Effects of oxysterols on cell survival and proliferation pathways in human endothelial cells.

Oxidized low density lipoproteins (ox-LDLs) are involved in the pathogenesis of atherosclerosis because of their cytotoxic and proapoptotic effect on vascular cells. However a dual effect of ox-LDL on endothelial cells (EC) has been demonstrated: they induce a proliferative effect at concentrations below 50 ?g/mL and a proapoptotic effect at concentrations above 50 ?g/mL. The cytotoxic effect of ox-LDLs has been linked to their content in oxysterols, in particular to 7beta-hydroxycholesterol and 7-ketocholesterol. These molecules have been shown to be cytotoxic to several cell lines, including EC at concentrations above 40 ?g/mL. This study investigates the effects of 7beta-hydroxycholesterol and 7-ketocholesterol at concentrations ≤ 20 ?g/mL on the viability of human umbilical vein endothelial cells (HUVEC) and the pathways involved in their biological effects. Both 7beta-hydroxycholesterol and 7-ketocholesterol exhibited a dual effect on HUVEC viability inducing cell death at the concentration of 20 ug/mL and increasing cell viability at concentrations below 20 ug/mL. The increase in cell viability was associated with augmented cell proliferation characterized by increased [3H]-thymidine uptake after 48 hours of treatment. In the presence of increasing concentrations of basic fibroblast growth factor (bFGF) (1-5 ng/mL), vascular endothelial growth factor(VEGF) (5-10 ng/mL) or epidermal growth factor (EGF) (5-10 ng/mL), 7beta-hydroxycholesterol had an additive effect on the proliferation induced by the GF alone. Moreover 7beta-hydroxycholesterol protected HUVEC from apoptosis induced by two different stimuli: bFGF-deprivation and staurosporine (50 nM), as shown by the decrease in annexin V/PI binding and caspase-3 activation. These effects were completely abolished when HUVEC were pretreated with PD98059 (25 uM) or UO126 (25 uM), two specific MEK inhibitors. On the other hand pretreatment of HUVEC with increasing concentrations (1-5 uM) of the JNK inhibitor SP600125 did not affect the increase in cell viability induced by 7betahydroxycholesterol. These results show that 7beta-hydroxycholesterol at low concentrations (1-10 ?g/mL) has a proliferative and an antiapoptotic effect in HUVEC and that this effect is mediated by the activation of the MEK/ERK but not JNK pathway.