Figure 1. Chronic L-arginine supplementation promotes endothelial senescence, inflammation and eNOS-uncoupling.

Confluent young HUVECs were L-arginine-starved overnight followed by treatment with 0.1 mmol/L or 0.5 mmol/L L-arginine for 7 days (7 d) with change of medium every 48 hours. Cells were serum starved overnight prior to experiment. (A)SA-β gal assay. (B)Western blot analysis of ICAM-1/VCAM-1. Tubulin served as loading control. (C) Detection of O₂^.- and NO by DHE and DAF-2DA staining, respectively. The eNOS inhibitor L-NAME (1 mmol/L) was added to the cells 2 hours prior to the DAF-2DA staining. (D) Western blot analysis of eNOS protein levels with tubulin as loading control. The Western blot analysis of expression of ICAM-1, VCAM-1 and eNOS shown in (B) and (D), respectively, was performed with the same membrane. The quantification of the signals is presented as graphics below the corresponding images or blots. n represents the number of the repeated independent experiments. *p < 0.05 vs 0.1 mmol/L; #p < 0.05 vs 0.5 mmol/L. Scale bar = 200 μm