996)and dogs (Clavell et al.,1993)thus supporting the thesis that CNP is an important physiological regulator of BP.These acute hypotensive effects of exogenous CNP are most likely due to a direct effect on vascular tone.However,long-term regulation of BP by CNP also has a central component through CNP-mediated suppression of ACTH(Guild& Cramb,1999)and A VP(vasopressin;Shirakami et al., 1993),and through inhibition of aldosterone synthesis (Guild&Cramb,1999).Surprisingly,CNP knockout mice are not hypertensive(Chusho et al.,2001)but conversely are mildly hypotensive due to compensatory increased ANP levels in these animals.6.C-type natriuretic peptideand ischaemia/reprefusion injuryIn addition to its dilator activity as a coronary EDHF, CNP also reduces the extent of infarction due to global ischaemia/reperfusion(I/R)in rat isolated hearts.Infusion of CNP,either prior to or following ischaemic insult,results in 30–50%reduction of infarction(Hobbs et al.,2004).This effect on tissue damage is accompanied by normalisation of left ventricular developed pressure(an index of cardiac contractility)and coronary perfusion pressure to pre-ischaemia levels.The efficacy of CNP post-ischaemia, indicates that it may be used to rescue the failing heart from I/R injury as well as a prophylactic treatment to prevent myocardial infarction(e.g.,pre-surgery).In accord with its dilator actions,the protective effects of CNP in the heart are mimicked by the NPR-C agonist cANF4–23, indicating that this receptor subtype mediates these actions of CNP and that targeting of CNP/NPR-C is beneficial in prevention of I/R injury.Since the CNP/EDHF/NPR-C pathway is not peculiar to the coronary circulation,it is tempting to speculate that CNP may also be used in treatment of other ischaemic diseases.Indeed,in a model of mouse hindlimb ischaemia(induced by femoral artery ligation),adenoviral-mediated CNP gene expression accel-erates angiogenesis and restoration of blood flow(Yamahara et al.,2003).The mechanism of protection against infarction is not fully understood but CNP also decreases heart rate through interaction with NPR-C(Rose et al.,2004)and therefore in combination with coronary dilatation CNP is likely to cause a profound increase in myocardial perfusion. The concentrations of exogenous CNP used in these studies are similar to those released from the coronary endothelium by endothelium-dependent dilators such as ACh(Hobbs etR.S.Scotland et al./Pharmacology&Therapeutics105(2005)85–9389al.,2004),suggesting that mediators such as bradykinin (Wennberg et al.,1999),or physical forces(i.e.,shear stress-regulated CNP expression;Chun et al.,1997),that the heart may be exposed to during ischaemia/reperfusion can stimulate sufficient CNP release from endothelial cells to exert a protective effect.Interestingly,hypoxia itself can stimulate CNP production,representing a further stimulus for CNP release during ischaemia that acts as a compensa-tory mechanism to limit damage caused by I/R P levels also appear to be raised in patients with chronic heart failure(Kalra et al.,2003;Wright et al.,2003),and may exert an analogous protective effect in these patients.7.C-type natriuretic peptide and vascular inflammationThe abundant localisation of CNP to endothelial cells places CNP in an ideal location to modulate the activity of circulating cells particularly immune cells and platelets. Moreover,inflammatory stimuli such as IL-1h,TNF,and lipopolysaccharide(Suga et al.,1993)stimulate CNP release from isolated endothelial cells.Indeed circulating CNP levels are significantly increased in patients with sepsis(Hama et al.,1994).Such modulation of CNP production may therefore reflect contribution to or attenuation of an inflammatory response.Current evidence indicates overwhelmingly that the latter is true.For example,CNP or the NPR-C ligand cANF4–23suppress the production of pro-inflammatory cyclo-oxygenase2-derived prostaglandin E2in isolated cells(Kiemer et al., 2002).More importantly,CNP in vivo also inhibits vascular inflammation associated with vein grafts.Several studies in mice(Schachner et al.,2004),rabbits(Ohno et al.,2002),and pigs(Rotmans et al.,2004)have demonstrated that overexpression of CNP by adenoviral gene delivery in veins dramatically reduces the luminal narrowing(neointimal hyperplasia)that develops when it is grafted to the carotid artery,thereby retaining patency of the graft.Again,these effects of CNP appear to be due to a direct local action on the blood vessel wall since adenoviral-driven CNP expression does not alter the level of circulating NP.More recently,studies in our own lab show a direct effect of CNP on inflammatory cells in vivo (unpublished observations).In these studies,application of CNP significantly reduces cytokine or histamine-induced leukocyte rolling in mouse mesenteric circulation,an effect that is mimicked by cANF4–23.Therefore like NO, endothelial CNP exerts a protective anti-inflammatory effect,probably through an NPR-C-mediated cGMP-independent mechanism.This inhibitory effect of CNP on leukocytes suggests that the peptide modulates the expression of adhesion molecules,either on the endothe-lium or leukocytes.This is supported by the observation that CNP expression in balloon injured arteries suppresses expression of vascular adhesion molecules VCAM-1and ICAM-1(Qian et al.,2002).Like its effects on leukocytes,CNP-mediated reduction of neointima formation in com-pressed arteries is also mediated by NPR-C(Brown& Chen,1995)supporting the hypothesis that CNP/NPR-C pathway has important cytoprotective effects in the vasculature.Atherosclerosis is an inflammatory disease of large arteries that is one of the principal causes of death in industrialised countries(for review,see Ross,1999).The anti-inflammatory actions of CNP suggest that it may also be protective against the development of atherosclerotic lesions.In human coronary arteries,expression of CNP and its receptors(NPR-B and NPR-C)are inversely correlated with severity of the lesion(Casco et al.,2002)but a causal link between CNP and lesion formation/regression has not been established.Direct suppression of leukocyte recruit-ment may explain,in part,some of the beneficial actions of CNP on cardiovascular inflammatory disorders.In addition, CNP is a potent inhibitor of oxidised LDL-stimulated vascular smooth muscle migration and proliferation(Kohno et al.,1997)that is an integral component of complex lesion formation.The effects of CNP on mitogenesis are consid-ered to be mediated by inhibition of the mitogen activated protein kinase(MAPK)cascade(Prins et al.,1996)and DNA synthesis(Cahill&Hassid,1994).It is not clear which receptor subtype is involved in these vascular effects of CNP since some of these studies report significant changes in cGMP(implicating NPR-B;Kohno et al.,1997), whilst others demonstrate that CNP or the NPR-C agonist cANF4–23can directly inhibit rat aortic smooth muscle migration and proliferation in vitro(Cahill&Hassid,1994). NPR-C activation has also been reported to mediatethe Fig.3.Schematic representation of the putative vasoprotective roles of endothelial CNP(endothelium-derived hyperpolarising factor[EDHF]). Activation of natriuretic peptide receptor(NPR)-C by endothelial CNP underlies a key mechanism regulating vascular tone and local blood flow (bottom).However,the importance of the CNP/NPR-C signalling pathway may extend to the prevention of leukocyte and platelet activation(top), thereby maintaining an important anti-atherogenic influence on the blood vessel wall.R.S.Scotland et al./Pharmacology&Therapeutics105(2005)85–93 90。