OBJECTIVES: We examined the hypothesis that low-density lipoprotein (LDL) that is both oxidized and glycosylated potently downregulates the expression of endothelial nitric oxide synthase III (NOSIII) in human coronary endothelial cells. BACKGROUND: Diabetes mellitus is accompanied by both oxidation and glycosylation of LDL, but the potential interaction of these processes or the pathophysiologic effects of these modified lipoproteins on arteries are poorly understood. METHODS: Low-density lipoprotein was glycoxidized in vitro, and Western and Northern blot analyses were used to investigate NOSIII expression in human coronary endothelial cells. Nitric oxide (NO) bioactivity was represented by both basal and bradykinin-stimulated cellular cyclic guanosine monophosphate accumulation and L-citrulline conversion from L-arginine. Nuclear run-on experiments were performed to study the transcription rate of nascent NOSIII messenger ribonucleic acid (mRNA). RESULTS: Data showed a significant decrease in NOSIII expression after 24-h treatment with glycosylated low-density lipoprotein (glycLDL) and oxidized low-density lipoprotein (ox-LDL). Accordingly, we observed a significant dose-dependent reduction in NO bioactivity (p <0.05 to p <0.001 vs. untreated cells, native low lipoprotein [nLDL], glycLDL, and oxLDL). Glyc-oxLDL did not reduce the half-life of NOSIII mRNA or significantly enhance L-citrulline conversion. Nuclear run-on experiments showed that high doses of glyc-oxLDL can reduce the transcription rate of nascent NOSIII mRNA (densitometric analysis revealed a reduction of 25% [p <0.05 vs. untreated cells, nLDL, and glycLDL] after treatment of cells with 300 μg/ml glyc-oxLDL). The effects of glyc-oxLDL are not related to the higher levels of oxidative compounds in comparison to those of oxLDL. CONCLUSIONS: These results indicate that glyc-oxLDL, per se, may influence signal transduction pathways involving NO-mediated regulatory signals and NOSIII activity in human endothelial cells. This phenomenon can adversely influence the evolution of clinical vascular complications, coronary heart disease, and atherogenesis in diabetic patients.
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