Effects of advanced glycation end products on caveolin-1 and endothelial nitric oxide synthase in endothelial cells

Abstract

Objective: Recent studies have suggested that advanced glycation end products (AGEs) may be one of the causes of endothelial dysfunction in diabetes mellitus. However, the underlying mechanism(s) are not fully understood. Since AGEs bind to the receptor for advanced glycation end products (RAGE) in caveolae, this study was performed to investigate whether AGEs influence endothelial nitric oxide synthase (eNOS) activity by affecting caveolin-1 which is involved in the post-translational regulation of eNOS. Methods: Caveolin-1 enriched membrane fractions were isolated from human aortic endothelial cells using a modified detergent-free extraction procedure. Isolated caveolae were subjected to Western blotting analyses with anti-caveolin-1, eNOS and RAGE antibodies. Nitric oxide (NO) production in response to AGEs was measured by colorimetric assay. Results: AGEs reduced NO production by endothelial cells in a dose-dependent manner despite no significant change in eNOS mRNA and protein in total cell lysate. Treatment with 200 mg/mL AGEs significantly increased protein levels of caveolin-1 (24.4%±3.7 vs. 13.5±5.6 in untreated control, p=0.03) and RAGE (30.0%±6.9 vs. 12.6±2.1, p=0.04) in caveolae compartment which was associated with a reduction in eNOS activity and NO production. This resulted in increased expression of adhesion molecules like vascular cell adhesion molecule-1 in the endothelial cells. Conclusion: Our results suggest that AGEs induce endothelial dysfunction by increasing caveolin-1 expression in endothelial cells and reduce eNOS activity and NO production. Whether the upregulation of caveolin-1 is mediated by the interaction of AGEs with RAGE warrants further investigations.