P163	Regulation of Nox isoforms by shear stress in human endothelial cells.
N.Duerrschmidt, C.Stielow, G.Müller, H.Morawietz
University of Technology Dresden, Dresden, DE.

Background: The endothelial cells are constantly exposed to shear stress by the flowing blood. An NAD(P)H oxidase complex has been identified as a major source of superoxide anion formation in endothelial cells. Several novel homologues of NADPH oxidase subunit gp91phox called the Nox family have been identified. Material and Methods: We analyzed the expression of different Nox isoformes and the effect of laminar shear stress on superoxide anion formation by cytochrome c reduction assay and on NAD(P)H oxidase subunit expression by standard calibrated competitive reverse transcription-polymerase chain reaction and Western blot in human endothelial cells. Primary cultures of human umbilical vein endothelial cells were exposed to laminar shear stress (1 to 50 dyne/cm2) in a cone-and-plate viscometer for up to 24 hours. Results: Endothelial cells mainly express the novel Nox isoform Nox4 and to a lower extend the isoform gp91phox/Nox2. Short-term application of shear stress caused induction of superoxide anion formation, but not of NAD(P)H oxidase expression. In contrast, superoxide anion formation and NAD(P)H oxidase subunits gp91phox/Nox2, Nox4 and p47phox are downregulated on mRNA level by long-term arterial laminar shear stress. These data could be confirmed with the available antibodies for gp91phox/Nox2 and p47phox on the protein level as well. Shear stress-dependent downregulation of superoxide anion formation and subunit expression of gp91phox and p47phox by shear stress can be blocked by NO synthase inhibitor L-NAME. NO donor DETA-NO downregulates superoxide anion formation, gp91phox and p47phox expression in static cultures. However, downregulation of Nox4 by shear stress does not involve NO. In conclusion, our data suggest a transient activation of the NAD(P)H oxidase complex by short-term laminar shear stress, but a downregulation of endothelial NAD(P)H oxidase in response to long-term arterial laminar shear stress. NAD(P)H oxidase subunits gp91phox/Nox2, Nox4 and p47phox seems to play an important role in this process. The NO-mediated downregulation might preferentially affect the gp91phox/Nox2-containing NAD(P)H oxidase complex. These regulatory mechanisms could contribute to the antiatherosclerotic and vasoprotective potential of laminar shear stress.

Copyright © 2005 S. Karger AG, Basel. Any further use of this abstract requires written permission from the publisher.