Ravi Pillai

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.

A Myocardial Nox2 Containing NAD(P)H Oxidase Contributes to Oxidative Stress in Human Atrial Fibrillation

Human atrial fibrillation (AF) has been associated with increased atrial oxidative stress. In animal models, inhibition of reactive oxygen species prevents atrial remodeling induced by rapid pacing, suggesting that oxidative stress may play an important role in the pathophysiology of AF. NAD(P)H oxidase is a major source of superoxide in the cardiovascular system; however, whether this enzyme contributes to atrial oxidative stress in AF remains to be elucidated. We investigated the sources of superoxide production (using inhibitors and substrates of a range of oxidases, RT-PCR, immunofluorescence, and immunoblotting) in tissue homogenates and isolated atrial myocytes from the right atrial appendage (RAA) of patients undergoing cardiac surgery (n=54 in sinus rhythm [SR] and 15 in AF). A membrane-bound gp91phox containing NAD(P)H oxidase in atrial myocytes was the main source of atrial superoxide production in SR and in AF. NADPH-stimulated superoxide release from RAA homogenates was significantly increased in patients with AF in the absence of changes in mRNA expression of the p22phox and gp91phox subunits of the NAD(P)H oxidase. In contrast with findings in SR patients, NO synthases (NOSs) contributed significantly to atrial superoxide production in fibrillating atria, suggesting that increased oxidative stress in AF may lead to NOS “uncoupling.” These findings indicate that a myocardial NAD(P)H oxidase and, to a lesser extent, dysfunctional NOS contribute significantly to superoxide production in the fibrillating human atrial myocardium and may play an important role in the atrial oxidative injury and electrophysiological remodeling observed in patients with AF.

5-methyltetrahydrofolate rapidly improves endothelial function and decreases superoxide production in human vessels: effects on vascular tetrahydrobiopterin availability and endothelial nitric oxide synthase coupling.

Background— The circulating form of folic acid, 5-methyltetrahydrofolate (5-MTHF), may have beneficial effects on endothelial function; however, its mechanisms of action remain uncertain. Decreased nitric oxide (NO) bioavailability and increased vascular superoxide production in vascular disease states are due in part to endothelial NO synthase (eNOS) uncoupling related to deficiency of the eNOS cofactor tetrahydrobiopterin (BH4), but whether this mechanism is important in human atherosclerosis and represents a rational therapeutic target remains unclear. We hypothesized that 5-MTHF would improve endothelial function by decreasing superoxide and peroxynitrite production and by improving eNOS coupling, mediated by BH4 availability. Methods and Results— Vascular superoxide/peroxynitrite production and vasomotor responses to acetylcholine and bradykinin were determined in saphenous veins and internal mammary arteries from 117 patients undergoing CABG. The effects of 5-MTHF were examined ex vivo (n=61) by incubating vessels with 5-MTHF (1 to 100 &mgr;mol/L) and in vivo by intravenous infusion of 5-MTHF or placebo before vessel harvest (n=56). 5-MTHF improved NO-mediated endothelium-dependent vasomotor responses and reduced vascular superoxide, both ex vivo and in vivo. These changes were not explained by direct superoxide scavenging by 5-MTHF in vitro or by changes in plasma total homocysteine in vivo. Rather, 5-MTHF was a strong peroxynitrite scavenger and increased vascular BH4 and the BH4/total biopterin ratio. Furthermore, 5-MTHF reversed eNOS uncoupling, as assessed by NG-nitro-l-arginine methyl ester–inhibitable superoxide production, increased the eNOS dimer:monomer ratio, and enhanced eNOS activity. Conclusions— 5-MTHF has beneficial effects on endothelial function and vascular superoxide production in human atherosclerosis, by preventing peroxynitrite-mediated BH4 oxidation and improving eNOS coupling.

Serum S100 protein: A potential marker for cerebral events during cardiopulmonary bypass

BACKGROUND There is no simple method to determine the incidence or severity of brain injury after a cardiac operation. A serum marker equivalent to cardiac enzymes is required. S100 protein leaks from the cerebrospinal fluid to blood after cerebral injury. We sought to determine the pattern of release after extracorporeal circulation (ECC). METHODS Thirty-four patients without neurologic problems underwent coronary bypass using ECC. Four had carotid stenoses. Nine others underwent coronary bypass without ECC. Serum S100 levels were measured before, during, and after the operation. RESULTS S100 was not detected before sternotomy. Postoperative levels of S100 were related to duration of perfusion (r = 0.89, p < 0.001). Patients who did not have ECC had undetectable or fractionally raised levels except in 1 who suffered a stroke. No patient in whom ECC was used suffered an event, but those with carotid stenosis had greater S100 levels. CONCLUSIONS S100 protein leaks into blood during ECC and may reflect both cerebral injury and increased permeability of the blood brain barrier. S100 is a promising marker for cerebral injury in cardiac surgery if elevated levels can be linked with clinical outcome.

Functional Effect of the C242T Polymorphism in the NAD(P)H Oxidase p22phox Gene on Vascular Superoxide Production in Atherosclerosis

BackgroundIncreased superoxide anion production increases oxidative stress and reduces nitric oxide bioactivity in vascular disease states. NAD(P)H oxidase is an important source of superoxide in human blood vessels, and some studies suggest a possible association between polymorphisms in the NAD(P)H oxidase CYBA gene and atherosclerosis; however, no functional data address this hypothesis. We examined the relationships between the CYBA C242T polymorphism and direct measurements of superoxide production in human blood vessels. Methods and ResultsVascular NAD(P)H oxidase activity was determined in human saphenous veins obtained from 110 patients with coronary artery disease and identified risk factors. Immunoblotting, reverse-transcription polymerase chain reaction, and DNA sequencing showed that p22phox protein, mRNA, and 242C/T allelic variants are expressed in human blood vessels. Vascular superoxide production, both basal and NADH-stimulated, was highly variable between patients, but the presence of the CYBA 242T allele was associated with significantly reduced vascular NAD(P)H oxidase activity, independent of other clinical risk factors for atherosclerosis. ConclusionsAssociation of the CYBA 242T allele with reduced NAD(P)H oxidase activity in human blood vessels suggests that genetic variation in NAD(P)H oxidase components may play a significant role in modulating superoxide production in human atherosclerosis.

Nitric oxide modulates superoxide release and peroxynitrite formation in human blood vessels.

Nitric oxide and superoxide have important roles as vascular signaling molecules. Nitric oxide (NO) reacts rapidly with superoxide, producing peroxynitrite. The relative balance between these radicals has important implications for vascular pathophysiology in hypertension and other vascular disease states. However, the relationships between superoxide, NO, and peroxynitrite formation in human blood vessels remain unclear. Accordingly, we systematically measured NO, superoxide, and peroxynitrite production from human internal mammary arteries, radial arteries, and saphenous veins from 78 patients undergoing coronary bypass surgery. Basal superoxide release was detected in all vessels at similar levels. However, endothelial removal or nitric oxide synthase inhibition increased mean superoxide release, with a corresponding reduction in peroxynitrite formation. Conversely, NO donors and superoxide scavengers both reduced superoxide release, whereas only NO donors increased peroxynitrite formation. These changes were much larger in arteries that in veins, but there were striking correlations between superoxide production, NO bioavailability, and peroxynitrite formation between the vessel types. Our findings provide direct evidence for coordinated vascular signaling mediated by interactions between NO, superoxide, and peroxynitrite and have important implications for studies of the functional effects of these radicals in human blood vessels.

Association of plasma asymmetrical dimethylarginine (ADMA) with elevated vascular superoxide production and endothelial nitric oxide synthase uncoupling: implications for endothelial function in human atherosclerosis

BACKGROUND Asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of endothelial nitric oxide synthase (eNOS), is considered to be a risk factor for atherosclerosis. However, the mechanisms relating ADMA with vascular function have been evaluated in vitro and in animal models, but its effect in human vasculature is unclear. AIMS We examined the impact of serum ADMA on endothelial nitric oxide (NO) bioavailability and vascular superoxide radical (O2-) production in patients with advanced atherosclerosis. METHODS AND RESULTS Paired samples of saphenous veins (SVs) and internal mammary arteries (IMAs) were collected from 201 patients undergoing coronary bypass surgery, and serum ADMA was measured pre-operatively. The vasomotor responses of SV segments to acetylcholine (ACh) and bradykinin (Bk) were evaluated ex vivo. Vascular O2- was measured in paired SV and IMA by lucigenin-enhanced chemiluminescence. The l-NAME-inhibitable as well as the NADPH-stimulated vascular O2- generation was also determined by chemiluminescence. High serum ADMA levels were associated with decreased vasorelaxation of SV to ACh (P < 0.05) and Bk (P < 0.05). Similarly, high serum ADMA was associated with higher total O2- production in both SVs and IMAs (P < 0.05) and greater L-NAME-inhibitable vascular O2- (P < 0.05). However, serum ADMA was not associated with NADPH-stimulated vascular O2-. In multivariable linear regression, serum ADMA was independently associated with vascular O2- in both SVs [beta (SE): 0.987 (0.412), P = 0.019] and IMAs [beta (SE): 1.905 (0.541), P = 0.001]. Asymmetrical dimethylarginine was also independently associated with maximum vasorelaxation in response to both ACh [beta (SE): 14.252 (3.976), P = 0.001] and Bk [beta (SE): 9.564 (3.762), P = 0.013]. CONCLUSION This is the first study that demonstrates an association between ADMA and important measures of vascular function, such as vascular O2- production and NO bioavailability directly in human vessels. Although serum ADMA has no effect on NADPH-stimulated superoxide in intact vessels, it is associated with greater eNOS uncoupling in the human vascular endothelium of patients with coronary artery disease.

Cytokine responses to cardiopulmonary bypass with membrane and bubble oxygenation

The systemic inflammatory response to cardiopulmonary bypass was assessed in 20 patients who underwent elective coronary artery bypass grafting with flat-sheet membrane oxygenation (group I; n = 10; age, 59 +/- 5 years) or bubble oxygenation (group II; n = 10; age, 62 +/- 8 years). The duration of cardiopulmonary bypass was 46 +/- 12 minutes in group I and 47 +/- 15 minutes in group II. Plasma interleukin-6, plasma interleukin-1 beta, transpulmonary leukocyte counts, pulmonary hemodynamic variables, and respiratory index were determined in all patients perioperatively. The plasma interleukin-6 response (median [range]) was similar in both groups at the end of the operation, peaked 4 hours postoperatively (99 [30 to 320] pg/mL in group I; 123 [21 to 300] pg/mL in group II; p greater than 0.05), and remained elevated 48 hours postoperatively (76 [9 to 140] pg/mL in group I; 65 [25 to 159] pg/mL in group II; p greater than 0.05). No significant interleukin-1 beta response was demonstrated. Pulmonary neutrophil and lymphocyte sequestration was observed on commencement of cardiopulmonary bypass in group II but did not occur in either group on discontinuation of cardiopulmonary bypass. Pulmonary vascular resistance at the end of the operation (82 [48 to 320] dynes.s.cm-5 in group I; 119 [54 to 385] dynes.s.cm-5 in group II; p greater than 0.05) was similar to preoperative values (151 [30 to 327] dynes.s.cm-5 in group I; 185 [62 to 291] dynes.s.cm-5 in group II; p greater than 0.05). The respiratory index at the end of the operation was similarly and significantly increased in both groups (1.26 [0.92 to 4.17] in group I; 1.44 [0.73 to 3.30] in group II).(ABSTRACT TRUNCATED AT 250 WORDS)

Association of Atrial Nicotinamide Adenine Dinucleotide Phosphate Oxidase Activity With the Development of Atrial Fibrillation After Cardiac Surgery

OBJECTIVES Our goal was to evaluate the role of myocardial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and plasma markers of oxidative stress in the pathogenesis of post-operative atrial fibrillation (AF). BACKGROUND Atrial fibrillation is a common complication of cardiac surgery, leading to increased morbidity and prolonged hospitalization. Experimental evidence suggests that oxidative stress may be involved in the pathogenesis of AF; however, the relevance of this putative mechanism in patients undergoing cardiac surgery is unclear. METHODS We measured basal and NADPH-stimulated superoxide production in right atrial appendage samples from 170 consecutive patients undergoing conventional coronary artery bypass surgery. Plasma markers of lipid and protein oxidation (thiorbabituric acid-reactive substances, 8-isoprostane, and protein carbonyls) were also measured in blood samples drawn from a central line before surgery and after reperfusion. RESULTS Patients who developed AF after surgery (42%) were older and had a significantly increased atrial NADPH oxidase activity than patients who remained in sinus rhythm (SR) (in relative light units/s/mug protein: 4.78 +/- 1.44 vs. 3.53 +/- 1.04 in SR patients, p < 0.0001). Plasma markers of lipid and protein oxidation increased significantly after reperfusion; however, neither pre-operative nor post-operative measurements differed between patients who developed AF and those who remained in SR after surgery. Multivariate analysis identified atrial NADPH oxidase activity as the strongest independent predictor of post-operative AF (odds ratio 2.41; 95% confidence interval 1.71 to 3.40, p < 0.0001). CONCLUSIONS Atrial NADPH oxidase activity is independently associated with an increased risk of post-operative AF, suggesting that this oxidase system may be a key mediator of atrial oxidative stress leading to the development of AF after cardiac surgery.

Does modern cardiac surgery require conventional intensive care

We considered that, with modern perfusion equipment and mildly hypothermic cardiopulmonary bypass, protracted post-operative ventilation in an intensive care unit (ITU) is no longer required after most cardiac operations. We used a three-bedded cardiac recovery area (CRA) within the operating suite for 1,000 patients between January 1990 and June 1991. Forty-five patients with special needs were managed in the ITU. The time to extubation (T50%; range) for coronary bypass, aortic valve, mitral valve, and double-valve patients was 2.0 (0-42), 2.5 (0-12), 3.0 (0-15), and 3.0 (1-36) hours, respectively. Recovery beds were re-used allowing 5-6 operations daily. The difference in nursing staff complement for a CRA versus ITU bed was 4.5/7.8. Patient management was by nurse specialists supported by cardiac surgeons. Intervention by cardiac anaesthetists or intensivists was limited to specific ventilatory problems or renal failure. The early extubation policy failed in ten patients (five coronary, three aortic, one mitral and one double-valve patient) through poor pre-operative respiratory function, left ventricular failure or intra-operative events. The overall mortality in CRA was 1.4%. The mean duration of post-operative stay was 7 days (range 5-12). We conclude that a CRA staffed by nurse practitioners provides a safe and effective alternative to the anaesthetist-managed ITU. A rapid turnover of CRA beds removes the constraints of ITU bed availability.