Suzuki et al. Particle and Fibre Toxicology (2016) 13:54
DOI 10.1186/s12989-016-0166-0

RESEARCH

Open AccessSingle- and double-walled carbon
nanotubes enhance atherosclerogenesis bypromoting monocyte adhesion to
endothelial cells and endothelial progenitor
cell dysfunction
Yuka Suzuki1, Saeko Tada-Oikawa1, Yasuhiko Hayashi2, Kiyora Izuoka1, Misa Kataoka1, Shunsuke Ichikawa1,
Wenting Wu3, Cai Zong3, Gaku Ichihara4 and Sahoko Ichihara1*

Abstract
Background: The use of carbon nanotubes has increased lately. However, the cardiovascular effect of exposure tocarbon nanotubes remains elusive. The present study investigated the effects of pulmonary exposure to single-walledcarbon nanotubes (SWCNTs) and double-walled carbon nanotubes (DWCNTs) on atherosclerogenesis using normal
human aortic endothelial cells (HAECs) and apolipoprotein E-deficient (ApoE−/−) mice, a model of human atherosclerosis.
Methods: HAECs were cultured and exposed to SWCNTs or DWCNTs for 16 h. ApoE−/− mice were exposed to SWCNTs
or DWCNTs (10 or 40 μg/mouse) once every other week for 10 weeks by pharyngeal aspiration.
Results: Exposure to CNTs increased the expression level of adhesion molecule (ICAM-1) and enhanced THP-1
monocyte adhesion to HAECs. ApoE−/− mice exposed to CNTs showed increased plaque area in the aorta by oil red O
staining and up-regulation of ICAM-1 expression in the aorta, compared with vehicle-treated ApoE−/− mice. Endothelial
progenitor cells (EPCs) are mobilized from the bone marrow into the circulation and subsequently migrate to
the site of endothelial damage and repair. Exposure of ApoE−/− mice to high-dose SWCNTs or DWCNTs
reduced the colony-forming units of EPCs in the bone marrow and diminished their migration function.
Conclusion: The results suggested that SWCNTs and DWCNTs enhanced atherosclerogenesis by promoting
monocyte adhesion to endothelial cells and inducing EPC dysfunction.
Keywords: Carbon nanotubes, Atherosclerosis, Endothelial cells, Adhesion, Endothelial progenitor cells

Background
Although humans have been exposed to airborne nanosized particles throughout their evolutionary stages, such
exposure has increased dramatically over the last century
due to anthropogenic sources. Engineered nanomaterials
and nanotechnologies are expected to have an impact onsociety and economy. There are also concerns that thesematerials may pose environmental and health risks dueto their unusual chemical and physical properties [1, 2].
* Correspondence: saho@gene.mie-u.ac.jp
1
Graduate School of Regional Innovation Studies, Mie University, 1577
Kurimamachiya-cho, Tsu 514-8507, Japan
Full list of author information is available at the end of the article

Thus, information about the safety and potential hazardsof nanomaterials is urgently needed, because various
engineered nanomaterials had already been incorporated
into various industrial processes and products [3].
Evidence based on epidemiological and toxicologicalstudies suggests that high concentrations of particles
measuring <2.5 μm in diameter (PM2.5) are associatedwith increased risk of pulmonary complications, cardiovascular events, and death from cardiovascular diseases[4–7]. Ambient particulate pollutants in the ultrafine
range have been also shown to enhance the early development of atherosclerosis [8]. It has been demonstrated thatcertain nanomaterials can generate reactive oxygen species© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Suzuki et al. Particle and Fibre Toxicology (2016) 13:54(ROS), resulting in induction of oxidative stress and inflammation [9, 10]. We have reported that zinc oxide(ZnO) nanoparticles can potentially enhance the migration
and adhesion of THP-1 monocytes to human umbilicalvein endothelial cells (HUVECs) and uptake of modified
LDL by THP-1 macrophages [11], suggesting that certain
nanoparticles can advance atherosclerogenesis.
Engineered carbon nanomaterials have many properties, such as large surface area, high electrical conductivity, and excellent strength. Carbon nanomaterials areused in many applications, such as electronic components and monitors, drug delivery, and hydrogen storage[12]. Carbon nanotubes are categorized as a single-layer;
single-walled carbon nanotubes (SWCNTs) and multiwalled carbon nanotubes (MWCNTs). The double-layer;
double-walled carbon nanotubes (DWCNTs) are a specific subset of MWCNTs. DWCNTs are suitable for use
as field-effect transistors and would be used for photoconversion and electrical energy storage that require
high technology [13]. Recent studies demonstrated that
exposure to SWCNTs is associated with increased ROS
production in cultured endothelial cells [14] and that exposure to MWCNTs have cytotoxic and genotoxic effects on HUVECs probably through oxidative damage[15]. However, the effects and the mechanisms of CNTs
on the cardiovascular system remain undefined.
Previous studies demonstrated the translocation andaccumulation of several types of nanoparticles in thebone marrow following their administration in laboratory animals [16, 17]. Since endothelial progenitor cells(EPCs) play an important role in facilitating vascular repair and tissue regeneration [18], we hypothesized thatnanomaterials have certain effects on the function of
EPCs. The present study investigated the effects of CNTs
on the adhesion of monocytes, which is an essential
process in atherosclerogenesis, using an in vitro set-up
of normal human aortic endothelial cells (HAECs) andhuman monocytic leukemia cells (THP-1). We also examined the effects of exposure to CNTs on the progression of atherosclerosis and analyzed their ex vivo role on
the function of isolated EPCs of bone marrow origin in
apolipoprotein E deficient (ApoE−/−) mice, a widely usedmodel of human atherosclerosis.

Page 2 of 11

Table 1 Physical characterization of CNTs
Materials

Hydrodynamic
size (nm)PdI

BET surface area
(m2/g)SWCNTs134.5 ± 1.886

0.270 ± 0.010

646.0 ± 4.688

DWCNTs128.6 ± 0.337*

0.218 ± 0.010*

527.5 ± 1.613*

Values are mean ± SD of 3–4 independent experiments
SWCNTs single-walled carbon nanotubes, DWCNTs double-walled carbon nanotubes, PdI polydispersity index*p < 0.05 vs. SWCNTs

agglomerated masses of CNTs were dispersed into
small-size clusters and CNTs bundles were separated
homogeneously by sonication (Fig. 1a). Transmission electron microscopy confirmed the presence of individual andbundled nanotubes in both sonicated CNTs (Fig. 1b).

Effects of CNTs on cell viability

HAECs were exposed to SWCNTs or DWCNTs at a concentration ranging from 0.1 to 20 μg/ml for 16 h. The cell
viability assay showed that incubation of HAECs in thepresence of SWCNTs or DWCNTs at 20 μg/ml reduced
cell viability (Fig. 2). When cell viability was determined
after incubation at the final concentrations of CNTs
ranged from 2.5 to 50 μg/ml for 16 h as a preliminary
study, exposure to 25 and more μg/ml of SWCNTs or

A
(a)

(b)

(c)

(d)

B

Results
Characterization of suspensions of CNTs

The intensity-weighted hydrodynamic average diameterof dispersed CNTs in the dispersion medium was measured by dynamic light scattering (DLS) technology. The
mean hydrodynamic diameter and polydispersity index(PdI) were significantly different between dispersed
SWCNTs and DWCNTs (Table 1). The Brunauer–
Emmett–Teller (BET) surface area of SWCNTs was significantly larger than that of DWCNTs (Table 1). Highly

Fig. 1 Optical microscope and TEM micrographs of CNT suspensions
at 1.0 mg/ml concentration. a Optical microscope before and after
dispersion by a cup-type sonicator at 100 W, 80 % pulse mode, for
10 min twice. b Transmission electron microscope (TEM) micrographsof SWCNTs and DWCNTs dispersed in the dispersion medium. The
areas, which include well-dispersed CNTs, were highlighted
within the dotted lines. SWCNT: single-walled carbon nanotube,
DWCNTs: double-walled carbon nanotube

Suzuki et al. Particle and Fibre Toxicology (2016) 13:54

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Effect on CNTs on expression of chemokine and adhesion
molecules

Fig. 2 Effects of SWCNTs and DWCNTs on cell viability. Cell viability
was measured by cell viability assay kit. HAECs were exposed to
CNTs at concentrations ranging from 0.1 to 20 μg/ml for 16 h. Data
are mean ± SD. (n = 8, *p < 0.05 vs. control; CTL) HAEC: normal
human aortic endothelial cell, SWCNT: single-walled carbon nanotube, DWCNT: double-walled carbon nanotube

DWCNTs reduced cell viability in a dose-dependent manner (Additional file 1: Figure S1).
Effects of CNTs on monocyte adhesion

We used the adhesion assay to test the effects of SWCNTs
or DWCNTs on the adhesion of THP-1 monocytes to
HAECs. The number of THP-1 cells that adhered to
HAECs was significantly increased in the presence of 1,
and 10 μg/ml of SWCNTs and 0.1, 1, and 10 μg/ml of
DWCNTs in a dose-dependent manner (Fig. 3).

A

Based on the above results, we examined the expressionof chemokines and integrin to determine their roles in
the adhesion of THP-1 monocytes. The expression of
monocyte chemotactic protein-1 (MCP-1) was significantly higher in THP-1 cells exposed to 10 μg/ml of
SWCNTs or DWCNTs than the control (Fig. 4a).
Furthermore, exposure to 10 μg/ml of DWCNTs resulted in upregulation of lymphocyte functionassociated antigen 1 (LFA-1) in THP-1 monocytes
compared with the control (Fig. 4b). We also examined the expression of adhesion molecules in HAECs.
Exposure to 10 μg/ml of SWCNTs or DWCNTs upregulated intracellular adhesion molecule 1 (ICAM-1)
expression in HAECs compared with the control(Fig. 4c, d).
Atherosclerogenesis in ApoE−/− mice

We assessed whether exposure to SWCNTs or DWCNTs
by pharyngeal aspiration induces atherosclerogenesis in
ApoE−/− mice. ApoE−/− mice were exposed chronically tothe dispersion medium or CNTs (SWCNTs or DWCNTs
at 10 or 40 μg/mouse) once every other week for 10 weeks.
After 10 weeks, lung weight was significantly higher in
ApoE−/− mice exposed to 40 μg DWCNTs than the control (Table 2). There were no significant differences inbody weight and weight of other organs (liver, kidney,spleen, and brain) between the groups. The percentage ofthe plaque area was determined. The extent of atherosclerosis was significantly larger in the thoracic aorta of

B

(a)

(b)

(c)
Fig. 3 Adhesion assay of THP-1 monocytes to HAECs. a Representative images of THP-1 monocyte adhesion to HAECs. Panel (a) represents image
under control conditions. (b) and (c) represent the adhesion of THP-1 cells to HAECs after exposure to 10 μg/ml of SWCNTs or DWCNTs for 16 h.
b Relative number of THP-1 monocytes adherent to HAECs after exposure to SWCNTs or DWCNTs (0.01- 10 μg/ml) for 16 h. Data are mean ± SD.
(n = 6,*p < 0.05 vs. control; CTL) SWCNT: single-walled carbon nanotube, DWCNT: double-walled carbon nanotube

Suzuki et al. Particle and Fibre Toxicology (2016) 13:54

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A

B

C

D

Fig. 4 Expression of MCP-1 and LFA-1 in THP-1 monocytes and ICAM-1 in HAECs. a MCP-1 and b LFA-1 relative mRNA expression levels in
THP-1 cells were determined 6 h after exposure to 0.1 or 10 μg/ml of SWCNTs or DWCNTs. Data are mean ± SD. (n = 8,*p < 0.05 vs. control; CTL) c
Representative images of western blot analysis of ICAM-1 in HAECs exposed to 0.1 or 10 μg/ml of SWCNTs or DWCNTs. d Relative expression
levels of ICAM-1 in HAECs exposed to SWCNTs or DWCNTs. Data are mean ± SD. (n = 6,*p < 0.05 vs. control; CTL) MCP-1: monocyte chemotactic
protein-1, LFA-1: lymphocyte function-associated antigen 1, ICAM-1: intracellular adhesion molecule 1, SWCNT: single-walled carbon nanotube,
DWCNT: double-walled carbon nanotube

ApoE−/− mice exposed to the high dose of SWCNTs or
DWCNTs than the control (Fig. 5a, b).
Based on the in vitro results of CNTs-enhanced adhesion of monocytes to endothelial cells, we examined theexpression of ICAM-1 in the aortas of ApoE−/− mice.
Exposure to the high dose of SWCNTs or DWCNTs significantly increased the expression level of ICAM-1 in
ApoE−/− mice than the control (Fig. 5c, d).
Effects of CNTs on number and function of EPCs

The atherosclerogenesis process is influenced by, in part,the number and functional status of EPCs [19]. Thus, we
examined the effects of CNTs on the number and function of EPCs, as expressed by the colony-forming units(CFU) and migration ability. The numbers of Flk-1/Sca1 positive cells in both the peripheral circulation andbone marrow were lower in ApoE−/− control mice thanwild-type control mice (Fig. 6a, b). Exposure to the highdose of SWCNTs and DWCNTs reduced the numbers of
Flk-1/Sca-1 positive cells in peripheral blood and bonemarrow of ApoE−/− mice, but there were no significant
differences between the groups (Fig. 6a, b). The numberof CFU was significantly higher in wild-type control micethan ApoE−/− control mice. Exposure to SWCNTs or

DWCNTs significantly reduced the number of CFU in
ApoE−/− mice (Fig. 6c). Furthermore, the number of
migratory EPCs in ApoE−/− control mice was lower
than in wild-type control mice, and exposure to
SWCNTs or DWCNTs significantly decreased thenumber of migratory EPCs in ApoE−/− mice (Fig. 6d).

Discussion
The present study demonstrated that SWCNTs and
DWCNTs enhanced the adhesion of THP-1 monocytes
to HAECs through the up-regulation of expression ofvarious adhesion molecules. We also demonstrated that
exposure to SWCNTs and DWCNTs increased atherosclerotic plaque progression in ApoE−/− mice. The results suggest that SWCNTs and DWCNTs enhanceatherosclerogenesis by promoting the adhesion of monocytes to endothelial cells and inducing EPCs dysfunction.
The present results showed that 20 μg/ml of SWCNTs
and DWCNTs each reduced HAECs cell viability. Previous studies reported that at 10 μg/ml or higher, MWCNTs
reduced HUVECs cell survival [15]. At lower concentrations (50 or 150 μg/106 cells, i.e., 1.5 or 4.5 μg/ml),
SWCNTs and MWCNTs also induced significant
LDH release which resulted in significant reduction inSuzuki et al. Particle and Fibre Toxicology (2016) 13:54

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Table 2 Body and organ weights of mice exposed to CNTs
C57BL/6 CTL

ApoE−/−CTL

SWCNTsDWCNTs

Low

High

Low

High

Body weight (g)26.3 ± 2.5

29.2 ± 2.3

28.3 ± 1.4

30.4 ± 1.5

29.6 ± 2.5

27.9 ± 2.6

Lung weight (g)0.16 ± 0.04

0.17 ± 0.02

0.18 ± 0.01

0.19 ± 0.02

0.20 ± 0.02

0.22 ± 0.01*

Liver weight (g)1.26 ± 0.12

1.40 ± 0.22

1.35 ± 0.10

1.43 ± 0.10

1.37 ± 0.20

1.35 ± 0.20

Kidney weight (g)0.16 ± 0.02

0.17 ± 0.02

0.16 ± 0.01

0.18 ± 0.02

0.17 ± 0.02

0.17 ± 0.02

Spleen weight (g)0.07 ± 0.01

0.08 ± 0.01

0.07 ± 0.01

0.09 ± 0.01

0.08 ± 0.02

0.09 ± 0.03

Brain weight (g)4.78 ± 0.10

4.74 ± 0.11

4.61 ± 0.07

4.67 ± 0.15

4.73 ± 0.10

4.72 ± 0.12

Values are mean ± SD of 5–7 independent experiments
SWCNTs single-walled carbon nanotubes, DWCNTs double-walled carbon nanotubes*p < 0.05 vs. control of ApoE−/− mice; CTL

cytotoxicity [20]. Recent studies described carboxylated MWCNTs-induced decrease in HUVECs viability,
associated with profound accumulation of autophagosomes [21]. Moreover, MWCNTs was also reported toincrease ROS production in HUVECs at 2–16 μg/ml

A[22] and in human microvascular endothelial cells(HMVECs) at 2.5 μg/ml [23]. These results suggestthat certain CNTs can induce damage of endothelial
cells. The use of CNTs at concentrations less than
10 μg/ml in our experiments was based on the above

B

D

C

Fig. 5 Plaque formation in the thoracic aorta of ApoE−/− mice. a Representative images of thoracic aortas stained with oil red O solution of ApoE−/−
mice exposed to SWCNTs or DWCNTs. b Plaque area in the thoracic aortas. c Representative images of western blot analysis of ICAM-1 in ApoE−/− micerepeatedly exposed to 40 μg of SWCNTs or DWCNTs. d Relative expression levels of ICAM-1 in the thoracic aorta of ApoE−/− mice. Data are mean ± SD.
(n = 5–7,*p < 0.05 vs. control of ApoE−/− mice; CTL) ICAM-1: intracellular adhesion molecule 1, SWCNT: single-walled carbon nanotube, DWCNT: doublewalled carbon nanotube

Suzuki et al. Particle and Fibre Toxicology (2016) 13:54

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A

C

B

D

Fig. 6 Number and function of EPCs in ApoE−/− mice exposed to SWCNTs or DWCNTs. a Representative flow cytometry plots of side scatter (SSC)and forward scatter (FSC) or FITC-Scan-1+/APC-Flk-1+ cells isolated from peripheral blood or bone marrow, b Relative number of Flk-1+/Sca-1+
cells in peripheral blood or bone marrow, c number of colony-forming units, and d number of migratory cells after exposure to 40 μg of SWCNTs
or DWCNTs. Data are mean ± SD. (n = 3,*p < 0.05 vs. control of ApoE−/− mice; CTL) SWCNT: single-walled carbon nanotube, DWCNT: doublewalled carbon nanotube

findings of significantly reduced HAECs cell viability
at 20 μg/ml.
SWCNTs and DWCNTs enhanced the adhesion of
THP-1 monocytes to HAECs in the present study. Furthermore, both CNTs significantly increased intracellularconcentrations of MCP-1 and LFA-1 in THP-1 monocytes. MCP-1 is known to play an important role in theearly recruitment of monocytes to atherosclerotic lesions[24] and LFA-1 is the main integrin in leukocytes and an
important molecule in firm adhesion and migration ofleukocytes to the inflammatory sites [25]. LFA-1 plays
pivotal roles as a signal transduction molecule by binding its ligand, namely, ICAM-1 [26]. In the present
study, CNTs also significantly up-regulated ICAM-1 expression in HAECs. These results are consistent withthose of previous studies that showed MWCNTs-

induced increase in ICAM-1 expression in endothelial
cells [22, 23]. ICAM-1 expression was also increased in
the aorta tissue of ApoE−/− mice exposed to SWCNTs
and DWCTs. Our results indicate that CNTs increased
intracellular concentration of LFA-1 in monocytes andICAM-1 in endothelial cells and induced adhesion of
monocytes to endothelial cells, which could be one of
the mechanisms responsible for the accelerated atherosclerogenesis induced by CNTs.
Our results demonstrated that SWCNTs and DWCNTs
increased the area stained with oil red in the thoracic
aorta in our mouse model of atherosclerosis. MWCNTs
have been shown to induce structural and functionalchanges in the endothelium of Sprague–Dawley rat modelof atherosclerosis [27]. Other studies in ApoE−/− mice on
high-fat diet also showed that instillation of SWCNTs

Suzuki et al. Particle and Fibre Toxicology (2016) 13:54

resulted in a slight increase in the plaque area in the aorta[28], and that ApoE−/− mice exposed to MWCNTs
showed accelerated plaque progression in the aorta tissue[22]. However, another study showed that these effects
were minimal [29]. Our results showed a significant increase in the plaque areas in mice treated with SWCNTs
or DWCNTs but there were no significant differences in
the plaque areas between the two CNTs. In this regard,
SWCNTs and MWCNTs are known to have direct effects
on endothelial cells and that these effects are dosedependent for both CNTs [20]. However, our study
showed that lower concentrations of DWCNTs, relative tothose of SWCNTs, induced significant increases in lungweight, THP-1 monocyte adhesion to HAECs and expression of adhesion molecules. Oxidative stress and inflammatory effects of MWCNTs were reported to be
associated with its surface area (BET) and length [30]. In
the present study, BET surface area of DWCNTs was significantly smaller than that of SWCNTs, while length of
DWCNTs was significantly longer than that of SWCNTs.
Therefore, it is difficult to determine which of surface areaor length is critical to biological responses of CNTs in thepresent study. Further studies are needed to understandthe cause(s) for the differences in the effects of SWCNTs
and DWCNTs on cardiovascular system.
EPCs represent one subset of progenitor cells that originate in the bone marrow and are mobilized to the circulation after birth. These cells play an importantrole in facilitating vascular repair and tissue regeneration [31, 32]. Previous clinical studies demonstrated
a clear association between reduced number or function of circulating EPCs and increased cardiovascularrisk [33, 34]. Furthermore, high concentrations of
PM2.5 were associated with high risk of cardiovascular
events and death from cardiovascular diseases [35]. Moreover, exposure to PM2.5 induced reversible vascular injury
by suppression of circulating EPC density in human [35].
Circulating EPCs were also decreased following exposure
to high levels of PM2.5 in mice [36]. Thus, exposure to air
pollutants seems to have the general property of reducing
circulating EPCs, but a significant reduction in EPC numbers was not observed in the present study following exposure to CNTs.
Given that several types of nanoparticles can translocate and accumulate in the bone marrow [16, 17], they
could be taken up by bone marrow-derived mononuclear
cells, thus explaining their direct effects on such cells.
Experiments in cultured cells showed that supermagnetic iron oxide nanoparticles impaired EPC migrationand promoted EPC adhesion [37]. We also demonstrated
recently that zinc oxide nano/micro particles suppressed
vasculogenesis in human endothelial colony-forming
cells [38]. Moreover, ex vivo functional assessments ofcultured EPCs from the bone marrow of mice exposed

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to nickel nanoparticle showed reduced EPC tube formation and chemotaxis [39]. We have not examined
whether CNTs were observed in the bone marrow or
not in the present study. However, the present study
demonstrated that SWCNTs and DWCNTs significantly
reduced the number of CFU and decreased the migration of EPCs in response to vascular endothelial growth
factor (VEGF) in ApoE−/− mice. Moreover, Patlolla et al.
[40] recently demonstrated the induction of oxidative
stress mediated genotoxicity in bone marrow collectedfrom the mice exposed to SWCNTs. Considered together, these results suggest that CNTs impair EPC functional activities. After vascular injury, EPCs are recruitedfrom the bone marrow to peripheral blood by VEGF.
Our findings suggest that CNTs enhanced atherosclerogenesis by, at least in part, reducing EPC function. However, the number of colony-forming units and migratory
cells were measured in bone marrow isolated from theonly three mice of each group. Further studies are needed
to identify the effects of CNTs on function of EPCs.
Nano-sized particles have a possibility to cross thepulmonary epithelial barrier and enter the bloodstream [41, 42]. After inhalation, MWCNTs translocated into the bloodstream and then accumulated inbody organs [43]. These results suggest that translocation to the peripheral circulation is a probablemechanism for the direct effect of these nanomaterials on the cardiovascular system. However, thepresent mice were exposed by pharyngeal aspiration
to CNTs one a week for 10 weeks. It is possible that
non-physiological phenomenon is induced by bolus
exposure to CNTs. Inhalation studies are ideally required to conclude the present results.
Our results showed significant increases in the plaquearea of mice exposed to SWCNTs or DWCNTs to thesame extent. However, some parameters, such as lungweight, THP-1 monocyte adhesion to HAECs, and expression of adhesion molecules, were significantly increased by exposure to DWCNTs at the concentration
lower than the concentration at which SWCNTs induced
the same effects. This difference between SWCNTs and
DWCNTs might be considered when establishing theexposure limit in occupational or environmental setting.

Conclusions
The present study investigated the effects of CNTs on
the adhesion of monocytes, an important process inatherosclerogenesis, using an in vitro set-up of HAECs
and THP-1 cells. We also examined the effects of CNTs
on atherosclerogenesis and analyzed their effects on thefunction of EPCs isolated from the bone marrow of
ApoE−/− mice, a model of human atherosclerosis. The
results suggested that SWCNTs and DWCNTs enhanced
atherosclerogenesis through the promotion of monocyte

Suzuki et al. Particle and Fibre Toxicology (2016) 13:54

Page 8 of 11

adhesion to endothelial cells and induction of EPCs
dysfunction.

cell proliferation was calculated as the percentage of inhibition of cell growth with respect to the controls.

Methods

Cell adhesion assay

CNTs preparation and characterization

Adhesion of THP-1 cells to HAECs was assessed as described in detail previously [13]. Briefly, HAECs (1.0 × 104
cells) were grown overnight in 96-well plates at 37 °C. The
cells were exposed to different concentrations (0.01, 0.1, 1,
or 10 μg/ml) of SWCNTs or DWCNTs for 16 h at 37 °C
and prior to the adhesion assay, washed three times with
Hank’s Balanced Salt Solution (HBSS) containing 0.1 %BSA. THP-1 cells were suspended at a density of 1.0 × 106
cells/ml of 0.1 % BSA/HBSS and labeled with 1 μM of
calcein-AM (BD Bioscience, Franklin Lakes, NJ) by
30 min incubation at 37 °C, followed by three washings
with 0.1 % BSA/HBSS. The labeled THP-1 cells were thenincubated with HAECs exposed to CNTs for 2 h at 37 °C.
Nonadherent cells were removed carefully by three-time
washings with 0.1 % BSA/HBSS. The adherence ofcalcein-labeled THP-1 cells was quantified by counting
the number of endothelial monolayers using a fluorescent
microscope (model FSX100, Olympus).

Single-walled carbon nanotubes (SWCNT; Nanocyl,
Sambreville, Belgium) with an average diameter of 2 nm
and length of several μm and double-walled carbon
nanotubes (DWCNT, Nanocyl) with an average diameterof 3.5 nm and length of 1–10 μm were used in thisstudy. CNTs were suspended in a dispersion medium
and dispersed using sonicator (BRANSON Sonifiermodel 450, Danbury, CT; 80 % pulsed mode, 100 W,
10 min, twice), as described previously [44]. The dispersion medium comprised Ca+2- and Mg+2-freephosphate-buffered saline (PBS, pH 7.4), supplemented
with 5.5 mM D-glucose and 0.6 mg/ml bovine serum albumin. The hydrodynamic sizes of the CNTs in themedium were measured four times after 1 h on standing
using the DLS technology with a Zetasizer Nano-S (Malvern Instruments, Worcestershire, UK). The dispersion
status was described by the intensity-weighted hydrodynamic average diameter (z-average) and PdI, which reflects the broadness of the size distribution (scale range
from 0 to 1, with 0 being monodispersion and 1 being
polydispersion) [45]. The BET surface area of CNTs wasmeasured three times using the surface area analyzer
with a BELSORP-mini II (Microtrac BEL, Osaka, Japan).
CNTs suspension was viewed using an Olympus BXJ1
optical microscope (Olympus, Tokyo, Japan) equippedwith a digital camera DP70, to capture images with the
DP controller software (Olympus). Dispersed CNTs werevisualized using a transmission electron microscope(TEM, JEM-1011; JEOL, Tokyo, Japan).
Cell culture

HAECs from Lonza Group (Basel, Switzerland) were
cultured in endothelial basal medium (EBM)-2 at 37 °C
in 5 % CO2. Experiments were performed using the cells
at passage 4 to 6. THP-1 cells from the American Type
Culture Collection (ATCC, Rockville, ML) were cultured
in RPMI 1640 medium (Life Technologies, Carlsbad,CA) containing 10 % FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), and 50 μM 2-mercaptoethanol at
37 °C in 5 % CO2.
Cell viability assay

HAECs were seeded at 1.0 × 104 cells per well on 96-wellplates overnight prior to the experiment. CNTs were dispersed in dispersion medium and the final concentrationsof CNTs ranged from 0.1 to 20 μg/ml. Cell viability wasdetermined after incubation of dispersed CNTs for 16 h as
indicated by the CellTiter-Glo™ Luminescent Cell Viability
Assay (Promega, Madison, WI). The effect of CNTs on

Measurement of expression of chemokines and adhesion
molecules

THP-1 cells were seeded at 2 × 105 cells/well onto 6-wellplates and exposed to 0.1 or 10 μg/ml of the dispersed
CNTs for 6 h. The cells were collected by centrifugation
at 1,000× rpm for 5 min at 4 °C. Total RNA from thecells was isolated by using ReliaPrep RNA cell miniprep
system according to the protocol provided by the manufacturer (Promega). The concentration of total RNA wasquantified by spectrophotometry (ND-1000; NanoDrop
Technologies, Wilmington, DE). RNA was reverse transcribed to single-strand cDNA using SuperScript III FirstStrand Synthesis System for RT-PCR (Life Technologies).
The cDNA was subjected to quantitative PCR analysis
with FastStart Universal Probe Master Mix (Roche, Basel,
Switzerland) and primers for MCP-1 and LFA-1 using an
ABI 7000 Real-Time PCR system (Life Technologies), as
described previously [38]. The gene expression level was
normalized to that of β-actin in the same cDNA.
For western blot analysis, HAECs were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitors (Santa Cruz, Dallas, TX). The
concentration of the extracted protein was measured intriplicate using the BCA Protein Assay Kit (Thermo
Fisher Scientific, Waltham, MA). Protein samples wereseparated by 12 % SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P,
Millipore, Billerica, MA). The membranes were incubated with a rabbit monoclonal antibody to ICAM-1(Abcam, Cambridge, MA) at a dilution of 1:500. Mouseanti-β-actin (ACTB) monoclonal antibody (Sigma-

Suzuki et al. Particle and Fibre Toxicology (2016) 13:54

Aldrich, St Louis, MO) at dilution 1:5,000 was used as aloading control. Immunoreactive bands were visualizedusing ECL-select chemiluminescence reagent (GEHealthcare–Amersham, Buckinghamshire, UK) and theintensity of the bands was quantified by Quantity One
v3.0 software (Bio-Rad Laboratories, Hercules CA). Protein expression levels were normalized relative to thelevel of β-actin protein in the same sample.
Animal studies

B6.129P2-Apoetm1Unc (ApoE−/−) mice were obtained
from Jackson Laboratory (Bar Harbor, ME). To evaluate
the effects of CNTs on atherosclerogenesis, ApoE−/− andwild-type mice (C57BL/6 J) (n = 5–7 in each group) were
exposed by pharyngeal aspiration to 10 or 40 μg of
SWCNTs or DWCNTs through multiple exposures(once a week) from 10 to 20 weeks of age (total amountadministered was 100 or 400 μg). Exposure to cumulative dose of 128 μg MWCNTs induced atherosclerosis in
one previous study, which examined plaque areas of
ApoE−/− exposed to MWCNTs, but exposure to cumulative dose of 640 μg MWCNTs did not in another study.
Therefore, the present study set 100 or 400 μg as a cumulative dose. Body weight was measured once a week.
All animal procedures were conducted in accordancewith the guidelines for the care and use of laboratory animals approved by Mie University.
Quantitative assessment of atherosclerosis

The thoracic aorta was harvested and fixed in PBS with
4 % paraformaldehyde and the adventitia was removed
under a microscope, as described in detail previously[46]. Then, the aortic arch and the thoracic aorta wereopened longitudinally, immersed for 1 min in 60 % isopropanol, and stained with oil red-O solution for 15 min
at 37 °C. All images were captured with a microscope
equipped with a camera (EZ4HD, Leica, Wetzlar,
Germany) and analyzed using Image J Software. Theedge of the aorta was traced using an automated featureand the extent of atherosclerosis was determined by
selecting threshold ranges in the three basic colors of
Image J Software. The total aortic surface area and thelesion area were then calculated. The extent of atherosclerosis was expressed as the percent of surface area of
the aorta covered by lesions.
Analysis of ICAM-1 production

The thoracic aorta was lysed in RIPA lysis buffer containing protease inhibitors. After measuring the concentrationof the extracted proteins, the protein samples were separated by 12 % SDS-PAGE and transferred onto PVDF
membranes. The membranes were incubated with rabbit
monoclonal antibody to ICAM-1 (Abcam) at a dilution of 1:500. Mouse anti-β-actin (ACTB) monoclonal

Page 9 of 11

antibody (Chemicon International, Billerica, MA) at
dilution 1:5,000 was used as the loading control. Immunoreactive bands were visualized using ECL-selectchemiluminescence reagent, as described above.
Measurement of number of EPCs in peripheral blood andbone marrow

Flow cytometry was applied for counting the number of
EPCs (Sca-1+ and Flk-1+) in peripheral blood and bonemarrow. Anticoagulated peripheral blood was obtained bydecapitation. Bone marrow cells were obtained by flushing
the tibias and femurs of mice with 2 % FBS/PBS. Next,
100 μl of peripheral blood or a volume of bone marrowsuspension containing 1 × 106 cells was immunolabeled with anti-Sca1-FITC (fluorescein isothiocyanateconjugated stem cell antigen-1; BD Pharmingen, Franklin,
NJ) and anti-Flk1-APC [allophycocyanin-conjugated fetalliver kinase-1 (VEGFR2, VEGF receptor 2), BD Pharmingen], as described previously [47]. Erythrocytes were lysedin FACS Lysing Solution (BD Pharmingen) and theremaining cells were analyzed by flow cytometry (FACS
Canto II, BD Biosciences).
Isolation of EPCs from bone marrow and colony-forming
assay

Low-density bone marrow mononuclear cells were isolated by density centrifugation Histopaque-1083 (SigmaAldrich). For analysis of endothelial cell-colony forming
units (EC-CFU), 2 × 106 bone marrow-derived mononuclear cells were isolated and sub-cultured for 7 daysin 20 % FBS/EBM-2 with supplements on human fibronectin pre-coated wells (including changing the culture
medium every second day), as described previously [44].
After 7-day culture, the adherent cells were identified as
EPCs by the uptake of 1,10-dioctadecyl-3,3,30,30-tetramethylindocarbocyanine-labeled acetylated LDL (DiLDL,
2.4 μg/mL; CellSystems, Troisdorf, Germany) and immunofluorescence staining of FITC-labeled Ulex europaeus agglutinin I (lectin, 10 μg/mL; Sigma-Aldrich).
The number of colonies per well was counted manually.
Migration assay

After counting the number of colonies, these cells wereused for migration assay, as described previously in detail [48]. Briefly, the cells were first trypsinized, and thenre-suspended in 20 % FBS/EBM-2. EPCs (2.0 × 103 cells/well) were placed on the upper chamber of Cell Culture
insert (8.0 mm pore size, 24-well plates; BD Falcon,
Franklin, NJ, n = 3). EBM-2 and recombinant murine
VEGF (50 ng/ml PeproTech, Rocky Hill, NJ) were harvested and used as the chemoattractant in the lower
chamber of Cell Culture inserts and incubated for 24 h at
37 °C 5 % CO2. Cells that had actively migrated throughthe membrane were fixed by 4 % paraformaldehyde andSuzuki et al. Particle and Fibre Toxicology (2016) 13:54

transmigration was quantified using a fluorescent microscope; FSX100 (Olympus).
Statistical analysis

All parameters were expressed as mean ± standard deviation (SD). Statistical analyses were performed using
one-way analysis of variance (ANOVA) followed byDunnett’s post hoc test. A p value less than 0.05 wasconsidered statistically significant.

Additional file
Additional file 1: Figure S1. Effects of SWCNTs and DWCNTs on cell
viability. (PPTX 70 kb)

Abbreviations
DWCNTs: Double walled carbon nanotubes; EPCs: Endothelial progenitor cells;
HAECs: Human aortic endothelial cells; ICAM1: Intracellular adhesion molecule 1;
LFA-1: Lymphocyte function-associated antigen-1; MCP-1: Monocyte
chemotactic protein-1; SWCNTs: Single walled carbon nanotubes
Acknowledgments
The authors thank Kumi Nakao for help in preparation of the manuscript andSatoshi Ogawa for help in electron microscope observation.
Funding
This work was supported in part by grants from the Japan Society for the
Promotion of Science (grants-in aid for Scientific Research #26293149,#15 K15236, and NEXT Program #LS056).
Availability of data and material
The data supporting our conclusions is included in the main body of themanuscript.
Authors’ contributions
YS performed the experiments, analyzed the data, and wrote the manuscript.
ST-O performed flow cytometry. YH and KI contributed to nanomaterial
characterization. MK and SI were involved in cell culture and sample preparation.
WW and CZ were involved in animal studies. GI reviewed the manuscript
and provided comments. SI designed the study, supervised the project, andcontributed to data interpretation and manuscript revision. All authors readand approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
No personal information is included in this study.
Ethics approval and consent to participate
All animal procedures described in this study were conducted in accordancewith the guidelines for the care and use of laboratory animals approved by
Mie University.
Author details
1
Graduate School of Regional Innovation Studies, Mie University, 1577
Kurimamachiya-cho, Tsu 514-8507, Japan. 2Graduate School of Natural
Science and Technology, Okayama University, Okayama, Japan. 3Departmentof Occupational and Environmental Health, Nagoya Univeristy Graduate
School of Medicine, Nagoya, Japan. 4Department of Occupational andEnvironmental Health, Tokyo Univeristy of Science, Noda, Japan.
Received: 28 June 2016 Accepted: 5 October 2016

Page 10 of 11

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