The adverse vascular effects of multi-walled carbon nanotubes (MWCNTs) to human vein endothelial cells (HUVECs) in vitro: role of length of MWCNTs

Abstract

Background: Increasing evidences indicate that exposure to multi-walled carbon nanotubes (MWCNTs) could induce adverse vascular effects, but the role of length of MWCNTs in determining the toxic effects is less studied. This study investigated the adverse effects of two well-characterized MWCNTs to human umbilical vein endothelial cells (HUVECs).

Methods: The internalization and localization of MWCNTs in HUVECs were examined by using transmission electron microscopy (TEM). The cytotoxicity of MWCNTs to HUVECs was assessed by water soluble tetrazolium-8 (WST-8), lactate dehydrogenase (LDH) and neutral red uptake assays. Oxidative stress was indicated by the measurement of intracellular glutathione (GSH) and reactive oxygen species (ROS). ELISA was used to determine the release of inflammatory cytokines. THP-1 monocyte adhesion to HUVECs was also measured. To indicate the activation of endoplasmic reticulum (ER) stress, the expression of ddit3 and xbp-1s was measured by RT-PCR, and BiP protein level was measured by Western blot.

Results: Transmission electron microscopy observation indicates the internalization of MWCNTs into HUVECs, with a localization in nuclei and mitochondria. The longer MWCNTs induced a higher level of cytotoxicity to HUVECs compared with the shorter ones. Neither of MWCNTs significantly promoted intracellular ROS, but the longer MWCNTs caused a higher depletion of GSH. Exposure to both types of MWCNTs significantly promoted THP-1 adhesion to HUVECs, accompanying with a significant increase of release of interleukin-6 (IL-6) but not tumor necrosis factor α (TNFα), soluble ICAM-1 (sICAM-1) or soluble VCAM-1 (sVCAM-1). Moreover, THP-1 adhesion and release of IL-6 and sVCAM-1 induced by the longer MWCNTs were significantly higher compared with the responses induced by the shorter ones. The biomarker of ER stress, ddit3 expression, but not xbp-1s expression or BiP protein level, was significantly induced by the exposure of longer MWCNTs.

Conclusions: Combined, these results indicated length dependent toxic effects of MWCNTs to HUVECs in vitro, which might be associated with oxidative stress and activation of ER stress.

Keywords: Endoplasmic reticulum (ER) stress; Human umbilical vein endothelial cell (HUVEC); Multi-walled carbon nanobute (MWCNT); Oxidative stress; Vascular effect.

Fig. 1

The Raman spectra XFM22 (the shorter MWCNT) and XFM19 (the longer MWCNT)

Fig. 2

The TEM pictures of XFM22 (the shorter MWCNT; a and c) and XFM19 (the longer MWCNT; b and d) before sonication

Fig. 3

TEM images of XFM22 (the shorter MWCNT; a and b) and XFM19 (the longer MWCNT; c and d) exposed HUVECs. HUVECs were exposed to MWCNTs for 24 h, and TEM was used to indicate the internalization and localization of MWCNTs

Fig. 4

The cytotoxicity of human umbilical vein endothelial cells (HUVECs) after exposure to XFM22 (the shorter MWCNT) and XFM19 (the longer MWCNT). HUVECs were exposed to various concentrations of XFM22 and XFM19 for 24 h, and WST-8 (a), LDH (b) and neutral red uptake assay (c) were used to indicate the cytotoxicity of MWCNTs. *p < 0.05, compared with control; ^#p < 0.05, comparison between XFM22 and XFM19 at the same concentration; ANOVA

Fig. 5

Oxidative stress in human umbilical vein endothelial cells (HUVECs) after exposure to XFM22 (the shorter MWCNT) and XFM19 (the longer MWCNT). HUVECs were exposed to various concentrations of XFM22 and XFM19 for 24 h, and intracellular GSH (a) and ROS (b) were measured to indicate oxidative stress. *p < 0.05, compared with control; ^#p < 0.05, comparison between XFM22 and XFM19 at the same concentration; ANOVA

Fig. 6

The release of inflammatory cytokines in human umbilical vein endothelial cells (HUVECs) after exposure to XFM22 (the shorter MWCNT) and XFM19 (the longer MWCNT). HUVECs were exposed to various concentrations of XFM22 and XFM19 for 24 h, and the release of TNFα (a) and IL-6 (b) was measured by ELISA to indicate inflammatory response. *p < 0.05, compared with control; ^#p < 0.05, comparison between XFM22 and XFM19 at the same concentration; ANOVA

Fig. 7

The release of soluble adhesion molecules in human umbilical vein endothelial cells (HUVECs) after exposure to XFM22 (the shorter MWCNT) and XFM19 (the longer MWCNT). HUVECs were exposed to various concentrations of XFM22 and XFM19 for 24 h, and the release of sICAM-1 (a) and sVCAM-1 (b) were measured by ELISA to indicate endothelial activation. ^#p < 0.05, comparison between XFM22 and XFM19 at the same concentration; ANOVA

Fig. 8

The adhesion of THP-1 monocytes to human umbilical vein endothelial cells (HUVECs) that have been exposed to XFM22 (the shorter MWCNT) and XFM19 (the longer MWCNT). HUVECs were exposed to various concentrations of XFM22 and XFM19 for 24 h (a), and THP-1 monocyte adhesion to HUVECs was determined by using a fluorescent probe. To induce an ER stress like condition, an ER stress inducer thapsigargin (TG) was used to co-exposure HUVECs before adhesion assay (b). *p < 0.01, compared with control; ^#p < 0.05, comparison between XFM22 and XFM19 at the same concentration; ANOVA

Fig. 9

The protein level of BiP (GRP78) in human umbilical vein endothelial cells (HUVECs) after exposure to XFM22 (the shorter MWCNT) and XFM19 (the longer MWCNT). HUVECs were exposed to 0 or 32 μg/mL XFM22 and XFM19 for 24 h, and Western blot was used to determine the protein level of BiP with α-Tubulin as the internal control

Fig. 10

The mRNA level of ddit3 (a) and xbp-1s (b) in human umbilical vein endothelial cells (HUVECs) after exposure to XFM22 (the shorter MWCNT) and XFM19 (the longer MWCNT). HUVECs were exposed to 0 or 32 μg/mL XFM22 and XFM19 for 24 h, and quantitative real-time RT-PCR was used to determine the mRNA level of ddit3 and xbp-1s. The mRNA level of gapdh was used as the internal control. *p < 0.05, compared with control; ^#p < 0.01, comparison between XFM22 and XFM19 at the same concentration; ANOVA