NADPH Oxidase 2 and Reactive Oxygen Species Mediate Pulmonary Arterial Smooth Muscle Cell Phenotypic Modulation Following Chronic Hypoxia

Abstract

Chronic hypoxia (CH), which is associated with chronic obstructive pulmonary disease and high‐altitude exposure, increases pulmonary vascular resistance and promotes pulmonary hypertension through mechanisms that stimulate vasoconstriction and arterial remodeling. Vascular remodeling is characterized in part by abnormal migration of pulmonary arterial smooth muscle cells (PASMCs). Based upon previous studies implicating NADPH oxidase 2 (NOX‐2) in the development of CH‐induced pulmonary hypertension, we hypothesized that NOX‐2‐generated reactive oxygen species (ROS) enhance PASMC migration following CH. To test this hypothesis, we harvested primary PASMCs from control and chronically hypoxic (4 wk at 0.5 atm) rats, cultured them in 5% serum DMEM F12 growth media for 3 days and in 0.3% serum media for 24 hr, and then plated the cells onto 8 μm polycarbonate membrane Transwell inserts (Costar). After allowing the PASMCs to adhere for 1 hr, they were treated with the general NOX inhibitor apocynin (30 μM), the specific NOX‐2 inhibitor GSK2795039 (500 nM), the superoxide dismutase/catalase mimetic EUK‐134 (10 μM), or a vehicle control (0.1% dimethyl sulfoxide) for 24 hr. PASMCs were fixed for 20 min in 4% paraformaldehyde, washed with phosphate‐buffered saline (PBS), stained with 1% Coomassie, and washed again with PBS. Transwell inserts were imaged with an upright brightfield microscope (Nikon) before and after the top layer of PASMCs was removed with a cotton swab. Cell migration was expressed as the percent of total cells that migrated through the Transwell membrane. Our results indicate that CH significantly (p < 0.05) enhances PASMC migration, while either inhibition of NOX‐2 or scavenging of ROS significantly blunts this response. We conclude that NOX‐2‐derived ROS contribute to enhanced PASMC migration following CH.Support or Funding InformationThis work was supported by NIH grants R01 HL132883 and T32 HL‐007736 (to T.C. Resta) and K12‐GM‐ 088021 (to A. Wandinger‐Ness).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.