Nitric oxide (NO) is one of the most important molecules released by endothelial cells, and its antiatherogenic properties support cardiovascular homeostasis. Diminished NO bioavailability is a common hallmark of endothelial dysfunction underlying the pathogenesis of the cardiovascular disease. Vascular NO is synthesized by endothelial nitric oxide synthase (eNOS) from the substrate L-arginine (L-Arg), with tetrahydrobiopterin (BH₄) as an essential cofactor. Cardiovascular risk factors such as diabetes, dyslipidemia, hypertension, aging, or smoking increase vascular oxidative stress that strongly affects eNOS activity and leads to eNOS uncoupling. Uncoupled eNOS produces superoxide anion (O₂⁻) instead of NO, thus becoming a source of harmful free radicals exacerbating the oxidative stress further. eNOS uncoupling is thought to be one of the major underlying causes of endothelial dysfunction observed in the pathogenesis of vascular diseases. Here, we discuss the main mechanisms of eNOS uncoupling, including oxidative depletion of the critical eNOS cofactor BH₄, deficiency of eNOS substrate L-Arg, or accumulation of its analog asymmetrical dimethylarginine (ADMA), and eNOS S-glutathionylation. Moreover, potential therapeutic approaches that prevent eNOS uncoupling by improving cofactor availability, restoration of L-Arg/ADMA ratio, or modulation of eNOS S-glutathionylation are briefly outlined.

Background— The prevalence of the endothelium-impaired function disorders, such as hypertension and diabetes mellitus, and the severity of their complications are considerably greater in blacks than whites. Evidence has accumulated that superoxide (O ₂ ⁻ ) production and its interaction with nitric oxide (NO), yielding the strong oxidant peroxynitrite (ONOO ⁻ ), play central roles in vascular pathophysiology. We hypothesized that the differences in endothelial NO/O ₂ ⁻ /ONOO ⁻ metabolism may highlight the potential predisposition to endothelial dysfunction and cardiovascular complications prevalent in blacks. Methods and Results— Highly sensitive tandem electrochemical NO/O ₂ ⁻ /ONOO ⁻ nanosensors were positioned in single human umbilical vein endothelial cells (HUVECs) isolated from blacks and whites, and the kinetics of NO/O ₂ ⁻ /ONOO ⁻ release were recorded in vitro. HUVECs were also analyzed by Western immunoblotting and enzyme activity assays for NAD(P)H-oxidase and endothelial NO synthase (eNOS). Compared with whites, HUVECs from blacks elicited reduced release of bioactive NO with an accompanying increase in the release of both O ₂ ⁻ and ONOO ⁻ . The greater potency of NO production because of eNOS upregulation in HUVECs from blacks is associated with a decrease in the NO bioavailability. This is due to increased NO degradation by excess O ₂ ⁻ produced primarily by 2 enzymatic sources: NAD(P)H-oxidase and uncoupled eNOS. Conclusions— Compared with whites, the steady-state NO/O ₂ ⁻ /ONOO ⁻ balance in endothelial cells from blacks is kept closer to the redox states characteristic for the endothelium-impaired function disorders. This may explain the differences in racial predisposition to the endothelium dysfunction during ongoing vascular disturbances with the hallmark of enhanced NO inactivation within the endothelium by oxidative stress.

Background— Nebivolol and carvedilol are third-generation β-adrenoreceptor antagonists, which unlike classic β-blockers, have additional endothelium-dependent vasodilating properties specifically related to microcirculation by a molecular mechanism that still remains unclear. We hypothesized that nebivolol and carvedilol stimulate NO release from microvascular endothelial cells by extracellular ATP, which is a well-established potent autocrine and paracrine signaling factor modulating a variety of cellular functions through the activation of P2-purinoceptors. Methods and Results— Contraction and relaxation of renal glomerular vasculature were measured by determination of intracapillary volume with [ ³ H]-inulin. Biologically active NO was measured with highly sensitive porphyrinic NO microsensors in a single glomerular endothelial cell (GEC). Extracellular ATP was measured by a luciferin-luciferase assay. Enzymatic degradation of extracellular ATP by apyrase and blockade of P2Y-purinoceptors by suramin or reactive blue 2 inhibited both β-blocker–induced glomerular vasorelaxations and β-blocker–stimulated NO release from GECs. Both β-blocker–induced vasorelaxations were in the micromolar concentration range identical to that required for the β-blocker stimulation of ATP and NO release from GECs. The maximum of NO release for nebivolol and carvedilol was very similar (188±14 and 226±17, respectively). Blockade of ATP release by a mechanosensitive ion channel blocker, Gd ³⁺ , inhibited the β-blocker–dependent release of ATP and NO from GECs. Conclusions— These results demonstrate for the first time that nebivolol and carvedilol induce relaxation of renal glomerular microvasculature through ATP efflux with consequent stimulation of P2Y-purinoceptor–mediated NO release from GECs.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide. The initial stage of CVDs is characterized by endothelial dysfunction, defined as the limited bioavailability of nitric oxide (NO). Thus, any factors that interfere with the synthesis or metabolism of NO in endothelial cells are involved in CVD pathogenesis. It is well established that hypoxia is both the triggering factor as well as the accompanying factor in cardiovascular disease, and diminished tissue oxygen levels have been reported to influence endothelial NO bioavailability. In endothelial cells, NO is produced by endothelial nitric oxide synthase (eNOS) from L-Arg, with tetrahydrobiopterin (BH₄) as an essential cofactor. Here, we discuss the mechanisms by which hypoxia affects NO bioavailability, including regulation of eNOS expression and activity. What is particularly important is the fact that hypoxia contributes to the depletion of cofactor BH₄ and deficiency of substrate L-Arg, and thus elicits eNOS uncoupling—a state in which the enzyme produces superoxide instead of NO. eNOS uncoupling and the resulting oxidative stress is the major driver of endothelial dysfunction and atherogenesis. Moreover, hypoxia induces impairment in mitochondrial respiration and endothelial cell activation; thus, oxidative stress and inflammation, along with the hypoxic response, contribute to the development of endothelial dysfunction.

The impact of statin therapy on plasma asymmetric dimethylarginine (ADMA) levels has not been conclusively studied. Therefore the aim of the meta-analysis was to assess the effect of statins on circulating ADMA levels. We searched selected databases (up to August 2014) to identify randomized controlled trials (RCTs) that investigate the effect of statins on plasma ADMA concentrations. A weighted meta-regression (WMD) using unrestricted maximum likelihood model was performed to assess the impact of statin dose, duration of statin therapy and baseline ADMA concentrations as potential variables on the WMD between statin and placebo group. In total, 1134 participants in 9 selected RCTs were randomized; 568 were allocated to statin treatment and 566 were controls. There was a significant reduction in plasma ADMA concentrations following statin therapy compared with placebo (WMD: − 0.104 μM, 95% confidence interval: − 0.131 to − 0.077, Z = − 7.577, p < 0.0001). Subgroups analysis has shown a significant impact of hydrophilic statins (WMD: − 0.207 μM, 95%CI: − 0.427 to + 0.013, Z = − 7.250, p < .0001) and a non-significant effect of hydrophobic statins (WMD: − 0.101 μM, 95%CI: − 0.128 to − 0.074, Z = − 1.845, p = 0.065). In conclusion, this meta-analysis of available RCTs showed a significant reduction in plasma ADMA concentrations following therapy with hydrophilic statins.

Background— Alterations in endothelial function may contribute to increased susceptibility of black Americans to cardiovascular disease. The ability to pharmacologically reverse endothelial dysfunction in blacks was tested with nebivolol, a β ₁ -selective agent with vasodilating and antioxidant properties. Methods and Results— The effects of nebivolol on endothelial nitric oxide (NO), superoxide (O ₂ ⁻ ), and peroxynitrite concentration (ONOO ⁻ ) release were studied in human umbilical vein endothelial cells and iliac artery endothelial cells isolated from age-matched black and white donors. Kinetics and concentrations of NO/O ₂ ⁻ /ONOO ⁻ were measured simultaneously with nanosensors from single cells and shown to have significant interracial differences. The rate of NO release was &5 times slower in blacks than in whites (94 versus 505 nmol · L ⁻¹ · s ⁻¹ ), whereas the rates of release were faster by &2 times for O ₂ ⁻ and &4 times for ONOO ⁻ (22.1 versus 9.4 nmol · L ⁻¹ · s ⁻¹ for O ₂ ⁻ and 810 versus 209 nmol · L ⁻¹ · s ⁻¹ for ONOO ⁻ ). Pretreatment with 1.0 to 5.0 μmol/L nebivolol restored NO bioavailability in endothelial cells from black donors with concurrent reductions in O ₂ ⁻ and ONOO ⁻ release, similar to levels in the endothelium of whites. The effects of nebivolol were dose-dependent and not observed with atenolol; similar effects were observed with apocynin, an NAD(P)H oxidase inhibitor. Conclusions— Reduced endothelial NO bioavailability in American blacks is mainly due to excessive O ₂ ⁻ and ONOO ⁻ generation by NAD(P)H and uncoupled endothelial NO synthase. Nebivolol decreased O ₂ ⁻ and ONOO ⁻ concentrations and restored NO bioavailability in blacks to the level recorded in cells from whites, independently of β ₁ -selective blockade.