Evidence for the Involvement of miRNA in Redox Regulated Angiogenic Response of Human Microvascular Endothelial Cells

Abstract

Objective— A Dicer knockdown approach was used to test the significance of miRNA in regulating the redox state and angiogenic response of human microvascular endothelial cells (HMECs). Methods and Results— Lowering of miRNA content by Dicer knockdown induced vascular endothelial growth factor expression but diminished the angiogenic response of HMECs as determined by cell migration and Matrigel tube formation. Such impairment of angiogenic response in the Matrigel was rescued by exogenous low micromolar H 2 O 2 . Dicer knockdown HMECs demonstrated lower inducible production of reactive oxygen species (ROS) when activated with either phorbol ester, tumor necrosis factor-α, or vascular endothelial growth factor. Limiting the production of ROS by antioxidant treatment or NADPH oxidase knockdown approaches impaired angiogenic responses. Experiments to identify how ROS production is limited by Dicer knockdown identified lower expression of p47phox protein in these cells. This lowering of cellular miRNA content induced expression of the transcription factor HBP1, a suppressor transcription factor that negatively regulates p47phox expression. Knockdown of HBP1 restored the angiogenic response of miRNA-deficient HMECs. Conclusion— This study provides the first evidence that redox signaling in cells is subject to regulation by miRNA. Specifically, p47phox of the NADPH oxidase complex has been identified as one target that regulates the angiogenic properties of endothelial cells.