title: NADPH oxidase 4 mediates insulin-stimulated HIF-1α and VEGF expression, and angiogenesis in vitro.
abstract: Acute intensive insulin therapy causes a transient worsening of diabetic retinopathy in type 1 diabetes patients and is related to VEGF expression. Reactive oxygen species (ROS) have been shown to be involved in HIF-1α and VEGF expression induced by insulin, but the role of specific ROS sources has not been fully elucidated. In this study we examined the role of NADPH oxidase subunit 4 (Nox4) in insulin-stimulated HIF-1α and VEGF expression, and angiogenic responses in human microvascular endothelial cells (HMVECs). Here we demonstrate that knockdown of Nox4 by siRNA reduced insulin-stimulated ROS generation, the tyrosine phosphorylation of IR-β and IRS-1, but did not change the serine phosphorylation of IRS-1. Nox4 gene silencing had a much greater inhibitory effect on insulin-induced AKT activation than ERK1/2 activation, whereas it had little effect on the expression of the phosphatases such as MKP-1 and SHIP. Inhibition of Nox4 expression inhibited the transcriptional activity of VEGF through HIF-1. Overexpression of wild-type Nox4 was sufficient to increase VEGF transcriptional activity, and further enhanced insulin-stimulated the activation of VEGF. Downregulation of Nox4 expression decreased insulin-stimulated mRNA and protein expression of HIF-1α, but did not change the rate of HIF-1α degradation. Inhibition of Nox4 impaired insulin-stimulated VEGF expression, cell migration, cell proliferation, and tube formation in HMVECs. Our data indicate that Nox4-derived ROS are essential for HIF-1α-dependent VEGF expression, and angiogenesis in vitro induced by insulin. Nox4 may be an attractive therapeutic target for diabetic retinopathy caused by intensive insulin treatment.
authors.affiliation: Department of Physiology and Pathophysiology, Fudan University Shanghai Medical College, Shanghai, China. dmeng@fudan.edu.cn
authors.forename: Dan
authors.initials: D
authors.lastname: Meng
authors.forename: Aihong
authors.initials: A
authors.lastname: Mei
authors.forename: Junxu
authors.initials: J
authors.lastname: Liu
authors.forename: Xueling
authors.initials: X
authors.lastname: Kang
authors.forename: Xianglin
authors.initials: X
authors.lastname: Shi
authors.forename: Ruizhe
authors.initials: R
authors.lastname: Qian
authors.forename: Sifeng
authors.initials: S
authors.lastname: Chen
mesh_terms: D001808:Blood Vessels / Q000187:drug effects* / Q000254:growth & development; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics* / Q000378:metabolism; D015151:Immunoblotting; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins / Q000378:metabolism; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014443:Tyrosine / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism
chemical_list: D007004:Hypoglycemic Agents; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D017382:Reactive Oxygen Species; D042461:Vascular Endothelial Growth Factor A; D014443:Tyrosine; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D011972:Receptor, Insulin; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases