The effects of oxysterols on cell viability in vascular endothelial and smooth muscle cells: role of reactive oxygen species

Oxidized low density lipoproteins (oxLDLs) are involved in the pathogenesis of atherosclerosis and the cytotoxicity of oxLDLs has been linked to the formation of oxysterols (Schroepfer et al, 2000). 7-ketocholesterol (7-KC) and 7- hydroxycholesterol (7-OHC) are the major oxysterols found in oxLDLs. High concentrations (> 20 ?g/ml) of 7-KC and 7-OHC have been shown to induce apoptosis in human endothelial cells (Lizard et al, 1999). Preliminary results obtained in our laboratory have shown that 7-OHC and 7-KC, at concentrations below 20 ?g/mL, induce an increase in cell viability. Furthermore, it has been recently reported that oxLDLs induce proliferation and apoptosis in vascular cells, depending on the concentration and that both effects are mediated by superoxide formation (Galle, et al, 2001). In fact, besides the known cytotoxicity of reactive oxygen species (ROS), evidences have been accumulated indicating that these molecules are involved in several signal transduction pathways leading to antiapoptotic and proliferative effects (Haendeler et al, 2004). The aim of this study was to investigate the role of ROS on cellular effects of 7-KC and 7-OHC in endothelial and vascular smooth muscle cells. Treatment of human umbilical vein endothelialcells (HUVEC) and A7r5 (rat vascular smooth muscle cells) for 24 and 48 hours with 7-KC or 7-OHC (1-10 μg/mL) increased cell viability, while a cytotoxic effect was induced at 20 ?g/mL. Both oxysterols induced an increase in intracellular ROS production in a time dependent manner. ROS production by 7-OHC was partially dependent on NADPH oxidase. Analysis of phosphatidylserine translocation and caspase-3 activation in HUVEC treated with 7-OHC showed an antiapoptotic effect of the oxysterol against bFGF deprivation and staurosporine treatment. Incubation of HUVEC with the NADPH oxidase inhibitor hydralazine or with the antioxidant N-acetilcysteine was not able to prevent the antiapoptotic effect of 7-OHC. Although 7-OHC did not induce an increase in ERK activation, treatments of HUVEC with two different MEK inhibitors (PD98059, U0126) antagonized the protective effect of the oxysterol. The results show that 7-OHC, at concentration below 20 ?g/mL is antiapoptotic by a mechanism independent on ROS production and dependent on activation of MEK/ERK pathway.