L-Hcy–increased O2− production in HCMECs. (A) Dose response. HCMECs were treated with L-Hcy at the indicated concentration for 48 hours and then treated with the indicated inhibitors for 60 minutes before the addition of DHE (3μM) for 30 minutes, including PEG-SOD (150 U/mL) and Tempol (1mM). (A-B) Photomicrographs of DHE-stained HCMECs. Images were acquired by 2-laser inverted confocal microscopy (Nikon TE3000). (C-D) Quantitative analysis of O2− production by DHE staining in panels A and B. Means ± SEM values from at least 3 separate experiments are shown. *P < .05 compared with untreated cells or vehicle in control mice. †P < .05 compared with vehicle in L-Hcy (500μM). HCMECs treated with angiotensin II (10μM) were used as a positive control. CT indicates the control group.
