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.

NADPH 氧化酶 4 (NOX4) 通过产生过氧化氢 (H ₂ O ₂ ) 和较小程度的 O ₂ ^•-来调节内皮炎症。NOX4 衍生的 H ₂ O ₂和 O ₂ ^•-的比率可以通过辅酶 Q (CoQ) 模拟物改变。因此，我们假设细胞色素b 5 还原酶 3 (CYB5R3) 是一种富含血管内皮细胞的 CoQ 还原酶，可调节炎症激活。为了在体内检查内皮 CYB5R3 ，我们创建了他莫昔芬诱导的内皮特异性Cyb5r3基因敲除小鼠 (R3 KO)。收缩压的无线电遥测测量显示脂多糖 (LPS) 攻击小鼠的全身性低血压，这在 R3 KO 小鼠中加剧。同时，LPS 处理在 R3 KO 小鼠中引起更大的内皮功能障碍，通过离体主动脉中乙酰胆碱诱导的血管舒张进行评估，并伴有血管粘附分子 1 ( Vcam-1 ) 的 mRNA 表达升高。类似地，在培养的人主动脉内皮细胞 (HAEC) 中，LPS 和肿瘤坏死因子 α (TNF-α) 诱导的 VCAM-1 蛋白表达被Cyb5r3 siRNA 增强，其通过沉默Nox4被消融同时基因。此外，超分辨率共聚焦显微镜表明 CYB5R3 和 NOX4 在 HAEC 中线粒体共定位。基于 APEX2 的电子显微镜和邻近生物素化也证明了 CYB5R3 在线粒体外膜上的定位及其与 NOX4 的相互作用，这进一步通过邻近连接测定得到证实。值得注意的是，Cyb5r3敲低的 HAEC 显示总 H ₂ O ₂较少，但线粒体 O ₂ ^•-较多。使用非活性或非膜结合活性 CYB5R3，我们发现 CYB5R3 活性和膜易位是 H ₂ O _{2的最佳生成所必需的}通过 NOX4。最后，缺乏 CoQ 合成酶 COQ6 的细胞显示 NOX4 衍生的 H ₂ O ₂减少，表明 NOX4 活性需要内源性 CoQ。总之，CYB5R3 通过 CoQ 协助 NOX4 依赖性 H ₂ O ₂生成来减轻内皮炎症激活。