O12	Reactive oxygen species upregulate NOX4, but not NOX2, in endothelial cells.
1A.Petry, 1T.Djordjevic, 1Chr.Bickel, 1S.Bonello, 1R.S.Belaiba, 2J.Pfeilschifter, 1J.Hess, 1A.Görlach
1Experimentelle Kinderkardiologie, Deutsches Herzzentrum an der TU München, München, DE; 2Pharmacenter Frankfurt, JWG Universität Frankfurt, Frankfurt/Main, DE.

Sustained generation of reactive oxygen species (ROS) has been involved in the pathogenesis of cardiovascular diseases including atherosclerosis and vascular remodeling. One of the main sources of ROS in the vasculature are NADPH oxidases, first described in phagocytic cells. The catalytic core of the phagocytic NADPH oxidase is composed of two subunits, p22phox and gp91phox. Recently, four additional homologues of gp91phox (now termed NOX2) have been identified. However, the role and regulation of these novel homologues in endothelial cells is not fully elucidated. Therefore, we investigated the regulation of p22phox, NOX2 and NOX4 expression in endothelial EaHy926 cells and their contribution to ROS production and proliferation. We confirmed by RT-PCR and Western blot analysis the expression of p22phox, NOX2 and NOX4 in endothelial cells. Bimolecular fluorescent complementation analysis and coimmunoprecipitation showed that p22phox is interacting with either NOX2 or NOX4. In unstimulated cells, the protein levels of p22phox and NOX4 were lower than those of NOX2. Stimulation with H2O2 (50 µM) enhanced p22phox and NOX4 protein levels peaking at 2 to 4 hours whereas NOX2 protein levels were only slightly enhanced after 8 hours of incubation. Co-expression of p22phox with NOX2 or NOX4 as well as stimulation with H2O2 resulted in increased ROS production and endothelial proliferation. Depletion of p22phox, NOX2 and NOX4 by siRNA prevented these responses. These data show that p22phox can physically interact with NOX2 as well as with NOX4 in endothelial cells. This interaction is required for fully activated NADPH oxidases to stimulate ROS production and proliferation of endothelial cells. Although forced expression of all subunits increased ROS production and cell proliferation to a similar extent, the finding that p22phox and NOX4 protein expression is inducible by H2O2 whereas NOX2 expression appears to be constitutively expressed in endothelial EaHy926 cells indicates that a positive feed back loop may exist promoting redox-sensitive upregulation of NOX4-containing NADPH oxidases. Such a pathway may contribute to the sustained elevation of ROS levels in various cardiovascular diseases.

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