Figure 7
Atorvastatin-induced CD59 and decay-accelerating factor in hypoxia enhance endothelial cell cytoprotection. (a) Human umbilical vein endothelial cells (HUVEC) were cultured under normoxic or hypoxic conditions with and without atorvastatin (0.25 μM) for 48 hours followed by 3 hours reoxygenation. Harvested endothelial cells (EC) were incubated with 20% C5-deficient (C5 D) serum (filled bars) or heat-inactivated (HI) normal human serum (NHS) (open bars) for 2 hours. C3 binding was analysed by flow cytometry and results are expressed as the percentage of C3 binding relative to that on EC exposed to C5 D in normoxia (shown as 100%). *P < 0.05 (n = 4), difference between levels of cell surface C3 deposition on EC cultured under hypoxic conditions in the presence or absence of atorvastatin (b) HUVEC were cultured under normoxic or hypoxic conditions with and without atorvastatin (0.5 μM) for 48 hours followed by 3 hours of reoxygenation. C9 binding was analysed by flow cytometry following incubation with 20% NHS (filled bars) or HI serum (open bars). Results are expressed as the percentage of C9 binding relative to that on EC exposed to NHS in normoxia (shown as 100%). *P < 0.05 (n = 4), difference between statin-treated and untreated EC in hypoxia.(c) HUVEC were incubated in 1% O2 with or without atorvastatin (At) 0.5 μM for 48 hours followed by 3 hours of reoxygenation. EC were preincubated with the inhibitory mAbs Bric229 (CD59) and 1H4 (decay-accelerating factor) (20 μg/ml) or veronal buffered saline + 1% gelatin at 4°C. EC were then incubated with 20% rabbit serum or 20% HI rabbit serum at 37°C for 1 hour and propidium iodide (PI) was added prior to analysis by flow cytometry. The percentage EC lysis was calculated as the number of PI-positive cells expressed as a percentage of the total number of cells. **P < 0.001 (n = 4), difference between statin-treated and untreated EC.