1. eNOS Impairment, Vascular Dysfunction and Cardiovascular Disease: Socioeconomic Impact and Molecular Triggers
1.1. Global Burden of Disease Study and Cardiovascular Risk Factors
Cardiovascular diseases/events (e.g., heart failure, myocardial infarction, acute coronary syndromes) represent significant health risk factors and they are major contributors to global deaths and chronic illness/disability [1]. The Global Burden of Disease Study is a comprehensive regional and global research program of disease burden that assesses mortality and disability from major diseases, injuries, and risk factors by a collaboration of over 1800 researchers from 127 countries.
According to the 2015 update of the Global Burden of Disease Study, these “classical” health risk factors increase the global mortality head-to-head with environmental risk factors, such as ambient particulate matter pollution and household air pollution from solid fuels, which ranked on the fifth and tenth position among all causes for global deaths [4]. Air pollution is indeed associated with a number of cardiovascular diseases [5] and environmental (traffic) noise exposure is regarded as a trigger of cerebro/cardiovascular and metabolic diseases [6], although noise-mediated cardiovascular adverse effects are less intensively characterized than those that are mediated by air pollution.
Finally, mental stress (including psychosocial origins) represents another important environmental risk factor that clearly contributes to cardiovascular risk and probably also mortality, as already reported by the INTERHEART Study in 2004, by the demonstration that psychosocial stress is associated with a higher risk for myocardial infarction [7,8].
1.2. Oxidative Stress as a Unifying Molecular Trigger of Endothelial Dysfunction, Atherosclerosis, and Cardiovascular Disease
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The functional correlate of eNOS dysfunction/uncoupling is endothelial dysfunction in coronary and peripheral vessels, which can be measured by acetylcholine-dependent or flow-mediated dilation (FMD) and it represents an early predictor of cardiovascular events via its direct connection to the process of atherosclerosis [26,27].
The close connection between oxidative stress and cardiovascular prognosis is supported by a number of small cohort clinical studies (e.g., by differential effects of vitamin C infusion on FMD in patients with high or low burden of ROS formation [26]). An example is based on the significantly impaired FMD and lower levels of reduced circulating glutathione in 52 smokers versus controls [34]. Additionally, a positive correlation between FMD and superoxide dismutase activity and negative correlations between FMD and oxidized low-density lipoprotein (oxLDL)/ADMA levels in 59 patients with chronic kidney disease versus controls were reported [35].
Also, large clinical trials exist that support a role of oxidative stress for cardiovascular prognosis. For example, the levels of glutathione peroxidase-1 showed a positive correlation with cardiovascular event-free survival in 636 individuals [37] and the oxidative stress serum markers D-ROM (=derivatives of reactive oxygen metabolites, indicating ROS levels) and TTL (=total thiol levels, representing the redox state) were independently and strongly associated with all-cause and cardiovascular mortality in 10,622 men (Figure 1) [38].
Despite the preclinical and clinical evidence for an important role of oxidative stress for the development and progression of cardiovascular disease, there are, to date, only a few examples of clinical studies demonstrating that targeted antioxidant drugs (e.g., xanthine oxidase inhibitors, meta-analysis in 10,684 individuals) or tight control of vitamin C plasma levels (e.g., EPIC-Norfolk trial, 19,496 individuals [39]) improve the prognosis of patients with cardiovascular disease. The majority of large clinical trials failed to show any health benefit for the treatment of cardiovascular disease with non-selective antioxidant drugs [40,41], and the synthetic antioxidant drug NXY-059, despite costly development/clinical testing, failed to demonstrate any benefit in 3195 stroke patients [42].
1.3. Role of Inflammation for Endothelial Dysfunction and Cardiovascular Disease
Also, low-grade inflammation is associated with most cardiovascular diseases [43,44] and it contributes significantly to oxidative stress [45]. Thereby, inflammation represents an independent cardiovascular risk factor (Figure 1) [46] that can be pharmacologically targeted [47]. Therefore, we will emphasize the importance of low-grade inflammation for the development of endothelial dysfunction and cardiovascular disease as well as its close interaction with oxidative stress, not only in vascular cells, but also in perivascular adipose tissue.
Systemic lupus erythematosus, rheumatoid arthritis, and also severe psoriasis represent chronic autoimmune diseases that are associated with an increased cardiovascular risk [59,60,61,62]. Psoriasis was even defined as an independent risk factor for cardiovascular disease [63] and the European League against Rheumatism guidelines even recommend cardiovascular therapy in patients with inflammatory arthritis [64].
1.4. Prognostic Value of Endothelial Dysfunction and Measurement in Human Subjects
Endothelial dysfunction is found in the presence of all classical cardiovascular risk factors, such as arterial hypertension [12,74], hypercholesterolemia [11], diabetes mellitus [75], obesity, and chronic smoking [76]. Endothelial dysfunction is also correlated with markers of chronic (low-grade) inflammation, such as C-reactive protein (CRP) [77] and cardiovascular risk predictors, such as adiponectin and brain natriuretic peptide (BNP) [78,79].
The presence of several risk factors produces synergistic effects on endothelial function as well as the associated cardiovascular prognosis [86]. Previous studies confirmed that hypercholesterolemia or chronic smoking lead to moderate impairment of endothelial function (reduction of the maximal acetylcholine-dependent vasodilation by ~30%), whereas the presence of both risk factors caused severe endothelial dysfunction (reduction of the maximal acetylcholine-dependent vasodilation by ~ 60%) [87].
A meta-analysis of 15 cohort studies reported a significant correlation of endothelial function measurements using FMD with diagnosed atherosclerosis in patients [89]. These reports are contrasted by the failure of endothelial function to predict cardiovascular events in mostly healthy subjects [90] and in individuals with intermediate cardiovascular risk [91]. According to another large population-based study non-invasive measurement of vascular function failed to add prognostic value to the European Society of Cardiology risk score [92]. Therefore, measurements of intima-media thickness [90] and stiffness index [93] may be recommended for more accurate determination of endothelial and vascular function, especially since intima-media thickness also correlates with redox state and early atherosclerosis [94].
1.5. Role of eNOS in Perivascular Adipose Tissue for Vascular Function
Soltis and Cassis have reported in 1991 that the presence of perivascular adipose tissue (PVAT) reduces the contractile response of rat aorta to noradrenaline [95]. It is now a widely accepted concept that PVAT plays an important role in regulating vascular function by releasing a large number of bioactive molecules, including NO [96].
It is still not completely understood how PVAT-derived NO regulates vascular tone. Available data suggest that NO produced by PVAT may induce vasorelaxation through the following mechanisms [96]: by diffusing into the adjacent smooth muscle cells and activating guanylyl cyclase, by stimulating adiponectin release from PVAT adipocytes [105], or by modulating BKCa (large-conductance calcium-activated potassium) channels in smooth muscle cells and potentiating hyperpolarization [106].
Dysfunction of PVAT eNOS can be reversed by pharmacological treatments [96,108]. In a recent study, we have shown that in vivo treatment of diet-induced obese mice with a standardized Crataegus extract completely restored the vascular function of PVAT-containing aorta [107].
2. “Redox Switches” in Endothelial Nitric Oxide Synthase (eNOS) and Associated Pathways for Therapeutic Targeting
There are classical regulatory mechanisms of eNOS activity, such as calcium/calmodulin, caveolin, HSP90, palmitoylation, and myristoylation, which also control the activating phosphorylation by protein kinase B (Akt) or AMP-activated protein kinase (AMPK) at Ser1177 (Ser1179) as well as the localization of eNOS. The non-classical regulation of eNOS activity is based on the formation of redox-active species that trigger adverse phosphorylation by redox-active kinases at Thr495/Tyr657 (e.g., kinases PKC and PYK-2), S-glutathionylation, oxidative tetrahydrobiopterin depletion, dysregulation of asymmetric dimethylarginine (ADMA) formation/degradation, and disruption of the zinc-sulfur-complex stabilizing the eNOS dimer (reviewed in [20,111]).
2.1. Oxidative Depletion of Tetrahydrobiopterin
Oxidative loss of tetrahydrobiopterin (BH4) as a trigger for eNOS uncoupling is the best characterized “redox switch” in eNOS and meanwhile well documented in hypertension, diabetes, and atherosclerosis [33,114,115,116,117,118]. Reviews on the role of BH4 deficiency in almost all cardiovascular diseases provide detailed insights on the mechanisms [23,119,120,121,122].
2.2. Oxidative Disruption of the Zinc-Sulfur-Complex (ZnCys4) in the Binding Region of the eNOS Dimer
Another direct redox-regulatory pathway for eNOS function is the oxidative disruption of the zinc-sulfur-complex (ZnCys4) in the binding region of the eNOS dimer, resulting in a loss of sodium dodecyl sulfate (SDS)-resistant eNOS dimers, which has been first described by Zou and coworkers for peroxynitrite-mediated oxidation of eNOS [136]. The reports on this “redox switch” reflected by a decreased eNOS dimer/monomer ratio were previously summarized until the year 2010 [20,23].
2.3. S-glutathionylation of the eNOS Reductase Domain
S-glutathionylation represents another important “redox switch” in eNOS. Zweier and coworkers showed that eNOS is adversely regulated and uncoupled (leading to superoxide formation) by S-glutathionylation at cysteine residues Cys689 and Cys908 in the reductase domain [157,158].
2.4. Phosphorylation at Thr495 and Tyr657
Regulation of eNOS is also mediated by phosphorylation via redox-sensitive kinases. Whereas, eNOS phosphorylation at Ser1177 via Akt pathway is of activating nature [177], phosphorylation at Tyr657 mediated by the protein tyrosine kinase-2 (PYK-2) is of inactivating nature [178].
2.5. ADMA Formation and Degradation by DDAH
Asymmetric dimethylarginine (ADMA) is a potent endogenous inhibitor of eNOS [194], which may trigger the uncoupling of eNOS [195]. ADMA serum/plasma levels have prognostic value for future cardiovascular events in patients at increased risk [196,197].
According to an overview on circulating biomarkers in diabetic patients, ADMA may be used as an indicator of endothelial dysfunction in diabetes [198].
Another study reported that coronary artery disease patients with too high ADMA serum concentrations are not protected anymore by simvastatin therapy against cardiovascular events, which however could be prevented by DDAH2 overexpression in a related experimental animal model [200].
2.6. L-Arginine Deficiency
L-Arginine is the endogenous substrate of eNOS and “L-arginine deficiency” may contribute to the uncoupling of eNOS [202]. However, the Km (the concentration of L-arginine that is necessary for half maximal saturation) of eNOS for L-arginine is approximately 2.9 µM, whereas intracellular L-arginine concentrations are usually in the mM range [20,23,43]. Therefore, L-arginine depletion as a regulator of eNOS was considered to be unlikely.
Nevertheless, a number of pre-clinical and clinical studies reported on highly beneficial effects by oral L-arginine supplementation and also increased superoxide formation by isolated NOS enzymes in the absence of L-arginine was observed. The direct antioxidant effects of the guanidino-group L-arginine may be one explanation for these observations [203]. Improved export of ADMA from endothelial cells by high dose L-arginine represents another explanation.
Referência :
(1) New Therapeutic Implications of Endothelial Nitric Oxide Synthase (eNOS) Function/Dysfunction in Cardiovascular Disease. Int J Mol Sci. 2019 Jan 7;20(1):187.
