P113	A NOX5 isoform is functionally active in human endothelial cells.
1A.Görlach, 1Chr.Bickel, 1A.Petry, 1T.Djordjevic, 1R.S.Belaiba, 1K.Diemer, 1S.Bonello, 2H.Acker, 1A.Pogrebniak, 1J.Hess
1Experimentelle Kinderkardiologie, Deutsches Herzzentrum München an der TU München, München, DE; 2Max-Planck-Institut für Molekulare Physiologie, Dortmund, DE.

Reactive oxygen species (ROS) generated by NADPH oxidases act as cellular signaling molecules in the vasculature. In leukocytes and endothelial cells, an NADPH oxidase containing gp91phox (NOX2) and p22phox, which form the catalytic core of the enzyme, has been described to produce ROS. Recently, several NADPH oxidase variants have been identified, which express different isoforms of gp91phox (NOX1 to NOX5). Whereas NOX1 and NOX4 have been described to contribute to vascular ROS production, the role of NOX5 in vascular cells is unclear. In lymphocytes, spermatozoa and pancreatic cells, several NOX5 isoforms have been identified which contain calcium-binding EF hands at the N-terminus. In this study we aimed to identify and characterize NOX5 in the vasculature. Using RT-PCR approaches we detected and cloned a NOX5 isoform (NOX5-S) in endothelial cells which does not contain calcium-binding EF hands as well as a splice variant (NOX5-SV) lacking exon 10 thus leading to a premature stop codon. Two photon confocal microscopy showed intracellular, calcium-independent ROS production associated with NOX5-S, but not with NOX5-SV. Depletion of NOX5-S by siRNA inhibited endothelial ROS production. NOX5-S, but not NOX5-SV, interacted with p22phox around the nucleus in an ER-like manner as was determined by bimolecular fluorescence complementation and confirmed by coimmunoprecipitation. Endothelial cells overexpressing NOX5-S showed enhanced proliferation and angiogeneis whereas depletion of NOX5-S by siRNA blunted these responses. Immunohistochemistry confirmed endogenous NOX5 expression in small vessels of human spleen. These findings show that endothelial cells express a functional, but distinct NOX5 isoform which is able to induce ROS production in a calcium-independent manner as well as to promote proliferation and angiogenesis.

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