Adverse effects of fullerenes on endothelial cells: Fullerenol C60(OH)24 induced tissue factor and ICAM-1 membrane expression and apoptosis in vitro

Monique P Gelderman1, Olga Simakova2, Jeffrey D Clogston3, Anil K Patri3, Sheena F Siddiqui1, Alexander C Vostal1, Jan Simak11CBER, FDA, Rockville, MD, USA; 2School of Medicine, USUHS, Bethesda, MD, USA; 3Nanotechnology Characterization Lab, Advanced Technology Program, SAIC-Frederick, Frederick, Maryland, USAAbstract: We studied the effects of a C60 water suspension at 4 µg/mL (nC60) and the water soluble fullerenol C60(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 C60(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 nC60 and C60(OH)24 caused G1 arrest of HUVECs and C60(OH)24 induced significant apoptosis (21 ± 2% TUNEL+ cells at 100 µg/mL of C60(OH)24 vs. 4 ± 2% TUNEL+ cells in control; p < 0.001). We also demonstrated that both nC60 and C60(OH)24 induced a rapid concentration dependent elevation of intracellular calcium [Ca2+]i. This could be inhibited by EGTA, suggesting that the source of [Ca2+]i in fullerene stimulated calcium flux is predominantly from the extracellular environment. In conclusion, fullerenol C60OH)24 had both pro-inflammatory and pro-apoptotic effects on HUVECs, indicating possible adverse effects of fullerenes on the endothelium.Keywords: endothelial cells, fullerenes, tissue factor, apoptosis, ICAM-1, flow cytometr