Abstract 2625: The Demethylating Agent 5-aza-2′deoxycytidine (Decitabine) Restores Superoxide Dismutase 2 (SOD2) Expression and Reactive Oxygen Species Production in the Pulmonary Arteries of Fawn-hooded Rats with Pulmonary Hypertension

Abstract

Introduction: Pulmonary arterial hypertension (PAH) is a lethal disease characterized by vascular obstruction. We evaluated the initiating mechanisms for PAH in Fawn-hooded rats (FHR), a strain that spontaneously develops PAH with age. Prior data (Circulation113:2630 – 41, 2006) show that FHR have reduced expression of superoxide dismutase 2 (SOD2), decreased levels of reactive oxygen species (ROS) measured by chemiluminescence, and normoxic activation of hypoxia-inducible factor (HIF-1α)-a pseudohypoxic phenotype. Hypothesis: We hypothesized that ROS are decreased in FHR pulmonary arteries (PA) due to the loss of SOD2 expression. Methods and Results: We measured ROS using Electron Paramagnetic Resonance (EPR) and cell-permeable spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH), a cyclic hydroxylamine derivative. Expression of SOD2 was measured using quantitative PCR. HIF-1α activation was measured using nuclear translocation of the protein on confocal microscopy. ROS levels are 7-fold lower in the pulmonary arteries of FHR (n=3) comparing to weight-matched Sprague-Dawley rats (n=5). The demethylating agent, 5-aza-2′deoxycytidine not only restored expression of SOD2 in FHR pulmonary artery smooth muscle cells (PASMC) but also tended to increase ROS production (3.4-fold increase) in the FHR pulmonary arteries. This agent also decreased activation of HIF-1α as evidenced by a reduced presence of nuclear HIF-1α protein measured by immuno-fluorescence. Conclusions: This spin-probe technique is useful for the measurement of ROS in blood vessels. The data support the hypothesis that methylation-induced silencing of SOD2 accounts for the reduced ROS production in FHR. Loss of ROS signaling molecules activates HIF-1α and may initiate PAH. Since human PAH is also associated with activation of HIF-1α this research may have clinical translational significance. Clinically, decitabine (a commercial name for 5-aza-2′deoxycytidine) has been safely used to treat myeloproliferative disorders in humans.