Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation

NADPH oxidase 4 (NOX4) regulates endothelial inflammation by producing hydrogen peroxide (H₂O₂) and to a lesser extent O₂^•-. The ratio of NOX4-derived H₂O₂ and O₂^•- can be altered by coenzyme Q (CoQ) mimics. Therefore, we hypothesize that cytochrome b5 reductase 3 (CYB5R3), a CoQ reductase abundant in vascular endothelial cells, regulates inflammatory activation. To examine endothelial CYB5R3 in vivo, we created tamoxifen-inducible endothelium-specific Cyb5r3 knockout mice (R3 KO). Radiotelemetry measurements of systolic blood pressure showed systemic hypotension in lipopolysaccharides (LPS) challenged mice, which was exacerbated in R3 KO mice. Meanwhile, LPS treatment caused greater endothelial dysfunction in R3 KO mice, evaluated by acetylcholine-induced vasodilation in the isolated aorta, accompanied by elevated mRNA expression of vascular adhesion molecule 1 (Vcam-1). Similarly, in cultured human aortic endothelial cells (HAEC), LPS and tumor necrosis factor α (TNF-α) induced VCAM-1 protein expression was enhanced by Cyb5r3 siRNA, which was ablated by silencing the Nox4 gene simultaneously. Moreover, super-resolution confocal microscopy indicated mitochondrial co-localization of CYB5R3 and NOX4 in HAECs. APEX2-based electron microscopy and proximity biotinylation also demonstrated CYB5R3's localization on the mitochondrial outer membrane and its interaction with NOX4, which was further confirmed by the proximity ligation assay. Notably, Cyb5r3 knockdown HAECs showed less total H₂O₂ but more mitochondrial O₂^•-. Using inactive or non-membrane bound active CYB5R3, we found that CYB5R3 activity and membrane translocation are needed for optimal generation of H₂O₂ by NOX4. Lastly, cells lacking the CoQ synthesizing enzyme COQ6 showed decreased NOX4-derived H₂O₂, indicating a requirement for endogenous CoQ in NOX4 activity. In conclusion, CYB5R3 mitigates endothelial inflammatory activation by assisting in NOX4-dependent H₂O₂ generation via CoQ.