Targeting Nitric Oxide With Drug Therapy

Abstract

Increasing knowledge of the role of nitric oxide (NO) in physiology and disease has stimulated efforts to target the NO pathway pharmacologically. These therapeutic strategies include NO donors that directly or indirectly release NO and agents that increase NO bioactivity. Traditional organic nitrates such as nitroglycerin, which indirectly release NO, were believed to have limited long‐term efficacy and tolerability, chiefly because of nitrate tolerance. Recent studies, however, suggest more effective ways of using these agents and new applications for them. Nicorandil, a hybrid organic nitrate that also activates potassium channels, has demonstrated significant benefits in acute coronary syndromes. Other nitrates are being investigated for use in neurodegenerative diseases. Direct NO donors include NO gas, which is useful in respiratory disorders, and the more recent classes of diazeniumdiolates, sydnonimines, and S‐nitrosothiols. Preliminary data suggest that these agents may be effective as anti‐atherosclerotic agents as well as in other disease states. In addition, hybrid agents that consist of an NO donor coupled with a parent anti‐inflammatory drug, including nonsteroidal anti‐inflammatory drugs, have demonstrated enhanced efficacy and tolerability compared with the anti‐inflammatory parent drug alone in diverse experimental models. Established drugs that enhance NO bioactivity include antihypertensive agents, particularly angiotensin‐converting enzyme inhibitors, calcium channel blockers, and newer vasodilating β‐blockers. In addition, 3‐methylglutaryl coenzyme A reductase inhibitors (statins) promote NO bioactivity, both through and independent of lipid lowering. The NO‐promoting actions of these established drugs provide some insight into their known benefits and suggest possible therapeutic potential.

Increased understanding of the essential role of nitric oxide (NO) in diverse physiologic processes and diseases has stimulated the development of multiple pharmacologic strategies to target the NO pathway. ¹ , ² , ³ , ⁴ These approaches include therapies that directly or indirectly release or “donate” NO, and the use of existing drugs, such as antihypertensive agents and 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitors (statins), that enhance NO bioactivity. ⁵ , ⁶ , ⁷ Reflecting the myriad biologic actions of NO, these therapeutic strategies designed to improve NO bioactivity are providing new insights into the pathogenesis and treatment of various diseases, including hypertension, atherosclerosis, cancer, Alzheimer's disease, colitis, and asthma. ¹ , ³

The therapeutic targeting of NO is not new, particularly in cardiovascular disease (CVD). A major mediator of endothelial function, NO plays a vital role in cardiovascular physiology, including facilitating endothelium‐dependent vasodilation; regulating blood pressure (BP), vascular tone, and compliance; and inhibiting platelet aggregation, vascular inflammatory factors, leukocyte adhesion, and smooth muscle cell proliferation. ⁸ NO donors such as sodium nitroprusside and organic nitrates, which include nitroglycerin and isosorbide dinitrate, are potent vasodilators and have been used for many years for the treatment of ischemic heart disease and heart failure (HF). ¹ , ² , ⁹ Unfortunately, the clinical utility of these drugs has long been hampered by drawbacks associated with their limited tolerability and long‐term efficacy. ¹ , ² Recent clinical study findings, however, offer therapeutic strategies and new uses for these traditional agents. ¹⁰ , ¹¹ , ¹²

Recently, several other classes of NO donors have been developed that may offer greater tolerability, long‐term efficacy, and therapeutic versatility than the older agents. ¹ , ² , ³ , ⁴ These include novel nitrates, direct NO donors, hybrid drugs combining NO release with other mechanisms of action, and agents consisting of an NO‐donating component with a parent agent of another drug class, including nonsteroidal, anti‐inflammatory drugs (NSAIDs). ³ Other agents, such as newer vasodilating β‐blockers, have also been found to increase NO bioactivity, in addition to their more conventional mechanisms of action. ¹³ , ¹⁴ This article reviews some of the emerging therapies that release NO or enhance its bioactivity for the potential treatment of 1 or more diseases.

INDIRECT NO DONORS

Pharmacologic agents that release NO indirectly—requiring metabolic activity, biotransformation, and redox activation—include sodium nitroprusside and the organic nitrates and nitrite esters. Sodium nitroprusside lowers BP acutely, and has primarily been used for treatment of hypertensive crises and to relieve symptoms of HF, but it is a toxic drug that raises levels of cyanide in the body. ² , ¹⁵ The oldest NO donors, used to treat ischemic heart disease for over a century, are the organic nitrates. ²

Organic Nitrates

The traditional organic nitrates include nitroglycerin, amyl nitrite, isosorbide dinitrate, and isosorbide 5‐mononitrate. ¹ , ² These agents mimic NO activity, inducing venous and arterial vasodilation in both the coronary and systemic blood vessels through guanylyl cyclase activation and inhibition of calcium channels in vascular smooth muscle cells. ¹ , ² Although they have been particularly useful for symptomatic relief of angina pectoris and HF, organic nitrates have until recently not demonstrated improved outcomes in patients with CVD. The large (N=58,050) Fourth International Study of Infarct Survival (ISIS‐4) trial ¹⁶ in hospitalized patients with suspected myocardial infarction showed that mono‐nitrate treatment had no effect on mortality. In the more recent African‐American Heart Failure Trial (A‐HeFT), ¹¹ in 1050 African Americans with HF, however, the combination of isosorbide dinitrate and hydralazine, when added to standard therapy, significantly reduced mortality by 43% (P=.01) and first hospitalization for HF by 33% (P=.001), compared with placebo; the study was stopped early due to demonstrated benefit after 18 months (mean follow‐up of 10 months).

The utility of organic nitrates has been limited by several drawbacks, including adverse events (AEs) associated with their abrupt vasodilatory action, most commonly headache and hypotension; a short half‐life; low bioavailability; and, most importantly, the onset of nitrate tolerance that occurs with long‐term usage. ¹ , ² , ¹⁷ Nitrate tolerance, which can occur within 24 hours with nitroglycerin, ¹⁸ is believed to stem in part from the action of organic nitrates in promoting an increase in superoxide radicals, which inactivate NO. ¹⁷ , ¹⁹ Organic nitrates may trigger increased production of superoxide radicals partly through their potent vasodilatory and hypotensive actions, which activate the renin‐angiotensin system that, in turn, promotes oxidative processes. ²⁰ In addition, organic nitrates may induce expression of a dysfunctional endothelial NO synthase (eNOS) gene that generates increased formation of superoxide rather than NO. ²¹ Administration of the antioxidant vitamins C or E with organic nitrate therapy has been shown to prevent nitrate tolerance. ²² , ²³ , ²⁴ It is hypothesized that in the A‐HeFT trial, the antioxidant actions of hydralazine may have prevented the nitrate tolerance usually associated with isosorbide dinitrate alone. ¹⁰ , ¹¹ , ¹⁹

In addition, isosorbide dinitrate and isosorbide mononitrate have produced significant reductions in systolic BP and augmentation index, which is a measure of pulse wave reflection, in elderly patients with systolic hypertension. ²⁵ , ²⁶ , ²⁷ These effects suggest that nitrates may ameliorate the effects of age‐related arterial stiffening and endothelial dysfunction in muscular arteries, which together result in increased pulse wave velocity and systolic BP. ²⁵ , ²⁷ Isolated systolic hypertension and elevated pulse pressure are major BP risk factors for stroke and HF in older patients, yet isolated systolic hypertension is also the most commonly untreated and uncontrolled subtype of hypertension. ²⁸ The efficacy of long‐term isosorbide mononitrate therapy also was not diminished by nitrate tolerance when used as adjuvant therapy in patients with treatment‐resistant systolic hypertension. ²⁶ Therefore, organic nitrates may play an important role in reducing systolic BP, although AEs such as headache may still limit the utility of these agents. ²⁷

Other Applications for Nitrates

NO may also play a role in inhibiting excessive bone resorption, a major mechanism of osteoporosis, and promoting bone formation. ²⁹ A large clinical trial (N=144) found that oral administration of isosorbide mononitrate significantly reduced markers of osteoporotic bone resorption and increased bone formation in healthy post‐menopausal women. ³⁰ Transdermal administration of isosorbide dinitrate and nitroglycerin has significantly reduced BP and vascular impedance, respectively, in women with preeclampsia. ³¹ , ³²

The role of NO in neurodegenerative diseases is controversial. Some data indicate that overproduction of inducible NO plays a pathogenic role, while other studies suggest NO may be neuroprotective through its antioxidant actions, inhibition of caspases and apoptosis, preservation of vascular integrity and tone, or other pathways. ¹² Several nitrates, called S‐nitrates, have demonstrated neuroprotection as well as memory and cognition enhancement in animal models, with potential application in Alzheimer's disease. ¹² , ³³

Nicorandil

Nicorandil, a newer nitrate, has a hybrid mechanism of action that combines NO release with adenosine triphosphate‐sensitive potassium channel activation. ³⁴ A nicotinamide ester with a nitrate‐like moiety, nicorandil is the only potassium channel activator with antianginal effects. ³⁵ , ³⁶ Nicorandil provides balanced peripheral and coronary vasodilation with reduction of both cardiac preload and afterload, and does not appear to cause nitrate tolerance, which may be due to its action as a potassium channel activator. ³⁴ , ³⁷ , ³⁸ The major AEs associated with nicorandil include headache and mouth ulcers. ³⁴

In major randomized controlled clinical trials, nicorandil has demonstrated multiple benefits in high‐risk patients with CVD, beyond relief of angina pectoris. ³⁴ In the Centralised European Studies in Angina Research (CESAR) 2 investigation, ³⁶ conducted in 188 patients with unstable angina, the addition of nicorandil to aggressive standard therapy significantly reduced transient myocardial ischemia (P=.003) and arrhythmia (P=.04) compared with placebo (Figure 1). The Impact of Nicorandil in Angina (IONA) study ³⁹ (N=5126) showed that nicorandil, in addition to standard antianginal therapy, significantly reduced the primary composite end point of coronary heart disease (CHD) death, nonfatal myocardial infarction, or unplanned hospitalization for angina pectoris (P=.01), acute coronary syndromes (P=.03), and all CVD events (P=.03), compared with placebo.

Figure 1.

Addition of nicorandil to standard therapy in 188 patients with unstable angina significantly reduced the incidence of silent and painful transient myocardial ischemia (TMI), compared with placebo. Reproduced with permission from Patel et al. ³⁶

DIRECT NO DONORS

In contrast to organic nitrates and nitrate‐like agents, direct NO donors release NO spontaneously, without the need for metabolism and bio‐transformation. ² Most of these agents are in the early stages of evaluation but have demonstrated potentially greater tolerability and a broader range of applications than the traditional NO donors.

NO Gas

NO gas has been useful for the treatment of multiple respiratory disorders and is the first vasodilator to produce selective pulmonary vasodilation. ⁴⁰ Inhaled NO gas has been effective in reducing pulmonary artery pressure and increasing arterial oxygenation in neonates and children with cardiorespiratory failure ⁴¹ ; neonates with pulmonary hypertension or hypoxic respiratory failure ⁴² , ⁴³ ; patients with adult respiratory distress syndrome ⁴⁴ ; and adults with HF, ⁴⁵ pulmonary hypertension, ⁴⁶ and other cardiorespiratory disorders or injury. ⁴⁰ , ⁴⁷ Inhaled NO with oxygen also appears to be safe and effective for the short‐ and long‐term treatment of chronic obstructive pulmonary disease. ⁴⁸ , ⁴⁹ Inhaled NO research is now expanding beyond its pulmonary effects to explore possible uses in such areas as attenuation of platelet activation and neutrophil‐mediated ischemia‐reperfusion injury. ⁴⁰

S‐nitrosothiols

S‐nitrosothiols are endogenous, naturally occurring moieties on proteins that form when a cysteine thiol reacts with NO in the presence of an electron acceptor to form an S‐NO bond. ⁵⁰ S‐nitrosothiols act as reservoirs of NO, and their degradation, which may occur through a number of enzyme systems, results in spontaneous NO release. ⁹

S‐nitrosothiols have demonstrated efficacy as platelet antiaggregation agents in various models of atherosclerosis and thrombosis. ¹ The endogenous S‐nitrosothiol, S‐nitrosoglutathione, significantly inhibits platelet activation in isolated human veins, compared with placebo, which could also be of benefit in preventing early vein graft failure (Figure 2). ⁵¹ Furthermore, S‐nitrosoglutathione is neuro‐protective in animal models of metabolic toxicity, mitochondrial dysfunction, and oxidative stress that are linked to neurodegenerative disease. ⁶ , ⁵² Preliminary data also suggest that S‐nitrosothiols may have antimicrobial or antiviral activity. ¹ , ⁵³

Figure 2.

. An in vivo study in platelet‐rich plasma in human vein grafts showed that the stable nitrosothiol, S‐nitrosoglutathione (GSNO) (40 nmol/min), diminished the expression of the platelet α granule membrane protein 140, a marker for platelet activation, compared with the absence of GSNO in control vein grafts, suggesting inhibitory effects on platelet activation of GSNO in human veins. Reproduced with permission from Salas et al. ⁵¹

Diazeniumdiolates

Diazeniumdiolates are complexes of NO bound to a nucleophile, with up to 40% NO content by weight. ⁵⁴ , ⁵⁵ These compounds, also called NONOates, are direct NO donors since they release NO at physiologic pH, and require no metabolism or redox activation. ⁵⁴ , ⁵⁵ Research on these agents is only in preliminary stages. In experimental studies, diazeniumdiolates inhibit aggregation in rat and human models. ⁵⁴ , ⁵⁶ In an interesting application of this benefit, the incorporation of a diazeniumdiolate‐modified NO‐producing peptide into polyurethane to enhance thrombotic resistance in small‐diameter vascular grafts dramatically decreased platelet adhesion, compared with control polyurethane. ⁵⁷ In addition, endothelial cell growth was increased and smooth muscle cell growth was inhibited with the diazeniumdiolate‐treated polyurethane.

Diazeniumdiolates also inhibit the 5‐hydroxy‐tryptamine transporter in human cells, which may be important in the treatment of pulmonary hypertension, since uptake of 5‐hydroxytryptamine by its transporter contributes to pulmonary vascular remodeling. ⁵⁸ In experimental studies, diazeniumdiolates have demonstrated antileukemic, antineoplastic, and neuroprotective effects. ⁵⁹ , ⁶⁰

Sydnonimines

The sydnonimine class of NO donors is largely represented by molsidomine and its active metabolite, linsidomine, which spontaneously release NO and induce vasodilation via the guanylyl cyclase‐3′,5′‐cyclic guanosine monophosphate pathway. Sydnonimines have hemodynamic effects on cardiac ischemia similar to those of nitrates. ⁵ , ⁶¹ , ⁶² , ⁶³ In the large Angioplastic Coronaire Corvasal Diltiazem (ACCORD) study, ⁶² conducted in 700 patients undergoing percutaneous transluminal coronary balloon angioplasty, intravenous pretreatment with linsidomine, followed by oral molsidomine for 6 months, significantly reduced restenosis (≥50% stenosis), compared with diltiazem (P=.03), although molsidomine had no effect on major clinical outcomes (death, nonfatal myocardial infarction, and coronary revascularization). ⁶²

HYBRID NO DONOR DRUGS

A promising application of NO donor therapy is through hybrid drugs synthesized via an ester linkage of an NO‐releasing moiety with conventional NSAIDs and other anti‐inflammatory agents. ³ Agents in the NO‐NSAID group include NO‐aspirin, NO‐flurbiprofen, NO‐naproxen, NO‐diclofenac or nitrofenac, and NO‐ibuprofen. Other hybrid drugs include sildenafil nitrate for the treatment of impotence, salbutamol nitrate for bronchodilation, and NO‐paracetemol for liver disease, as well as NO‐prednisolone and NO‐mesalamine, which enhance the tolerability of the parent drug. ³ Although NSAIDs are the primary treatment for inflammation, their use is limited by serious gastrointestinal AEs, which have been attributed to inhibition of gastric cyclooxygenase‐1 activity. ³ In the gastrointestinal tract, NO is a vital mediator of mucosal defense and repair, and a modulator of smooth muscle tone that enhances motility and sphincter tone. ⁴ Therefore, the NO component added to an NSAID may hypothetically protect against NSAID‐associated AEs. ³ , ⁴

Numerous animal studies, extensively reviewed elsewhere, have shown that acute or chronic NO‐NSAID administration is associated with greatly reduced gastrointestinal toxicity, compared with NSAID monotherapy. ³ , ⁴ Moreover, NO‐NSAIDs appear to reduce gastrointestinal damage and counteract the impairment of ulcer healing caused by toxic agents or endogenous stimuli. ⁶⁴ , ⁶⁵ , ⁶⁶

Other notable effects of NO‐NSAID therapies demonstrated in animal models include neuroprotection against inflammation of the brain and cerebral ischemic injury, which could indicate possible therapeutic value of these agents in Alzheimer's disease ³ , ⁶⁷ , ⁶⁸ ; antinociceptive action in pain reduction ³ ; antithrombotic effects and reduction of restenosis ³ , ⁶⁹ ; and enhancement of the tolerability and antineoplastic efficacy of NSAIDs in cancer treatment. ³

NONPHARMACOLOGIC THERAPIES

A variety of nonpharmacologic interventions, including foods, dietary supplements, and exercise, that have been associated with reduced risks for CVD may also promote endothelial function and NO bioactivity. ⁷⁰ , ⁷¹ Nutritional or hormonal substances that have demonstrated these beneficial effects in experimental or clinical studies include estrogen in women, ⁷² dark chocolate, ⁷³ , ⁷⁴ omega‐3 fish oils, ⁷⁵ , ⁷⁶ red wine, ⁷⁷ vitamin E, ⁷⁸ folate, ⁷⁹ , ⁸⁰ and vitamin C. ⁸¹ While exercise appears to promote NO bioactivity through antioxidant effects, ⁷¹ the mechanisms of nutritional substances in this regard are quite varied and complex, including nonantioxidant effects. ⁷⁰ , ⁷⁴ Supplemental folic acid, for example, may both promote the bioavailability of tetrahydrobiopterin, an essential cofactor of NO synthase, and reduce nitrate tolerance. ⁸⁰

ANTIHYPERTENSIVE DRUGS

Although BP reduction alone may indirectly improve endothelial function, some antihypertensive agents appear to directly enhance NO bioactivity. ⁷ , ⁸² These actions include increasing endogenous production of NO synthase and release of NO; promoting expression of cofactors of NO such as tetrahydrobiopterin or the NO substrate L‐arginine; and preventing the oxidative processes that contribute to the breakdown and impairment of NO. ⁸² Since the different classes of antihypertensive drugs vary with regard to these therapeutic actions, they have exhibited disparate effects on NO bioactivity. ⁸³

Angiotensin‐Converting Enzyme Inhibitors

Angiotensin‐converting enzyme (ACE) inhibitors exhibit several mechanisms that enhance NO release and bioactivity. ⁸² By preventing the breakdown and promoting the preservation of brady‐kinin, ACE inhibitors stimulate bradykinin β ₂ receptors, which release vasoactive substances, including NO. ⁸² , ⁸⁴ ACE inhibitors may also protect NO against oxidative damage and impairment by suppressing production of angiotensin II, which contributes to oxidation and atherosclerosis by activating nicotinamide adenine dinucleotide phosphate (reduced) (NAD[P]H) oxidase and thus promoting NAD(P)H oxidase‐mediated production of superoxide anion. ⁸⁴

Multiple clinical studies have demonstrated that ACE inhibitors significantly improve endothelium‐dependent vasodilation in patients with hypertension and endothelial dysfunction. ⁸³ , ⁸⁵ , ⁸⁶ , ⁸⁷ , ⁸⁹ In the Trial on Reversing Endothelial Dysfunction (TREND), ⁹⁰ the ACE inhibitor quinapril significantly improved endothelium‐dependent vasodilation in coronary artery target segments (P=.002) and all segments (P=.02), compared with placebo, in normotensive patients with CHD, but without severe dyslipidemia or HF. ⁹⁰ The ACE inhibitor ramipril also improved endothelial function significantly in patients with CHD (P<.01) (Figure 3). ⁹¹ In several comparison studies with agents from other antihypertensive drug classes, including calcium channel blockers (CCBs), diuretics, β‐blockers, and angiotensin II receptor blockers (ARBs), ACE inhibitors were the only agents to significantly improve endothelial function. ⁸³ , ⁸⁶ , ⁸⁷ , ⁸⁸ , ⁸⁹ Other studies with ACE inhibitors, however, have failed to demonstrate effects on endothelial function. ⁹² , ⁹³ The inconsistency of these data has caused uncertainty as to whether ACE inhibitors improve endothelial function primarily by promoting NO bioactivity or through another pathway, which may be related to hyperpolarization. ⁷ Findings that ACE inhibitors also improve endothelial function in high‐risk patients with diabetes mellitus, ⁵ , ⁹⁴ hypercholesterolemia, and CHD ⁹⁵ , ⁹⁶ suggest that these agents may have pleiotropic vasculoprotective effects that may improve endothelial function through multiple pathways.

Figure 3.

. In a study in 35 patients with angiographically documented coronary heart disease, 4‐week therapies with the angiotensin‐converting enzyme inhibitor ramipril and the angiotensin II receptor blocker losartan both significantly improved flow‐dependent endothelium‐mediated vasodilation, as measured by radial artery diameter. The intra‐arterial infusion of the nitric oxide synthase antagonist N^G‐monomethyl‐l‐arginine (L‐NMMA) significantly reduced the improvement in endothelium‐dependent vasodilation with both agents. *P<.01 vs before l‐NMMA. Reproduced with permission from Hornig et al. ⁹¹

Calcium Channel Blockers

CCBs also appear to promote NO bioactivity primarily through antioxidant actions. ⁷ Dihydropyridine CCBs, including amlodipine and nisoldipine, exhibit antioxidant effects similar to those of ACE inhibitors in human endothelial cells. ⁹⁷ , ⁹⁸ CCBs may also directly induce eNOS production by increasing Ca²⁺ in endothelial cells, in contrast to the defining action of CCBs in blocking L‐type Ca²⁺ channels in smooth muscle, ⁹⁹ and through kinin preservation and stimulation of bradykinin β₂ receptors, as observed with ACE inhibitors. ¹⁰⁰ Other CCBs that have demonstrated stimu2lation of eNOS apparently through kinin‐mediated mechanisms include benidipine and clevidipine. ¹⁰⁰

In multiple clinical placebo‐controlled studies, CCBs significantly improved endothelium‐dependent vasodilation, reversing endothelial dysfunction in patients with hypertension ¹⁰¹ , ¹⁰² , ¹⁰³ , ¹⁰⁴ and in patients with hypercholesterolemia. ¹⁰⁵ , ¹⁰⁶ The Evaluation of Nifedipine and Cerivastatin On Recovery of Coronary Endothelial function (ENCORE I) study ¹⁰⁷ in 343 patients undergoing percutaneous coronary intervention showed that nifedipine significantly improved endothelium‐dependent vasodilation in the most constricted coronary segment, compared with placebo (P<.05), and by 11% in all coronary segments (P<.05). A number of other studies have nonetheless failed to demonstrate any beneficial effects of CCBs on endothelial function in hypertensive patients. ⁸³ , ⁸⁷ , ⁸⁸ , ⁸⁹ In one study, however, enalapril and amlodipine demonstrated similar significant improvement of endothelial function in patients with hypertension. ¹⁰¹ As BP reduction per se can indirectly improve endothelial function, it may be difficult to distinguish between direct and indirect effects of antihypertensive agents on endothelial function.

Angiotensin II Receptor Blockers

ARBs suppress the effects of angiotensin II, as do ACE inhibitors, and may thereby provide antioxidant protection against impairment of NO. ⁷ ARBs do not, however, increase bradykinin levels, an important mechanism through which ACE inhibitors promote NO release. Two studies in bovine pulmonary artery endothelial cells showed that the ARBs losartan and candesartan significantly increased levels of eNOS (P<.05 for both studies), which was abolished by an angiotensin II type 2 receptor antagonist. ¹⁰⁸ , ¹⁰⁹ These results suggest that ARBs, which block only the angiotensin II type 1 receptor, may promote an angiotensin II type 2 receptor‐mediated upregulation of eNOS protein. ¹⁰⁸ , ¹⁰⁹ A clinical trial in patients with hypertension, however, found that neither losartan nor hydrochlorothiazide administered for 4 weeks increased levels of NO urinary metabolites. ¹¹⁰

Clinical studies of the effects of ARBs on endothelial function yield conflicting results. In several studies, ARBs demonstrated significant improvement of endothelium‐dependent vasodilation in patients with hypertension compared with placebo or comparator agents. ⁷ , ¹¹¹ , ¹¹² , ¹¹³ The use of losartan has significantly improved endothelial function in patients with atherosclerosis and CHD, ⁹¹ , ¹¹⁴ and candesartan improved forearm blood flow significantly in patients with hypercholesterolemia‐associated endothelial dysfunction. ¹¹⁵ In other clinical studies in diverse populations, however, ARBs have demonstrated no significant effect on NO production or bioactivity. ⁸³ , ⁸⁹ , ¹¹⁶ Still, further comparison studies have shown similar effects of ARBs and ACE inhibitors on endothelial function in patients with hypertension ¹¹⁷ and with CHD (Figure 3). ⁹¹ Overall, more clinical data are needed to assess the effects of ARBs on NO bioactivity. ⁷

Vasodilating β‐Blockers

The use of conventional β‐blockers has failed to demonstrate significant direct effects on endothelial function and NO bioactivity, aside from their antihypertensive actions, in several studies. ⁸³ , ⁸⁶ , ⁸⁸ , ¹¹² Some newer β‐blockers distinguished primarily by their vasodilating action have, however, demonstrated direct effects on those parameters in experimental studies. ¹¹⁸

Carvedilol. Carvedilol produces peripheral vasodilation primarily through α₁‐adrenergic receptor blockade ¹¹⁹ ; improvement of endothelial vasodilatory and anti‐inflammatory and antiplatelet anti‐aggregation functions has been demonstrated in experimental studies and in patients with diabetes and hypertension. ¹²⁰ , ¹²¹ , ¹²² Carvedilol may achieve these effects primarily through antioxidant actions, thereby preserving NO bioactivity. ¹²⁰ , ¹²¹ , ¹²² An in vitro study, however, found that carvedilol scavenged NO in a cell‐free system and quenched NO to prevent nitrosylhemoglobin formation in vascular endothelial cells, suggesting that despite its favorable antioxidant actions, carvedilol had anti‐NO effects when reacting directly with NO. ¹²³ Thus, the overall effects of carvedilol on NO bioactivity remain uncertain.

Nebivolol. Nebivolol induces endothelium‐dependent vasodilation mediated via the l‐arginine/NO pathway. ¹⁴ , ¹²⁴ , ¹²⁵ , ¹²⁶ For example, the effects of brachial artery infusion of nebivolol and atenolol on forearm blood flow in 40 healthy subjects were assessed using venous occlusion plethysmography. ¹²⁴ Compared with baseline, nebivolol infusion increased forearm blood flow by 80% (P<.01), whereas atenolol had no significant effect; the vasodilatory response to nebivolol was reduced by infusion of the NO inhibitor N^G‐monomethyl‐l‐arginine, proving that it is mediated by endothelium‐dependent NO. Similarly, in hypertensive patients, nebivolol with bendrofluazide significantly improved forearm blood flow compared with baseline (P<.001); atenolol plus bendrofluazide showed no effect. ¹⁴

Nebivolol also appears to exert antithrombotic and antiplatelet antiaggregation effects associated with enhanced NO bioactivity, as demonstrated in an experimental rabbit model of hyperlipidemia. ¹²⁷ In a clinical study (N=550) comparing the effects of nebivolol, celiprolol, and carvedilol in patients with hypertension, nebivolol and celiprolol significantly reduced plasma levels of all 3 markers of thrombosis measured (homocystine, fibrinogen, and plasminogen activator inhibitor‐1) in the entire cohort, with nebivolol inducing greater reductions than celiprolol, whereas carvedilol had no significant effect on any of these measures. ¹²⁸ Among the subgroup who were smokers (one third of the subjects), all 3 drugs significantly reduced homocystine levels, but only nebivolol and celiprolol significantly reduced the other thrombotic factors.

In addition, an experimental study showed that nebivolol may promote NO bioactivity in African Americans, a group at high risk for CVD. ¹²⁹ Pretreatment with nebivolol improved NO bioactivity and reduced the rates of superoxide anion and peroxynitrite release in cells from black donors (Figure 4). Nebivolol has also demonstrated NO‐dependent benefits in ameliorating arterial stiffness, which is a key factor in systolic hypertension and an important independent predictor of mortality in patients with hypertension. ¹³⁰ A reduction in pulse wave velocity was observed with nebivolol, but not atenolol; this was attenuated with N^G‐monomethyl‐l‐arginine, proving that the effects of nebivolol were mediated by NO. ¹³⁰

Figure 4.

An in vitro study evaluated the effects of pretreatment with the vasodilating β‐blocker nebivolol (at doses of1.0 and 5.0 μmol/L) in human umbilical vein endothelial cells (HUVECs) and iliac artery endothelial cells (IAECs) from black and white donors. The cells from black donors exhibited significantly reduced nitric oxide (NO) bioactivity, compared with cells from white donors. Treatment with nebivolol restored NO bioavailability in a dose‐dependent manner to the level observed in cells from white donors. Atenolol, by contrast, had no effect on NO bioactivity in either the black or white cell samples. Reproduced with permission from Mason et al. ¹²⁹

STATINS

Along with other risk factors for atherosclerosis, hypercholesterolemia is associated with impaired endothelial function and reduced NO bioactivity. ¹³¹ , ¹³² Biochemical studies have shown that low‐density lipoprotein cholesterol (LDL‐C), particularly when oxidized, promotes production of superoxide anion, as well as other oxidative and inflammatory factors, and thus impairs NO bioactivity. ¹³³ , ¹³⁴ , ¹³⁵ Therefore, it is reasonable to expect that LDL‐C reduction with statins could enhance NO bioactivity and endothelial function by reducing the oxidative stress associated with hypercholesterolemia. ¹³⁵ Multiple clinical studies have demonstrated that various statins have significantly improved endothelium‐dependent vasodilation in coronary or brachial arteries and in patients with hypercholesterolemia and other associated risk factors such as diabetes mellitus, nephropathy, and CHD. ¹³⁶ , ¹³⁷ , ¹³⁸ , ¹³⁹ , ¹⁴⁰ , ¹⁴¹ , ¹⁴³

The decrease of oxidative stress associated with LDL‐C reduction is one mechanism by which statins improve endothelial function. ¹³⁵ , ¹³⁹ Indeed, statins have been shown to significantly reduce vascular prothrombotic and inflammatory factors in patients with CHD. ¹³⁷ , ¹³⁸ Experimental data suggest, however, that statins also promote NO bioactivity by several other actions independent of lipid reduction, such as increasing the expression of eNOS by stabilizing eNOS messenger RNA and by posttranscriptional modification of a variety of proteins (Table). ¹³⁵ , ¹³⁸ , ¹⁴³ In clinical studies, atorvastatin has significantly improved endothelium‐dependent vasodilation in normocholesterolemic smokers and postmenopausal women with average cholesterol levels, independent of changes in lipid levels. ¹⁴⁴ , ¹⁴⁵ A study in patients with stable angina pectoris showed that statin therapy significantly improved coronary endothelium‐dependent vasodilation after 24 hours, before any effect on cholesterol levels was observed (Figure 5). ¹⁴⁰ Another trial in patients with hypercholesterolemia found that statin therapy induced significant improvement in endothelial function after 3 days, at a more rapid rate than the accompanying LDL‐C reduction. ¹⁴⁶ , ¹⁴⁷

Table.

Effects of Statins Promoting Nitric Oxide Bioactivity and Endothelial Function

Effect	Mediator	Benefit
↓ NAD(P)H oxidase activity	Rac1	↓ Oxidative stress
↓ Synthesis of endothelin‐1	Rho	↑ Endothelial function
↓ Expression of AT1 receptor	Rho	↑ Endothelial function
↓ Expression of tissue type plasminogen activator	Rho	↓ Thrombosis
↑ Expression of plasminogen activator inhibitor‐1	Rho	↓ Thrombosis
↓ Expression of adhesion molecules	LFA‐1, Rho	↓ Inflammation
↑ eNOS activity	Rho, Akt	↑ Endothelial function
↑ Number and differentiation of circulating endothelial cells	Akt	↑ Neovascularization and re‐endothelialization
↓ Apoptosis	Akt	↑ Cell survival
NAD(P)H indicates nicotinamide adenine dinucleotide phosphate (reduced); AT1, angiotensin II type 1; LFA, leukocyte function antigen; eNOS, endothelial nitric oxide synthase; and Akt, protein kinase B.

Figure 5.

A clinical study evaluated the effects of pravastatin compared with placebo in 27 patients with stable angina pectoris, average low‐density lipoprotein cholesterol levels (138±9 mg/dL), and abnormal vasoconstriction in response to acetylcholine, indicating endothelial dysfunction. After 24 hours, patients given placebo (A) (n=14) exhibited no improvement in vasodilatory response to acetylcholine, as expressed in coronary luminal diameter, compared with baseline, while those given a single dose of pravastatin 40 mg (B) (n=13) showed significantly increased coronary luminal diameter. No significant changes were observed after 24 hours in serum lipid levels or high‐sensitivity C‐reactive protein. Negative values indicate vasoconstriction. *P<.05, before vs after treatment. Reproduced with permission from Wassmann et al. ¹⁴⁰

CONCLUSIONS

The diverse beneficial actions of NO in human physiology make it a potential therapeutic target in various disease states. Traditional nitrates, long considered limited in application, are being reappraised for more extensive use, and a number of new NO donors have been developed that directly or indirectly increase NO release. These include hybrid agents that possess multiple mechanisms of action and NO donors coupled with other types of drugs. In addition, well‐established drugs such as statins and antihypertensive agents are being evaluated for their possible effects in enhancing NO bioactivity and for the clinical implications of these effects.