Increased generation of reactive oxygen species in pulmonary arterial smooth muscle cells from monocrotaline-treated rats

Abstract

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 the NADPH-oxidase (NOX) subunits play an important role in this remodeling process. Objectives: We investigated whether similar mechanisms are active for non-hypoxia induced PH, namely monocrotaline (MCT) induced PH in rats. The aim was to examine ROS production and their possible source. Further we adressed the expression of NOX subunits, antioxidative enzymes SOD1, SOD2, and catalase in pulmonary arterial smooth muscle cells (PASMC), isolated from MCT-treated rats. Methods/Results: Comparing PASMC from MCT-treated and non-treated rats by real-time PCR analysis revealed an upregulation of NOX1 and p22phox (n=6). Upregulation of NOX1 was confirmed by western blot. RT-PCR and immunofluorescence showed an increased SOD2 expression after MCT treatment. ROS measurements using ESR spectroscopy showed an increased intracellular production of ROS in total, but also especially superoxide in PASMC after MCT treatment in comparison to healthy controls. Stimulation with the NOX activator phorbol myristate acetate (PMA) induced an additional ROS increase. Results were validated by dihydroethidium staining. Conclusion: Our results show an increased ROS production in PASMC of MCT-treated rats which may contribute to vascular remodeling. Data from 1) enhanced ROS production upon PMA stimulation, 2) increased expression of NOX1 and 3) the increased SOD2 expression suggests a contribution of both, mitochondria and NOX1 as possible sources of ROS release in MCT treated rats.