The importance of the various NOX homologues for vascular function is still an open question. This present study showed that endothelial cells express at least four of the five known NOX homologues as we could show that these cells express in addition to NOX2 and NOX4 also NOX1 and NOX5, and that coexpression of various NOX proteins in one single cell can take place. However, the contribution of NOX1 to endothelial basal ROS generation and proliferation seems to be limited. Still, NOX1 could be important under specific conditions of endothelial cell activation, the elucidation of which needs further investigations.
The other NOX homologues NOX2, NOX4 and NOX5 contribute equally to basal endothelial ROS generation, proliferation and angiogenesis. There are differences in the intracellular localization of the NOX homologues in endothelial cells. NOX2, NOX4 and NOX5 are mainly localized in the ER, but NOX2 is also partially localized to the plasma membrane while the precise localization of NOX1 is still unclear. Stress factors such as thrombin and ER-stress are able to induce ROS generation. Here we showed that thrombin induces NOX2 and NOX5 expression and that NOX2 and NOX5 contribute to endothelial ROS generation, proliferation and angiogenesis upon thrombin stimulation. Differences in the fine tuning of NOX2 and NOX5 activation could be relevant and the exact role of ROS generation by either NOX2 or NOX5 is still not precisely known. However, these novel findings mark NOX2, NOX5 as well as ROS in general as novel targets for antithrombotic research in the future.
Furthermore, the identification of calcium-dependent and –independent NOX5 variants in endothelial cells is a novel finding. However, activation and embedment of NOX5 in calcium-related pathways remains still unclear and constitutes a promising topic for future research.
The present study also shows for the first time that p22phox, which is required for the function of most NOX homologues except NOX5, is induced by ER-stress, and that the UPR genes ATF4 and XBP1 enhance ROS generation by upregulating p22phox. Although, little is known about the involvement of NADPH oxidases in the UPR, these findings suggest that NADPH oxidases may play a role in the this stress response. In summary, the results of this study reveal new insights in the role of ROS and novel NADPH oxidases in endothelial cells under basal and stress conditions which can serve as new background for further research in this field.
5 Summary
Increased levels of reactive oxygen species (ROS) contribute to vascular diseases like pulmonary hypertension and atherosclerosis. Although a NOX2-containing NADPH oxidase similar to the neutrophil one has been described to be active in endothelial cells, the contribution of newly discovered NOX homologues (NOX1- NOX5) was still unclear. Therefore, the overall aim of this study was to better characterize the expression, regulation and function of NOX homologues in different endothelial cell models.
First, we could demonstrate the presence of NOX1, NOX2, NOX4, NOX5 including NOX5S as well as p22phox mRNA and protein levels in Ea.Hy926 or HMEC-1 cells. Furthermore, NOX5 protein was also present in endothelial and smooth muscle cells in the vascular wall of spleen and lung tissue. We found that NOX2, NOX4 and NOX5 were present in an intracellular perinuclear compartment, whereby NOX2 and NOX4 could be localized simultaneously in one cell. NOX2, NOX4, NOX5 were able to interact with p22phox and overexpression of NOX2, NOX4 and NOX5 increased ROS generation, although NOX5-dependent ROS generation did not require the presence of p22phox. NOX2, NOX4 and NOX5 also increased endothelial proliferation while depletion of NOX2, NOX4 and NOX5 decreased ROS generation, proliferation and tube forming ability indicating angiogenic activity under basal conditions. NOX2- and NOX4-induced proliferation was mediated by p38 MAP kinase.
Although NOX1 expression as well as the expression of its regulatory subunits NOXO1 and NOXA1 was detectable in endothelial cells, depletion of NOX1 did not significantly affect basal ROS generation or proliferation of endothelial cells.
Second, we could demonstrate the upregulation of NOX2, NOX5 and NOX5S after thrombin stimulation in endothelial cells and the modulation of p22phox expression in an ATF4- and XBP1-dependent manner under ER-stress conditions. Cellular stress either by thrombin or UPR also induced ROS generation of endothelial cells. In addition, thrombin induced proliferation and enhanced the tube forming ability of endothelial cells. Thrombin-induced ROS generation, proliferation and tube forming ability were diminished by silencing NOX2 or NOX5, whereas UPR induced ROS generation was inhibited by silencing p22phox as well as by silencing ATF4 or XBP1. In summary, this work provides evidence that in endothelial cells, NOX2, NOX4 and NOX5, but not NOX1, contribute to basal ROS generation, proliferation and angiogenesis and that the NOX proteins NOX2 and NOX5 as well as p22phox play an important role in the response to thrombin and ER-stress providing new insights in endothelial function and redox signaling.