Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation
Shuai Yuan,
Scott A. Hahn,
Megan P. Miller,
Subramaniam Sanker,
Michael J. Calderon,
Mara Sullivan,
Atinuke M. Dosunmu-Ogunbi,
Marco Fazzari,
Yao Li,
Michael Reynolds,
Katherine C. Wood,
Claudette M. St Croix,
Donna Stolz,
Eugenia Cifuentes-Pagano,
Placido Navas,
Sruti Shiva,
Francisco J. Schopfer,
Patrick J. Pagano,
Adam C. Straub
Affiliations
Shuai Yuan
Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA
Scott A. Hahn
Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA
Megan P. Miller
Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA
Subramaniam Sanker
Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA
Michael J. Calderon
Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, PA, USA
Mara Sullivan
Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, PA, USA
Atinuke M. Dosunmu-Ogunbi
Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA; Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA
Marco Fazzari
Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA
Yao Li
Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA
Michael Reynolds
Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA
Katherine C. Wood
Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA
Claudette M. St Croix
Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, PA, USA
Donna Stolz
Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, PA, USA
Eugenia Cifuentes-Pagano
Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA; Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA
Placido Navas
Centro Andaluz de Biología del Desarrollo and CIBERER, Instituto de Salud Carlos III, Universidad Pablo de Olavide-CSIC-JA, Sevilla, Spain, Spain
Sruti Shiva
Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA; Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA
Francisco J. Schopfer
Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA; Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA; Pittsburgh Liver Research Center (PLRC), University of Pittsburgh, Pittsburgh, PA, USA
Patrick J. Pagano
Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA; Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA
Adam C. Straub
Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA; Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA; Center for Microvascular Research, University of Pittsburgh, Pittsburgh, PA, USA; Corresponding author. Pharmacology and Chemical Biology Director of the Center for Microvascular Research University of Pittsburgh School of Medicine, Department of Pharmacology and Chemical Biology, The Heart, Lung, Blood and Vascular Medicine Institute, E1254 Biomedical Science Tower, 200 Lothrop St, Pittsburgh, PA, 15216, USA.
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.
