The NADPH oxidase 4 protects vascular endothelial cells from copper oxide nanoparticles-induced oxidative stress and cell death.

Nanoparticles (NPs) exposure is associated with increased risk of cardiovascular diseases, but the underlying mechanism is still obscure. In this study, we investigated the role of NADPH oxidase 4 (NOX4) in copper oxide nanoparticles (CuONPs)-induced cytotoxicity in human umbilical vein endothelial cells (HUVECs). Morphology changes were examined under the microscope. Cell viability was determined by MTS assay and Calcein AM assay. Apoptosis and the levels of superoxide anion (O2-) and hydrogen peroxide (H2O2) were measured by fluorescence activated cell sorting (FACS). Oxidative stress was detected by assaying the levels of glutathione/glutathione disulfide (GSH/GSSG) and malondialdehyde (MDA). Protein expression levels were determined by western blotting. We revealed that O2- rather than H2O2 was the major component of reactive oxygen species (ROS) in CuONPs-treated HUVECs. Meanwhile, CuONPs downregulated expression of O2--eliminating enzyme NOX4 both at mRNA and protein levels, but did not affect the expression of SOD2 and catalase. NOX4 knockdown caused more accumulation of O2-, and a further decrease of H2O2 in CuONPs-treated HUVECs, suggesting that NOX4 regulates the conversion of O2- to H2O2 in CuONPs-treated HUVECs. Furthermore, we revealed that NOX4 knockdown aggravated CuONPs-induced oxidative stress, characterized by a decrease of GSH/GSSG ratio, an increase of MDA level, and upregulation of HSPA5 and γH2AX. Finally, we showed that NOX4 knockdown exacerbated CuONPs-induced apoptotic cell death in HUVECs, indicating that NOX4 could protect ECs from CuONPs-induced cell death. Our study provides the evidence that NOX4 protects vascular endothelial cells from CuONPs-induced oxidative stress and cell death.