Adrian Devine

Functional consequences of endothelial nitric oxide synthase uncoupling in congestive cardiac failure.

Background—Impaired endothelium-mediated vasodilatation (EMVD) in congestive cardiac failure (CCF) has been linked to decreased nitric oxide (NO) bioavailability because of its interaction with vascular superoxide (O2·−), derived predominantly from NAD(P)H-dependent oxidases. When uncoupled from essential cofactors, endothelial nitric oxide synthase (eNOS) produces O2·−. We studied the functional consequences of eNOS uncoupling in relation to EMVD in patients with CCF. Methods and Results—We employed the platelet as a compartmentalized ex-vivo model to examine O2·− and NO production. When eNOS is functioning normally, incorporation of N&ohgr;-Nitro-l-Arginine methyl ester (L-NAME, 1 mmol/L), results in increased O2·− detection, as inhibition of NO production prevents NO scavenging of O2·−. This was observed in controls and 9 of the CCF patients, in whom O2·− detection increased by 63% and 101%, respectively. In the remaining 9 CCF patients, incorporation of L-NAME reduced O2·− production by 39%, indicating O2·− production by eNOS uncoupling. Detection of platelet-derived NO was significantly greater in eNOS-coupled platelets compared with the uncoupled group (2.8±1.4 versus 0.9±0.4 pmol/108 platelets, P =0.04). Endothelium-dependent and -independent vasodilator responses to acetylcholine and sodium nitroprusside recorded using venous occlusion plethysmography were significantly impaired in patients exhibiting eNOS uncoupling. Conclusions—This study provides first evidence that platelet eNOS can become uncoupled in human CCF. Impaired endothelium-dependent and -independent vasodilator responses and diminished platelet-derived NO production occurred in association with enzyme uncoupling.

A randomised interventional trial of ω-3-polyunsaturated fatty acids on endothelial function and disease activity in systemic lupus erythematosus

Objective: To determine the clinical effect of dietary supplementation with low-dose ω-3-polyunsaturated fatty acids on disease activity and endothelial function in patients with systemic lupus erythematosus. Methods: A 24-week randomised double-blind placebo-controlled parallel trial of the effect of 3 g of ω-3-polyunsaturated fatty acids on 60 patients with systemic lupus erythematosus was performed. Serial measurements of disease activity using the revised Systemic Lupus Activity Measure (SLAM-R) and British Isles Lupus Assessment Group index of disease activity for systemic lupus erythematosus (BILAG), endothelial function using flow-mediated dilation (FMD) of the brachial artery, oxidative stress using platelet 8-isoprostanes and analysis of platelet membrane fatty acids were taken at baseline, 12 and 24 weeks. Results: In the fish oil group there was a significant improvement at 24 weeks in SLAM-R (from 9.4 (SD 3.0) to 6.3 (2.5), p<0.001); in BILAG (from 13.6 (6.0) to 6.7 (3.8), p<0.001); in FMD (from 3.0% (−0.5 to 8.2) to 8.9% (1.3 to 16.9), p<0.001) and in platelet 8-isoprostanes (from 177 pg/mg protein (23–387) to 90 pg/mg protein (32–182), p = 0.007). Conclusions: Low-dose dietary supplementation with ω-3 fish oils in systemic lupus erythematosus not only has a therapeutic effect on disease activity but also improves endothelial function and reduces oxidative stress and may therefore confer cardiovascular benefits.

Platelet nitric oxide and superoxide release during the development of nitrate tolerance: effect of supplemental ascorbate

Background—The therapeutic benefits that accompany the continuous administration of organic nitrates are attenuated by the development of tolerance to the compounds. Altered superoxide production and NO bioavailability have been implicated in contributing to the development of tolerance, an effect that may be ameliorated by the administration of antioxidants. Methods and Results—We studied the effect of 3 days of continuous transdermal administration of nitroglycerin (NTG) (10 mg/24 hours) on platelet free radical (NO and superoxide anion [O2·−] activity) with and without coadministration of supplemental ascorbate (2.4 g/24 hours). NAD(P)H oxidase activity, nitric oxide synthase (NOS) activity, and cyclic guanosine monophosphate (cGMP) content were also assessed. Radial artery pressure pulse waveforms were used to track the hemodynamic actions of NTG. Three days of NTG/placebo was associated with a significant increase in platelet NO and O2·− production from 1.0±1.17 to 2.52±0.88 pmol/108 platelets and 13.2±4.8 to 72.5±34.4 pmol/108 platelets, respectively (P <0.01 for both). These changes were accompanied by increased platelet NADH oxidase activity from 47.9±11.0 to 65.3±13.6 pmol O2·− min/mg protein and cGMP content from 0.60±0.10 to 0.89±0.16 pmol/109 platelets (P <0.05 for both). Administration of NTG/ascorbate attenuated both NO and O2·− release in platelets. Conclusions—Three days of continuous transdermal administration of NTG was accompanied by increased platelet NO and O2·− production and NADH oxidase activity that was suppressed by coadministration of oral ascorbate. Although a significant degree of tolerance would be expected during continuous nitrate administration, a residual hemodynamic action could be identified by arterial pulse contour analysis.

Microcirculatory hemodynamics and endothelial dysfunction in systemic lupus erythematosus.

Objective—Impaired flow-mediated dilation (FMD) occurs in disease states associated with atherosclerosis, including SLE. The primary hemodynamic determinant of FMD is wall shear stress, which is critically dependent on the forearm microcirculation. We explored the relationship between FMD, diastolic shear stress (DSS), and the forearm microcirculation in 32 patients with SLE and 19 controls. Methods and Results—DSS was calculated using (mean diastolic velocity×8×blood viscosity)/baseline brachial artery diameter. Doppler velocity envelopes from the first 15 seconds of reactive hyperemia were analyzed for resistive index (RI), and interrogated in the frequency domain to assess forearm microvascular hemodynamics. FMD was significantly impaired in SLE patients (median, 2.4%; range, −2.1% to 10.7% versus median 5.8%; range, 1.9% to 14%; P<0.001). DSS (dyne/cm2) was significantly reduced in SLE patients (median, 18.5; range, 3.9 to 34.0 versus median 21.8; range, 14.1 to 58.7; P=0.037). A strong correlation between FMD and DSS, rs=0.65, P=0.01 was found. Postischemic RI was not significantly different between the 2 groups; however, there were significant differences in the power-frequency spectrums of the Doppler velocity envelopes (P<0.05). Conclusions—These data suggest that in SLE, altered structure and function of the forearm microcirculation contributes to impaired FMD through a reduction in shear stress stimulus.

Differential Expression of Components of the Cardiomyocyte Adrenomedullin/Intermedin Receptor System following Blood Pressure Reduction in Nitric Oxide-Deficient Hypertension

Adrenomedullin (AM) and intermedin (IMD; adrenomedulln-2) are vasodilator peptides related to calcitonin gene-related peptide (CGRP). The actions of these peptides are mediated by the calcitonin receptor-like receptor (CLR) in association with one of three receptor activity-modifying proteins. CGRP is selective for CLR/receptor activity modifying protein (RAMP)1, AM for CLR/RAMP2 and -3, and IMD acts at both CGRP and AM receptors. In a model of pressure overload induced by inhibition of nitric-oxide synthase, up-regulation of AM was observed previously in cardiomyocytes demonstrating a hypertrophic phenotype. The current objective was to examine the effects of blood pressure reduction on cardiomyocyte expression of AM and IMD and their receptor components. Nω-nitro-l-arginine methyl ester (l-NAME) (35 mg/kg/day) was administered to rats for 8 weeks, with or without concurrent administration of hydralazine (50 mg/kg/day) and hydrochlorothiazide (7.5 mg/kg/day). In left ventricular cardiomyocytes from l-NAME-treated rats, increases (-fold) in mRNA expression were 1.6 (preproAM), 8.4 (preproIMD), 3.4 (CLR), 4.1 (RAMP1), 2.8 (RAMP2), and 4.4 (RAMP3). Hydralazine/hydrochlorothiazide normalized systolic blood pressure (BP) and abolished mRNA up-regulation of hypertrophic markers sk-α-actin and BNP and of preproAM, CLR, RAMP2, and RAMP3 but did not normalize cardiomyocyte width nor preproIMD or RAMP1 mRNA expression. The robust increase in IMD expression indicates an important role for this peptide in the cardiac pathology of this model but, unlike AM, IMD is not associated with pressure overload upon the myocardium. The concordance of IMD and RAMP1 up-regulation indicates a CGRP-type receptor action; considering also a lack of response to BP reduction, IMD may, like CGRP, have an anti-ischemic function.

The Pleiotropic Effects of Simvastatin on Retinal Microvascular Endothelium Has Important Implications for Ischaemic Retinopathies

Background Current guidelines encourage the use of statins to reduce the risk of cardiovascular disease in diabetic patients; however the impact of these drugs on diabetic retinopathy is not well defined. Moreover, pleiotropic effects of statins on the highly specialised retinal microvascular endothelium remain largely unknown. The objective of this study was to investigate the effects of clinically relevant concentrations of simvastatin on retinal endothelium in vitro and in vivo. Methods and Findings Retinal microvascular endothelial cells (RMECs) were treated with 0.01–10 µM simvastatin and a biphasic dose-related response was observed. Low concentrations enhanced microvascular repair with 0.1 µM simvastatin significantly increasing proliferation (p<0.05), and 0.01 µM simvastatin significantly promoting migration (p<0.05), sprouting (p<0.001), and tubulogenesis (p<0.001). High concentration of simvastatin (10 µM) had the opposite effect, significantly inhibiting proliferation (p<0.01), migration (p<0.01), sprouting (p<0.001), and tubulogenesis (p<0.05). Furthermore, simvastatin concentrations higher than 1 µM induced cell death. The mouse model of oxygen-induced retinopathy was used to investigate the possible effects of simvastatin treatment on ischaemic retinopathy. Low dose simvastatin(0.2 mg/Kg) promoted retinal microvascular repair in response to ischaemia by promoting intra-retinal re-vascularisation (p<0.01). By contrast, high dose simvastatin(20 mg/Kg) significantly prevented re-vascularisation (p<0.01) and concomitantly increased pathological neovascularisation (p<0.01). We also demonstrated that the pro-vascular repair mechanism of simvastatin involves VEGF stimulation, Akt phosphorylation, and nitric oxide production; and the anti-vascular repair mechanism is driven by marked intracellular cholesterol depletion and related disorganisation of key intracellular structures. Conclusions A beneficial effect of low-dose simvastatin on ischaemic retinopathy is linked to angiogenic repair reducing ischaemia, thereby preventing pathological neovascularisation. High-dose simvastatin may be harmful by inhibiting reparative processes and inducing premature death of retinal microvascular endothelium which increases ischaemia-induced neovascular pathology. Statin dosage should be judiciously monitored in patients who are diabetic or are at risk of developing other forms of proliferative retinopathy.

Reduced Nitro-oxidative Stress and Neural Cell Death Suggests a Protective Role for Microglial Cells in TNFα−/− Mice in Ischemic Retinopathy

PURPOSE Neovascularization occurs in response to tissue ischemia and growth factor stimulation. In ischemic retinopathies, however, new vessels fail to restore the hypoxic tissue; instead, they infiltrate the transparent vitreous. In a model of oxygen-induced retinopathy (OIR), TNFalpha and iNOS, upregulated in response to tissue ischemia, are cytotoxic and inhibit vascular repair. The aim of this study was to investigate the mechanism for this effect. METHODS Wild-type C57/BL6 (WT) and TNFalpha(-/-) mice were subjected to OIR by exposure to 75% oxygen (postnatal days 7-12). The retinas were removed during the hypoxic phase of the model. Retinal cell death was determined by TUNEL staining, and the microglial cells were quantified after Z-series capture with a confocal microscope. In situ peroxynitrite and superoxide were measured by using the fluorescent dyes DCF and DHE. iNOS, nitrotyrosine, and arginase were analyzed by real-time PCR, Western blot analysis, and activity determined by radiolabeled arginine conversion. Astrocyte coverage was examined after GFAP immunostaining. RESULTS The TNFalpha(-/-) animals displayed a significant reduction in TUNEL-positive apoptotic cells in the inner nuclear layer of the avascular retina compared with that in the WT control mice. The reduction coincided with enhanced astrocytic survival and an increase in microglial cells actively engaged in phagocytosing apoptotic debris that displayed low ROS, RNS, and NO production and high arginase activity. CONCLUSIONS Collectively, the results suggest that improved vascular recovery in the absence of TNFalpha is associated with enhanced astrocyte survival and that both phenomena are dependent on preservation of microglial cells that display an anti-inflammatory phenotype during the early ischemic phase of OIR.

Docosahexaenoic Acid Improves the Nitroso-Redox Balance and Reduces VEGF-Mediated Angiogenic Signaling in Microvascular Endothelial Cells

PURPOSE Disturbances to the cellular production of nitric oxide (NO) and superoxide (O(2)(-)) can have deleterious effects on retinal vascular integrity and angiogenic signaling. Dietary agents that could modulate the production of these signaling molecules from their likely enzymatic sources, endothelial nitric oxide synthase (eNOS) and NADPH oxidase, would therefore have a major beneficial effect on retinal vascular disease. The effect of ω-3 polyunsaturated fatty acids (PUFAs) on angiogenic signaling and NO/superoxide production in retinal microvascular endothelial cells (RMECs) was investigated. METHODS Primary RMECs were treated with docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) for 48 hours. RMEC migration was determined by scratch-wound assay, proliferation by the incorporation of BrdU, and angiogenic sprouting using a three-dimensional model of in vitro angiogenesis. NO production was quantified by Griess assay, and phospho-eNOS accumulation and superoxide were measured using the fluorescent probe dihydroethidine. eNOS localization to caveolin-rich microdomains was determined by Western blot analysis after subfractionation on a linear sucrose gradient. RESULTS DHA treatment increased nitrite and decreased superoxide production, which correlated with the displacement of eNOS from caveolar subdomains and colocalization with the negative regulator caveolin-1. In addition, both ω-3 PUFAs demonstrated reduced responsiveness to VEGF-stimulated superoxide and nitrite release and significantly impaired endothelial wound healing, proliferation, and angiogenic sprout formation. CONCLUSIONS DHA improves NO bioavailability, decreases O(2)(-) production, and blunts VEGF-mediated angiogenic signaling. These findings suggest a role for ω-3 PUFAs, particularly DHA, in maintaining vascular integrity while reducing pathologic retinal neovascularization.

Differential effects of an anti-oxidant intervention on cardiomyocyte expression of adrenomedullin and intermedin and their receptor components in chronic nitric oxide deficiency.

AbstractBackground: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial oxidative stress and hypertrophic remodeling. Up-regulation of the cardiomyocyte adrenomedullin (AM) / intermedin (IMD) receptor signaling cascade is also apparent in NO-deficient cardiomyocytes: augmented expression of AM and receptor activity modifying proteins RAMP2 and RAMP3 is prevented by blood pressure normalization while that of RAMP1 and intermedin (IMD) is not, indicating that the latter is regulated by a pressure-independent mechanism. Aims: to verify the ability of an anti-oxidant intervention to normalize cardiomyocyte oxidant status and to investigate the influence of such an intervention on expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes. Methods: NO synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 35mg/kg/day) was given to rats for 8 weeks, with/without con-current administration of antioxidants (Vitamin C (25mg/kg/day) and Tempol (25mg/kg/day)). Results: In left ventricular cardiomyocytes isolated from L-NAME treated rats, increased oxidative stress was indicated by augmented (3.6 fold) membrane protein oxidation, enhanced expression of catalytic and regulatory subunits of pro-oxidant NADPH oxidases (NOX1, NOX2) and compensatory increases in expression of anti-oxidant glutathione peroxidase and Cu/Zn superoxide dismutases (SOD1, SOD3). Vitamin C plus Tempol did not reduce systolic blood pressure but normalized augmented plasma levels of IMD, but not of AM, and in cardiomyocytes: (i) abolished increased membrane protein oxidation; (ii) normalized augmented expression of prepro-IMD and RAMP1, but not prepro-AM, RAMP2 and RAMP3; (iii) attenuated (by 42%) increased width and normalized expression of hypertrophic markers, skeletal-α-actin and prepro-endothelin-1 similarly to blood pressure normalization but in contrast to blood pressure normalization did not attenuate augmented brain natriuretic peptide (BNP) expression. Conclusion: normalization specifically of augmented IMD/RAMP1 expression in NO-deficient cardiomyocytes by antioxidant intervention in the absence of blood pressure reduction indicates that these genes are likely to be induced directly by myocardial oxidative stress. Although oxidative stress contributed to cardiomyocyte hypertrophy, induction of IMD and RAMP1 is unlikely to be secondary to cardiomyocyte hypertrophy.

Expression of the counter-regulatory peptide intermedin is augmented in the presence of oxidative stress in hypertrophied cardiomyocytes.

Background: Intermedin (IMD), a novel cardiac peptide related to adrenomedullin (AM), protects against myocardial ischemia-reperfusion injury and attenuates ventricular remodelling. IMD’s actions are mediated by a calcitonin receptor-like receptor in association with receptor activity modifying proteins (RAMPs 1-3). Aim/method: using the spontaneously hypertensive rat (SHR) and normotensive Wistar Kyoto (WKY) rat at 20 weeks of age, to examine (i) the presence of myocardial oxidative stress and concentric hypertrophy; (ii) expression of IMD, AM and receptor components. Results: In left and right ventricular cardiomyocytes from SHR vs. WKY cell width (26% left, 15% right) and mRNA expression of hypertrophic markers ANP (2.7 fold left, 2.7 fold right) and BNP (2.2 fold left, 2.0 fold right) were enhanced. In left ventricular cardiomyocytes only (i) oxidative stress was indicated by increased membrane protein carbonyl content (71%) and augmented production of O2- anion (64%); (ii) IMD (6.8 fold), RAMP1 (2.5 fold) and RAMP3 (2.0 fold) mRNA was increased while AM and RAMP2 mRNA was not altered; (iii) abundance of RAMP1 (by 48%), RAMP2 (by 41%) and RAMP3 (by 90%) monomers in cell membranes was decreased. Conclusion: robust augmentation of IMD expression in hypertrophied left ventricular cardiomyocytes indicates a prominent role for this counter-regulatory peptide in the adaptation of the SHR myocardium to the stresses imposed by chronic hypertension. The local concentration and action of IMD may be further enhanced by down-regulation of NEP within the left ventricle.