Denise McDonald

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.

Truncated estrogen receptor alpha 46-kDa isoform in human endothelial cells: relationship to acute activation of nitric oxide synthase.

Background—Estrogen acutely activates endothelial nitric oxide synthase (eNOS). However, the identity of the receptors involved in this rapid response remains unclear. Methods and Results—We detected an estrogen receptor &agr; (ER&agr;) transcript in human endothelial cells that encodes a truncated 46-kDa ER&agr; (&Dgr;1a-hER&agr;-46). A corresponding 46-kDa ER&agr; protein was identified in endothelial cell lysates. Transfection of cDNAs encoding the full-length ER&agr; (ER&agr;-66) and &Dgr;1a-hER&agr;-46 resulted in appropriately sized recombinant proteins identified by anti-ER&agr; antibodies. Confocal microscopy revealed that a proportion of both ER&agr;-66 and hER&agr;-46 was localized outside the nucleus and mediated specific cell-surface binding of estrogen as assessed by FITC-conjugated, BSA-estrogen binding studies. Both ER&agr; isoforms colocalized with eNOS and mediated acute activation of eNOS in response to estrogen stimulation. However, estrogen-stimulated transcriptional activation mediated by &Dgr;1a-hER&agr;-46 was much less than with ER&agr;-66. Furthermore, &Dgr;1a-hER&agr;-46 inhibited classical hER&agr;-66–mediated transcriptional activation in a dominant-negative fashion. Conclusions—These findings suggest that expression of an alternatively spliced, truncated ER&agr; isoform in human endothelial cells confers a unique ability to mediate acute but not transcriptional responses to estrogen.

Inhibition of Tumor Necrosis Factor-α Improves Physiological Angiogenesis and Reduces Pathological Neovascularization in Ischemic Retinopathy

The present study was undertaken to test whether inhibition of the proangiogenic inflammatory cytokine tumor necrosis factor (TNF)-alpha can modulate retinal hypoxia and preretinal neovascularization in a murine model of oxygen-induced retinopathy (OIR). OIR was produced in TNF-alpha-/- and wild-type (WT) control C57B6 neonatal mice by exposure to 75% oxygen between postnatal days 7 and 12 (P7 to P12). Half of each WT litter was treated with the cytokine inhibitor semapimod (formerly known as CNI-1493) (5 mg/kg) by daily intraperitoneal injection from the time of reintroduction to room air at P12 until P17. The extent of preretinal neovascularization and intraretinal revascularization was quantified by image analysis of retinal flat-mounts and retinal hypoxia correlated with vascularization by immunofluorescent localization of the hypoxia-sensitive drug pimonidazole (hypoxyprobe, HP). HP adducts were also characterized by Western analysis and quantified by competitive enzyme-linked immunosorbent assay. TNF-alpha-/- and WT mice showed a similar sensitivity to hyperoxia-induced retinal ischemia at P12. At P13 some delay in early reperfusion was evident in TNF-alpha-/- and WT mice treated with semapimod. However, at P17 both these groups had significantly better vascular recovery with less ischemic/hypoxic retina and preretinal neovascularization compared to untreated retinopathy in WT mice. Immunohistochemistry showed deposition of HP in the avascular inner retina but not in areas underlying preretinal neovascularization, indicating that such aberrant vasculature can reduce retinal hypoxia. Inhibition of TNF-alpha significantly improves vascular recovery within ischemic tissue and reduces pathological neovascularization in OIR. HP provides a useful tool for mapping and quantifying tissue hypoxia in experimental ischemic retinopathy.

GTP cyclohydrolase I gene transfer augments intracellular tetrahydrobiopterin in human endothelial cells: effects on nitric oxide synthase activity, protein levels and dimerisation

OBJECTIVES Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) activity. BH4 levels are regulated by de novo biosynthesis; the rate-limiting enzyme is GTP cyclohydrolase I (GTPCH). BH4 activates and promotes homodimerisation of purified eNOS protein, but the intracellular mechanisms underlying BH4-mediated eNOS regulation in endothelial cells remain less clear. We aimed to investigate the role of BH4 levels in intracellular eNOS regulation, by targeting the BH4 synthetic pathway as a novel strategy to modulate intracellular BH4 levels. METHODS We constructed a recombinant adenovirus, AdGCH, encoding human GTPCH. We infected human endothelial cells with AdGCH, investigated the changes in intracellular biopterin levels, and determined the effects on eNOS enzymatic activity, protein levels and dimerisation. RESULTS GTPCH gene transfer in EAhy926 endothelial cells increased BH4 >10-fold compared with controls (cells alone or control adenovirus infection), and greatly enhanced NO production in a dose-dependent, eNOS-specific manner. We found that eNOS was principally monomeric in control cells, whereas GTPCH gene transfer resulted in a striking increase in eNOS homodimerisation. Furthermore, the total amounts of both native eNOS protein and a recombinant eNOS-GFP fusion protein were significantly increased following GTPCH gene transfer. CONCLUSIONS These findings suggest that GTPCH gene transfer is a valid approach to increase BH4 levels in human endothelial cells, and provide new evidence for the relative importance of different mechanisms underlying BH4-mediated eNOS regulation in intact human endothelial cells. Additionally, these observations suggest that GTPCH may be a rational target to augment endothelial BH4 and normalise eNOS activity in endothelial dysfunction states.

Functional comparison of the endothelial nitric oxide synthase Glu298Asp polymorphic variants in human endothelial cells

The G894T endothelial nitric oxide synthase (eNOS) polymorphism results in a Glu to Asp substitution at position 298. This position is located externally on the protein and as the regulation of eNOS is dependent on its subcellular localization and interaction with modulatory proteins, we aimed to address whether the substitution of Asp at 298 had any effect on these mechanisms. Initially, we developed a novel method to accurately determine molar quantities of each variant by expressing them as green fluorescent protein (GFP) fusion proteins and using recombinant adenoviruses to facilitate transient infection of human microvascular endothelial cells. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting of eNOSAsp revealed a 135-kDa proteolytic fragment which was not present with eNOSGlu. This proteolysis was prevented by using LDS buffer confirming that this differential cleavage is an artefact of sample preparation and unlikely to occur intracellularly. Nitric oxide was measured following stimulation with calcium ionophore or oestrogen in the presence of varying sepiapterin concentrations. GFP fluorescence was used to quantify the amount of fusion protein and calculate intracellular specific activity. There was no significant difference in intracellular specific activity between Glu and Asp eNOS in response to calcium ionophore or oestrogen. Tetrahydrobiopterin supplementation increased eNOS activity of both variants in an identical manner. The presence of the GFP also facilitated the visualization of the variants by confocal microscopy and demonstrated that both localized to the plasma membrane and the Golgi. These findings demonstrate that the Asp substitution at 298 does not have a major effect in modulating eNOS activity in vivo.

Coxsackie and adenovirus receptor (CAR)-dependent and major histocompatibility complex (MHC) class I-independent uptake of recombinant adenoviruses into human tumour cells.

The role of two receptors, previously proposed to mediate the entry of adenoviruses into human cells, the coxsackie and adenovirus receptor (CAR) and the major histocompatibility complex (MHC) class I heavy chain has been investigated. The expression of MHC class I in many tumours is reduced or absent, therefore if this were a means by which adenoviruses gained entry into cells, it would have important implications for their application in cancer treatment. In order to determine if MHC class I heavy chain is involved in adenovirus type 5 (Ad5) uptake, the binding of recombinant Ad5 fibre knob domain (which mediates viral attachment) to human cell lines that had greatly different levels of surface MHC class I was studied. We also created derivatives of a non-permissive Chinese hamster ovary (CHO) cell line that expressed human class I (HLA-A2) and found that these cells did not bind fibre or take up virus. In addition, the extracellular domain of CAR was expressed in E. coli and used to generate a polyclonal anti-CAR antibody. This antibody blocked both 125I labelled fibre knob binding and virus uptake. Thus CAR, and not MHC class I, is a receptor for human adenoviruses in cultured tumour cells. Tissue CAR levels may therefore be an important factor in the efficiency of adenovirus-mediated gene therapy.

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.

The gastrointestinal peptide obestatin induces vascular relaxation via specific activation of endothelium‐dependent NO signalling

BACKGROUND AND PURPOSE Obestatin is a recently discovered gastrointestinal peptide with established metabolic actions, which is linked to diabetes and may exert cardiovascular benefits. Here we aimed to investigate the specific effects of obestatin on vascular relaxation.

Platelet Redox Balance in Diabetic Patients With Hypertension Improved by n-3 Fatty Acids

OBJECTIVE Patients with type 2 diabetes mellitus (T2DM) are at increased risk of developing cardiovascular disease, largely as a result of defective production of cardioprotective nitric oxide and a concomitant rise in oxidative stress. Dietary interventions that could reverse this trend would be extremely beneficial. Here we investigated whether dietary n-3 polyunsaturated fatty acid (n-3 PUFA) supplementation positively affected platelet nitroso-redox imbalance. RESEARCH DESIGN AND METHODS We randomized hypertensive T2DM patients (T2DM HT; n = 22) and age-and-sex matched hypertensive study participants without diabetes (HT alone; n = 23) in a double-blind, crossover fashion to receive 8 weeks of n-3 PUFAs (1.8 g eicosapentaenoic acid and 1.5 g docosahexaenoic acid) or identical olive oil capsules (placebo), with an intervening 8-week washout period. Platelet nitrite and superoxide were measured and compared before and after treatment; 8-isoprostane was determined by ELISA and subcellular compartmentalization of the NAD(P)H oxidase subunit p47-phox examined by Western blotting. RESULTS The n-3 PUFA supplementation reduced 8-isoprostane and superoxide levels in platelets from T2DM HT, but not HT alone, participants, without effect on nitrite production. This coincided with a significant decrease in p47-phox membrane localization and a similar reduction in superoxide to that achieved with apocynin. At baseline, a subcohort of T2DM HT and HT alone participants showed evidence of nitric oxide synthase (NOS)–derived superoxide production, indicating defective enzymatic activity. This was reversed significantly in T2DM HT participants after treatment, demonstrating improved NOS function. CONCLUSIONS Our finding that n-3 PUFAs diminish platelet superoxide production in T2DM HT patients in vivo suggests a therapeutic role for these agents in reducing the vascular-derived oxidative stress associated with diabetes.