Molecular Mechanism and Functional Role of Hypoxia‐induced Reactive Oxygen Species in Pulmonary Arterial Endothelial Cells

Abstract

Hypoxia‐induced (Group 3) pulmonary hypertension (PH) is a common devastating lung disease. Pulmonary artery endothelial cells (PAECs) are a major contributor in hypoxic PH due to possible roles of reactive oxygen species (ROS). However, the production up‐and‐down, molecular mechanisms and functional roles of hypoxia‐induced ROS in PAECs are not well established. In this study, we first used Amplex UltraRed reagent to assess hydrogen peroxide (H2O2) generation. The result indicated that hypoxic exposure resulted in a significant increase in Amplex UltraRed‐derived fluorescence (i.e., H2O2 production) in human PAECs. To complement this result, we employed lucigenin as a probe to detect superoxide (O2−) production. Our assays showed that hypoxia largely increased O2− production. Hypoxia also enhanced H2O2 production in isolated mitochondria from PAECs. In support, using the genetically encoded H2O2 sensor HyPer, we revealed that hypoxia caused a large increase in HyPer‐derived fluorescence (ROS production) in PAECs, which was blocked by the mitochondrial inhibitor rotenone or myxothiazol. Hypoxia largely increased proliferation of PAECs, determined using Ki67 staining and direct cell number accounting. The hypoxic cell proliferation was blocked by rotenone or myxothiazol. Like rotenone and myxothiazol, lentiviral shRNA‐mediated knockdown of Rieske iron‐sulfur protein nicotine (RISP), known to be a primary molecule in mitochondrial ROS generation, inhibited the hypoxic proliferation as well. Hypoxic exposure evoked a significant increase in migration of PAECs, evaluated by wound healing assay. Pharmacological and genetic inhibition of mitochondrial ROS generation with myxothiazol and RISP knockdown both diminished the hypoxic cell migration. Taken together, we conclude that hypoxia significantly increases intracellular ROS generation in human PAECs, the hypoxia‐induced intracellular ROS generation primarily occur in mitochondria, and RISP serves as an essential molecule in controlling the hypoxic mitochondrial ROS generation. Equally Importantly, and RISP‐dependent mitochondrial proliferation that is likely to be ROS‐dependent. Conceivably, hypoxia remarkably causes proliferation and migration of human PAECs, which is predominantly mediated by RISP‐dependent mitochondrial ROS, thereby making an essential contribution to hypoxic pulmonary hypertension.