1 - 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. 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) under static and unidirectional shear stress conditions. Shear stress was induced using the ibidi pump system and cone-and-plate viscometer. Levels 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 caspase 3/7 activity. Human umbilical veins as an ex vivo model were exposed to CSE under static conditions. Results Exposure to CSE induced the expression of BACH1 and reduced total heme levels in HUVEC. It also reduced miR-125b expression in HUVEC and umbilical veins, whereas the expression of miR-125b target AhR repressor (AHRR) increased. Inhibiting BACH1 increased miR-125b expression and reduced AHRR expression. Acute exposure to CSE led to a high oxidative stress load. This observation was supported by a decrease in glutathione levels, an increase in nuclear:cytosolic ratio of the upstream transcription factor Nrf2 and an increase in expression of HMOX1 and NQO1. CSE-induced apoptosis was reversible by preconditioning with N-acetyl cysteine or overexpression of miR-125b. Conclusions cigarette smoke-induced Bach1 impairs the anti-oxidative response through inhibiting miR-125b. Reduced heme levels could be involved in the upregulation of Bach1 activity.