nucleosides formed on (deoxy)cytidine, (deoxy)adenosine and (deoxy)guanosine with macrophages, which play a key role in lesion development. Exposure of macrophages to 5-chloro-cytidine, 8-chloro-adenosine and 8-chloro-guanosine resulted in incorporation of these compounds into cellular RNA, with incorporation of 5-chloro-deoxycytidine, but not 8-chloro-deoxyguanosine or 8-chloro-deoxyadenosine, seen in cellular DNA. Treatment of cells with chlorinated nucleosides also resulted in changes to cellular viability and proliferation, and alterations in the expression of pro-inflammatory cytokines, including interleukin 1 (IL-1 ) and interleukin 18 (IL-18), which have been implicated in atherosclerosis. Increased IL-1 and IL-18 expression was associated with nuclear translocation of the NF- B sub-unit p65 and activation of caspase 1, consistent with inflammasome activation. These results highlight a potential role for chlorinated nucleosides in the propagation of inflammation during the pathogenesis of atherosclerosis.

86 Role of Myeloperoxidase in the Modulation of Cellular Lysosomal Cathepsin Protease Function: A Contributing Factor to Macrophage Dysfunction in Atherosclerosis? Fahd Ismael1,2, Tessa Barrett1,2, Diba Sheipouri1, Bronwyn Brown1,2, Michael Davies1,2,3, and Clare Hawkins1,2 1Heart Research Institute, Australia, 2University of Sydney, Australia, 3University of Copenhagen, Denmark Atherosclerosis is characterized by the accumulation of lipids within macrophages in the artery wall, resulting in the formation of

-density lipoprotein (LDL) is the major source of these lipids, owing to the recognition and uptake of oxidized LDL by scavenger receptors. Elevated myeloperoxidase (MPO) is found in human atherosclerotic lesions, and this enzyme has been identified as both a risk factor and prognostic factor for the development of atherosclerosis and cardiovascular complications. MPO forms reactive oxidants including hypochlorous and hypothiocyanous acids (HOCl and HOSCN), which have been linked to tissue damage. Lysosomal proteases contribute to the metabolism of LDL after uptake by macrophages. In this study, we examined the effect of LDL pre-treated with HOCl and HOSCN, and the oxidants themselves on the function of lysosomal enzymes in murine macrophage-like J774A.1 cells. Exposure of J774A.1 cells (1 x 106 cells/mL) to HOCl or HOSCN (0 160 μM) resulted in a similar, rapid, dose-dependent decrease in the activity of the thiol-dependent cathepsins B and L, whereas no loss of the aspartate-dependent cathepsin D activity was observed. A decrease in cathepsin B and L activity was also observed on treating the macrophages with LDL pre-treated (for 24 h) with HOCl or HOSCN (50 500 μM). The decrease in cathepsin B and L activity was more marked with HOSCN-LDL compared to HOCl-LDL. Exposure of the cells to HOSCN-LDL, but not HOCl-LDL also resulted in a small, but significant, decrease in cathepsin D activity. The ability of LDL-modified by MPO oxidants, particularly HOSCN, and the individual oxidants to impair the activity of lysosomal proteases, is likely to perturb the cellular processing of LDL, which may contribute to macrophage dysfunction in atherosclerosis. These results may be particularly significant for smokers, who have elevated plasma thiocyanate, the precursor to HOSCN.

87 Mechanism Involved in the Blunted Anti-Oxidant Response in the Frataxin-Deficient Heart: Increased Nrf2 Degradation via both the Keap1 and GSK3 Pathway Amy Anzovino1, Clare L. Hawkins2, Michael L.H. Huang1, and Des R. Richardson1 1University of Sydney, Australia, 2Heart Research Institute, Australia Oxidative stress is emerging as a key contributor to the

Biochemical hallmarks of this disorder, such as dysregulated iron metabolism, mitochondrial iron accumulation and decreased iron sulphur cluster enzyme activity, may cause chronic levels of oxidative stress in affected tissue. While previous studies in neuronal models of FA have reported a diminished antioxidant response due to low expression of the transcription factor Nrf2, these studies have not fully elucidated the mechanism responsible. Furthermore, no studies of the antioxidant response have been conducted in the FA heart, which develops a severe hypertrophic cardiomyopathy and is the leading cause of death in patients. Using the muscle creatine kinase conditional frataxin knockout (KO) mouse, which mimics the progression of cardiomyopathy in FA patients, we found pathological levels of ROS formation in the KO heart. In agreement with previous studies in neuronal models of FA, we also report decreased antioxidant levels in the KO heart due to low Nrf2 mRNA and protein levels. In order to clarify the reason for the reduced antioxidant response in the KO heart, we investigated the Nrf2 signalling pathway. Our results show that the expression of key players involved in regulating Nrf2 activity are drastically altered in the KO heart, and that decreased expression of Nrf2 may be associated with increased Keap1-mediated degradation. Moreover, examination of the Keap1-

of Nrf2 also appears to be increased. These together suggest the blunted antioxidant response in the frataxin-deficient heart is due to reduced Nrf2 activity caused by increased Nrf2 degradation via

88 HSF-1 Deletion Attenuates Atherosclerosis by Enhancing CYP7A1 and Multidrug Transporter (MDR1) Gene Expressions Krishanamoorthy Karthikeyean1 and Govindasamy Ilangovan1 1The Ohio State University, USA Stress response, in terms of activation of stress factors, is known to cause obesity and coronary heart disease such as atherosclerosis in human. However, the underlying mechanism(s) of these pathways are not known. Here we investigated the effect of heat shock factor-1 (HSF-1) on atherosclerosis. Both, HSF-1 and low density lipoproteins receptor (LDLr) double knockoutmice (HSF-1 / /LDLr / ) and LDLr-/- mice, were fed with atherogenic western diet (WD) for 12 weeks. WD-induced weight gain and atherosclerotic lesion in aortic arch and carotid regions, were reduced in HSF-1-/-/LDLr-/- mice, compared to LDLr / mice. Also, repression of PPAR- 2 and AMPK expression in adipose tissue, low hepatic steatosis, and lessened plasma adiponectins and lipoproteins were observed. Furthermore, reduced heat shock proteins and their mRNA levels in atherosclerotic lesions corroborated with reduction in lesion burden. In HSF-1 / /LDLr / liver, higher cholesterol 7 hydroxylase (CYP7A1) and multidrug

SFRBM 2014S46

doi: 10.1016/j.freeradbiomed.2014.10.401

doi: 10.1016/j.freeradbiomed.2014.10.402

doi: 10.1016/j.freeradbiomed.2014.10.400