terminally acetylated pentapeptide (Ac-FHACA), glutathione and an amidated hexapeptide (SLIGKV-NH2). When DHA was preincubated with homocysteine thiolactone and after addition of the pentapeptide Ac-FHACA, S-homocysteinylation increased continuously as a function of time. In addition, adducts of DHA and an unknown adduct (+56) was also observed. In the absence of DHA, S-homocysteinylation was not detected. Tandem mass

cysteinyl residue. Similar results were observed with glutathione. As expected, when the hexapeptide SLIGKV-NH2 was incubated with homocysteine thiolactone, N-homocysteinylation increased as a function of time with optimal modification at pH 6.8. Interestingly, in presence of DHA N-homocysteinylation was absent. In conclusion, oxidized vitamin C induces S-

The biological relevance of these modifications in diseases associated with oxidative stress remains to be confirmed.

92 Upregulation of Endothelial Nitric Oxide Synthase in Thoracic Aortic Aneurysms Associated with Bicuspid Aortic Valve Mary P Kotlarczyk1, Eric E Kelley1, Sruti Shiva1, Julie A Phillippi1, and Thomas G Gleason1

1University of Pittsburgh, USA Bicuspid aortic valve (BAV), the most common congenital cardiac defect, confers an increased risk of aortic dissection and thoracic aortic aneurysm (TAA).The mechanisms underlying the susceptibility of BAV patients to TAA are not entirely understood, but likely involve both molecular and hemodynamic influences. Endothelial dysfunction, particularly in nitric oxide signaling, is widely reported in cardiovascular diseases. Mice lacking endothelial nitric oxide synthase (eNOS-/-) can display BAV, implicating eNOS as a contributor to BAV-associated aortopathy. In this study, eNOS was profiled in human ascending thoracic aortic tissue acquired from aneurysmal patients with BAV or normal tricuspid aortic valve (TAV). Tissue was classified into three circumferential regions corresponding to anatomical position of the right, left, and non-coronary sinuses. Analyses were performed on adventitial-stripped specimens containing media and intima only. Real-time PCR and Western blot analyses revealed upregulation of eNOS gene expression in the right region of BAV patients (n=10-11, p<0.01) and a concomitant increase in total eNOS protein (n=9-13, p<0.05) compared to TAV-TAA specimens. Overall NOS activity remained unchanged as measured by conversion of [3H]-arginine to [3H]-citrulline, despite increased eNOS phospho-Ser1177 (n=9-13, p<0.001) in BAV-TAA samples. Interestingly, eNOS gene expression varied regionally in BAV patients, with higher levels in the greater curvature of the aorta. Conversely, TAV patients exhibited consistent eNOS gene expression in all circumferential regions. The heterogeneity of eNOS expression in BAV patients challenges the notion of downregulated eNOS expression with BAV phenotype and indicates instabilities of the eNOS/NO signaling pathway. Methods to restore eNOS function hold promise for non-surgical management of TAA risk in patients with BAV.

93 Impairment of Macrophage Reverse Cholesterol Transport by StAR-Dependent Cholesterol Hydroperoxide Trafficking: Implications for Atherogenesis under Oxidative Stress Witold Kowytowski1, Jared C Schmitt1, Kararzyna Wawak2, Pawel Pabisz2, and Albert W Girotti1

1Medical College of Wisconsin, USA, 2Jagiellonian University, Poland Oxidative stress associated with cardiovascular disease can produce a large variety of oxidized lipids, including cholesterol ring oxides such as 7-hydroperoxide (7-OOH), 7-hydroxide (7-OH), and 7-ketone (7=O). Unlike 7=O and 7-OH, 7-OOH is redox-active, giving rise to the others via potentially toxic free radical reactions. Consequently, 7=O and 7-OH are usually found at much higher levels than 7-OOH in vascular lesions and have garnered more interest than the latter concerning involvement in atherogenesis. Our objective in this study was to test the novel hypothesis that under oxidative stress conditions, StAR family proteins not only deliver cholesterol to/into mitochondria (Mito) of vascular macrophages, but also 7-hydroperoxycholesterol (7-OOH), which induces peroxidative damage that impairs early stage reverse cholesterol transport (RCT). Stimulation of human monocyte-derived THP-1 macrophages with dibutyryl-cAMP resulted in substantial upregulation of Western blot-assessed StarD1 and ABCA1 proteins, but not StarD4 or CYP27A1. siRNA-based StarD1 knockdown (kd) prior to stimulation had no effect on StarD4, but increased CYP27A1 and strongly reduced ABCA1 upregulation, linking the latter to StarD1 functionality. Mito in stimulated StarD1-kd cells internalized 7-OOH slower than non-stimulated controls and underwent less 7-OOH-induced lipid peroxidation and loss of membrane potential, as probed with C11-BODIPY and JC-1, respectively. A major functional consequence was a significantly reduced level of cholesterol-exporting ABCA1 and also 27-hydroxycholesterol, which signals for ABCA1 transcription. Consistent with this, stimulated THP-1 cells that were loaded with [14C]cholesterol in the presence of 7-OOH exported cholesterol more slowly than non-7-OOH-exposed controls. Thus, macrophage RCT was significantly impaired by 7-OOH delivery to mitochondria via a natural trafficking pathway. Moreover, StarD1-dependent 7-OOH transport was highly cytotoxic, whereas 7=O or 7-OH had no effect over the same concentration range. This study describes a previously unrecognized mechanism by which RCT can be incapacitated under oxidative stress disorders such as chronic obesity and hypertension. Our findings provide new insights into the role of macrophage redox damage/dysfunction in atherogenesis. Supported by NIH grant HL85677 (AWG) and Polish National Science Center grant 2011/01/B/NZ3/02167 (WK).

94 Induction of Phase II Enzymes by Quercetin via P38 MAPK and Nrf2: A Potential Mechanism for Quercetin s Antioxidant and Anti-Inflammatory Effects in Human Aortic Endothelial Cells Chuan Li1,2, Wei-jian Zhang1,2, and Balz Frei1,2

1Linus Pauling Institute, Oregon State University, USA, 2Department of Biochemistry and Biophysics, Oregon State University, USA Atherosclerosis, the underlying cause of ischemic heart disease and stroke, is an inflammatory disease of arteries in a hyperlipidemic milieu. Expression of adhesion molecules, such as

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homocysteinylation while protecting against N-homocysteinylation.

E-selectin and intercellular adhesion molecule 1 (ICAM-1), on endothelial cell surfaces plays a critical role in the initiation and progression of atherosclerosis. Quercetin has been reported to inhibit expression of cellular adhesion molecules, but the underlying mechanisms are incompletely understood. In our study we found that quercetin up to 20 μM dose-dependently inhibited lipopolysaccharide-induced mRNA and protein expression of E-selectin and ICAM-1 in human aortic endothelial cells (HAEC). Administration of quercetin also significantly reduced levels of reactive oxygen species (ROS) in HAEC. Further study revealed that quercetin caused Nrf2 activation and increased transcription of heme oxygenase 1 (HO-1), NAD(P)H dehydrogenase, quinone 1 (NQO1) and glutamate-cysteine ligase catalytic (GCLC) and regulatory (GCLM) subunits in a time- and dose-dependent manner. Such induction of phase II enzymes was inhibited by the p38 MAPK inhibitor, SB203580, whereas the protein kinase C (PKC) inhibitor, staurosporine, inhibited quercetin-induced NQO-1 transcription but increased HO-1 transcription. These results indicate that quercetin induces transcription of HO-1, NQO1, GCLC and GCLM by activating p38 MAPK and Nrf2, whereas the role of PKC varies, dependent on each specific phase II enzyme. Our findings also suggest that ROS formation may be involved in adhesion molecule expression in HAEC, and suppression of ROS formation by inducing phase II enzymes may explain, in part, the inhibitory effects of quercetin on endothelial adhesion molecule expression.

95 The Myeloperoxidase-Derived Oxidant HOSCN Induces Mitochondrial Dysfunction in Macrophages Dominic T Love1, David I Pattison1, Michael J Davies1, and Clare L Hawkins1 1The Heart Research Institute, Australia Myeloperoxidase (MPO) has been linked to tissue damage in

s, MPO forms reactive oxidants by catalysing the reaction of halide and pseudo-halide ions with H2O2. With physiological levels of halide and pseudo-halide ions, similar amounts of hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN) are produced by MPO. Although it is well established that HOCl is a potent oxidant that causes significant cellular damage, less is known about the role of HOSCN in biological damage and disease. In this study, we examined the ability of HOSCN to inhibit mitochondrial respiration and induce mitochondrial dysfunction in the murine macrophage-like cells, J774A.1. The cells were treated with varying concentrations of HOSCN and mitochondrial function was assessed by extracellular flux analysis. Results show that HOSCN treatment leads to the attenuation of basal respiration, electron transport chain accelerator response, ATP coupler response and ATP coupling efficiency. Treatment of the cells with HOSCN also resulted in the depolarization and increased permeability of mitochondria, as assessed by JC-1 staining and flow cytometry. The alteration of mitochondrial respiration and permeability seen on exposure of the macrophages to HOSCN at physiologically and pathophysiologically relevant concentrations, results in a decrease in ATP production, triggering cell death by apoptosis. These data support a role for HOSCN as a modulator of redox signalling pathways, and has potential implications for smoking-related pathologies owing to the elevation of thiocyanate (SCN-), the precursor to HOSCN in the circulation of smokers.

96 Role of the Myeloperoxidase-Derived Oxidant HOSCN in Glycolytic Inhibition in Macrophages Dominic T Love1, Tessa J Barrett1, David I Pattison1, and Clare L Hawkins1 1The Heart Research Institute, Australia Myeloperoxidase (MPO), released by inflammatory phagocytes forms reactive oxidants by catalyzing the reaction of H2O2 with halides and pseudo-halide ions. under physiological conditions, similar levels of hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN) are produced. HOCl is a potent oxidant that has been strongly linked with tissue damage during inflammatory disease. In contrast, the role of HOSCN in disease is less well defined, though there is increasing evidence for the initiation of cellular damage via selective thiol oxidation. In this study, we examined the mechanisms and cellular targets of HOSCN with macrophage-like cells (J774A.1). A proteomics approach with the thiol-specific probe, 5-iodoacetamidofluorescein, showed that multiple proteins involved in metabolism and glycolysis, including fructose bisphosphate aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and creatine kinase were modified in macrophages treated with HOSCN. Thiol modification occurred in a reversible manner, and involved the formation of sulfenic acids, and glutathionylation. Exposure of macrophages to HOSCN and thiol modification resulted in a loss of enzymatic activity, and a decrease in basal glycolysis, glycolytic reserve, glycolytic capacity and extracellular lactate release, which may contribute to the initiation of cell death via both apoptotic and necrotic pathways. These data may have important implications for the development of inflammatory pathologies in people with elevated plasma levels of the thiocyanate, the precursor of HOSCN, arising from cigarette smoking.

97 Carbamylation Decreases PON1 Enzyme Activity in Plasma Jonathon Pullara1 and Sean M Lynch1 1Midwestern University, Downers Grove, Illinois, USA Chemical modification of HDL, including carbamylation, has the potential to cause loss of -protective paraoxonase-1 (PON1) antioxidant enzyme activity. Previous studies in our laboratory reported decreased PON1 activity in isolated HDL subjected to carbamylation in vitro (Kosmach et al. FRBM 2010;49:S26; Devine & Lynch. JAOA 2014;In press). In the study reported here, we extended these studies to examine whether carbamylation might decrease PON1 activity within the physiological milieu of plasma. In our experiments, 5-fold diluted plasma was initially incubated overnight with KOCN (0-100 mM). Excess KOCN was then removed from the plasma using PD-10 desalting columns, and total protein carbamylation and PON1 enzyme activities measured by standard spectrophotometric assays. Using this experimental system, we found no significant increase in plasma total protein carbamylation during incubation with KOCN until the concentration exceeded 10 mM. under these same conditions, we noted a progressive (but not statistically significant) decline in PON1 enzyme activity. Thus, plasma PON1 phosphotriesterase activity declined to 68 % of normal, while its lactonase activity declined to 78 % of normal, as the KOCN concentration increased from 0.1 to 10 mM. At the highest concentration of KOCN tested (100 mM), consistent with the observed increase in plasma total protein carbamylation, both PON 1 activities were significantly (P<0.05) impaired (decreased

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