Endothelial nitric oxide synthase (eNOS) serves as a critical enzyme in maintaining vascular pressure by producing nitric oxide (NO); hence, it has a crucial role in the regulation of endothelial function. The bioavailability of eNOS-derived NO is crucial for this function and might be affected at multiple levels. Uncoupling of eNOS, with subsequently less NO and more superoxide generation, is one of the major underlying causes of endothelial dysfunction found in atherosclerosis, diabetes, hypertension, cigarette smoking, hyperhomocysteinemia, and ischemia/reperfusion injury. Therefore, modulating eNOS uncoupling by stabilizing eNOS activity, enhancing its substrate, cofactors, and transcription, and reversing uncoupled eNOS are attractive therapeutic approaches to improve endothelial function. This review provides an extensive overview of the important role of eNOS uncoupling in the pathogenesis of endothelial dysfunction and the potential therapeutic interventions to modulate eNOS for tackling endothelial dysfunction.

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The use of doxorubicin (DOXO) as a chemotherapeutic drug has been hampered by cardiotoxicity leading to cardiomyopathy and heart failure. Folic acid (FA) is a modulator of endothelial nitric oxide (NO) synthase (eNOS), which in turn is an important player in diseases associated with NO insufficiency or NOS dysregulation, such as pressure overload and myocardial infarction. However, the role of FA in DOXO‐induced cardiomyopathy is poorly understood. The aim of this study was to test the hypothesis that FA prevents DOXO‐induced cardiomyopathy by modulating eNOS and mitochondrial structure and function. Male C57BL/6 mice were randomized to a single dose of DOXO (20 mg/kg intraperitoneal) or sham. FA supplementation (10 mg/day per oral) was started 7 days before DOXO injection and continued thereafter. DOXO resulted in 70% mortality after 10 days, with the surviving mice demonstrating a 30% reduction in stroke volume compared with sham groups. Pre‐treatment with FA reduced mortality to 45% and improved stroke volume (both P < 0.05 versus DOXO). These effects of FA were underlain by blunting of DOXO‐induced cardiomyocyte atrophy, apoptosis, interstitial fibrosis and impairment of mitochondrial function. Mechanistically, pre‐treatment with FA prevented DOXO‐induced increases in superoxide anion production by reducing the eNOS monomer:dimer ratio and eNOS S‐glutathionylation, and attenuated DOXO‐induced decreases in superoxide dismutase, eNOS phosphorylation and NO production. Enhancing eNOS function by restoring its coupling and subsequently reducing oxidative stress with FA may be a novel therapeutic approach to attenuate DOXO‐induced cardiomyopathy.

TO THE EDITOR: He et al. (3) presented in the American Journal of Physiology -Endocrinology and Metabolism that chronic hyperhomocysteinemia (HHcy; plasma level of homocysteine Ͼ15 mol/l) impairs coronary endothelial function and that plasma levels of nitric oxide (NO) and tetrahydrobiopterin (BH4) are positively correlated and significantly decreased in patients with HHcy compared with controls (99.54 Ϯ 32.23 vs. 119.50 Ϯ 37.68 mol/l and 1.43 Ϯ 0.46 vs. 1.73 Ϯ 0.56 pmol/ml, all P Ͻ 0.05). Moreover, coronary flow velocity reserve (CFVR) was significantly lower (ϳ10%) in the HHcy patients than in the control group. In addition, plasma level of homocysteine was negatively correlated with CFVR and plasma level of NO. He et al. suggested that chronic HHcy decreased plasma NO and BH4, leading to endothelial nitric oxide synthase (eNOS) uncoupling and, consequently, coronary artery endothelium dysfunction.While the results of He et al. are intriguing, the methodologies used are controversial, and subsequent conclusions are too preliminary. First, the authors used a nitrite/nitrate assay based on the Griess reagent to determine plasma NO levels. Nitrite and nitrate are the degradation products of NO. Unfortunately, this methodology carries some disadvantages when used in serum, as it does not allow an exact differentiation of nitrite and biogenic amines that are physiologically present in plasma and is, hence, not a fully reliable marker to indicate decreases in NO levels or, for that matter, uncoupling of eNOS.Second, the ratio of BH4 and its oxidized form dihydrobiopterin (BH2) should have been determined instead of plasma BH4 levels alone. A decrease in plasma BH4 levels alone is insufficient to induce and indicate eNOS uncoupling. Indeed, the ratio of reduced and oxidized biopterins (i.e., the BH4/BH2 ratio), and not only the absolute amount of BH4, plays an important role in eNOS coupling in vivo (2). Although BH2 does not have an NOS cofactor property, it inhibits BH4 competitively (5) and binds eNOS with an affinity equal to that of BH4 (1); therefore, BH2 should have been measured.Third, although transthoracic Doppler echocardiography using adenosine-induced hyperemia is a well-studied method to evaluate coronary flow reserve (CFR), there are several factors influencing CFR. Normal CFR indicates a normal two-compartment system: a patent epicardial vessel supplying a normal myocardial bed. Abnormal CFR may be due to multiple factors such as abnormal epicardial vessels (i.e., flow limiting stenosis), abnormal microvasculature, and factors influencing blood composition and endothelial dysfunction. In addition, there is a large body of evidence showing great variations in CFR due to different medical conditions such as heart failure, hypertension, or even dietary intake (4). In the study of He et al., there is no evidence of absence of flow-limiting coronary artery disease, left ventricular hypertrophy, heart failure, or other factors influencing CFR. As these factors all influence CFR, CFR should not be used as a...

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