Increased levels of reactive oxygen species (ROS) in the vessel wall contribute to atherogenesis and restenosis (Ross, 1999). The stress protein heme oxygenase-1 (HO-1) is induced by ROS and catabolises heme to generate the antioxidant biliverdin and vasodilator carbon monoxide, which protects against oxidative injury (Siow et al. 1999). Transforming growth factor-β1 (TGF-β1) promotes migratory and proliferative responses in smooth muscle cells (SMC) during vascular remodelling and can induce HO-1 expression (Kutty et al. 1994). We have investigated whether oxidative stress agents hydrogen peroxide, generated by glucose oxidase (GO), or diethylmaleate (DEM), an electrophilic agent that depletes glutathione, can induce TGF-β1 generation and HO-1 expression in SMC, and studied the effects of adenoviral overexpresion of smad7, an endogenous inhibitor of TGF-β1 signalling (Nakao et al. 1997).
Human aortic SMC obtained with local ethics approval were cultured from explants and transfected with adenoviruses co-ordinating expression of either smad7 or β-galactosidase as a control. Cells were then treated for 24 h with GO (10 mU ml-1) or DEM (100 µM). TGF-β1 production by SMC was determined by an enzyme-linked immunosorbant assay of the conditioned culture medium and HO-1, smad7 and phosphorylated smad2 protein expression by Western blot analysis.
Expression of HO-1 was markedly induced in SMC treated with the stress agents, concomitant with a significant (P < 0.01, Student’s unpaired t test) increase in medium TGF-β1 levels (n = 4, mean ± S.E.M., pg ml-1) from 120 ± 10 in control cells to 375 ± 18 and 280 ± 15 in DEM and GO-treated cells, respectively. Adenoviral overexpression of smad7, but not β-galactosidase, attenuated DEM or GO-mediated HO-1 induction, but did not alter TGF-β1 generation by SMC. Inhibition of TGF-β1 signalling in cells by smad7 overexpression was confirmed by a decrease in smad2 phosphorylation induced by recombinant TGF-β1. We have demonstrated for the first time that TGF-β1 signalling can modulate HO-1 induction by ROS, thus providing further insights into mechanisms involved in SMC dysfunction in vascular diseases.
This work was supported by the British Heart Foundation.