Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, PC188
Transforming growth factor-??1 enhances expression of oxidative stress proteins in human aortic adventitial fibroblasts
T. Mughal1, G. E. Mann1, R. C. Siow1
1. Cardiovascular Division, King's College London, London, United Kingdom.
Transforming growth factor-β1 (TGF-β1) enhances vascular remodelling through activation of adventitial fibroblast (AF) migration and proliferation [1,2], mediated in part by enhanced generation of reactive oxygen species (ROS) [3,4]. The transcription factor Nrf2 mediates induction of cytoprotective oxidative stress genes such as heme oxygenase-1 (HO-1) and NADPH:quinone oxidoreductase 1 (NQO-1) via activation of antioxidant response elements in their promoters . HO-1 catabolises the pro-oxidant heme to generate the vasodilator carbon monoxide and antioxidants biliverdin while NQO-1 acts as a phase II detoxification enzyme. To address whether TGF-β1 elicits oxidative stress responses in AF, cultured primary human aortic AF (Lonza, passage 4-8) were equilibrated (18 h) in medium containing 1% fetal calf serum prior to treatments (0-24 h) with either TGF-β1 (5 ng/ml), the hydrogen peroxide generator glucose oxidase (GOx, 2.5 mU/ml) or the electrophilic stress agent diethylmaleate (DEM, 100 μM). HO-1 and NQO-1 protein expression were determined by western blot analyses using specific primary antibodies (Santacruz). Protein bands were quantified by densitometry with α-tubulin used as a reference protein. Knock down of Nrf2 prior to treatments was achieved following transfection with Nrf2 specific siRNA (Santacruz) using FuGene (Roche). Treatment of cells with TGF-β1, GOx and DEM (4-8 h) elicited significant increases in HO-1 and NQO-1 expression (n=3, p<0.05, Student's unpaired t-test). Knock down of Nrf2 using specific siRNA, but not control scrambled siRNA, significantly attenuated the increases in HO-1 and NQO-1 protein levels in response to DEM treatment (8 h). These findings demonstrate for the first time that TGF-β1, GOx and DEM can elicit induction of oxidative stress response genes in human aortic AF mediated by Nrf2 transcriptional activity. Altered AF redox signalling elicited by TGF-β1 and ROS may thus contribute to enhanced vascular remodelling in atherosclerosis and restenosis through activation of AF and their phenotypic modulation to myofibroblasts.
Where applicable, experiments conform with Society ethical requirements