Superoxide generation by NOX1 contributes to abnormal migration and proliferation of pulmonary arterial smooth muscle cells in rats

Rationale: Chronic alveolar hypoxia induces pulmonary hypertension (PH), concomitant with pulmonary vascular remodeling. Reactive oxygen species (ROS) are thought to play a major role in this process. Our recent findings suggest that ROS production by NAD(P)H-oxidase 4 (NOX4) is important in this remodeling process.

Objectives: We investigated whether similar mechanisms are true for non-hypoxia induced PH, namely PH induced by monocrotaline (MCT) in rats. The aim was to examine ROS production, their possible sources and their impact on cell function and signaling pathways. Furthermore we investigated the expression of NOX subunits, of the antioxidative enzymes SOD1, SOD2 and catalase in pulmonary arterial smooth muscle cells (PASMC), isolated from MCT-treated rats.

Methods/Results: ROS measurements using electronspinresonance spectroscopy and dihydroethidium staining showed an increased superoxide production in PASMC after MCT treatment. Real-time PCR analysis revealed an upregulation of NOX1 and SOD2 in MCT-treated PASMC. Both could be verified by western blot, as well as expression of cyclin D1 and status of phosphorylation of cofilin and SSH-1L. Knockdown of NOX1 by RNA interference lead to normalized intracellular superoxide production, proliferation, and migration of MCT treated PASMC. Pathway analysis has so far revealed increased expression of cyclin D1 and decreased phosphorylation of cofilin and SSH-1L

Conclusions: 1) enhanced intracellular superoxide production in PASMC of MCT-treated rats, 2) the increased SOD2 expression, and increased expression of NOX1 in PASMC of MCT-treated rats, 4) the normalized proliferation, migration and superoxide generation after NOX1 knockdown, 5) the increased expression of cyclin D1 expression, and the decreased phosphorylation of cofilin ad SSH-1L, suggests a contribution of ROS generated by NOX1, but not NOX4, in the vascular remodeling process in the microvascular parts of the pulmonary artery after MCT treatment.