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nox enzyMes and tHe develoPMent oF Heart FailUreAjay Shah, King’s College London, UKCardiac failure occurs as a result of the failure of the heart to adapt to chronic stresses such as pressure overload or ischaemia. Increased production of reactive oxygen species (ROS) is implicated in cardiac stress responses and the development of heart failure but the roles and effects of different ROS sources are unclear. NADPH oxidases (Noxs) are important sources of ROS that appear to be particularly important in modulating signal transduction pathways. Two Nox isoforms, Nox2 and Nox4, are co-expressed in the heart. Previous studies have shown that Nox2 promotes the development of cardiac hypertrophy, fibrosis and contractile dysfunction but the role of Nox4 is unclear. Unlike other Nox proteins, Nox4 activity is regu-lated mainly by its expression level which is low in the healthy adult heart but increases sig-nificantly in response to pressure overload, hypoxia or ischaemia. To investigate the functional role of increases in Nox4 during the cardiac response to stress, we have recently studied mice with either a complete deletion of Nox4 or a cardiomyocyte-targeted overexpression of Nox4. Surprisingly, Nox4 transgenic mice are protected against pressure overload-induced contrac-tile dysfunction and failure whereas Nox4-null mice develop significantly worse dysfunction and failure compared to wild-type littermates. An investigation of the mechanisms underlying this protective effect of Nox4 indicates that cardiomyocyte Nox4 enhances the preservation of myocardial capillary density in response to chronic pressure overload. This effect involves an increase in the activation of Hif1 and the release of VEGF, and results in increased paracrine angiogenic activity. These data indicate that cardiomyocyte Nox4 is an inducible regulator of myocardial angiogenesis that can facilitate cardiac adaptation to overload stress and as such differs significantly from Nox2 in its effects on the heart.

doi:10.1016/j.freeradbiomed.2010.10.669

regUlation oF tHioredoxin-1 in endotHelial cellsJudith Haendeler, University of Dӥsseldorf, GermanyThioredoxin-1 (Trx-1) is one of the major antioxidative enzymes in endothelial cells. Trx-1 has been shown to be required for apoptosis inhibition in endothelial cells. Apoptosis induction is dependent on cytoskeletal changes resulting in formation of stress fibers, which are bundles of actin. Therefore, the aim of this project was to elucidate whether a direct connection exists between Trx-1 and actin. First, we immunoprecipitated Trx-1 out of human primary endo-thelial cells, and γ-actin was identified as a new binding partner using a mass spectrometric approach. This interaction was confirmed by co-immunoprecipitation experiments. Since the induction of stress fibers is directly linked to the phosphorylation and thereby activation of the Focal Adhesion Kinase (FAK), inhibition of FAK phosphorylation with PF-573228, a known FAK inhibitor, reduced H2O2-induced formation of stress fibers after preincubation. Interest-ingly, preincubation with exogenous Trx-1 also inhibited phosphorylation of FAK and stress fiber formation. However, we did not find an additive effect of PF-573228 and exogenous Trx-1, suggesting a common signaling pathway. To further investigate whether stress fiber formation is indeed required for H2O2-induced apoptosis, we treated endothelial cells with H2O2. Increased phosphorylation of FAK, induced stress fiber formation, reduced Trx-1 protein levels and increased apoptosis were observed. Preincubation with PF -573228 inhibited phos-phorylation of FAK, reduction of Trx-1 protein levels and apoptosis induction. On the contrary, incubation with PF-573228 1h after H2O2 treatment did not inhibit formation of stress fibers and degradation of Trx-1 protein and, thus, did not abrogate apoptosis induction by H2O2. These data demonstrate that stress fiber formation is indeed a prerequisite for apoptosis induction in endothelial cells. Furthermore, the interaction of Trx-1 and γ-actin protects Trx-1 from degradation, a process required for endothelial cell death.

doi:10.1016/j.freeradbiomed.2010.10.670

Judith Haendeler

Ajay Shah