cells to H2O2.

372 The Myeloperoxidase-Derived Oxidant Hypochlorous Acid Promotes Extracellular Trap Formation and Inflammatory Cytokine Expression in Human Monocyte-Derived Macrophages: A Potential Mechanism of Atherosclerotic Lesion Development Benjamin S. Rayner1,2, Leila Reyes1,2, Victoria C. Cogger2,3, and Clare L. Hawkins1,2 1Heart Research Institute, Australia, 2University of Sydney, Australia, 3ANZAC Research Institute, Australia The infiltration of macrophages at sites of inflammation within the arterial wall induces the development of the atherosclerotic lesion. Additionally, the formation and presence of the myeloperoxidase (MPO)-derived oxidant hypochlorous acid (HOCl) within atherosclerotic lesions has long been considered a major factor in the vascular cell damage, dysfunction and death associated with the progression and severity of atherosclerosis. The precise mechanisms involved in the promotion of atherosclerotic lesion development, however, remain poorly understood. In this study we demonstrate that exposure of human monocyte-derived macrophages (HMDM) to HOCl leads to the extrusion of cellular components, resulting in the formation of extracellular traps that contain nuclear histones, DNA and MPO. Concurrent with these events, HMDM exposed to HOCl also demonstrate the increased production of pro-inflammatory chemokines and cytokines including the pro-fibrotic plasminogen activator inhibitor (PAI-1) and tissue factor (TF), as well as chemoattractant molecules including interleukin 8 (IL8) and monocyte chemoattractant protein 1 (MCP-1). There is increasing evidence in the literature confirming the presence of extracellular traps within atherosclerotic lesions and supporting the role of the inflammatory response within the pathogenesis of atherosclerosis. Taken together, therefore, our data indicate a novel role for HOCl both within the cellular signalling mechanisms that underlie the induction of the inflammatory response, and potentially within the processes involved in atherosclerotic lesion development.

373 Differential Effect of Nicotinamide on MMP-9 Expression Induced by High Glucose or Hydrogen Peroxide in Cultured Mouse Blastocysts Alejandra Sánchez-Santos1, María Guadalupe Martínez-Hernández1, Alejandra Contreras-Ramos2, Clara Ortega-Camarillo3, and Luis Arturo Baiza-Gutman1 1Unidad de Morfofisiología, FES-Iztacala, UNAM, Mexico, 2Departamento de Biología del Desarrollo y Teratogénesis Experimental, Hospital Infantil de México Federico Gómez., Mexico, 3Unidad de Investigación Médica en Bioquímica, Hospital de Especialidades, CMN Siglo XXI, IMSS, México, Mexico Increased reactive oxygen species (ROS) has been associated with chronic exposition to high glucose concentration. ROS regulate the degradation of extracellular matrix by altering the expression of proteases like MMP-9; since, the promoter of the gene for MMP-9 contains binding elements, to transcription factors affected by oxidative stress. However, factors that regulate MMP-9 expression in early mouse embryo are poorly understood.

In this study, the effect of nicotinamide on the action of high glucose or H2O2 on oxidative stress and MMP-9 expression during the in vitro development of mouse blastocysts was evaluated. Gestational day (GD) 4 blastocysts were cultured in HAM-F10 and after 3 days of culture (GD7), treated for 48 h with glucose 25 mmol/L or H2O2 de (NAM, 15 mmol/L) was added to some groups of both treatments. ROS and

- -dichloro dihydroflurescein and MitoTracker Red. MMP-9 mRNA was evaluated by real time RT-PCR and MMP9 levels were analyzed by zymography using SDS-polyacrylamide gels co-polymerized with gelatin. High glucose and H2O2 induced similar changes, including increased concentration of intracellular ROS, higher levels of MMP-9 mRNA and protein. However higher proportion of ROS induced by high glucose presented a non-mitochondrial localization; ROS induced by high glucose were prevented by apocynin or rotenone. NAM prevented the action of high glucose on ROS and MMP-9 mRNA, but not when these changes are induced by H2O2. Our results suggested that high glucose induced the generation of ROS derived of mitochondria and NADPH oxidase activities and that the expression of MMP-9 during trophoblast differentiation and the invasion of endometrium may be regulated by glucose and oxidative stress. Supported by PAPIT, DGAPA, UNAM, grant IN223014.

374 The Defensive Role of Cumulus Cells against Reactive Oxygen Species Insult in Metaphase II Mouse Oocytes Faten Shaeib1, Sana Khan1, Iyad Ali2, Jashoman Banarjee1, Ghassan Saed1, and Husam M Abu-Soud1 1Wayne State University, USA, 2Najah University, USA

species insult in metaphase II mouse oocytes Recently, we have shown that in the absence of cumulus cells, cells surrounding the oocyte, reactive oxygen species (ROS) deteriorate oocyte quality through a mechanism that involves alteration in the microtubule morphology (MT) and chromosomal alignment (CH). Here, we investigated the capacity of cumulus

ROS such as hydrogen peroxide (H2O2), hydroxyl radical ( OH), and hypochlorous acid (HOCl). Metaphase II mouse oocytes with (n = 1634) and without cumulus cells (n = 1633) were treated with increasing concentration of ROS and the deterioration in oocyte quality was assessed by the changes in the MT and CH. Oocyte viability and cumulus cell number were assessed by indirect immunofluorescence and the trypan blue staining. All the treated oocytes showed decreased quality as a function of increasing concentrations of ROS as compared to untreated controls.

lower concentrations, but this protection was lost at higher concentrations (>

protection to the oocyte, both cumulus cell number and viability were decreased. Therefore, the deterioration in oocyte quality that is linked with poor reproductive outcomes may be caused by one or more of the following: a decrease in the antioxidant machinery of the oocyte cumulus complex by the loss of cumulus cells, the lack of scavengers for specific ROS, and/or the ability of the ROS to overcome these defenses.

S152 SFRBM 2014

doi: 10.1016/j.freeradbiomed.2014.10.567

doi: 10.1016/j.freeradbiomed.2014.10.568

doi: 10.1016/j.freeradbiomed.2014.10.570

doi: 10.1016/j.freeradbiomed.2014.10.569

The defensive role of cumulus cells against reactive oxygen

cells to protect the oocyte against increasing concentrations of

Cumulus cells show protection against H2O2 and •OH insult at

signifi cant protection against HOCl at any concentration (10-100