Our study showed that fullerenol induced pro-inflammatory activation of cultured HUVECs, which was indicated by the increase of CD54 (ICAM-1) and CD142 (TF) surface expression. The intercellular adhesive molecule 1 (ICAM-1), also referred to as CD54, is an adhesive receptor of the immunoglobulin gene superfamily. It is constitutively expressed at low levels on most types of vascular endothelial cells, and is highly upregulated on pro-inflammatory cytokine activated endothelium (Mutin et al 1997a
). Tissue factor (TF), also referred to as CD142, is a cellular receptor and cofactor of activated factor VIIa. Tissue factor initiates activation of the plasma coagulation system, which leads to the generation of thrombin activity (Steffel et al 2006
). Although functional TF is not exposed in significant levels on resting HUVECs, its expression is greatly increased after stimulation with pro-inflammatory cytokines and some other stimuli, similarly to ICAM-1 (Mutin et al 1997b
). In addition, the observed increased exposure of phosphatidylserine (PS) on the plasma membrane of HUVECs indicates a disturbance of the membrane asymmetry, likely due to pro-apoptotic stimulation or other types of cell activation. Although the exposure of PS on cells incubated with the fullerenol vehicle (the Milli-Q water) was higher than expected, the difference in PS-surface exposure between vehicle- treated and fullerenol-treated cells was highly significant (P < 0.001). PS is an essential cofactor for activation complexes in the plasma coagulation system. Also, it is a signaling molecule for phagocytes (Zwaal et al 2005
). Thus, our results show that the phenotype of endothelial cells treated with fullerenol become pro-inflammatory and pro-coagulant. It is important to further investigate whether fullerenes exhibit similar effects on the endothelium in vivo
in animal models.
Our cell cycle analysis showed that both nC60
induced G1 arrest in HUVECs. Polyhydroxylated fullerenol-1 was reported to inhibit the proliferative responses in a variety of cells, including smooth muscle cells and human lymphocytes, in a concentration dependent manner. The tested concentration range was of 10-6
M, which corresponds to approximately 1 μg/mL to 10 mg/mL. It was also shown that fullerenol inhibited cytosolic protein kinase C activity. Therefore, it was suggested that the anti-proliferative effect of fullerenol-1 on vascular smooth muscle cells may partly be mediated through the inhibition of protein tyrosine kinase (Lu et al 1998
We found that C60
induced apoptosis in HUVECs. This was clearly detected by TUNEL assay, which is a common method for detecting DNA fragmentation. In addition, the observed increase in PS exposure of fullerenol treated cells can be considered as supporting evidence of pro-apoptotic activation of HUVECs. Our finding contradicts the results of a recent study by Yamawaki and Iwai (Yamawaki and Iwai 2006
). These authors concluded that fullerenol did not cause apoptosis based on the failure to demonstrate cleavage of caspase 3 and PARP. However, their study showed autophagic cell death in fullerenol treated HUVECs. We may therefore speculate that both apoptotic and autophagic cell death may occur in fullerenol treated endothelium.
To investigate a possible mechanism of fullerene action on endothelium, we demonstrated that both nC60 and C60(OH)24 induced influx of extracellular Ca2+ in HUVECs. This suggests that fullerenes may either activate some of the Ca2+ channels on the cell membrane, or that they may create new channels/pores to facilitate Ca2+ influx, or both. In either case, it results in an increase in intracellular Ca2+, which may induce activation and/or apoptotic changes of the cells.
In conclusion, the results of this pilot study show possible adverse effects of fullerenes on the endothelium. The hydroxyfullerene C60(OH)24, had both pro-inflammatory and pro-apoptotic effects on endothelial cells. In addition, cell cycle arrest and increase of intracellular Ca2+ was observed in cells treated with hydroxyfullerene C60(OH)24 and also with a low concentration of nC60. With the wide potential applications of fullerenes in mind, the safety hazards of these materials need to be thoroughly evaluated. We suggest that additional in depth studies should be performed to investigate possible adverse effects of fullerenes on the cardiovascular system in exposed populations.