Abstract
Objective: Endothelial cells (EC) produce a variety of reactive oxygen species (ROS) which act as mediators of signaling for proliferation, migration, differentiation and cell death. The major source of ROS in EC at rest is the NADPH oxidase enzyme complex. We aimed to determine the roles of the Nox2 and Nox4 catalytic subunits of NADPH oxidase in proliferation and survival of human microvascular endothelial cells (HMEC-1). Methods: Intracellular superoxide was measured by dihydroethidium fluorescence and lucigenin chemiluminescence, and total ROS by dichlorodihydrofluorescein-diacetate assay. Nox4 and Nox2 protein expression was measured by Western blot, and cell proliferation was quantitated by trypan blue exclusion and a tetrazolium-based MTS assay. EC survival was measured by caspase3/7 activity. Results: The expression of Nox4 (but not Nox2) protein, superoxide and total ROS, were all significantly higher (P < 0.05, N = 4) under proliferative conditions (sparse culture or serum addition) than in quiescent (dense culture or serum-starved) cells. Suppression of NADPH oxidase-derived ROS with specific siRNA significantly reduced cell proliferation as shown below. Under proliferative conditions phosphorylation of ERK and Akt were increased. Suppression of Nox4-derived ROS production reduced the phosphorylation of both ERK and Akt, whereas suppression of Nox2 reduced only Akt phosphorylation. Further, suppression of Nox2, but not Nox4, significantly activated caspase3/7 (P < 0.05, N = 4). Thus Nox4-derived ROS act to promote proliferation of EC via ERK and Akt signaling, whereas Nox2 prevents apoptotic signaling via Akt and caspase3/7. Conclusion: Nox isoform-derived ROS act via distinct signaling pathways to promote EC proliferation and survival. Selective modulation of Nox-type NADPH oxidase could be a useful approach for blocking angiogenesis or for therapeutic angiogenesis in chronic ischemia or tissue engineering.
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