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Drug-eluting stents are commonly utilized for the treatment of coronary artery disease, where they maintain vessel patency and prevent restenosis. However, problems with prolonged vascular healing, late thrombosis, and neoatherosclerosis persist; these could potentially be addressed via the local generation of nitric oxide (NO) from endogenous substrates. Herein, we develop amine-functionalized graphene as a NO-generating coating on polylactic acid (PLA)-based bioresorbable stent materials. A novel catalyst was synthesized consisting of polyethyleneimine and polyethylene glycol bonded to graphene oxide (PEI-PEG@GO), with physicochemical characterization using x-ray diffraction, Raman spectroscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. In the presence of 10 μM S-nitrosoglutathione (GSNO) or S-nitroso-N-acetylpenicillamine (SNAP), PEI-PEG@GO catalyzed the generation of 62% and 91% of the available NO, respectively. Furthermore, PEI-PEG@GO enhanced and prolonged real-time NO generation from GSNO and SNAP under physiological conditions. The uniform coating of PEI-PEG@GO onto stent material is demonstrated via an optimized simple dip-coating method. The coated PLA maintains good biodegradability under accelerated degradation testing, while the PEI-PEG@GO coating remains largely intact. Finally, the stability of the coating was demonstrated at room temperature over 60 days. In conclusion, the innovative conjugation of polymeric amines with graphene can catalyze the generation of NO from S-nitrosothiols at physiologically relevant concentrations. This approach paves the way for the development of controlled NO-generating coatings on bioresorbable stents in order to improve outcomes in coronary artery disease. [ABSTRACT FROM AUTHOR]

5,6,7,8-Tetrahydrobiopterin ([BH.sub.4]) is an essential cofactor of nitric oxide synthases (NOSs). Oxidation of [BH.sub.4], in the setting of diabetes and other chronic vasoinflammatory conditions, can cause cofactor insufficiency and uncoupling of endothelial NOS (eNOS), manifest by a switch from nitric oxide (NO) to superoxide production. Here we tested the hypothesis that eNOS uncoupling is not simply a consequence of [BH.sub.4] insufficiency, but rather results from a diminished ratio of [BH.sub.4] vs. its catalytically incompetent oxidation product, 7,8-dihydrobiopterin ([BH.sub.2]). In support of this hypothesis, [[sup.3]H][BH.sub.4] binding studies revealed that [BH.sub.4] and [BH.sub.2] bind eNOS with equal affinity ([K.sub.d] [approximately equal to] 80 nM) and [BH.sub.2] can rapidly and efficiently replace [BH.sub.4] in preformed eNOS-[BH.sub.4] complexes. Whereas the total biopterin pool of murine endothelial cells (ECs) was unaffected by 48-h exposure to diabetic glucose levels (30 mM), [BH.sub.2] levels increased from undetectable to 40% of total biopterin. This [BH.sub.2] accumulation was associated with diminished calcium ionophore-evoked NO activity and accelerated superoxide production. Since superoxide production was suppressed by NOS inhibitor treatment, eNOS was implicated as a principal superoxide source. Importantly, [BH.sub.4] supplementation of ECs (in low and high glucose-containing media) revealed that calcium ionophore-evoked NO bioactivity correlates with intracellular [BH.sub.4]: [BH.sub.2] and not absolute intracellular levels of [BH.sub.4]. Reciprocally, superoxide production was found to negatively correlate with intracellular [BH.sub.4]:[BH.sub.2]. Hyperglycemia-associated [BH.sub.4], oxidation and NO insufficiency was recapitulated in vivo, in the Zucker diabetic fatty rat model of type 2 diabetes. Together, these findings implicate diminished intracellular [BH.sub.4]: [BH.sub.2], rather than [BH.sub.4] depletion per se, as the molecular trigger for NO insufficiency in diabetes. nitric oxide; diabetes; endothelial dysfunction

Diminished nitric oxide (NO) bioactivity and enhanced peroxynitrite formation have been implicated as major contributors to atherosclerotic vascular dysfunctions. Hallmark reactions of peroxynitrite include the accumulation of 3-nitrotyrosine (3-NT) in proteins and oxidation of the NO synthase (NOS) cofactor, tetrahydrobiopterin (B[H.sub.4]). The present study sought to 1) quantify the extent to which 3-NT accumulates and [BH.sub.4] becomes oxidized in organs of apolipoprotein E-deficient ([ApoE.sup.-/-]) atherosclerotic mice and 2) determine the specific contribution of inducible NOS (iNOS) to these processes. Whereas protein 3-NT and oxidized B[H.sub.4] were undetected or near the detection limit in heart, lung, and kidney of 3-wk-old [ApoE.sup.-/-] mice or [ApoE.sup.-/-] mice fed a regular chow diet for 24 wk, robust accumulation was evident after 24 wk on a Western (atherogenic) diet. Since 3-NT accumulation was diminished 3- to 20-fold in heart, lung, and liver in [ApoE.sup.-/-] mice missing iNOS, iNOS-derived species are involved in this reaction. In contrast, iNOS-derived species did not contribute to elevated protein 3-NT formation in kidney or brain, iNOS deletion also afforded marked protection against B[H.sub.4] oxidation in heart, lung, and kidney of atherogenic [ApoE.sup.-/-] mice but not in brain or liver. These findings demonstrate that iNOS-derived species are increased during atherogenesis in [ApoE.sup.-/-] mice and that these species differentially contribute to protein 3-NT accumulation and B[H.sub.4] oxidation in a tissue-selective manner. Since B[H.sub.4] oxidation can switch the predominant NOS product from NO to superoxide, we predict that progressive NOS uncoupling is likely to drive atherogenic vascular dysfunctions. inducible nitric oxide synthase; high-fat diet

Aims Systemic inflammation and increased activity of atrial NOX2-containing NADPH oxidases have been associated with the new onset of atrial fibrillation (AF) after cardiac surgery. In addition to lowering LDL-cholesterol, statins exert rapid anti-inflammatory and antioxidant effects, the clinical significance of which remains controversial. Methods and results We first assessed the impact of cardiac surgery and cardiopulmonary bypass (CPB) on atrial nitroso-redox balance by measuring NO synthase (NOS) and GTP cyclohydrolase-1 (GCH-1) activity, biopterin content, and superoxide production in paired samples of the right atrial appendage obtained before (PRE) and after CPB and reperfusion (POST) in 116 patients. The effect of perioperative treatment with atorvastatin (80 mg once daily) on these parameters, blood biomarkers, and the post-operative atrial effective refractory period (AERP) was then evaluated in a randomized, double-blind, placebo-controlled study in 80 patients undergoing cardiac surgery on CPB. CPB and reperfusion led to a significant increase in atrial superoxide production (74% CI 71–76%, n  = 46 paired samples, P  < 0.0001) and a reduction in atrial tetrahydrobiopterin (BH4) (34% CI 33–35%, n  = 36 paired samples, P  < 0.01), and in GCH-1 (56% CI 55–58%, n  = 26 paired samples, P  < 0.001) and NOS activity (58% CI 52–67%, n  = 20 paired samples, P  < 0.001). Perioperative atorvastatin treatment prevented the effect of CPB and reperfusion on all parameters but had no significant effect on the postoperative right AERP, troponin release, or NT-proBNP after cardiac surgery. Conclusion Perioperative statin therapy prevents post-reperfusion atrial nitroso-redox imbalance in patients undergoing on-pump cardiac surgery but has no significant impact on postoperative atrial refractoriness, perioperative myocardial injury, or markers of postoperative LV function. Clinical Trial Registration https://clinicaltrials.gov/ct2/show/NCT01780740 [ABSTRACT FROM AUTHOR]

Aims When activated, Na+/H+ exchanger-1 (NHE1) produces some of the largest ionic fluxes in the heart. NHE1-dependent H+ extrusion and Na+ entry strongly modulate cardiac physiology through the direct effects of pH on proteins and by influencing intracellular Ca2+ handling. To attain an appropriate level of activation, cardiac NHE1 must respond to myocyte-derived cues. Among physiologically important cues is nitric oxide (NO), which regulates a myriad of cardiac functions, but its actions on NHE1 are unclear. Methods and results NHE1 activity was measured using pH-sensitive cSNARF1 fluorescence after acid-loading adult ventricular myocytes by an ammonium prepulse solution manoeuvre. NO signalling was manipulated by knockout of its major constitutive synthase nNOS, adenoviral nNOS gene delivery, nNOS inhibition, and application of NO-donors. NHE1 flux was found to be activated by low [NO], but inhibited at high [NO]. These responses involved cGMP-dependent signalling, rather than S-nitros(yl)ation. Stronger cGMP signals, that can inhibit phosphodiesterase enzymes, allowed [cAMP] to rise, as demonstrated by a FRET-based sensor. Inferring from the actions of membrane-permeant analogues, cGMP was determined to activate NHE1, whereas cAMP was inhibitory, which explains the biphasic regulation by NO. Activation of NHE1-dependent Na+ influx by low [NO] also increased the frequency of spontaneous Ca2+ waves, whereas high [NO] suppressed these aberrant forms of Ca2+ signalling. Conclusions Physiological levels of NO stimulation increase NHE1 activity, which boosts pH control during acid-disturbances and results in Na+-driven cellular Ca2+ loading. These responses are positively inotropic but also increase the likelihood of aberrant Ca2+ signals, and hence arrhythmia. Stronger NO signals inhibit NHE1, leading to a reversal of the aforementioned effects, ostensibly as a potential cardioprotective intervention to curtail NHE1 overdrive. [ABSTRACT FROM AUTHOR]