Hypoxia-Induced Mitochondrial ROS and Function in Pulmonary Arterial Endothelial Cells.

Pulmonary artery endothelial cells (PAECs) are a major contributor to hypoxic pulmonary hypertension (PH) due to the possible roles of reactive oxygen species (ROS). However, the molecular mechanisms and functional roles of ROS in PAECs are not well established. In this study, we first used Amplex UltraRed reagent to assess hydrogen peroxide (HO) generation. The result indicated that hypoxic exposure resulted in a significant increase in Amplex UltraRed-derived fluorescence (i.e., HO production) in human PAECs. To complement this result, we employed lucigenin as a probe to detect superoxide (O) production. Our assays showed that hypoxia largely increased O production. Hypoxia also enhanced HO production in the mitochondria from PAECs. Using the genetically encoded HO sensor HyPer, we further revealed the hypoxic ROS production in PAECs, which was fully blocked by the mitochondrial inhibitor rotenone or myxothiazol. Interestingly, hypoxia caused an increase in the migration of PAECs, determined by scratch wound assay. In contrast, nicotine, a major cigarette or e-cigarette component, had no effect. Moreover, hypoxia and nicotine co-exposure further increased migration. Transfection of lentiviral shRNAs specific for the mitochondrial Rieske iron-sulfur protein (RISP), which knocked down its expression and associated ROS generation, inhibited the hypoxic migration of PAECs. Hypoxia largely increased the proliferation of PAECs, determined using Ki67 staining and direct cell number accounting. Similarly, nicotine caused a large increase in proliferation. Moreover, hypoxia/nicotine co-exposure elicited a further increase in cell proliferation. RISP knockdown inhibited the proliferation of PAECs following hypoxia, nicotine exposure, and hypoxia/nicotine co-exposure. Taken together, our data demonstrate that hypoxia increases RISP-mediated mitochondrial ROS production, migration, and proliferation in human PAECs; nicotine has no effect on migration, increases proliferation, and promotes hypoxic proliferation; the effects of nicotine are largely mediated by RISP-dependent mitochondrial ROS signaling. Conceivably, PAECs may contribute to PH via the RISP-mediated mitochondrial ROS.