Oxidized Low-density Lipoprotein Decreases the Expression of Endothelial Nitric Oxide Synthase

The atherogenic effects of low-density lipoprotein (LDL) may be mediated, in part, by its effect(s) on endothelial-derived nitric oxide (NO). To determine whether LDL can modulate NO production by changing NO synthase expression, we treated human saphenous vein endothelial cells with increasing concentrations of native or oxidized LDL (0-100 μg/ml) for various durations (0-72 h). Oxidized, but not native LDL caused a time-dependent decrease in steady-state NO synthase mRNA levels. This coincided with a maximal 56% decrease in NOS activity as determined by 3Harginine to 3Hcitrulline conversion. In the presence of actinomycin D, treatment with oxidized LDL reduced the half-life of NO synthase mRNA from 36 to 10 h. This decrease in NO synthase mRNA correlated with the degree of LDL oxidation and was attenuated by pretreatment with cycloheximide. Nuclear run-off studies showed a biphasic transcriptional pattern of NO synthase gene with an initial 25% decrease during the first 6 h followed by a maximal 2.2-fold increase over baseline during the subsequent 18 h. These results indicate that oxidized LDL regulates endothelial NOS expression through a combination of early transcriptional inhibition and post-transcriptional mRNA destabilization. The atherogenic effects of low-density lipoprotein (LDL) may be mediated, in part, by its effect(s) on endothelial-derived nitric oxide (NO). To determine whether LDL can modulate NO production by changing NO synthase expression, we treated human saphenous vein endothelial cells with increasing concentrations of native or oxidized LDL (0-100 μg/ml) for various durations (0-72 h). Oxidized, but not native LDL caused a time-dependent decrease in steady-state NO synthase mRNA levels. This coincided with a maximal 56% decrease in NOS activity as determined by 3Harginine to 3Hcitrulline conversion. In the presence of actinomycin D, treatment with oxidized LDL reduced the half-life of NO synthase mRNA from 36 to 10 h. This decrease in NO synthase mRNA correlated with the degree of LDL oxidation and was attenuated by pretreatment with cycloheximide. Nuclear run-off studies showed a biphasic transcriptional pattern of NO synthase gene with an initial 25% decrease during the first 6 h followed by a maximal 2.2-fold increase over baseline during the subsequent 18 h. These results indicate that oxidized LDL regulates endothelial NOS expression through a combination of early transcriptional inhibition and post-transcriptional mRNA destabilization.