Abstract
Directed migration of endothelial cells (ECs) is an important process during both physiological and pathological angiogenesis. The binding of vascular endothelial growth factor (VEGF) to VEGF receptor-2 (VEGFR-2) on the EC surface is necessary for directed migration of these cells. Here, we used TAXIScan, an optically accessible real-time horizontal cell dynamics assay approach, and demonstrate that reactive oxygen species (ROS)-producing NADPH oxidase 4 (NOX4), which is abundantly expressed in ECs, mediates VEGF/VEGFR-2-dependent directed migration. We noted that a continuous supply of endoplasmic reticulum (ER)-retained VEGFR-2 to the plasma membrane is required to maintain VEGFR-2 at the cell surface. siRNA-mediated NOX4 silencing decreased the ER-retained form of VEGFR-2, resulting in decreased cell surface expression levels of the receptor. We also found that ER-localized NOX4 interacts with ER-retained VEGFR-2 and thereby stabilizes this ER-retained form at the protein level in the ER. We conclude that NOX4 contributes to the directed migration of ECs by maintaining VEGFR-2 levels at their surface.
Highlights
Angiogenesis is the formation of new capillary blood vessels to supply oxygen and nutrients to tissues [1] and is fundamental to the process of recovery from tissue ischemia in adults [2]
Use of a mouse model overexpressing WT Nox4 showed that Nox4 plays a positive role in cardiac adaptation to pressure overload [25]
The addition of exogenous H2O2 did not restore the expression level of VEGF receptor-2 (VEGFR-2) (Fig. 6C, lane 3). These results indicate that the stabilizing effect of endoplasmic reticulum (ER)-retained VEGFR-2 by Nox4 required the production of H2O2 in the closed space between Nox4 and VEGFR-2
Summary
Angiogenesis is the formation of new capillary blood vessels to supply oxygen and nutrients to tissues [1] and is fundamental to the process of recovery from tissue ischemia in adults [2]. The present study investigated whether Nox4 is involved in VEGF/VEGFR-2-dependent directed migration using TAXIScan, which visualizes the migration of cells using various parameters. Nox4 regulates the directed migration of ECs by maintaining surface VEGFR-2 levels. Because Nox4 knockdown cells showed reduced directional migration, we investigated the expression pattern of VEGFR-2.
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