NADPH oxidase 4 promotes cardiac microvascular angiogenesis after hypoxia/reoxygenation in vitro.
Microvascular endothelial cell dysfunction plays a key role in myocardial ischemia/reperfusion (I/R) injury, wherein reactive oxygen species (ROS)-dependent signaling is intensively involved. However, the roles of the various ROS sources remain unclear. This study sought to investigate the role of NADPH oxidase 4 (Nox4) in the cardiac microvascular endothelium in response to I/R injury. Adult rat cardiac microvascular endothelial cells (CMECs) were isolated and subjected to hypoxia/reoxygenation (H/R). Our results showed that Nox4 was highly expressed in CMECs, was significantly increased at both mRNA and protein levels after H/R injury, and contributed to H/R-stimulated increase in Nox activity and ROS generation. Downregulation of Nox4 by small interfering RNA transfection did not affect cell viability or ROS production under normoxia, but exacerbated H/R injury as evidenced by increased apoptosis and inhibited cell survival, migration, and angiogenesis after H/R. Nox4 inhibition also increased prolyl hydroxylase 2 (PHD2) expression and blocked H/R-induced increases in HIF-1α and VEGF expression. Pretreatment with DMOG, a specific competitive PHD inhibitor, upregulated HIF-1α and VEGF expression and significantly reversed Nox4 knockdown-induced injury. However, Nox2 was scarcely expressed and played a minimal role in CMEC survival and angiogenesis after H/R, though a modest upregulation of Nox2 was observed. In conclusion, this study demonstrated a previously unrecognized protective role of Nox4, a ROS-generating enzyme and the major Nox isoform in CMECs, against H/R injury by inhibiting apoptosis and promoting migration and angiogenesis via a PHD2-dependent upregulation of HIF-1/VEGF proangiogenic signaling.
Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, China.
Jinyi
J
Wang
Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, China.
Zhibo
Z
Hong
Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, China.
Chao
C
Zeng
Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, China. Electronic address: yuqiujun@gmail.com.
Qiujun
Q
Yu
Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, China. Electronic address: wanghc@fmmu.edu.cn.
Haichang
H
Wang
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D015687:Cell Hypoxia / Q000235:genetics; D002470:Cell Survival; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology; D005786:Gene Expression Regulation; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D008297:Male; D055806:Microvessels / Q000254:growth & development / Q000473:pathology; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000096:biosynthesis* / Q000235:genetics; D018919:Neovascularization, Physiologic / Q000235:genetics*; D011393:Procollagen-Proline Dioxygenase / Q000096:biosynthesis; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D015427:Reperfusion Injury / Q000235:genetics* / Q000473:pathology; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis
C497444:Hif1a protein, rat; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017382:Reactive Oxygen Species; D042461:Vascular Endothelial Growth Factor A; C467498:vascular endothelial growth factor A, rat; D011393:Procollagen-Proline Dioxygenase; C580731:Egln1 protein, rat; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human
Angiogenesis; Cardiac microvascular endothelial cells; Free radicals; Ischemia/reperfusion; NADPH oxidase 4; Reactive oxygen species