Abstract 282: 17β-Estradiol and 16α-Hydroxyestrone Increase Oxidative Stress Through Nrf2 Dysfunction In Human Pulmonary Artery Smooth Muscle Cells - Implications in Pulmonary Hypertension.

Abstract

Gender differences in pulmonary arterial hypertension (PAH) may be, in part, due to increased formation of the deleterious estrogen metabolite, 16α-hydroxyestrone (16αOHE1). Oxidative stress and Noxs have been implicated in the pathogenesis of PAH. We hypothesised that 17β-estradiol (E2) and 16αOHE1, specifically in human pulmonary artery smooth muscle cells (PASMCs), leads to Nox-induced oxidative stress, which promotes PASMC damage. Cultured human PASMCs were stimulated with either E2 (1nM) or 16αOHE1 (1nM). ROS production was assessed by chemiluminescence (O2-) and Amplex Red (H2O2); antioxidants, regulators of Nrf2, and PCNA (marker of growth) expression by immunoblotting; and Nrf2 activity by ELISA. E2 increased superoxide (219%) and H2O2 (52%) in PASMCs (p<0.05 vs vehicle). E2 induced ROS was blocked by PHTPP (ERβ antagonist), tempol (SOD mimetic), ML171 (Nox1 inhibitor) and GKT137831 (Nox1/4 inhibitor). Thioredoxin (71%), NQ01 (78%) and peroxiredoxin1 (69%) protein levels were decreased by E2, even though Nrf2 activity was increased (38%), p<0.05 vs vehicle. 16αOHE1 exhibited a rapid (5 min) and exaggerated increase in superoxide (355%) and a decrease in H2O2 (65%) production, p<0.05. 16αOHE1-induced ROS was blocked by MPP (ERα antagonist), G15 (GPR30 antagonist), tempol and ML171. 16αOHE1 increased BACH1 (129%; p<0.05), a competitor of Nrf2, which was decreased (92%). E2 stimulation resulted in decreased PCNA expression (30%), while 16αOHE1 increased PCNA levels (150%); p<0.05. E2 and 16αOHE1 induced a rapid and sustained ROS generation in PASMCs derived from PAH subjects. E2 and 16αOHE1 did not increase superoxide production in VSMCs from resistance arteries of healthy subjects. In conclusion, E2 induces ROS production through ERβ-Nox-dependent mechanisms, while 16αOHE1 increases superoxide through an ERα/GPR30-Nox-dependent manner. The effects of E2 and 16αOHE1 on oxidative stress is present in PASMCs but not in VSMCs from peripheral arteries. and seems to be related to a dysregulation of the Nrf2 pathway. These processes may impact on molecular processes contributing to vascular remodeling in PAH.