Oxidative stress in hypoxic signal transduction

Chronic exposure of the lung vasculature to hypoxia causes remodeling of the pulmonary artery and leads to pulmonary hypertension. Recent work has identified evidence of increased intracellular reactive oxygen species (ROS) formation during hypoxia in non-vascular cell types. This project first sought to determine if hypoxia induced ROS in pulmonary artery smooth muscle cells (PASMC). PASMC exposed to exogenous xanthine oxidase or hypoxia demonstrated increased production of ROS using 2,7-dichlorofluorescein (DCF) as a fluorescent marker. The fluorescence was attenuated by the addition of the antioxidant dimethylthiourea (DMTU) and electron transport chain inhibitors. Targets of oxidative stress were analyzed after exposure to xanthine oxidase or hypoxia to determine the effects of hypoxia-induced ROS production in the cell. Unlike xanthine oxidase, hypoxia caused no detectable cell death or lipid peroxidation. However, time- and location-dependent oxidative DNA damage occurred after xanthine oxidase and hypoxic exposure. Hypoxia-induced damage could be blocked by co-incubation of DMTU. Surprisingly, no DNA damage could be detected in the mitochondrial DNA after exposure to hypoxia when averaged over most of the genome, unlike xanthine oxidase treatment. For further characterization, LM-PCR was used to map the location of DNA damage in the hypoxia-responsive region of the vascular endothelial growth factor (VEGF) promoter and in a fragile site of the mitochondrial genome. DNA damage was seen in both genomic targets and were often clustered in "hot spots," i.e. nucleotides that were regularly damaged upon exposure to hypoxia. Many of these hot spots occurred in response elements in the VEGF gene and in the mitochondrial break site. Given demonstration of hypoxia-induced DNA damage, additional studies were directed towards possible functional consequences. Hypoxia caused a temporary accumulation of cycling PASMC in the G1 phase of the cell cycle and consequential decreased thymidine uptake. Hypoxic-mediated delay in cell cycling could also be attenuated by DMTU. This work is the first to describe hypoxic-induced ROS production and DNA damage in PASMC, a key player in the development of vascular remodeling. This work also proposes oxidative stress is a component of hypoxic signal transduction in vascular smooth muscle.