Adverse effects of fullerenes on endothelial cells: Fullerenol C(60)(OH)(24) induced tissue factor and ICAM-1 membrane expression and apoptosis in vitro

We studied the effects of a C(60) water suspension at 4 μg/mL (nC(60)) and the water soluble fullerenol C(60)(OH)(24) at final concentrations of 1(–)100 μg/mL on human umbilical vein endothelial cells (HUVECs) in culture. We found that a 24 hr treatment of HUVECs with C(60)(OH)(24) at 100 μg/mL significantly increased cell surface expression of ICAM-1(CD54) (67 ± 4% CD54(+) cells vs. 19 ± 2 % CD54(+) cells in control; p < 0.001). In addition, this treatment induced the expression of tissue factor (CD142) on HUVECs (54 ± 20% CD142(+) cells vs 4 ± 2% CD142(+) cells in control; p = 0.008) and increased exposure of phosphatidylserine (PS) (29 ± 2% PS(+) cells vs. 12 ± 5% PS(+) cells in control; p < 0.001). Analysis of cell cycle and DNA fragmentation (TUNEL) showed that both nC(60) and C(60)(OH)(24) caused G1 arrest of HUVECs and C(60)(OH)(24) induced significant apoptosis (21 ± 2% TUNEL(+) cells at 100 μg/mL of C(60)(OH)(24) vs. 4 ± 2% TUNEL(+) cells in control; p < 0.001). We also demonstrated that both nC(60) and C(60)(OH)(24) induced a rapid concentration dependent elevation of intracellular calcium [Ca(2+)](i). This could be inhibited by EGTA, suggesting that the source of [Ca(2+)](i) in fullerene stimulated calcium flux is predominantly from the extracellular environment. In conclusion, fullerenol C(60)(OH)(24) had both pro-inflammatory and pro-apoptotic effects on HUVECs, indicating possible adverse effects of fullerenes on the endothelium.