Shafi Mussa

Mechanisms of Increased Vascular Superoxide Production in Human Diabetes Mellitus Role of NAD(P)H Oxidase and Endothelial Nitric Oxide Synthase

Background—Increased superoxide production contributes to reduced vascular nitric oxide (NO) bioactivity and endothelial dysfunction in experimental models of diabetes. We characterized the sources and mechanisms underlying vascular superoxide production in human blood vessels from diabetic patients with coronary artery disease compared with nondiabetic patients. Methods and Results—Vascular superoxide production was quantified in both saphenous veins and internal mammary arteries from 45 diabetic and 45 matched nondiabetic patients undergoing coronary artery bypass surgery. NAD(P)H-dependent oxidases were important sources of vascular superoxide in both diabetic and nondiabetic patients, but both the activity of this enzyme system and the levels of NAD(P)H oxidase protein subunits (p22phox, p67phox, and p47phox) were significantly increased in diabetic veins and arteries. In nondiabetic vessels, endothelial NO synthase produced NO that scavenged superoxide. However, in diabetic vessels, the endothelium was an additional net source of superoxide production because of dysfunctional endothelial NO synthase that was corrected by intracellular tetrahydrobiopterin supplementation. Furthermore, increased superoxide production in diabetes was abrogated by the protein kinase C inhibitor chelerythrine. Conclusions—These observations suggest important roles for NAD(P)H oxidases, endothelial NO synthase uncoupling, and protein kinase C signaling in mediating increased vascular superoxide production and endothelial dysfunction in human diabetes mellitus.

Tetrahydrobiopterin-dependent preservation of nitric oxide–mediated endothelial function in diabetes by targeted transgenic GTP–cyclohydrolase I overexpression

Increased production of reactive oxygen species and loss of endothelial NO bioactivity are key features of vascular disease states such as diabetes mellitus. Tetrahydrobiopterin (BH4) is a required cofactor for eNOS activity; pharmacologic studies suggest that BH4 may mediate some of the adverse effects of diabetes on eNOS function. We have now investigated the importance and mechanisms of BH4 availability in vivo using a novel transgenic mouse model with endothelial-targeted overexpression of the rate-limiting enzyme in BH4 synthesis, guanosine triphosphate-cyclohydrolase I (GTPCH). Transgenic (GCH-Tg) mice demonstrated selective augmentation of endothelial BH4 levels. In WT mice, induction of diabetes with streptozotocin (STZ) increased vascular oxidative stress, resulting in oxidative loss of BH4, forming BH2 and biopterin. Endothelial cell superoxide production in diabetes was increased, and NO-mediated endothelium-dependent vasodilatation was impaired. In diabetic GCH-Tg mice, superoxide production from the endothelium was markedly reduced compared with that of WT mice, endothelial BH4 levels were maintained despite some oxidative loss of BH4, and NO-mediated vasodilatation was preserved. These findings indicate that BH4 is an important mediator of eNOS regulation in diabetes and is a rational therapeutic target to restore NO-mediated endothelial function in diabetes and other vascular disease states.

Endothelial nitric oxide synthase dysfunction in diabetic mice: importance of tetrahydrobiopterin in eNOS dimerisation.

Aims/hypothesisImpaired nitric oxide (NO) bioactivity and increased superoxide (SO) production are characteristics of vascular endothelial dysfunction in diabetes. The underlying mechanisms remain unknown. In this regard, we investigated the role of tetrahydrobiopterin (BH4) bioavailability in regulating endothelial nitric oxide synthase (eNOS) activity, dimerisation and SO production in streptozotocin-induced diabetic mice.MethodsMouse aortas were used for assays of the following: (1) aortic function by isometric tension; (2) NO by electronic paramagnetic resonance; (3) SO by lucigenin-enhanced chemiluminescence and dihydroethidine fluorescence; (4) total biopterin and BH4 by high-performance liquid chromatography; and (5) eNOS protein expression and dimerisation by immunoblotting.ResultsIn diabetic mouse aortas, relaxations to acetylcholine and NO levels were significantly decreased, but SO production was increased, in association with reductions in total biopterins and BH4. Although total eNOS levels were increased in diabetes, the protein mainly existed in monomeric form. Conversely, specifically augmented BH4 in diabetic endothelium preserved eNOS dimerisation, but the expression remained unchanged.Conclusions/interpretationOur results demonstrate that BH4 plays an important role in regulating eNOS activity and its functional protein structure, suggesting that increasing endothelial BH4 and/or protecting it from oxidation may be a rational therapeutic strategy to restore eNOS function in diabetes.

Comparative efficacies and durations of action of phenoxybenzamine, verapamil/nitroglycerin solution, and papaverine as topical antispasmodics for radial artery coronary bypass grafting.

OBJECTIVE Radial arteries are increasingly used as conduits for coronary artery bypass grafts, but perioperative graft vasospasm continues to be a concern. Phenoxybenzamine, verapamil/nitroglycerin solution, and papaverine have been advocated as topical antispasmodic agents. We compared the relative efficacies and durations of action of these agents. METHODS Isometric tension developed in response to clinically important vasoconstrictors was measured in 100 radial artery rings (from patients undergoing coronary artery bypass grafting, n = 25) after 15 minutes of ex vivo incubation with phenoxybenzamine, verapamil/nitroglycerin solution, papaverine, or vehicle (control). Duration of action was assessed by measuring responses to vasoconstrictors in antispasmodic pretreated and control rings at intervals through 5 hours. RESULTS Verapamil/nitroglycerin solution reduced vasoconstriction in response to epinephrine, angiotensin II, prostaglandin F(2alpha), and phenylephrine but its effect had almost completely waned after 5 hours. Phenoxybenzamine prevented vasoconstriction in response to epinephrine, dopamine, and phenylephrine, with its effect lasting at least 5 hours. Papaverine had limited antispasmodic efficacy and prevented vasoconstriction in response to potassium (60 mmol/L) and phenylephrine for only 1 hour at the longest. CONCLUSIONS Verapamil/nitroglycerin solution has a broad efficacy against a range of vasoconstrictors but a limited duration of action. Papaverine has the shortest duration of action. Phenoxybenzamine is an effective agent with a prolonged duration of action, specifically preventing catecholamine mediated vasospasm of radial artery conduits.

Duplex ultrasonography predicts safety of radial artery harvest in the presence of an abnormal Allen test

BACKGROUND The Allen test is commonly used to assess collateral hand circulation before radial artery harvest for coronary artery bypass grafting. However there is no consensus as to whether an abnormal Allen test is an absolute or relative contraindication to radial artery harvesting. We assessed the safety of harvesting the radial artery using arterial duplex ultrasonography in patients with an abnormal Allen test. METHODS Two hundred and eighty-seven consecutive patients scheduled for total arterial coronary revascularisation underwent preoperative Allen tests over a 34-month period. Patients with an abnormal Allen test underwent duplex ultrasonography preoperatively to assess the adequacy of the ulnar collateral supply and the suitability of the radial artery for harvesting. RESULTS Two hundred and forty-four patients (85%) had a normal left Allen test and proceeded directly to radial artery harvest. Forty-three patients (15%) with an abnormal left Allen test underwent duplex ultrasonography scans and of those, 5 patients had an abnormal scan. These patients underwent scanning of the contralateral forearm. Three patients had a normal right forearm arterial duplex scan and the right radial artery was harvested. The mean diameter of the radial and ulnar arteries was not significantly different between the patients with normal and abnormal duplex ultrasonograms. There were no ischemic hand complications in this series. CONCLUSIONS The Allen test is a quick, easy, and reliable screening test before radial artery harvesting in the majority of patients. Duplex ultrasonography predicts safe radial artery harvest in the majority of patients with an abnormal Allen test.

Relation of preoperative radial artery flow-mediated dilatation to nitric oxide bioavailability in radial artery grafts used in off-pump coronary artery bypass grafting.

The radial artery is prone to vasospasm after coronary bypass surgery, and endothelial dysfunction is likely to be a key factor. We investigated whether endothelial dysfunction in radial artery conduits is present, and can be identified, preoperatively using a simple noninvasive ultrasound test of radial artery endothelial response, flow-mediated dilatation (FMD). The study population consisted of 126 patients scheduled for coronary artery bypass grafting. The afternoon before operation, patients had noninvasive ultrasound assessment of endothelial function in the left radial artery by FMD, which measures change in arterial size after an increase in flow-an endothelial-dependent response. Surplus graft segments were obtained at operation and nitric oxide bioavailability within the vessels determined from ex vivo responses to acetylcholine. Preoperative FMD in the radial artery was associated with vasorelaxations to acetylcholine in radial artery grafts (p<0.001 for both dose-response curves and maximum relaxations), although there was weak borderline association between FMD and vasorelaxations of saphenous vein grafts (p=0.07 for dose-response curves and p<0.05 for maximum relaxations). In multivariate analysis including cardiac risk factors, FMD was a predictor of vasorelaxations of radial artery grafts (beta=0.020, SE=0.009, p=0.030), independent of classic risk factors for atherosclerosis. In conclusion, there is significant interindividual variation in the endothelial function of vessels used for coronary artery bypass surgery, particularly the radial artery. These differences are present and can be identified preoperatively by FMD.

Myocardial and Cerebral Injury After Off-Pump Coronary Artery Surgery

To the Editor: Van Dijk and colleagues1 are to be congratulated on the contribution of their randomized trial to the on-pump versus off-pump coronary artery bypass surgery debate and, in particular, the discussion of the high proportion of patients undergoing total arterial revascularization. Most of the pertinent points are covered in Dr Yacoub’s balanced accompanying editorial.2 Two methodological flaws may, however, invalidate the authors’ conclusions regarding myocardial and cerebral injury. First, the reduction in creatine kinase (CK)-MB release in the off-pump group compared with the on-pump group is based on measurements taken within the first 20 hours postoperatively. Release of biochemical markers of myocardial injury over this period may reflect turnover of cytosolic pools …