Abstract 16728: Cigarette Smoke Impairs the Anti-Oxidative Response by Reducing Heme Levels and Inducing Bach1

Abstract

Introduction: Cigarette smoke is a major risk factor of cardiovascular diseases. Heme regulates the activity of enzymes and transcription factors including Bach1. Bach1 competes with Nrf2 transcriptional activity and inhibits the expression of anti-oxidative enzymes. However, the molecular mechanism of Bach1 activation and its impact on the anti-oxidative response after exposure to cigarette smoke is not clear. Hypothesis: We aim to understand the mechanism through which cigarette smoke impairs the anti-oxidative response. Methods: Primary human umbilical vein endothelial cells (HUVEC) were exposed to a cigarette smoke extract (CSE) (3 mg/ml nicotine) under static and unidirectional shear stress conditions. Shear stress was induced using the ibidi pump system and cone-and-plate viscometer. Llevels of reduced glutathione (GSH) were measured using the fluorescent probe o-phthalaldehyde. Heme levels were measured after oxalic acid extraction. Gene and miR expression was quantified by RT-qPCR. Immunofluorescence imaging was performed on fixed HUVEC. MiR-125b overexpression was achieved by transfecting mimics. Inhibition of Bach1 was done using siRNA transfection. Apoptosis was assessed using the caspase 3/7 assay. Human umbilical veins as an ex vivo model were exposed to CSE under static conditions. Results: Exposure to CSE induced the expression of BACH1 (1.90±0.18; p<0.05) and reduced total heme levels (0.30±0.04; p<0.001) in HUVEC. It also reduced miR-125b expression in HUVEC (0.39±0.09; p<0.001) and umbilical veins (0.72±0.12; p<0.05), whereas the expression of miR-125b target AhR repressor ( AHRR ) increased (14.93±4.08; p<0.05). Inhibiting BACH1 rescued miR-125b expression (1.02±0.45; p>0.05) and reduced AHRR expression (0.66±0.12; P<0.05). Acute exposure to CSE led to a high oxidative stress load. This observation was supported by a 2-fold reduction in glutathione levels, an increase in nuclear:cytosolic ratio of the upstream transcription factor Nrf2 (1.33±0.05; p<0.01) and an increase in expression of anti-oxidative HMOX1 (18.18±1.8; p<0.001) and NQO1 (16.12±1.99; p<0.001). CSE-induced apoptosis (1.23±0.04; p<0.001) was reversible by pre-conditioning with 1 mM N-acetyl cysteine (0.99±0.02; p>0.05) or overexpression of miR-125b (0.99±0.07; p>0.05). Conclusions: These results show that cigarette smoke-induced Bach1 impairs the anti-oxidative response through inhibiting miR-125b in the human endothelium. Reduced heme levels could be involved in the upregulation of Bach1 activity.