Heart 2001;85:342–350

which was subsequently identified as nitric GENERAL CARDIOLOGY oxide.23

L-: pathway Endothelial function and nitric oxide: derived nitric oxide is synthesised clinical relevance from the L-arginine by the endothelial isoform of , 342 yielding L- as a byproduct.4 Nitric Patrick Vallance, Norman Chan oxide is labile with a short half life (< 4 seconds Centre for Clinical Pharmacology, University College London, in biological solutions). It is rapidly oxidised to London, UK and then by oxygenated haemo- globin before being excreted into the urine.4 Several co-factors are required for nitric oxide he vascular endothelium is a monolayer biosynthesis. These include nicotinamide of cells between the vessel lumen and adenine dinucleotide phosphate (NADPH), Tthe vascular smooth muscle cells. Far flavin mononucleotide, flavin adenine dinucle- from being inert, it is metabolically active and otide, (BH4), and cal- produces a variety of vasoactive mediators. modulin. Once synthesised, the nitric oxide Among these mediators, endothelial derived diVuses across the endothelial membrane nitric oxide is essential in the maintenance of and enters the vascular smooth muscle cells vascular homeostasis, and defects in the where it activates , leading to L-arginine: nitric oxide pathway have been an increase in intracellular cyclic guanosine- implicated in a variety of cardiovascular 3’,5-monophosphate (cGMP) concentrations4 diseases. (fig 1). As a second messenger, cGMP mediates many of the biological eVects of nitric Historic perspectives oxide including the control of vascular tone and platelet function. In addition, nitric oxide has other molecular targets which include From EDRF to nitric oxide haem or other iron centred , DNA, and In 1980, Furchgott and Zawadzki showed that thiols. These additional reactions may mediate the presence of vascular endothelial cells is changes in functions of certain key or essential for to induce relaxation ion channels. Nitric oxide also interacts with of isolated rabbit aorta.1 If the vascular enzymes of the respiratory chain including Correspondence to: endothelium was removed, the complex I and II, and aconitase, and through Professor Patrick failed to relax in response to acetylcholine but these eVects alters tissue mitochondrial respi- Vallance, Centre of Clinical Pharmacology, still responded to glyceryl trinitrate. This ration. Interaction of nitric oxide with super- Gower Street Campus, endothelium dependent relaxation of vascular oxide anion can attenuate physiological re- 3rd Floor, Rayne Institute, smooth muscle to acetylcholine is mediated by sponses mediated by nitric oxide and produce 5 University Street, irreversible inhibitory eVects on mitochondrial London WC1E 6JJ, UK an endogenous mediator initially named en- 1 [email protected] dothelium derived relaxing factor (EDRF), function as a result of the formation of (ONOO−), a powerful oxidant Endothelial cell Vascular smooth muscle cell species.

Nitric oxide synthase isoforms Three isoforms of nitric oxide synthase (NOS) GTP cGMP O2 Citrulline have been identified: the endothelial isoform (eNOS), neuronal isoform (nNOS), and mac- L-arginineNO NO-haem GC rophage or inducible isoform (iNOS). All three NOS isoforms play distinct roles in the regula- NOS tion of vascular tone (fig 2). The genes encod- ing eNOS, nNOS, and iNOS are located on chromosome 7, 12, and 17, respectively.5 Figure 1. The L-arginine: nitric oxide pathway. NO, nitric oxide; NOS, nitric oxide Whereas eNOS and nNOS are normal con- synthase; GC, guanylate cyclase; cGMP, cyclic guanosine-3’,5-monophosphate; stituents of healthy cells, iNOS is not usually GTP, expressed in vascular cells and its expression is seen mainly in conditions of infection or AB C inflammation.

Biological effects of nitric oxide

Nitric oxide and the vasculature Endothelium derived nitric oxide is a potent vasodilator in the vasculature, and the balance between nitric oxide and various endothelium Figure 2. (A) In most, if not all, vessels nitric oxide is synthesised within the derived vasoconstrictors and the sympathetic endothelium. (B) In certain vessels (for example, cerebral vessels) nitric oxide is maintains blood vessel tone. also synthesised by nerves in the adventitia (nitrogenic nerves). (C) After exposure to endotoxin or , iNOS is expressed throughout the vessel wall In addition, nitric oxide suppresses platelet and produces large amounts of nitric oxide. aggregation, leucocyte migration, and cellular

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Basal nitric oxide release The L-arginine: nitric oxide pathway The synthesis of nitric oxide in vascular endothelial cells in culture or intact vascular x Nitric oxide is synthesised from L-arginine tissue can be inhibited by NG monomethyl-L- to yield citrulline arginine (L-NMMA), an analogue of L-arginine in which one of the guanidino Co-factors required for nitric synthesis: x nitrogen atoms is methylated. This inhibitory – nicotinamide adenine dinucleotide eVect of L-NMMA is readily reversed by 343 phosphate (NADPH) 7 – flavin mononucleotide L-arginine. L-NMMA and similar substrate – flavin adenine dinucleotide based inhibitors have been used to examine the role of nitric oxide in various vascular beds in – tetrahydrobiopterin (BH4) – vitro and in both human and animal models. In rings of rabbit aorta, L-NMMA causes x Enzymes responsible for nitric oxide significant endothelium dependent contrac- synthesis: tion. Intravenous infusion of L-NMMA into – endothelial nitric oxide synthase experimental animals induces a dose related (expressed in normal cells) (eNOS) increase in which is reversed by – neuronal nitric oxide synthase intravenous administration of L-arginine, and (expressed in normal cells) (nNOS) in the human forearm vasculature infusion of – inducible nitric oxide synthase L-NMMA into the brachial causes sub- (expressed during infection/ stantial dose dependent vasoconstriction. Thus inflammation) (iNOS) continuous generation of nitric oxide is crucial in maintaining peripheral vasodilatation in x Nitric oxide activates guanylate cyclase in 7 vascular smooth muscle cells to synthesise humans (fig 3). This basal nitric oxide cyclic guanosine-3’,5-monophosphate mediated dilatation has been seen in every (cGMP) which causes many of its other arterial bed studied including cerebral, biological eVects pulmonary, renal, and coronary vasculature. In contrast, in the venous system inhibitors of NOS do not lead to an increase in basal tone in adhesion to the endothelium, and attenuates a variety of venous preparations from animals 8 vascular smooth muscle cell proliferation and or humans, suggesting that basal nitric oxide migration. Furthermore, nitric oxide can in- production does not have a major role in the hibit activation and expression of certain adhe- maintenance of the resting tone in most veins. sion molecules, and influence production of In conduit vessels, there is some basal nitric anion. Loss of endothelium derived oxide mediated dilatation but it appears to be nitric oxide would be expected to promote a less than that seen in resistance vessels. vascular phenotype more prone to atherogen- esis, a concept supported by studies in experi- Agonist stimulated nitric oxide release mental animals.6 Many chemical substances such as acetylcho- line, bradykinin, , and substance P are Nitric oxide release from the vascular able to induce endothelium dependent va- endothelium sodilatation. In rings of rabbit aorta, endothe- There is a continuous basal synthesis of nitric lium dependent relaxation induced by acetyl- oxide from the vascular endothelium to main- , ionophore (A23187) or tain resting vascular tone. A number of chemi- substance P is inhibited by L-NMMA. This cal and physical stimuli may activate eNOS and provides in vitro evidence that vasorelaxation lead to increased nitric oxide production. induced by endothelium dependent agonists is nitric oxide mediated. L-NMMA also attenu- ates the hypotensive eVect of acetylcholine in vivo in animals. However, the blockade is far 5 from complete and there is now growing evidence for additional mechanisms underlying endothelium dependent responses to acetyl- choline, particularly in resistance vessels. Simi- larly in humans, L-NMMA inhibits agonist 7 3 stimulated relaxation in resistance, conduit, and venous vessels in vivo. However, the degree of inhibition to agonist dependent dilatation varies between vascular beds, and mechanisms (for example, prostaglandins and endothelium

Forearm blood flow Forearm blood derived hyperpolarising factors) other than that (ml/100 ml forearm/min) 1 L-NMMA mediated by nitric oxide appear to be involved. 124 (µmol/min) Physical forces and nitric oxide release Haemodynamic shear stress exerted by the 010 30 50 70 viscous drag of flowing blood is an important physiological stimulus in the regulation Minutes after cannulation of nitric oxide release from the endothelium. Figure 3. Inhibition of endogenous nitric oxide by L-NMMA produces a dose The mechanisms of shear stress induced dependent reduction in forearm blood flow. nitric oxide release is complex, involving

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(1) extremely rapid initiation via ion channel supported by studies of long term NOS inhibi- activation, and (2) subsequent events related to tion. Aortic rings from rabbits fed with choles- signalling pathway activation such as phospho- terol rich diet showed impaired endothelial rylation of eNOS and increased dependent vasorelaxation to acetylcholine. expression of eNOS mRNA and protein. These Furthermore, blockade of nitric oxide with complex events allow rapid and short lasting as nitro-L-arginine methylester (L-NAME) well as slow onset and sustained vasodilatation causes structural changes with development of 344 in response to changes in shear stress. greater lesion surface area in the aorta of hypercholesterolaemic rabbits. Ultra-quick: ion channels Numerous in vitro studies have provided strong evidence that ion channels—including in established certain calcium, potassium, and chloride ion channels—open seconds after exposure to haemodynamic shear. Application of shear stress to bovine aortic endothelial cells by fluid perfusion led to an immediate large increase in Impaired endothelium dependent vasorelaxa- intracellular free calcium within one minute tion to acetylcholine occurs in experimental followed by a rapid decline. Notably, the models of atherosclerosis. In these studies, increase in intracellular calcium occurs only in relaxation to endothelium independent nitric response to pulsatile flow and not to steady oxide donors such as glyceryl trinitrate and flow. The detection of potassium selective cur- was unaVected, indicat- rent with whole cell patch clamp recordings of ing selective impairment of the L-arginine– arterial endothelial cells suggests activation of a nitric oxide pathway rather than a generalised distinct potassium channel in response to shear reduced vascular smooth muscle cell response stress. Recently, a flow activated, chloride to nitric oxide. Similar findings were also con- selective membrane current in vascular en- firmed in human coronary and peripheral 10 dothelial cells, distinct from the potassium cur- circulation in vivo. In patients with early cor- rent, was demonstrated. The balance between onary artery disease, abnormal responses to anionic and cationic current determines the net acetylcholine were found even in angiographi- membrane potential and the subsequent cally normal segments of coronary artery. change in calcium that alters eNOS activation Similarly, in patients with established coronary and nitric oxide output. artery disease, endothelial dysfunction in the peripheral vessels as assessed by flow mediated Quick: phosphorylation dilatation is impaired. This impairment corre- Mechanical activation of eNOS as induced by lates with the extent of the coronary artery dis- shear stress occurs also via phosphorylation ease. and the eVect is independent of intracellular calcium concentrations. It has been shown that Chronic heart failure in response to shear stress, the serine/threonine Chronic heart failure (CHF) is characterised protein B (Akt) directly phosphorylates by a reduced vasodilator response to exercise and activates eNOS with a maximal increase and increased vasoconstriction; this is prima- up to sixfold after one hour of exposure to rily a result of an imbalance between endothe- shear stress. The stimulation of Akt phosphor- lium derived vasodilator and constrictor sub- ylation by shear stress appears to be mediated stances. There is general agreement that in by phosphoinositide 3-OH kinase. CHF synthesis of -1, a potent vaso- constrictor, is greatly increased and this may Slow: increased transcription contribute to the characteristic haemodynamic Shear stress also stimulates eNOS gene tran- abnormalities. However, it remains unclear scription to maintain long term nitric oxide whether nitric oxide synthesis is decreased in production. Application of shear stress for CHF. The fact that endothelium dependent three hours resulted in an induction of eNOS vasodilator response to acetylcholine, metha- mRNA in a dose dependent manner in both choline, and serotonin11 is attenuated in bovine and human aortic endothelial cells. peripheral resistance vessels suggests a reduc- These in vitro experimental data demon- tion in agonist stimulated nitric oxide release. strate the complexity of short medium and long In contrast, response to L-NMMA appears to term regulation of nitric oxide release in be either unchanged or even paradoxically response to shear stress. exaggerated in CHF. This could be partially explained by an increased basal nitric oxide Loss of nitric oxide and predisposition to synthesis in the face of increased vasoconstric- atherogenesis tor generation. Interestingly, endothelium in- A reduction in nitric oxide activity (manifested dependent vasodilator response to nitric oxide as impaired endothelial dependent vasodilata- donors may also be attenuated,11 suggesting tion) occurs very early in experimental and that vascular smooth muscle sensitivity to nitric human hypercholesterolaemia, even before any oxide might be reduced, with the degree of structural changes in the vascular wall. In some attenuation correlating with the severity of cases, the impaired endothelial dependent CHF. It is possible that the underlying vasodilatation appears to be reversed with mechanism relates to increased vascular gen- 9 − L-arginine. The action that nitric oxide eration of superoxide anion (O2 ) that inacti- normally has as an antiatherogenic factor is vates nitric oxide.

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Treatment of CHF with con- rather than the loss of nitric oxide causing verting (ACE) inhibitors has been essential . This notion is sup- shown to improve endothelium dependent ported by the observation that impaired vasodilatation induced by cholinergic stimuli. endothelium dependent vasodilatation in es- Furthermore, in a randomised, placebo con- sential hypertension can be restored with anti- trolled study of patients with moderate to hypertensive treatment,13 and that endothelium severe CHF, spironolactone increased vasodi- dependent vasodilatation is impaired following lator response to acetylcholine with an associ- acute elevation of blood pressure in normoten- 345 ated increase in vasoconstriction to L-NMMA, sive subjects. suggesting enhanced basal nitric oxide medi- ated dilatation following spironolactone treat- Diabetes mellitus ment. Whether these beneficial eVects on There is considerable controversy regarding endothelial nitric oxide contribute to the the extent of endothelial dysfunction that favourable outcome remains to be determined. occurs in type 1 diabetes mellitus, as endothe- lial function studies in both animal and human models of type 1 diabetes have produced con- 14 Endothelial dysfunction and risk flicting results. For example, agonist stimu- factors for coronary heart disease lated, endothelium dependent vasodilatation has been found to be either impaired or unchanged. Endothelial function may be Hyperlipidaemia modulated by several factors associated with Experimentally induced hyperlipidaemia by diabetes such as the degree of acute hypergly- either a fatty meal or intralipid infusion impairs caemia, chronicity of hyperglycaemia (disease flow mediated dilatation (FMD). In patients duration), accumulation of advanced glyco- with , endothelium de- sylated end products, concentrations, pendent vasodilatation in both coronary and and diabetic complications such as autonomic peripheral vessels is impaired before the devel- neuropathy and microalbuminuria. Variation 912 opment of clinical atherosclerosis. Restora- in these factors between studies may in part tion of normal or near normal endothelium explain the conflicting results. At present, there dependent vasodilatation in the forearm resist- is no clear consensus about the level at which ance vessels of hypercholesterolaemic subjects the disease might alter nitric oxide signalling. can be achieved after six months of lipid lower- Most data would be consistent with a reduced ing treatment. Interestingly, reduced vasodila- responsiveness to nitric oxide in type 1 tation in response to acetylcholine has also diabetes, and large scale definitive studies in been seen in patients with raised triglyceride the future would be required to show whether but normal low density lipoprotein (LDL) this is the case. cholesterol concentrations. In patients with Vascular studies in type 2 diabetes have gen- familial combined hyperlipidaemia, lipid low- erally shown an impaired muscarinic, agonist ering treatment improved forearm blood flow stimulated, endothelium dependent response in response to serotonin. Finally, raised Lp(a) as well as an impaired endothelium independ- lipoprotein appears to enhance acetylcholine ent response, although the impaired endothe- and cold pressor coronary constrictor responses lium independent response was not confirmed in patients with normal coronary on by other investigators. Interestingly, it has and impairs basal nitric oxide recently been shown that even normoglycaemic production in forearm resistance vessels. Thus, subjects who are prone to develop type 2 there is compelling evidence to indicate that diabetes and insulin resistance syndrome, such endothelial function is impaired by hyperlipi- as those characterised by previous gestational daemia. However, recently the reversibility of diabetes and low birth weight, exhibit impaired this defect in coronary arteries has been called FMD. This has led to the suggestion that into question. endothelial dysfunction may precede the devel- opment of type 2 diabetes. Hypertension Substantial evidence from animal and human Hyperhomocysteinaemia studies indicates that acetylcholine induced Raised plasma homocysteine is thought to be relaxation is impaired in patients with hyper- an independent risk factor for coronary heart tension. However, no diVerence was found in disease, and this may be mediated by endothe- endothelium dependent vasodilator response lial dysfunction. Acute induced to acetylcholine or carbachol between patients hyperhomocysteinaemia impairs endothelium with essential hypertension and matched nor- dependent FMD in healthy subjects which can motensive controls in at least one study. This be reversed by folic acid supplementation. may be because of diVerences in methodology Impaired FMD has also been found in chronic and in population subgroups in this heteroge- hyperhomocysteinaemia and homocysteinuric neous condition. There is, however, evidence children. The concentration of plasma homo- that basal nitric oxide synthesis is reduced in associated with endothelial dysfunc- essential hypertension and that vasoconstrictor tion in these in vivo human studies were several response to L-NMMA is reduced in untreated folds higher than the normal range. Recently, it hypertension, although again this has not been has been shown that even mild physiological a universal finding.13 Current data would be increments in plasma homocysteine concentra- most consistent with hypertension causing a tions were suYcient to impair endothelial decrease in nitric oxide mediated dilatation function.

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Links between risk factors and atherogenesis (DDAH I and II) have been identified, In addition to various disease states, endothe- sequenced, and cloned.16 Circulating ADMA is lium dependent vasodilatation is also impaired increased in certain disease states. This in- in old age,15 and in young healthy subjects with cludes animal models of hypertension, a family history of premature coronary heart diabetes, hypercholesterolaemia, and athero- disease and cigarette smoking. The age related sclerosis. In humans, raised ADMA concentra- endothelial dysfunction may partially explain tions were found in chronic renal failure,17 346 the increased cardiovascular risk in the elderly. childhood hypertension, pre-eclampsia, throm- In asymptomatic young smokers, impairment botic microangiopathy, hypercholesterolaemia, of endothelium dependent vasodilatation is and atherosclerosis. The mechanism whereby reversible with smoking cessation. It may be various disease states are associated with that has a direct toxic eVect on the increased ADMA concentrations remains un- vascular endothelium. Additionally, there is clear but may involve alteration in DDAH growing evidence of a link between infection/ activity, and this may be an important enzyme inflammation and risk of coronary heart in atherogenesis. Accumulation of ADMA disease. A recent study showed that acute sys- would be expected to enhance atherogenesis temic inflammation induced by vaccination through loss of nitric oxide. causes impaired endothelium dependent vaso- dilatation in both resistance and conduit Reduced nitric oxide bioavailability vessels. Thus various eVects on the L-arginine: Role of nitric oxide pathway exerted by hypertension, Even with adequate production, nitric oxide diabetes, hyperlipidaemia, hyperhomocystein- may not reach its biological targets (vascular aemia, infection/inflammation, aging, cigarette smooth muscle and circulating cells) to exert smoking, and family history of coronary heart its eVect because of the lack of its bioavailabil- disease may form a link between risk factors ity. For example, in hyperlipidaemia, excess and predisposition to atherogenesis or acute LDL synthesis increases the formation of events. oxidised LDL. The resultant increase of oxida- tive stress enhances nitric oxide destruction, thereby reducing its biological eVects. In Alteration of the L-arginine: nitric atherosclerotic rabbit aorta, despite a threefold oxide pathway in diseases: potential increase in total nitric oxide synthesis com- mechanisms pared to normal rabbits, there is notably impaired endothelium dependent vasodilata- tion. This impaired vascular response can be The involvement of the L-arginine: nitric oxide partially restored following treatment with pathway in disease states is complex and it can , suggesting that superox- be altered in several ways. ide induced nitric oxide inactivation plays a major role. In humans, hypertriglyceridaemia Reduced nitric oxide production with or without diabetes may have greater Deficiency of NOS co-factor potential than cholesterol to increase superox- Several conditions are associated with NOS ide production by leucocytes. Other athero- co-factor deficiency which may contribute to genic factors such as free fatty acids and low endothelial dysfunction. For instance, insulin concentrations of high density lipoprotein also resistance is associated with deficiency of the increase oxidative stress, contributing to re- essential co-factor BH4, resulting in impaired duced nitric oxide bioavailability. In addition to vascular relaxation. Additionally, chronic ciga- the associated atherogenic phenotype and oxi- rette smoking contributes to depletion of BH4 dative stress, hyperglycaemia per se increases resulting in decreased nitric oxide synthesis, free production through increased and supplementation of this co-factor restores arachidonic acid . endothelial function in chronic smokers. In In human aortic endothelial cells, although hypercholesterolaemia, impaired endothelium prolonged exposure to high glucose concentra- dependent vasodilatation can also be restored tion causes increased eNOS expression, it also with BH4 supplementation, suggesting that

BH4 deficiency plays an important role in impaired vascular function in these conditions. L-arginine Atherosclerosis

Role of endogenous inhibitors of NOS Hypertension Overproduction of endogenous inhibitors of NOS in certain disease states may contribute to ADMA Diabetes reduced nitric oxide synthesis (fig 4). Asym- Chronic renal failure metric and symmetric dimethylarginine (ADMA and SDMA) have been identified in DDAH Hypercholesterolaemia human plasma. ADMA has properties similar NO to L-NMMA. It is synthesised by the human endothelial cells from arginine and is metabo- lised to citrulline before excretion into the Citrulline urine (fig 4). The enzyme responsible for Figure 4. Asymmetric dimethylarginine (ADMA) is synthesised from L-arginine by ADMA metabolism in the human vascular protein methylase I and subsequently metabolised by dimethylarginine dimethylaminohydrolase (DDAH) yielding citrulline. ADMA acts as an endogenous endothelial cells is dimethylarginine dimethyl- inhibitor of nitric oxide (NO) synthesis and its concentration is increased in certain aminohydrolase of which two isoforms disease states, possibly as a result of decreased DDAH actions.

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leads to a concomitant increase in superoxide Under normal physiological conditions, iNOS anion production (probably from NADH/ is not expressed in the vasculature. Exposure to NADPH oxidase) resulting in nitric oxide bacterial lipopolysaccharide or proinflamma- inactivation. Oxidative stress may also be a key tory cytokines stimulate iNOS expression. In mechanism for endothelial dysfunction in experimental animals, administration of hyperhomocysteinaemia. Studies in vitro and tumour necrosis factor á (TNFá) in doses in animals suggest that elevation of homo- similar to those produced endogenously during cysteine enhances lipid peroxidation, which endotoxaemia rapidly results in a fall in arterial 347 may contribute to impaired endothelium de- pressure, and longer exposure to TNFá and pendent vasodilatation. This potential mech- interleukin-1â (IL-1â) in vitro leads to hypo- anism is supported by the findings that the responsiveness to vasopressors. antioxidants folic acid and C rapidly Several animal studies provide evidence for reverse the impaired endothelium dependent iNOS involvement in the pathogenesis of sepsis. vasodilatation without reducing the raised Administration of bacterial lipopolysaccharide homocysteine concentrations. Similar im- to mice causes an increase in nitrite concentra- provement in endothelial function in type 2 tion, which is attenuated by a selective iNOS diabetes with antioxidants such as vitamin E inhibitor. Moreover, selective inhibition of iNOS have also been observed, suggesting yet again greatly increases vascular catecholamine reactiv- the central role of oxidative stress in endothelial ity in experimental mice with sepsis but not in dysfunction under various pathophysiological control mice. Studies in mice genetically engi- conditions. neered to lack the gene for iNOS also confirm the role of this isoform in septic vasodilatation. Advanced glycation endproducts In wild type mice, treatment of carotid arteries Accumulation of advanced glycation endprod- with lipopolysaccharide leads to impaired con- ucts (AGEs)—the product of non-enzymatic strictor responses which is improved with selec- glycation and crosslinking of collagen protein tive iNOS inhibitors. In contrast, lipopolysac- in sustained hyperglycaemia—may lead to charide treatment causes no impairment of quenching (or inactivation) of nitric oxide in vasoconstrictor responses in carotid arteries diabetes. In experimentally induced diabetic from iNOS deficient mice. rats, there is in vitro and in vivo evidence that In human sepsis the evidence for iNOS reactive intermediates resulting from glycation involvement has been less consistent. Some quench nitric oxide rapidly. Furthermore, studies have suggested an increased iNOS impairment of endothelium dependent vaso- activity—for example, in urinary leucocytes dilatation in diabetic rats can be partially from patients with urinary tract infection, in restored by aminoguanidine, an inhibitor of alveolar macrophages from patients with acute AGEs formation respiratory distress syndrome following sepsis, and in peripheral blood mononuclear cells and Diminished vascular smooth muscle macrophages isolated from putrescent muscle sensitivity areas in patients with cellulitis. However, other Vascular smooth muscle sensitivity may be studies have suggested the involvement of decreased even with adequate nitric oxide sup- eNOS rather than iNOS in human sepsis. In an ply. In human vascular studies, nitrovasodila- in vitro study using human umbilical vein tors or nitric oxide donors (such as glyceryl endothelial cells cultured with IL-1â and trinitrate or sodium nitroprusside) have fre- TNFá, the resultant increase in nitric oxide quently been used as a control for agonist production was shown to originate from eNOS stimulated endothelium dependent vasodilata- as a result of activation of guanosine triphos- tion. These agents act directly upon vascular phate cyclohydrolase (GTPCH-I), the rate smooth muscle and resultant vasodilatation is limiting enzyme responsible for the synthesis of endothelium independent. There is evidence to BH4. Similar findings have recently been shown suggest that vascular smooth muscle sensitivity in human veins in vivo. While it is clear that overproduction of nitric oxide contributes to to nitric oxide is reduced in diabetes and 18 hyperglycaemia interferes with nitric oxide vasodilatation in human sepsis, the molecular induced guanylate cyclase activation in vitro. mechanisms and isoform of NOS activated are Consistent with this finding, impaired vascular unclear. It is also not known whether inhibition response to nitric oxide donors in vivo have of nitric oxide generation is beneficial. Expres- been demonstrated in patients with type 1 sion of iNOS in active atherosclerotic plaques diabetes. has also been detected. It is possible that this iNOS contributes to tissue damage or other features of plaque development or stability. Overproduction of nitric oxide in sepsis Therapeutic possibilities Bacterial endotoxin and certain proinflamma- tory cytokines can lead to profound vaso- L-arginine dilatation and decreased vasopressor Supplementation of the nitric oxide substrate responsiveness—the main clinical features of L-arginine has beneficial eVects in certain con- . These cardiovascular eVects ditions in laboratory animals and humans. result from excessive nitric oxide production Dietary L-arginine for 10 weeks has been thought to be caused by induction of iNOS. shown to prevent intimal thickening in the

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coronary arteries and attenuates platelet reac- function in some conditions merits further tivity in hypercholesterolaemic rabbits. Fur- investigation. thermore, oral L-arginine administration re- duces neointimal formation following balloon /nitric oxide donors catheter induced injury in both hypercholes- Nitrovasodilators such as , glyceryl terolaemic and normocholesterolaemic rabbit trinitrate, sodium nitroprusside, and molsid- models. In humans, dietary L-arginine supple- omine are all pro- and exert their 348 mentation reduces the increased platelet reac- pharmacological eVects after metabolism to tivity in hypercholesterolaemic subjects. Addi- nitric oxide. Hence they are termed “nitric tionally, intravenous L-arginine infusion oxide donor”. Based on their venodilatory reduces peripheral and properties, nitrovasodilators have convention- decreases both systolic and diastolic blood ally been used for the treatment of cardiac fail- pressure, at least in some studies, improves ure and . Nitrosoglutathione, a com- endothelium dependent coronary vasodilata- pound in the class of nitrosothiols, has been tion in response to intracoronary acetylcholine studied extensively in humans. It has profound in hypercholesterolaemic subjects,9 and im- antiplatelet eVects and more balanced arterial proves blood flow in critical lower limb ischae- and venous vasodilatory eVects than organic mia. . Nitrosoglutathione inhibits platelet activation in the coronary artery following angioplasty and in coronary bypass grafts. The arginine paradox Hence, nitrosothiols are potential pharmaco- The beneficial eVects of exogenous L-arginine logical agents in the treatment of nitric oxide to the vasculature in various disease states with deficient conditions, and it is possible that increased plasma nitrate and cGMP concentra- novel nitric oxide donors could be developed tions during L-arginine administration suggest that diVer greatly from existing drugs. provision of excess L-arginine supply can stimulate NOS activity. However, it is perplex- Inhalation of nitric oxide ing that extracellular arginine administration Administration of nitric oxide as inhalation should drive nitric oxide production since the treatment has been shown to improve several intracellular arginine concentrations are always conditions aVecting the pulmonary vascula- available in great excess of the needs of NOS, a ture, including persistent pulmonary hyper- phenomenon known as the “arginine para- tension of the newborn, pulmonary hyper- dox”. This was first demonstrated in hypercho- tension secondary to chronic hypoxia, and lesterolaemic rabbits19 and has also been adult respiratory distress syndrome (ARDS). observed in patients with pulmonary hyper- In ARDS, selective delivery of nitric oxide to tension. Several explanations have been pro- the pulmonary vasculature reduces the pulmo- posed to account for this paradox. Firstly, it is nary arterial pressure and increases arterial possible that the endogenous inhibitor of NOS, oxygenation by improving the matching of ADMA, might antagonise the normal intra- ventilation with perfusion. As the nitric oxide is cellular concentrations of L-arginine, and rapidly inactivated by haemoglobin, inhalation additional arginine supplementation is re- of nitric oxide gas does not cause systemic quired to overcome a functional defect of NOS vasodilatation. However, whether this form of substrate. Secondly, since eNOS is preferen- nitric oxide treatment will improve survival in tially localised to specific intracellular sites patients with ARDS remains to be determined known as caveolae, local concentration of by randomised controlled clinical trials. It does L-arginine in this microenvironment may diVer have potential adverse eVects such as pulmo- considerably from that within the endothelial nary oedema and methaemoglobinaemia. cell. It remains unclear how specific localisa- There are as yet no clear guidelines regarding tion of eNOS by caveolae might aVect local the eVective dose of inhaled nitric oxide for the substrate availability, but the mechanism may above pulmonary conditions. involve the co-localisation of eNOS with certain arginine transporters (for example, Antioxidants cationic amino acid transporter-1). The forma- Since oxidative stress has been strongly impli- tion of such a caveolar complex seems to facili- cated in endothelial dysfunction, numerous tate arginine delivery to eNOS. studies have examined the role of antioxidants It is also important to recognise that the in vascular function and in the prevention of vasodilatory eVects of L-arginine are not all cardiovascular disease. Intrabrachial adminis- mediated directly by nitric oxide. L-arginine tration of ascorbic acid () improves may inhibit peripheral sympathetic tone lead- endothelium dependent vasodilatation in pa- ing to vasodilatation via its metabolite, agma- tients with type 2 diabetes, smokers, patients

tine, which stimulates central á2 adrenoceptors. with hypercholesterolaemia, and patients with Additionally, arginine also stimulates the re- heart failure. Similarly, oral administration of lease of several other hormones such as gluca- ascorbic acid in patients with coronary heart gon, prolactin, and growth hormone which disease also improves flow mediated vasodilata- may account for its vasodilatory action. Fur- tion. This beneficial eVect has been shown to thermore, many of the vascular and other occur rapidly after two hours and can be actions of L-arginine are shared by its stereo- sustained for 30 days. Furthermore, intra- , D-arginine, which is not a substrate for venous infusion of ascorbic acid improved NOS. The complex mechanisms whereby endothelium dependent vasodilatation in epi- L-arginine appears to improve cardiovascular cardial arteries in hypertensive patients without

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Intravenous administration of the arginine Summary analogue L-NMMA reverses the decrease in peripheral vascular resistance and fall in x Endothelium derived nitric oxide has important antiatherogenic arterial blood pressure in endotoxaemic dogs. properties: The same eVects with L-NMMA have also – enhancement of vasodilatation been confirmed in humans in a randomised, – inhibition of platelet aggregation double blind, placebo controlled trial.18 A – inhibition of leucocyte migration potential pitfall in this therapeutic strategy is 349 – inhibition of smooth muscle proliferation and migration that none of the currently available NOS – inhibition of adhesion molecule activation and expression – anti-oxidant inhibitors are specific for the excess nitric oxide causing pathophysiology, and may therefore x Three distinct nitric oxide synthase isoforms exist which all have a lead to potential hazardous eVects, by also role in vascular control under diVerent conditions inhibiting “physiological” nitric oxide. Indeed, both pulmonary hypertension and reduced x Reduced nitric oxide synthesis or defective nitric oxide activities cardiac output have been reported following predispose to atherosclerosis NOS inhibition. Future development in this area should focus on NOS isoform specifi- x DiVerent disease processes and risk factors aVects the city as well as the degree of inhibition required L-arginine–nitric oxide pathway via diVerent mechanisms: in order to produce an optimal pharmacologi- – decreased synthesis cal eVect. – decreased co-factor availability – decreased enzyme expression/activity – increased endogenous nitric oxide synthase inhibitors Conclusions – decreased nitric oxide bioavailability – decreased vascular smooth muscle sensitivity to nitric oxide Nitric oxide is a major player in cardiovascular x Understanding these mechanisms in diseases may help therapeutic physiology and pharmacology. Its release from strategies in prevention of atherosclerosis endothelium exerts a tonic hypotensive, anti- platelet, antiatherogenic influence in the arte- rial tree. Several common cardiovascular dis- coronary heart disease. In patients with coron- ease states or risk factors impair nitric oxide ary heart disease, intracoronary co-infusion of synthesis, increase nitric oxide destruction or vitamin C with L-arginine causes greatly aVect the ability of cells to respond to this increased vasodilatation. Despite these obser- mediator. The net eVect is to enhance vascular vations, not all experiments have shown tone and reactivity and predispose to athero- beneficial eVects and further studies are sclerosis. Clinicians already use drugs that required. In addition, agents such as vitamin C either mimic nitric oxide (nitric oxide donors) can produce pro-oxidant eVects in some or enhance its endogenous production (for circumstances. example, by lowering blood pressure or serum cholesterol). In contrast, overproduction of Gene transfer nitric oxide is an important mechanism of The direct transfer of NOS isoform genes to inflammatory vasodilatation. Over the next few the vessel wall alters vasomotor function and years a new generation of drugs will emerge may have a role in the treatment of cardiovas- that specifically modify nitric oxide production cular diseases.20 This approach has been shown or mimic its action. Indeed, the first such drug to be eVective in a variety of animal models of is sildenafil which blocks the phosphodieste- . Vascular gene therapy of rase that degrades cGMP and thereby acts as eNOS delivered to specific tissues enhances an amplifier of nitric oxide signalling (fig 1). nitric oxide production at the site of interest. In Diagnostic tests based on early assessment of 1995, NOS gene transfer was first reported endothelial function may also become part of when eNOS cDNA, in a complex with the hae- cardiovascular risk stratification or assessment magglutinating virus of Japan, was delivered to of novel therapeutics. rat carotid arteries intraluminally following balloon injury. This resulted in a pronounced This work is supported by the British Heart Foundation. reduction in neointima formation at 14 days. Shortly after, similar results were confirmed by 1. Furchgott RF, Zawadski JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle other research groups in iliac and coronary by acetylcholine. Nature 1980;288:373–6. arteries and vein grafts. Gene therapy has also • The original paper describing the importance of endothelium in acetylcholine induced vasorelaxation. been extended from local to systemic delivery. Furchgott was the recipient of the 1998 Nobel prize in Delivery of a single injection of naked eNOS medicine. cDNA into the systemic circulation through 2. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of the tail vein of spontaneously hypertensive rats endothelial-derived relaxing factor. Nature 1987;327:524–6. resulted in increased production and excretion • Novel finding that endothelial derived relaxing factor is of cGMP and nitrite/nitrate, and was associ- nitric oxide. 3. Ignarro LJ, Buga GM, Wood KS, et al. ated with a significant reduction in systolic Endothelium-derived relaxing factor produced and released blood pressure lasting up to 12 weeks. from artery and vein is nitric oxide. Proc Natl Acad Sci USA 1987;84 9265–9. • Novel finding that endothelial derived relaxing factor is NOS inhibitors nitric oxide. Published in the same year as reference 2. Inhibition of iNOS has been shown to improve 4. Moncada S, Higgs EA. The L-arginine-nitric oxide nitric oxide mediated haemodynamic changes pathway. N Engl J Med 1993;329:2002–12. 18 • Review article describing in detail the biochemical nitric in experimental models of septic shock. oxide pathway.

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5. Xu W, Charles IG, Moncada S, et al. Mapping of the 13. Calver A, Collier J, Moncada S, et al. Effect of local genes encoding human inducible and endothelial nitric oxide intra-arterial NG-monomethyl-L-arginine in patients with synthase (NOS2 and NOS3) to the pericentric region of hypertension: the nitric oxide dilator mechanism appears chromosome 17 and to chromosome 7, respectively. abnormal. J Hypertens 1992;10:1025–31. Genomics 1994;21:419–22. • Vascular study using plethysmography demonstrating • Identification of chromosomes encoding nitric oxide impaired vasoconstrictor response to L-NMMA in forearm synthase isoforms. resistance vessels of hypertensives. 6. Cayatte AJ, Palacino JJ, Horten K, et al. Chronic 14. Chan NN, Vallance P, Colhoun HM. Nitric oxide and inhibition of nitric oxide production accelerates neointima vascular responses in type 1 diabetes. Diabetologia formation and impairs endothelial function in 350 hypercholesterolemic rabbits. Arterioscler Thromb 2000;43:137–47. 1994;14:753–9. • A recent review of the conflicting evidence regarding nitric • The original animal study which demonstrated the oxide release and response in type 1 diabetes. Potential antiatherogenic role of endogenous nitric oxide. mechanisms of defective L-arginine: nitric oxide pathway in diabetes are also discussed. 7. Vallance P, Collier J, Moncada S. Effects of endothelium-derived nitric oxide on peripheral arterial tone in 15. Celermajer DS, Sorensen KE, Spiegelhalter DJ, et al. man. Lancet 1989;ii:997–1000. Aging is associated with endothelial dysfunction in healthy • First experimental evidence showing the key role of nitric men years before the age-related decline in women. JAm oxide in maintaining the resting arterial tone in humans. Coll Cardiol 1994;24:471–6. 8. Vallance P, Collier J, Moncada S. Nitric oxide • This study showed that endothelial function as assessed synthesised from L-arginine mediates by flow-mediated dilatation declines with aging, particularly endothelium-dependent dilatation in human veins in vivo. in males. Cardiovasc Res 1989;23:1053–7. 16. Leiper JM, Santa Maria J, Chubb A, et al. Identification • Experiment demonstrating the dilatatory property of nitric of two human dimethylarginine dimethylaminohydrolases oxide is not only confined to the arteries. with distinct tissue distributions and homology with microbial 9. Drexler H, Zeiher AM, Meinzer K, et al. Correction of arginine deaminases. Biochem J 1999;343:209–14. endothelial dysfunction in coronary microcirculation of • Novel discovery of dimethylarginine hypercholesterolemic patients by L-arginine. Lancet dimethylaminohydrolases, enzymes responsible for 1991;338:1450–6. metabolism of the endogenous nitric oxide synthase • Early experiment in humans showing that endothelial inhibitor, asymmetrical dimethylarginine (ADMA). dysfunction is reversible with L-arginine supplementation. This paper provides important insight to potential 17. Vallance P, Leone A, Calver A, et al. Accumulation of therapeutic strategies targeting the nitric oxide pathway. an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet 1992;339:572–5. 10. Ludmer P, Selwyn A, Shook TL, et al. Paradoxical • Identification of ADMA as an endogenous inhibitor of nitric vasoconstriction induced by acetylcholine in atherosclerotic oxide synthase. coronary arteries. N Engl J Med 1986;315:1046–51. • Angiographic evidence that atherosclerotic coronary 18. Petros A, Bennett D, Vallance P. Effects of nitric oxide arteries constrict in response to the conventional synthase inhibitors on in patients with septic vasodilator, acetylcholine. shock. Lancet 1991;338:1557–8. 11. Maguire SM, Nugent AG, McGurk C, et al. Abnormal • Evidence of cardiovascular effects of NOS inhibition in vascular responses in human chronic cardiac failure is both treatment in human septic shock. endothelium dependent and endothelium independent. Heart 19. Girerd Xj, Hirsch AT, Cooke JP, et al. L-Arginine 1998; 80:141–5. augments endothelium-dependent vasodilatation in • Evidence of impaired endothelium dependent and independent vasodilatation as assessed by venous cholesterol-fed rabbits. Circ Res 1995;67:1301–8. website occlusion plethysmography in non-ACE inhibitor treated • Early animal evidence that arginine supplementation chronic heart failure. improves nitric oxide production in hypercholesterolaemic extra rabbits. 12. Chowienczyk PJ, Watts GF, Cockcroft JR, et al. Additional references 20. Kibbe M, Billiar T, Tzeng E. Nitric oxide synthase gene Impairment of endothelium-dependent vasodilatation of appear on the forearm resistance vessels in hypercholesterolaemia. Lancet transfer to the vessel wall. Curr Opin Nephrol Hypertens 1992;340:1430–2. 1999;8:75–81. Heart website • This paper provides early evidence that raised cholesterol • Excellent review of the potential role of nitric oxide gene impairs endothelial function. transfer in the treatment of cardiovascular disorders. www.heartjnl.com