Nitric Oxide-Mediated Coronary Flow Regulation in Patients with Coronary Artery Disease: Recent Advances

Abstract

Nitric oxide (NO) formed via endothelial NO synthase (eNOS) plays crucial roles in the regulation of coronary blood flow through vasodilatation and decreased vascular resistance, and in inhibition of platelet aggregation and adhesion, leading to the prevention of coronary circulatory failure, thrombosis, and atherosclerosis. Endothelial function is impaired by several pathogenic factors including smoking, chronic alcohol intake, hypercholesterolemia, obesity, hyperglycemia, and hypertension. The mechanisms underlying endothelial dysfunction include reduced NO synthase (NOS) expression and activity, decreased NO bioavailability, and increased production of oxygen radicals and endogenous NOS inhibitors. Atrial fibrillation appears to be a risk factor for endothelial dysfunction. Endothelial dysfunction is an important predictor of coronary artery disease (CAD) in humans. Penile erectile dysfunction, associated with impaired bioavailability of NO produced by eNOS and neuronal NOS, is also considered to be highly predictive of ischemic heart disease. There is evidence suggesting an important role of nitrergic innervation in coronary blood flow regulation. Prophylactic and therapeutic measures to eliminate pathogenic factors inducing endothelial and nitrergic nerve dysfunction would be quite important in preventing the genesis and development of CAD.

Coronary blood flow is regulated through complex adjustments in the arteriolar tone and resistance of the microcirculation. Impairment of microvascular function leads to organ dysfunction in any body system including the heart. Recent evidence supports the concept that the impairment of endothelial function is an upstream event in the pathophysiology of atherosclerosis, CAD, and myocardial infarction (MI). Nitric oxide (NO) liberated as a paracrine relaxant from the vascular endothelium is known to play a pivotal role in the modulation of microvascular tone and regional blood flow.¹ In addition, NO inhibits platelet aggregation and adhesion, inhibits leukocyte adhesion and migration, and reduces vascular smooth muscle proliferation, thus leading to prevention of atherosclerosis. NO produced via neuronal NO synthase (nNOS) is released from parasympathetic postganglionic (nitrergic) neurons and participates in vasodilatation, decreasing vascular resistance, and increasing blood flow.^2,3

Abundant and varied data from animal and human studies, performed over the course of more than two decades, indicate that depression of synthesis and bioavailability of NO in the endothelium participates in many cardiovascular diseases, including atherosclerosis,⁴ coronary heart diseases,⁵ stroke,³ renal failure,⁶ and hypertension^5,7 and also in insulin resistance and diabetes mellitus.⁸ Mechanisms underlying impairment of NO-mediated vasodilatation and blood flow increase include the downregulation of endothelial NOS (eNOS) and nNOS expressions, generation of NOS inhibitors and NO scavengers, and upregulation of vasoconstrictor substances, such as endothelin-1 (ET-1), vasoconstrictor prostanoids, and Rho/Rho-kinase.

The literature since the discovery of endothelium-derived relaxing factor by Furchgott and Zawadzki⁹ contains numerous reports about the interactions between NO and coronary arteries/arterioles or blood flow in health and disease. The present review covers recent advances in these investigations, including those published during these several years, on the roles of endothelial and neurogenic NO in the regulation of coronary circulation in patients with CAD and in some healthy subjects.

SYNTHESIS, DEGRADATION, AND ACTIONS OF NO

NO is produced when L-arginine is transformed to L-citrulline via catalysis by NO synthase (NOS) in the presence of oxygen and cofactors, including calmodulin, tetrahydrobiopterin (BH4), nicotinamide adenine dinucleotide phosphate (NADPH), heme, FAD, and FMN. Calcium ion (Ca²⁺) is required for the activation of nNOS (NOS I) and eNOS (NOS III) but not inducible NOS (iNOS, NOS II). nNOS is constitutively expressed in the brain, peripheral nerves,¹⁰ and kidneys, and eNOS is constitutively expressed mainly in endothelial cells.¹¹ iNOS is not constitutively expressed but is induced mainly in macrophages by bacterial lipopolysaccharide and cytokines.

eNOS binds to caveolin-1 in the caveolae, microdomains of the plasma membrane. Caveolin-1 inhibits eNOS activity, and this interaction is regulated by Ca²⁺/calmodulin.¹² The eNOS intracellularly migrates in response to increased cytosolic Ca²⁺ in the presence of calmodulin (Fig. 1) and is activated for NO synthesis. The transmembrane influx of Ca²⁺ and its mobilization from intracellular storage sites are caused via stimulation of drug receptors located on the endothelial cell membrane by acetylcholine (ACh), bradykinin (BK), and adenosine diphosphate (ADP) or via mechanical stimuli such as shear stress and vascular smooth muscle stretch. In human conduit coronary arteries, ACh causes contraction rather than relaxation, whereas substance P and histamine induce relaxations mediated by endothelial NO.¹³ On the other hand, shear stress, BK, or insulin induce the phosphorylation of Ser1177/1179 of eNOS through phosphatidylinositol 3-kinase (PI3K) and the downstream serine/threonine protein kinase Akt, resulting in enhanced NO formation.¹⁴ This mechanism does not require the increase in intracellular Ca²⁺ for NO production (Fig. 1). The alternative pathway through extracellular signal-regulated kinases also plays a role in eNOS activation.¹⁵

Figure 1.

Information pathways via NO liberated from endothelial cells and nitrergic neurons to vascular smooth muscle cells. On the endothelial membrane, receptors (R) responding to chemical and physical stimuli; ADMA, asymmetric dimethylarginine; Akt, serine/threonine protein kinase Akt; CaM, calmodulin; cGMP, cyclic GMP; CV, caveolin-1; DDAH, dimethylarginine dimethylaminohydrolase; eNOS*, activated eNOS; GC, soluble guanylyl cyclase; L-Arg., L-arginine; L-Citru., L-citrulline; nNOS*, activated nNOS; O2-, superoxide anion; ODQ, 1H[1,2,4]oxadiazolo [4,3-a]quinoxalin-o1-one; ONOO-, peroxynitrite; oxyHb, oxyhemoglobin; PDE-5, phosphodiesterase-5; PI3K, phosphatidyl inositol 3-kinase pool, Ca²⁺ storage site; SMTC, S-methyl-L-thiocitrulline; SOD, superoxide dismutase. Solid lines denote stimulation; dotted lines denote inhibition.

Endothelial NO causes vasodilatation, increased blood flow, lowered blood pressure, inhibition of platelet aggregation and adhesion, inhibition of leukocyte adhesion, and reduced smooth muscle proliferation; and it acts to prevent atherosclerosis. These NO actions are mediated by cyclic guanosine monophosphate (cyclic GMP) from GTP synthesized through soluble guanylyl cyclase. Nonadrenergic noncholinergic inhibitory responses to parasympathetic nerve stimulation are mainly mediated through NO synthesized by nNOS; NO plays a crucial role as a neurotransmitter from the peripheral efferent nerves in the blood vessel.^2,16

The synthesis of NO by NOS isoforms is inhibited by L-arginine analogs, including NG-monomethyl-L-arginine (L-NMMA), NG-nitro-L-arginine (L-NA), and L-NA methylester (L-NAME). The endogenous NOS inhibitor asymmetric dimethylarginine (ADMA)¹⁷ plays a pathogenic role, particularly in the circulation. 7-Nitroindazol (7-NI)¹⁸ and S-methyl-L-thiocitrulline (SMTC)¹⁹ are promising specific inhibitors of nNOS. Nitro compounds, such as nitroglycerin (GTN) and sodium nitroprusside (SNP), are capable of liberating NO. 1H[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one²⁰ decreases the synthesis of cyclic GMP by inhibiting guanylyl cyclase activity. Insufficiency of BH4 makes NOS uncoupled, which consequentially results in superoxide anions being produced instead of NO. Superoxide anions are also generated by NADPH oxidase and xanthine oxidase. Superoxide dismutase (SOD), catalase, and dimethyl sulfoxide scavenge free radicals. NO reacts with superoxide anions, generating highly toxic compounds such as peroxynitrite (ONOO⁻).

ROLE OF NO IN CORONARY CIRCULATION

Role of eNOS-Derived NO

Endothelial NO functions as a critical modulator of coronary blood flow by inhibiting smooth muscle contraction and platelet aggregation, and also contributes to angiogenesis and cytoprotection in the heart. The Dietary Approaches to Stop Hypertension (DASH) diet lowers blood pressure and substantially reduces the risk of coronary heart disease.²¹ In hypertensive and obese hypertensive patients, endothelial function is improved by the DASH diet.^22,23

Katz et al²⁴ provided evidence that endothelial dysfunction in patients with chronic heart failure, as assessed by flow-mediated dilatation in the brachial artery and exhaled NO production during submaximal exercise, is associated with an increased mortality risk in subjects with both ischemic and nonischemic chronic heart failure. In patients with slow coronary flow, thrombolysis in myocardial infarction (TIMI) frame count²⁵ was higher, the plasma NO level was lower, and brachial artery endothelium-dependent flow-mediated dilatation was smaller than in subjects with normal coronary flow.²⁶ Endothelial dysfunction appears to contribute to the pathogenesis of slow coronary flow. In patients with normal coronary angiograms, flow-mediated endothelium-dependent vasodilatation in the brachial artery had a negative relation with the intima + media area in coronary artery.²⁷ As the pathogenesis of acute coronary syndrome has been reported to involve plaque rupture even in the patients with normal coronary angiograms, it may be necessary to monitor patients with impaired flow-mediated vasodilatation even if their coronary angiograms show no abnormalities. Flow-mediated brachial artery dilatation, but not GTN-induced vasodilatation, was decreased in children of hypertensive patients as compared with controls, suggesting that children of hypertensive parents appear to have endothelial dysfunction, which may be an early marker for the development of CAD.²⁸ Forearm blood flow increase in response to ACh was smaller in patients with CAD who had periodontitis than in the nonperiodontitis group, SNP-induced vasodilatation was similar in both groups, and circulating levels of C-reactive protein (CRP) and interleukin-6 (IL-6) were higher in the periodontitis group; periodontal therapy reduced serum concentrations of CRP and IL-6, and augmented ACh-induced vasodilatation in patients with periodontitis.²⁹ Periodontitis appears to be associated with endothelial dysfunction in patients with CAD through a decrease in NO bioavailability. In patients without significant CAD, the gradient of F2-isoprostanes between arterial levels and coronary sinus correlated with the change in coronary artery diameter in response to ACh; isoprostanes net production across the left anterior descending (LAD) artery territory correlated with a decrease in SOD activity and a decrease in coronary artery diameter in response to L-NMMA.³⁰ These authors concluded that coronary endothelial dysfunction in humans may be characterized by local enhancement of oxidative stress without a decrease in basal NO release.

In patients with paroxysmal atrial fibrillation, in whom the atrial fibrillation was induced by burst atrial pacing, local (coronary sinus sample) cardiac platelet activation and thrombin generation increased and NO production decreased. However, there was no change in inflammatory markers, suggesting that atrial fibrillation may contribute to the hypercoagulable state within minutes.³¹ Guazzi and Arena³² summarize evidence that atrial fibrillation is a risk factor for endothelial dysfunction as documented by impaired ACh-induced blood flow increase, reduced plasma nitrite/nitrate levels, and additive impairment of flow-mediated vasodilatation by morbidities causing endothelial dysfunction.

Nitrite levels were increased in the anterior interventricular vein after an anastomosis between the left internal mammary artery and the LAD artery compared with those before the anastomosis, suggesting that the increased production of NO by the internal mammary graft may provide a perpetual vasodilator response.³³ The effect of L-NMMA on coronary blood flow, coronary artery diameter, and coronary vascular resistance was attenuated in cyclosporine-treated heart transplant recipients with normal coronary angiograms compared with controls.³⁴ Cardiac allograft epicardial coronary endothelial function is abnormal and may have an impaired endogenous NOS pathway and reduced endothelial NO production in transplant recipients. In cardiac transplant recipients with diabetes mellitus, postprandial hyperglycemia acutely doubled circulating levels of the oxidation product malondialdehyde, but did not affect the ability of ACh to dilate conduit coronary artery segments or accelerate coronary blood flow, suggesting that the oxidative stress associated with an acute episode of hyperglycemia affects neither ACh-mediated coronary endothelial NO release nor subsequent bioavailability.³⁵

Progressive worsening of functional coronary circulatory abnormalities of NO-mediated, endothelium-dependent vasodilatation occurs with increasing severity of insulin resistance and carbohydrate intolerance.³⁶ The levels of homocysteine, ET-1, and circulating endothelial cell in patients with coronary lesions were increased in comparison with patients with no recognizable plaque and/or stenosis, whereas the NO level was lower in those with coronary lesions, suggesting that homocysteine appears to be a predictor for preliminary or active coronary lesion.³⁷

In studies on patients with chronic CAD, it was noted that macrophage-colony stimulating factor (MCSF) and CRP levels were increased in those with T-786C at the promotor region of eNOS or variable nucleotide tandem repeat (VNTR) allele; patients with the combination of VNTR and T-786C had higher MCSF and CRP levels than patients with one or none of these alleles; patients with MCSF >262 pg/mL had lower flow-mediated dilatation of the brachial artery.³⁸ The intron 4-VNTR and T-786C mutation of eNOS appear to enhance the inflammatory process in patients with chronic CAD.

In summary, endothelial dysfunction is a predictor and also one of the important risk factors for CAD. Impaired endothelial function in coronary vasculatures is recognized by attenuated endothelium-dependent vasodilatation induced by chemical stimulation (ACh, BK, and ADP) and physical stimuli, such as flow and shear stress, without affecting the response to NO donors GTN and SNP. Forearm blood flow responses to chemical or physical stimuli and plasma nitrate/nitrite levels appear to reflect coronary arterial/arteriolar endothelial functioning. Decreased production of NO in endothelial cells would be associated with reduced eNOS protein expression and/or activity that might result from pathogenic factors, including smoking,³⁹ chronic alcohol intake,⁴⁰ high salt intake,⁴¹ hyperhomocysteinemia,⁴² diabetes mellitus,⁸ hypertension,⁴³ and increased production of endogenous NOS inhibitors such as ADMA. Increased activations of NADPH oxidase and xanthine oxidase, eNOS uncoupling due to BH4 depletion, and SOD deprivation appear to participate in generation of oxidative stress. Table 1 summarizes the synthesis and actions of NO and the possible mechanisms underlying endothelial dysfunction in patients with CAD. Atrial fibrillation may be a risk factor for coronary endothelial dysfunction.

Table 1.

Endothelial Dysfunction in Patients with Coronary Artery Disease

Author, Year	Disease	Change in Responses	Mechanism
Katz et al, 2005	Chronic heart failure	FMD↓ exhaled NO↓	E-dysfunction
Sezgin et al, 2005	Slow coronary flow	FMD↓ plasma NO↓	E-dysfunction
Khalil et al, 2008	Child of HT parents	FMD↓ GTN-D→	E-dysfunction, No change in NO response
Higashi et al, 2009	Periodontitis	ACh-D↓ SNP-D→ CRP↑	E-dysfunction, No change in NO response
Levy et al, 2009	Coronary artery dis.	ACh-R↓ SOD↓	E-dysfunction, Oxidative stress↑
Guazzi and Arena, 2009	Atrial fibrillation	ACh-D↓ plasma NO↓	E-dysfunction
Selcuk et al, 2007	Slow coronary flow	ADMA↑ L-arg./ADMA↓	E-dysfunction, NOS inhibition by ADMA↑
Okyay et al, 2007	Syndrome X	ADMA↑ L-arg.→	E-dysfunction, NOS inhibition by ADMA↑
Tang et al, 2009	Obst. coronary dis.	L-arginine availability↓ L-citrulline↑	Global arginine availability ratio↓

FMD, flow-mediated vasodilatation; NO, nitric oxide; E-dysfunction, endothelial dysfunction; HT, hypertension; GTN-D, glyceryl trinitrate-induced dilatation; ACh-D, acetylcholine-induced dilatation; SNP-D, sodium nitroprusside-induced dilatation; →, no change; CRP, C-reactive protein; dis., disease; Ach-R, acetylcholine receptor; SOD, superoxide dismutase; ADMA, asymmetric dimethylarginine; L-arg., L-arginine; NOS, nitric oxide synthase; obst., obstructive.

Involvement of ADMA in Blunted NO Availability

ADMA, an endogenous NOS inhibitor, has been known to be a risk factor for cardiovascular diseases¹⁷ through impairment of NO synthesis by eNOS and nNOS. Plasma ADMA is accumulated, because the degradation of ADMA through dimethylarginine dimethylaminohydrolase (DDAH)⁴⁴ and alanine-glyoxylate aminotransferase 2 (AGXT2)⁴⁵ is reduced.

Patients with slow coronary flow were detected to have higher levels of plasma ADMA and lower L-arginine/ADMA ratio compared with participants with normal coronary flow; both ADMA and L-arginine/ADMA ratio were correlated with coronary flow, as assessed by the TIMI frame count methods, suggesting that endothelial dysfunction may be an important factor in the pathogenesis of slow coronary flow.⁴⁶ The level of ADMA is suggested to predict survival in patients with chronic heart failure.⁴⁷ The plasma ADMA levels were higher in patients with typical exertional angina, positive exercise test, and normal coronary arteries diagnosed as cardiac syndrome X than in the control group, whereas plasma L-arginine levels were similar in both groups; patients with abnormal myocardial tissue perfusion had increased plasma ADMA levels compared with those with normal tissue perfusion.⁴⁸ In the patients with cardiac syndrome X, increased plasma ADMA levels may be associated with impaired myocardial tissue perfusion. Wang et al⁴⁹ provided evidence suggesting that ADMA, symmetric dimethylarginine (SDMA), and the integrated quantification of arginine methylation provided independent risk prediction for both obstructive CAD and incident major adverse cardiac events in stable patients undergoing cardiac evaluation, and that factors beyond direct NOS inhibition contribute to the clinical association between methylarginines and CAD outcome. Patients with obstructive CAD had a lower global arginine bioavailability ratio (defined as arginine/[ornithine + citrulline] versus plasma L-arginine levels) than those without obstructive CAD.⁵⁰ After adjusting for Framingham risk score, the lower global arginine availability ratio (but not L-arginine levels) and higher L-citrulline levels remained associated with the prevalence of obstructive CAD; global arginine availability ratio and ADMA showed a negative correlation. The global arginine availability ratio appears to serve as a more comprehensive concept of reduced NO synthetic capacity compared with systemic L-arginine levels. Therefore, Tang et al⁵⁰ suggested that diminished arginine bioavailability ratio and high citrulline levels are associated with development of obstructive CAD and heightened long-term risk for major adverse cardiovascular events.

Studies on high-risk diabetic men with CAD indicated that plasma ADMA levels were a strong and independent predictor of all-cause mortality; in addition, baseline ADMA values were also an independent predictor of the outcome of all-cause mortality for MI, suggesting that elevated baseline levels of ADMA are an independent predictor of cardiovascular outcomes in patients with diabetes mellitus.⁵¹ Coronary flow reserve was reduced in patients with early rheumatoid arthritis compared with that in healthy volunteers; higher levels of plasma ADMA were associated with decreased coronary flow reserve; common carotid intima-media thickness was negatively associated with coronary flow reserve.⁵²

In patients with stable angina, plasma levels of ADMA were related to the severity of CAD and correlated inversely with the number of circulating endothelial progenitor cells (EPCs) and endothelial colony forming units; ADMA repressed in vitro differentiation of EPCs and reduced EPC incorporation into endothelial tube-like structures, suggesting that ADMA is an endogenous inhibitor of mobilization, differentiation, and function of EPCs.⁵³ Surdacki et al⁵⁴ obtained evidence suggesting that elevated ADMA and EPC deficiency may synergistically contribute to accelerated renal dysfunction and that impairment of the EPC-dependent endothelial renewal may be associated with decreased bioavailability of NO.

Coronary angiogenesis and collateral growth are chronic adaptations to myocardial ischemia to restore coronary blood flow, and increased plasma ADMA levels are related with poor coronary collateral development.⁵⁵ Collateral development was lower in patients with the Asp variant.⁵⁶ This may be explained by the decreased eNOS activity in patients with this variant. Patients without CAD, who underwent coronary angiography alone, responded to the angiography with an increase in plasma ADMA, SDMA, and L-ornithine levels, whereas the stent implantation to diseased coronary artery, independent of the stent type used, reduced plasma ADMA levels.⁵⁷ Plasma ADMA activity in patients, who had diseased saphenous vein grafts was higher than in those with nondiseased saphenous vein grafts; mean platelet volume was also higher in patients with diseased vein grafts, suggesting that increased ADMA activity leads to the acceleration of saphenous vein graft disease and that ADMA may be a precious marker for detecting late saphenous vein graft patency.⁵⁸

Role of nNOS-Derived NO

Despite the fact that extensive studies have been performed to determine the functional role of autonomic, nitrergic nerves innervating cerebral,^2,3 renal,⁵⁹ and systemic vasculatures,⁶⁰ there is still a paucity of information concerning the role of nitrergic nerves in the regulation of coronary arterial and arteriolar tone and coronary hemodynamics.

Nerve cell bodies and perivascular neurons containing nNOS immunoreactivity or NADPH diaphorase have been reported in the heart of several species including the rat,^61,62,63 guinea-pig,⁶² and dog.⁶⁴ However, functional roles of neurogenic NO in the regulation of coronary arterial tone have not been determined. Isolated dog conduit coronary arteries respond to transmural field stimulation and nicotine with relaxations that are abolished by treatment with β-adrenoceptor antagonists. This is in contrast to the findings obtained from isolated cerebral arteries, which respond to electrical and chemical stimulations with relaxations that are sensitive to NOS inhibitors but resistant to β-adrenoceptor blockers. Despite the histological demonstration of NOS-containing neurons in the adventitia of large coronary arteries, evidence for the functional role of nitrergic neurons has not been provided⁶⁴; however, possible roles of nitrergic nerves in the regulation of coronary arteriolar tone and vascular resistance have been suggested.²

In patients with angiographically normal coronary arteries, intracoronary infusion of the nNOS-selective inhibitor SMTC reduced basal coronary blood flow and epicardial coronary diameter but had no effect on increases in flow evoked by intracoronary substance P that stimulated the release of NO from the endothelium, whereas L-NMMA infusion reduced basal coronary flow and inhibited substance P-induced increases in flow.⁶⁵ Local nNOS-derived NO, possibly from nitrergic neurons innervating coronary arteries and arterioles (Fig. 1), appears to regulate basal coronary blood flow in humans. Recent studies on anesthetized pigs treated with selective NOS isomer blockade⁶⁶ and on mice deficient in eNOS, nNOS, and iNOS genes⁶⁷ provided evidence that NO derived from nNOS alone or in combination with eNOS plays a role in protecting against fatal coronary circulatory disorders, whereas iNOS-derived NO appears to participate in impaired cardiac perfusion and contractility. Whether nNOS involved in the beneficial action is from autonomic nitrergic neurons or other organs and tissues remains to be determined.

Apart from nNOS in nitrergic neurons, Han et al⁶⁸ provided evidence suggesting that estrogen opens Ca²⁺-activated K⁺ channels in human coronary artery smooth muscle cells by stimulating nNOS via a transduction sequence involving PI3K and Akt. This may be a mechanism underlying the estrogen-induced enhancement of coronary blood flow in patients with diseased or damaged coronary arteries.

Role of iNOS-Derived NO

Activation of iNOS during immunological reactions and NO overproduction cause circulatory shock and neurotoxic actions.

Dover et al⁶⁹ provided evidence that selective iNOS inhibition by intrabrachial infusion of 1400 W {N-[3-(aminomethyl)benzyl]acetamidine} did not influence forearm blood flow in patients with New York Heart Association class II–V heart failure. iNOS activity does not seem to participate in peripheral vascular tone in patients with symptomatic heart failure. On the other hand, patients with heart failure from idiopathic dilated cardiomyopathy, who suffered from adverse events, had a diminished forearm blood flow response to ACh, compared with patients without adverse events. Intrabrachial infusion of aminoguanidine (another selective iNOS inhibitor) decreased forearm blood flow in patients with adverse events, but not in patients without adverse events, indicating that congestive heart failure patients with vascular iNOS activation, as evidenced by a greater vasoconstrictor response to aminoguanidine, had poor outcomes.⁷⁰ Whether the discrepancy in the actions of iNOS inhibitors is due to different severity or etiology of heart failure, different selectivity of so-called selective iNOS inhibitors used, and different doses of the NOS inhibitor used remains to be determined.

Kawasaki Disease and NO

In Kawasaki disease, a systemic vasculitis of unknown etiology, the intense inflammatory process has a predilection for the coronary arteries and abnormalities of myocardial blood flow appears to be associated with endothelial dysfunction.

Intracoronary infusion of ACh increased the LAD coronary artery area to a lesser extent in Kawasaki disease patients with a normal left coronary artery and patients with a persistent or regressed aneurysm than control subjects, whereas increases in coronary blood flow were similar in these groups, suggesting a persistent endothelial dysfunction in the epicardial but not resistance coronary arteries in patients with Kawasaki disease.⁷¹ Long-term coronary artery lesions, even after aneurysm regression, in patients with Kawasaki disease have impaired endothelial function.⁷² Kurio et al⁷³ obtained evidence suggesting that the endothelial injury in Kawasaki disease is confined to the endothelium of medium-sized arteries and that microvascular endothelial cells are normal after acute Kawasaki disease.

The number of EPCs was higher, the migratory response of EPCs was decreased, and the proliferative and adhesive activities were decreased in patients with Kawasaki disease compared with those in controls: the plasma NO, tumor necrosis factor-α (TNF-α), and high sensitivity CRP levels in the Kawasaki disease group were higher.⁷⁴ The number of circulating EPCs positively correlated with the level of NO, and the functions of EPCs negatively correlated with the levels of TNF-α and CRP. The two-way regulation of circulating EPCs (an increase in the number and a decrease in the function) in Kawasaki disease patients may be associated with the disorders of cytokines or messengers in these patients.

Neutrophils from patients with the early phase of Kawasaki disease produced higher amount of NO compared with controls; the amount of NO produced by neutrophils in the patients decreased after immunoglobulin treatment; increased production of reactive oxygen species (ROS) was found in both Kawasaki disease and non-Kawasaki disease febrile children.⁷⁵ The abnormal immune system in Kawasaki disease may be characterized by an overproduction of NO.

PENILE ERECTILE AND CORONARY ENDOTHELIAL DYSFUNCTION

There is growing evidence that erectile dysfunction is a sentinel for future CAD. Erectile dysfunction is associated with impairment of nitrergic neuronal and endothelial functions.^76,77 Therefore, interactions between vasculogenic erectile dysfunction and coronary endothelial dysfunction/hemodynamic disorder are inferred.

Significant correlation has been demonstrated between erectile function and the number of occluded coronary vessels in patients with ischemic heart disease in early studies by Greenstein et al.⁷⁸ The prevalence of erectile dysfunction was relatively high in patients with CAD, and it was related to the extent of CAD.⁷⁹ The authors suggest that erectile dysfunction may occur before CAD. There was a positive correlation between the severity of erectile dysfunction and coronary artery calcification in men with erectile dysfunction.^80,81 As compared with patients without erectile dysfunction, those with erectile dysfunction exhibited higher probability of having coronary atherosclerosis, higher number of coronary stenoses, and higher prevalence of a triple-vessel disease, suggesting that the coincidence of CAD and erectile dysfunction identifies patients at increased risk of severe forms of CAD.⁸² Erectile dysfunction is also strongly predictive of atherosclerotic cardiovascular events; this is even more striking when erectile dysfunction presents at a younger age.⁸³ According to Böhm et al,⁸⁴ erectile dysfunction is a potent predictor of all-cause death and the composite of cardiovascular death, MI, and heart failure in men. Vlachopoulos et al⁸⁵ summarized the pathophysiologic links between erectile dysfunction, endothelial dysfunction, and CAD.

The possible beneficial effect of PDE-5 inhibitors, regarded as promising therapeutics for male sexual dysfunction, in conditions such as MI and endothelial dysfunction has been reviewed by Kapur et al.⁸⁶ Clinical evidence supports the use of PDE-5 inhibitors as first-line therapy in men with CAD.⁸⁷

Taken together, pathogenic mechanisms underlying endothelial dysfunction in the corpus cavernosum appear to contribute to impairment of coronary arterial endothelial function; therefore, early signs of penile erectile dysfunction are regarded as an important predictor of CAD. It is hypothesized that neurogenic NO derived from parasympathetic nitrergic nerves plays a pivotal role in the intracavernous pressure increase and immediate penile erection, and endothelially generated NO and neurogenic NO act together to maintain penile erection.^76,77 Despite this fact, only little information is available about the role of nitrergic nerves in the regulation of coronary blood flow and its dysfunction in human subjects.

THERAPEUTIC MEASURES

L-Arginine and BH4

In patients with CAD, the intracoronary application of L-arginine (150 μmol/min) increased the luminal diameter of the stenotic segment without affecting other coronary artery segments and also increased the poststenotic coronary blood flow; the NO donor isosorbide dinitrate dilated all segments with a predominance of the stenotic coronary artery segment, suggesting a therapeutic potential of L-arginine in patients with coronary stenosis.⁸⁸ Compared with that in preischemia, the endothelium-dependent vasodilatation induced by ACh was reduced by reperfusion when saline was infused, but not following intrabrachial infusion of L-arginine (20 mg/min) and BH4 (500 μg/min) in patients with type II diabetes mellitus and CAD; vasodilatation induced by SNP was unaffected by ischemia/reperfusion, suggesting that L-arginine and BH4 supplementation may be a novel treatment strategy to limit ischemia/reperfusion injury in these patients.⁸⁹ On the other hand, in patients with noncritical CAD or following percutaneous coronary intervention, coronary microvascular endothelial function, as assessed by intracoronary infusions of ACh, was not improved by administration of BH4 (250 and 500 μg/min).⁹⁰

Abnormal zone myocardial blood flow reserve treated with adenosine and sildenafil exceeded that with adenosine and placebo, suggesting that PDE-5 inhibition appears to improve the myocardial blood flow response to adenosine in abnormal zones, possibly by augmenting NO-mediated increase in cyclic GMP.⁹¹

Antioxidants

Inhibition of xanthine oxidase activity by oxypurinol attenuated ACh-induced coronary vasoconstriction and increased coronary blood flow in patients with CAD compared with patients with preserved coronary endothelial function; flow-mediated dilatation of the brachial artery was also increased.⁹² Xanthine oxidase-derived ROS may contribute to impaired coronary NO bioavailability in CAD. In patients with chronic heart failure, allopurinol lowered ROS and ADMA concentrations and improved postischemic vasodilatation and endothelium-dependent vasodilatation.⁴⁷ Recent studies by Noman et al⁹³ on patients with chronic stable angina showed that high-dose (600 mg/d) alloprinol increased the mean time to ST depression, median total exercise time, and the time to chest pain from the baseline. Alloprinol seems to be a useful, inexpensive, well-tolerated, and safe anti-ischemic drug for patients with chronic stable angina.

Folic Acid

According to Moat et al,⁹⁴ both 400 μg/d and 5 mg/d of folic acid (for 6 weeks) increased plasma folate and decreased plasma homocysteine in patients with CAD; flow-mediated vasodilatation of the brachial artery was improved after treatment with 5 mg/d folic acid, but this did not correlate with the reduction of homocysteine; there was no change in flow-mediated vasodilatation in the 400 μg/d folic acid or placebo group; folic acid promoted eNOS dimerization in cultured porcine aortic endothelial cells. Folic acid appears to improve endothelial function in CAD via a promotion of eNOS dimerization but not through a mechanism dependent on homocysteine lowering. On the other hand, Shirodaria et al⁹⁵ obtained evidence that low-dose folic acid treatment (400 μg/d for 7 weeks) improved vascular function via an increase in enzymatic coupling of eNOS through availability of BH4 and a decrease in vascular oxidative stress in patients with CAD undergoing coronary artery bypass grafting surgery and that high-dose (5 mg/d) treatment provided no additional benefit. Despite the similar study designs used by these groups (Moat et al⁹⁴ versus Shirodaria et al⁹⁵), there is quite a difference in the effective doses of folic acid. Whether this is due to the use of patients with bypass grafting by the latter group remains to be determined. According to Dragoni et al,⁹⁶ treatment of healthy volunteers with folic acid (10 mg/d for 7 days) did not protect the vascular endothelium from ischemia/reperfusion injury.

3-Hydroxy-3-Methylglutaryl Coenzyme: A Reductase Inhibitor (Statin)

Beneficial pleiotropic effects of statins including improvement of endothelial dysfunction, increased NO bioavailability, antioxidant properties, and stabilization of atherosclerotic plaques have been summarized in previous review articles.^97,98 In patients with nonischemic chronic heart failure, the area under the curve ratio during ACh infusion increased in resistance vessels to a greater extent with atorvastatin treatment (40 mg/d for 6 weeks) compared with that without treatment; in conduit arteries, flow-mediated vasodilatation increased more with statin.⁹⁹ Young et al¹⁰⁰ noted that short-term (6 weeks) atorvastatin treatment in patients with nonischemic chronic heart failure improved endothelial function but had no effect on ADMA or the L-arginine/ADMA ratio. In 35 out of 46 patients with CAD, EPCs and EPC colony-forming units increased after a cardiac rehabilitation program and treatment for 1 month with statin compared with before the program and therapy, but the remaining 11 patients had no increase in either measure; those patients whose EPCs increased from baseline showed increases in plasma nitrite and decreases in annexin-V staining, a marker of apoptosis, in EPCs; over the course of the program, EPCs increased before the nitrite increase in the blood.¹⁰¹ Most, but not all, patients responded to the cardiac rehabilitation and statin therapy with increases in EPC number, EPC survival, and endothelial differentiation potential, possibly associated with increased NO in the blood. In patients with established CAD, abrupt discontinuation of simvastatin treatment led to a rebound of serum total cholesterol and low-density lipoprotein (LDL) cholesterol levels and decreased endothelial dependent flow-mediated dilatation of the brachial artery; in human umbilical vein endothelial cells, the NO production and eNOS expression were decreased after stopping statin treatment.¹⁰² Abrupt withdrawal of simvastatin treatment appears to not only abrogate its beneficial effects on endothelial function but also induce further vascular injury.

Angiotensin II Type 1 Receptor Blocker (ARB)

In patients with type II diabetes, serum ADMA concentrations decreased and coronary flow velocity reserve increased after a 4-week treatment with temocapril, an angiotensin-converting enzyme inhibitor.¹⁰³ Decrease in ADMA may be related to improvement of coronary circulation. Oral administration of the ARB losartan tended to improve endothelium-dependent brachial artery flow-mediated vasodilatation compared with the baseline (although statistically insignificant), while combination therapy with losartan and intravenous L-arginine significantly improved flow-mediated vasodilatation; urinary NO excretion after losartan alone and combined therapy was correlated with improved hemodynamic variables.¹⁰⁴ According to Koh et al,¹⁰⁵ ARBs decrease the incidence of CAD, because they inhibit angiotensin II-induced increases in superoxide anion generation and oxidative stress, leading to activation of nuclear transcription factor and endothelial dysfunction.

Vasodilating β-Adrenoceptor Blockers (β-Blockers of the Third Generation)

β-Adrenoceptor blockers with vasodilatory action associated with the release of NO (celiprolol, nebivolol, and nipradilol) or the suppression of ROS (carvedilol) are expected to counteract the possible β-blockade-induced coronary vasoconstriction.¹⁰⁶ Celiprolol was suggested to be potentially useful in patients with angina pectoris and hypertension, complicated by other conditions associated with advanced age, impaired glucose tolerance or diabetes mellitus, peripheral vascular disease, and hyperlipidemia.^107,108 Coronary flow reserve at rest was less in patients with CAD than in control individuals; intracoronary administration of nebivolol increased coronary flow reserve both in the controls and patients; collateral flow index decreased with nebivolol and correlated to changes in heart rate.¹⁰⁹ It appears that intracoronary nebivolol is associated with an increase in coronary flow reserve due to an increase in maximal coronary flow and that the collateral flow index decreases with nebivolol parallel to the reduction in myocardial oxygen consumption. Changes of coronary flow reserve due to vasodilator β-blockers improve microvascular angina pectoris or silent ischemia in patients without epicardial artery stenosis.¹¹⁰ Akçay et al¹¹¹ provided evidence suggesting that nebivolol is beneficial for improving oxidative stress parameters in patients with slow coronary flow.

Herbal Agents

During aged-garlic extract supplementation, flow-mediated endothelium-dependent brachial artery dilatation increased from the baseline in patients with CAD that were currently being treated with aspirin and a statin; markers of oxidative stress (plasma oxidized LDL and peroxides), systemic inflammation, and endothelial activation did not change during the study.¹¹² Intravenous administration of Ginkgo biloba extract to patients with CAD increased LAD coronary artery blood flow and brachial artery flow-dependent dilatation.¹¹³ In addition, plasma NO increased and ET-1 decreased after 2 weeks of ginkgo extract treatment; a linear correlation was obtained between the percentage change in LAD coronary artery blood flow and in NO, ET-1, or NO/ET-1 ratio following extract treatment, suggesting that the ginkgo extract led to an increase in coronary blood flow, which may be related to improvement of NO/ET-1 imbalance.¹¹⁴

Exercise and External Counterpulsation

After 8 weeks of exercise training in patients with congestive heart disease, forearm blood flow responses to ACh and SNP increased as compared with the control group (usual living); the clearance of L-arginine also increased in the training group.¹¹⁵ The authors suggested that an increase in the transport of L-arginine may contribute to the augmentation of endothelial function by exercise. In addition, increased NO actions appear to be involved, since endothelium-independent NO-mediated vasodilatation was also augmented. According to Duncker and Bache,¹¹⁶ exercise training augments endothelium-dependent vasodilatation through the coronary microcirculation, possibly through an increased expression of NOS; during exercise, endothelium-derived NO, prostanoids, and β-adrenergic activity exert vasodilator influences on coronary collateral vessels.

Enhanced external counterpulsation (EECP) is a noninvasive, pneumatic technique that provides beneficial effects for patients with chronic, symptomatic angina pectoris. EECP elicited increases in intracoronary pressure with a decrease in systolic pressure, intracoronary Doppler flow velocity, and coronary flow, as assessed by TIMI frame count, suggesting that EECP may serve as a potential mechanical assist device.¹¹⁷ During the course of EECP therapy in patients with CAD, plasma nitrate/nitrite progressively increased and plasma ET-1 decreased, suggesting that EECP improves endothelial function.¹¹⁸ In symptomatic patients with CAD, EECP increased flow-mediated vasodilatation and plasma levels of nitrate/nitrite and 6-keto-prostaglandin F_1α, whereas it decreased plasma levels of ET-1, ADMA, and proinflammatory cytokines.¹¹⁹

Miscellaneous

Treatment with the α-adrenoceptor antagonist urapidil improved coronary flow, myocardial perfusion, and left ventricular function following percutaneous coronary intervention in patients with ST-elevation acute coronary syndrome; myocardial NO concentrations in the urapidil group was higher than that of the control group.¹²⁰ These beneficial effects appear to be associated with an enhanced biosynthesis of NO. Patients with coronary vasospasm had lower endothelium-dependent flow-mediated vasodilatation as compared with normal individuals; benidipine, but not diltiazem and verapamil, increased flow-mediated vasodilatation and plasma cyclic GMP levels; none of the treatments affected GTN-induced vasodilatation, suggesting that upregulation of the NO-cyclic GMP system by benidipine may partly contribute to the improvement of endothelial dysfunction.¹²¹ Short-term (4 weeks) trimetazidine therapy improved heart rate variability parameters and endothelial products such as NO and ET-1 as well as anginal symptom in patients with slow coronary artery flow; this improvement was correlated with increased NO and decreased ET-1 levels.¹²² The Rho kinase inhibitor fasudil increased endothelium-dependent vasodilatation in patients with CAD but not in healthy controls and also reduced Rho kinase activity in the patients, suggesting that inhibition of the Rho/Rho kinase pathway appears to provide a useful strategy to restore NO bioavailability in humans with atherosclerosis.¹²³

In patients with symptomatic coronary disease and long-term aspirin therapy, vascular function tests showed improvement of ACh-induced vasodilatation and L-NMMA responses in the clopidogrel-added group, while SNP-induced vasodilatation was not altered; urinary excretion of 8-iso-prostaglandin F_2α and plasma levels of inflammation products were reduced in patients on additional treatment with clopidogrel but not in patients on placebo.¹²⁴ Beyond inhibition of platelet aggregation, adenosine diphosphate–receptor blockade may have promising vasoprotective effects, such as improvement of endothelial NO bioavailability and diminishment of biomarkers of oxidative stress and inflammation in these patients. The increase in leukocyte-derived myeloperoxidase plasma content on bolus heparin was higher in patients with CAD; heparin treatment improved endothelial NO bioavailability, as evidenced by flow-mediated vasodilatation and by ACh-induced increase in forearm blood flow, suggesting that mobilization of vessel-associated myeloperoxidase may represent a mechanism by which heparins exert anti-inflammatory effects and increase vascular NO bioavailability.¹²⁵

Aldehyde dehydrogenase-2 may confer cardioprotection through metabolism of reactive aldehydes and through its role in the bioconversion of nitrates to NO. Therefore, Budas et al¹²⁶ suggest that aldehyde dehydrogenase-2 is a key mediator of endogenous survival signaling in the heart and its agonists, such as an aldehyde dehydrogenase-2 activator 1, may lead to novel therapeutics, which limit injury during MI or bypass surgery.

SUMMARY

This review article summarizes information concerning recent advances in research on coronary blood flow regulation by NO generated mainly through eNOS and also nNOS or iNOS in patients with CAD. The mechanisms underlying endothelial dysfunction in CAD cannot be fully discussed because of limited information from studies on healthy and diseased individuals, in which ethical problems must be avoided. Endothelial dysfunction is undoubtedly one of the important risk factors for CAD. In addition, the imbalance between vasodilator factors, such as NO, endothelium-derived hyperpolarizing factor, and prostacyclin, and vasoconstrictors, including ET-1, thromboxane A₂, Rho-Rho-kinase, and endothelium-derived contracting factors, must be kept in mind for treatment of CAD. Studies on the physiological role of nitrergic neurons in the coronary blood flow regulation in humans are still insufficient; however, together with neurogenic coronary vasodilatation mediated by the β-adrenergic mechanism, the nitrergic vasodilatation is expected to play a role in the control of coronary circulation. Penile erectile dysfunction of both nitrergic neural and endothelial origins is an independent predictor of coronary insufficiency and the severity of erectile dysfunction appears to reflect the extents of coronary dysfunction and histological damage. Maintenance of healthy endothelial cells through controlled daily life, including quitting smoking, balanced diet, decreasing body weight to a healthy level, and adequate physical exercise, together with prophylactic and therapeutic measures to augment constitutive NOS expression, increase NO availability, degrade oxygen radicals, and inhibit the production of endogenous NOS inhibitors would provide us with an important way to prevent or treat impairments of endothelial and nitrergic neural functions and then CAD.