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

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 C₆₀(OH)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

Notes

The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.