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 Simak¹

¹CBER, FDA, Rockville, MD, USA; ²School of Medicine, USUHS, Bethesda, MD, USA; ³Nanotechnology Characterization Lab, Advanced Technology Program, SAIC-Frederick, Frederick, Maryland, USA

Abstract: 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 cytometry