7-Ketocholesterol enhances leukocyte adhesion to endothelial cells via p38MAPK pathway
7-Ketocholesterol enhances leukocyte adhesion to endothelial cells via p38MAPK pathway
(grant no. 1
to MT) 1
Grant-in-Aid for Exploratory Research (grant no. 1
to MY 1
Astellas Pharma Inc (MT 1
Takeda Pharmaceutical Company Limited 1
Daiichi Sankyo Company Limited 1
MSD K.K. 1
Boehringer Ingelheim Japan 1
Mariko Tani 0 1
Yuko Kamata 1
Michiyo Deushi 1
Mizuko Osaka 0 1
Masayuki Yoshida 1
0 Department of Nutrition in Cardiovascular Disease, Tokyo Medical and Dental University , Yushima, Bunkyo-ku, Tokyo , Japan , 2 Department of Life sciences and Bioethics, Tokyo Medical and Dental University , Yushima, Bunkyo-ku, Tokyo , Japan
1 Editor: Partha Mukhopadhyay, National Institutes of Health , UNITED STATES
7-Ketocholesterol is a major dietary cholesterol oxidation product found in high concentrations in atherosclerotic plaques, which contribute to the development of atherosclerosis. This study aimed to investigate the effects of 7-ketocholesterol on endothelial inflammation, as well as the underlying mechanisms. Pretreatment of human umbilical vein endothelial cells (HUVEC) with 7-ketocholesterol significantly enhanced the total interactions between human monocytic cells (THP-1 cell line) and TNFα-activated HUVECs under physiological flow conditions, compared to pretreatment with cholesterol (TNFα+50 μM cholesterol: 13.1 ± 0.54 cells/CPF, TNFα+50 μM 7-ketocholesterol: 18.9 ± 0.35 cells/CPF, p < 0.01). 7-Ketocholesterol enhanced the expression of E-selectin, ICAM-1, and VCAM-1 proteins. It also activated p38 mitogen-activated protein kinase (MAPK), and treatment with a p38 MAPK inhibitor inhibited both E-selectin expression via ATF-2 activation and 7-ketocholesterolinduced THP-1 adhesion to HUVECs. These findings suggest that 7-ketocholesterol enhances leukocyte±endothelial interactions by upregulating the expression of adhesion molecules, presumably via the p38 MAPK-dependent pathway.
Data Availability Statement; All relevant data are within the paper
High levels of oxysterols, a product of cholesterol oxidation, are found in typical
cholesterolrich foods such as dairy, egg and meat products that have been heated or stored for long
periods (Br J Nutr 2002). Both dietary oxysterols and cholesterols are absorbed in the intestine by
the cholesterol transporter Niemann-Pick C1-like 1 (NPC1L1). 7-Ketocholesterol is a major
dietary oxysterol component. Specifically, this oxysterol is a product of cholesterol
autoxidation that forms via two known non-enzymatic mechanisms: singlet oxygen, which requires a
photosensitizing agent, and free radicals, which require a transition metal catalyst.
7-Ketocholesterol exhibits both proinflammatory and cytotoxic properties that lead to
atherosclerosis. Additionally, it enhances the expression of vascular endothelial growth factor
] and inflammatory cytokines . Several studies have also shown that
7-ketocholesterol decreases NO-induced vascular relaxation [3±5] and induces apoptosis in smooth muscle
AstraZeneca (MY). The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript. MSD
K.K. and Mitsubishi Tanabe Pharma Corporation
provided support in the form of salaries for author
MT, but did not have any additional role in the
study design, data collection and analysis, decision
to publish, or preparation of the manuscript. The
specific roles of these authors are articulated in the
`author contributions' section.
Competing interests: MSD K.K. and Mitsubishi
Tanabe Pharma Corporation provided support in
the form of salaries for author MT. Research grant
for this work was provided by Astellas Pharma Inc
(MT and MY), Takeda Pharmaceutical Company
Limited (MY), Daiichi Sankyo Company Limited
(MY), MSD K.K. (MY), Boehringer Ingelheim
Japan, Inc. (MY), and AstraZeneca (MY). The
sponsors had no control over the study design,
data collection, interpretation, writing, or
publication of this work. The authors have no
conflicts of interest directly relevant to the content
of this article. This does not alter our adherence to
PLOS ONE policies on sharing data and materials.
7-Ketocholesterol has also been detected at high concentrations in oxidized low-density
lipoprotein (LDL)  within high concentrations in atherosclerotic plaques, which contribute
to the development of atherosclerosis . Notably, patients with coronary artery disease have
significantly higher serum 7-ketocholesterol levels than those with normal coronary artery
The progression of atherosclerosis begins with interactions between leukocytes and
endothelial cells in a process known as endothelial inflammation . Continuous exposure to risk
factors induce endothelial cells to express adhesion molecules, such as intercellular cell
adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and members of
the selectin family (e.g., E-selectin) and also to secrete chemotactic substances that promote
leukocyte recruitment, adhesion, and transmigration into the vessel wall. Subsequently,
monocytes differentiate into macrophages and internalize modified lipoproteins, resulting in foam
cell formation [11±12].
To better understand the adverse effects of 7-ketocholesterol on atherogenicity, the present
study aimed to test the hypothesis that leukocyte-endothelial interactions mediated by this
oxysterol play a key role in this process and also to determine the underlying inflammatory
Materials and methods
7-Ketocholesterol (5-cholesten-3β-ol-7-one) was purchased from Sigma-Aldrich (St. Louis,
MO, USA). The chemical in powder form was dissolved in ethanol for the following
experiments. In atherosclerotic plaques, 7-ketocholesterol can reach concentrations exceeding
100 μM . A MTT assay was performed to evaluate cell viability within physiological
concentrations of this oxysterol (data not shown). Accordingly, 50 μM was identified as the
appropriate working dose (i.e., did not cause) excessive cytotoxicity for subsequent experiments. A
Micro BCA Protein Assay Kit was obtained from Thermo Fisher Scientific (Rockford, IL,
USA). RPMI-1640 medium was purchased from Wako (Tokyo, Japan). Fetal bovine serum
(FBS) and penicillin/streptomycin were obtained from GIBCO (Life Technologies, Grand
Island, NY, USA). The p38 mitogen-activated protein kinase (MAPK) phosphorylation
inhibitor SB203580 was purchased from Calbiochem (San Diego, CA, USA).
Human umbilical vein endothelial cells (HUVECs) (Lonza, Walkersville, MD, USA) were
cultured in RPMI-1640 medium supplemented with 20% fetal bovine serum (FBS), 10 ng/ml
human fibroblast growth factor (hFGF), 5 units/ml novoheparin, and 50 units/ml penicillin
and streptomycin at 37ÊC in an atmosphere containing 5% CO2. All experiments used
HUVECs from passages 3 and 4. The human monocytic cell line THP-1 was obtained from
the RIKEN CELL BANK (Tsukuba, Japan) and cultured in RPMI-1640 supplemented with 50
units/ml penicillin and streptomycin and 10% FBS.
Non-static monocyte adhesion assay
HUVECs were pretreated with 50 μM of cholesterol or 7-ketocholesterol or ethanol alone for
18 h and then stimulated with/without tumor necrosis factor (TNF)-α for 4 h. THP-1
monocytic cells were labeled with BCECF-AM (Merck KGaA, Darmstadt, Germany) via incubation
for 20 min at 37ÊC. Subsequently, HUVECs were incubated with labeled THP-1 cells on a flat
rotator (64 revolutions/min) for 10 min. The cells were then incubated with 1 ml of EDTA/
2 / 13
EGTA/HBSS(-) buffer for 3 min at room temperature to release adherent THP-1 cell±HUVEC
clusters. The collected THP-1 cells and HUVECs were lysed with 50 mM Tris±HCI + 0.1%
SDS (pH 8.0). The fluorescence intensity was measured using a spectrofluorometer at
excitation and emission wavelengths of 485 and 535 nm, respectively.
Adhesion assay under flow condition
The protocols of the adhesion assays conducted under flow conditions were previously
described in detail . Briefly, HUVEC monolayers grown on coverslips were pretreated
with 50 μM cholesterol or 7-ketocholesterol for 18 h, after which 0.1 ng/ml TNFα was added
prior to an additional 4-h incubation. Next, the HUVECs were positioned in a flow chamber
mounted on an inverted microscope (IX70, Olympus, Tokyo, Japan). The monolayers were
perfused with perfusion medium for 5 min, after which the THP-1 cells (106/mL) were drawn
through the chamber with a syringe pump (PHD2000, Harvard Apparatus) for 10 min at a
controlled flow rate to generate a shear stress of 1.0 dyne/cm2. The entire period of perfusion
was recorded on videotape, transferred to a personal computer and subjected to an image
analysis to determine the numbers of rolling and adherent THP-1 cells on the HUVEC monolayers
in 10 randomly selected 20 microscope fields.
Expression of adhesion molecules by western blotting
HUVEC were pretreated with 50 μM cholesterol or 7-ketocholesterol for 18 h, followed by
0.1 ng/ml TNF-α for an additional 4 h. Fresh total protein extracts were isolated from
HUVECs using RIPA lysis buffer supplemented with protease and phosphatase inhibitors.
The proteins were quantified using a DC™ Protein assay (BioRad, Hercules, CA, USA).
Except for E-selectin, lysates for all protein analyses were run on 10% polyacrylamide gels
under reducing conditions. Lysates for E-selectin were run under non-reducing conditions.
Protein extracts (10 μg) were transferred to PVDF membranes, which were and blocked
with 5% milk powder in 0.2% Tween-Tris-buffered saline (TBS) for non-phosphorylated
proteins or with 2% bovine serum albumin (BSA) in 0.2% Tween-TBS for phosphorylated
proteins. The membranes were incubated overnight at 4ÊC with primary antibodies against
E-selectin (clone 7A9), ICAM-1, and VCAM-1 (Santa Cruz Biotechnology, Dallas, TX,
USA). Immunoreactive proteins were detected using a luminol-based enhanced
chemiluminescence (ECL) kit (Thermo Scientific). All signals were detected on a LAS-1000 device
(Fujifilm, Tokyo, Japan), and Multi Gauge software version 3.0 was used to perform the
Real-time quantitative PCR for inflammatory cytokines
HUVECs were pretreated with cholesterol or 7-ketocholesterol for 18 h, followed 0.1 ng/ml
TNF-α for an additional 2 h. Total RNA was isolated with an RNeasy mini column kit (Qiagen,
Hilden, Germany). RNA purity and concentration were determined by measuring the
absorbances at 260 and 280 nm, respectively. cDNA was produced from 0.5 μg of RNA using a
PrimeScript RT-PCR reagent kit (TAKARA BIO Inc., Kyoto, Japan). Real-time quantitative
RT-PCR to quantitate the mRNA expression of IL-8, MCP-1 and 18rs in HUVECs was
performed using a Thermal Cycler Dice (TAKARA BIO Inc., Kyoto). Quantitative RT-PCRs used
a KAPA SYBR1 FAST universal qPCR Master Mix (2x) kit (Sigma-Aldrich) with 18rs as an
3 / 13
Effect of 7-ketocholesterol on NF-kB activity
HUVECs were treated with 50 μM cholesterol or 7-ketocholesterol in RPMI medium
supplemented with 1% FBS for the indicated time intervals, followed by stimulation with 0.1 ng/ml
TNF-α for an additional 15 min. Western blotting was performed as described above.
Antibodies against inhibitor of κB (IκB)-α, nuclear factor (NF)-kB p65 subunit and
phospho-NFkB p65 subunit were purchased from Santa Cruz Biotechnology.
The nuclear translocation of NF-kB was monitored by indirect immunofluorescence using
a monoclonal antibody specific for p65 (Santa Cruz Biotechnology). HUVECs were plated in a
fibronectin-coated Nunc Lab-Tek™II Chamber Slide™ System (Thermo Fisher Scientific). After
stimulation as described above, HUVECs were fixed with 4% paraformaldehyde at room
temperature for 10 min. After washing with PBS (+), HUVECs were blocked with 2% BSA in
PBS-Tween at room temperature for 30 min. Subsequently, HUVECs were incubated at room
temperature for 1 h with a primary antibody specific for the NF-kB p65 subunit diluted in 2%
BSA in PBS. A FITC-labeled goat anti-mouse antibody was used as the secondary antibody
(incubation: 30 min). The nuclei were stained with 0.1 μg/ml DAPI for 10 seconds before
mounting the cells on microscope slides. The cells were examined using an OLYMPUS IX70
Effect of 7-ketocholesterol on MAPK and ATF-2 activity
HUVECs were treated with 50 μM cholesterol or 7-ketocholesterol in RPMI medium
supplemented with 1% FBS for 30, 60 or 120 min, followed by stimulation with 0.1 ng/ml TNF-α for
an additional 15 min. Total proteins were isolated and analyzed by western blotting as
described above. Nuclear ATF-2 protein was extracted using a NE-PER Nuclear and
Cytoplasmic Extraction Kit (Thermo Scientific). Antibodies against p38 MAPK, phospho-p38
MAPK,ATF-2 (F2BR-1) and phospho-ATF-2 (F-1) were purchased from Santa Cruz
Biotechnology. Antibodies against c-jun N-terminal kinase (JNK) and phospho-JNK were purchased
from Cell Signaling (Danvers, MA, USA). An antibody specific for lamin A/C, was used as an
internal control of nuclear extraction (clone 14; Upstate Biotechnology, Waltham, MA, USA).
All results are expressed as means ± standard errors of the means (SEM), and differences
between groups were analyzed using a one-way analysis of variance (ANOVA), followed by
Tukey's test. Differences were considered significant at a p value < 0.05. Statistical analyses
were performed with GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA).
7-Ketocholesterol enhanced the adhesion of monocytes to HUVECs under
physiological flow conditions
To investigate the effects of 7-ketocholesterol on monocyte±endothelial interactions, we
performed an adhesion assay under non-static conditions. As shown in Fig 1A, TNF-α
stimulation significantly increased the adhesion of THP-1 cells to HUVECs, compared with
unstimulated cells. 7-Ketocholesterol significantly enhanced the adhesion of THP-1 cells to
HUVECs when compared with cholesterol. To further verify this phenomenon under more
physiological conditions, we performed an adhesion assay under laminar flow. The
pretreatment of HUVECs with 7-ketocholesterol significantly enhanced the total interaction of THP-1
cells with TNFα-activated HUVECs under flow conditions, compared to pretreatment with
cholesterol (TNFα+50 μM cholesterol: 13.1 ± 0.54 cells/CPF, TNFα+50 μM 7-ketocholesterol:
4 / 13
Fig 1. 7-Ketocholesterol enhances monocyte adhesion to human umbilical vascular endothelial cells (HUVECs).
HUVECs were pretreated with 50 μM 7-ketocholesterol or cholesterol or ethanol alone for 18 h, followed by
stimulation with or without 0.1 ng/ml tumor necrosis factor (TNF)-α for an additional 4 h. A non-static adhesion assay
was subsequently performed. Fluorescently labeled THP-1 cells were added to the HUVECs and allowed to adhere for
10 min under rotating conditions. Data are shown as means ± standard errors of the means (SEM). p < 0.01 by a
one-way analysis of variance (ANOVA) followed by Tukey's test. (B) A monocyte adhesion assay was performed under
laminar flow condition. Representative photos from three independent experiments are shown. (C) A monocyte
adhesion assay was performed under laminar flow condition. Cells were perfused over activated HUVEC monolayers
at a flow rate of 1.0 dyne/cm2 as described in the Materials and Methods. Adherent and rolling cells were counted as
described in the Materials and Methods. Data are representative of the results of three separate experiments and are
shown as means ± SEM. p < 0.01, p < 0.001 by one-way ANOVA followed by Tukey's test.
18.9 ± 0.35 cells/CPF, p < 0.01) (Fig 1B and 1C). Particularly, the numbers of rolling THP-1
cells on TNFα-activated HUVECs increased significantly in the presence of 7-ketocholesterol
(TNFα+50 μM cholesterol: 6.4 ± 0.88 cells/CPF, TNFα+50 μM 7-ketocholesterol: 10.3 ± 0.86
cells/CPF, p < 0.01) (Fig 1C).
7-Ketocholesterol increases the expression of adhesion molecules and
chemokines in HUVECs
To investigate the mechanism by which 7-ketocholesterol induces monocyte±endothelial
interactions, we evaluated the expression levels of adhesion molecules and inflammatory
chemokines in cell cultures. Western blotting analysis revealed that 7-ketocholesterol upregulated
5 / 13
Fig 2. 7-Ketocholesterol increases the expression of adhesion molecules and cytokines in human umbilical
vascular endothelial cells (HUVECs). (A) HUVECs were pretreated with 50 μM 7-ketocholesterol or cholesterol or
ethanol alone for 18 h, followed by stimulation with or without 0.1 ng/ml tumor necrosis factor (TNF)-α for an
additional 4 h. The levels of E-selectin, ICAM-1, and VCAM-1 protein expression were analyzed by western blotting.
Representative blots from three independent experiments are shown. (B) HUVECs were pretreated with 50 μM
7-ketocholesterol or cholesterol or ethanol alone for 18 h, followed by stimulation with or without 0.1 ng/ml TNF-α for
an additional 2 h. IL-8 and MCP-1 mRNA levels were analyzed by RT-qPCR. Data are shown as mean ± standard
errors of the means. p < 0.05, p < 0.01 by one-way analysis of variance followed by Tukey's test.
the expression of adhesion molecules, particularly E-selectin, on HUVECs (Fig 2A).
7-Ketocholesterol also enhanced the expression of IL-8 mRNA, but had no effect on MCP-1
expression (Fig 2B).
Effect of 7-ketocholesterol on TNF-α-induced NF-κB activity in HUVECs
To characterize the mechanisms by which 7-ketocholesterol enhances pro-inflammatory
responses, we next investigated whether 7-ketocholesterol induced the nuclear translocation of
the p65 subunit of NF-κB. HUVECs were treated with 7-ketocholesterol for 30 min to 2 h,
followed by TNF-α for an additional 15 min. The immunoblot results presented in Fig 3A show
that both the phosphorylation of NF-κB and the degradation of IκB-α, a suppressor of NF-κB,
increased slightly following exposure to 7-ketocholesterol. Furthermore, an
immunofluorescence microscopy assay showed that p65 translocated from the cytoplasm to the nucleus (Fig
3B). These results suggest that 7-ketocholesterol partially enhances NF-κB activation in
HUVECs by suppressing the degradation of IκB and nuclear translocation of NF-κB.
Effect of 7-ketocholesterol on TNFα-induced MAPK pathway activity in
To further elucidate the molecular targets of 7-ketocholesterol in inflammatory signaling
pathways, we examined the effects of 7-ketocholesterol on the activities of MAPKs such as JNK and
p38 MAPK, which regulate the induction of several genes encoding inflammatory factors. As
indicated in Fig 4A, the stimulation of HUVECs with 7-ketocholesterol enhanced the
TNFα6 / 13
Fig 3. Effect of 7-ketocholesterol on tumor necrosis factor (TNF)-α-induced nuclear factor (NF)-kB activity in
human umbilical vascular endothelial cells (HUVECs). (A) HUVECs were treated with 50 μM cholesterol or
7-ketocholesterol for each indicated time point, followed by stimulation with 0.1 ng/ml TNF-α for an additional 15 min.
IκB-α, NF-kB p65 and phospho-NF-kB p65 protein expression was analyzed via western blotting as described in the
Materials and Methods. Representative blots from three independent experiments are shown. (B) 7-Ketocholesterol
stimulates the nuclear translocation of the p65 subunit. Immunofluorescent images depict HUVECs stained with a
mouse monoclonal antibody specific for p65 (green) and FITC-labeled goat anti-mouse secondary antibody. Nuclei were
stained with 0.1 μg/ml DAPI (red) for 10 seconds before the cells were mounted on microscope slides. Representative
merged photos from three independent experiments are shown.
induced activation of p38 MAPK but not JNK, with peak MAPK phosphorylation occurring
30±120 min after the addition of 7-ketocholesterol; no changes were seen in the levels of
corresponding unphosphorylated proteins. As shown in Fig 4B, although 7-ketocholesterol
enhanced the ability of TNFα to induce THP-1 cell adhesion to HUVECs, this effect was
7 / 13
Fig 4. Effects of 7-ketocholesterol on tumor necrosis factor (TNF)-α-induced mitogen-activated protein kinase
(MAPK) activity in human umbilical vascular endothelial cells (HUVECs). (A) HUVECs were treated with 50 μM
cholesterol or 7-ketocholesterol for each indicated time interval, followed by stimulation with 0.1 ng/ml TNF-α for an
additional 15 min. Western blotting was used to evaluate the levels of p38, phospho-p38, JNK and phospho-JNK
proteins as described in the Materials and Methods. Representative blots from three independent experiments are
shown. (B) HUVECs were pretreated with 50 μM of cholesterol or 7-ketocholesterol for 18 h, followed by incubation
with 5 μM p38MAPK inhibitor (SB203580) for 30 min and stimulation with TNF-α for an additional 4 h. A non-static
adhesion assay was performed. Fluorescently labeled THP-1 cells were added to the HUVECs and allowed to adhere
for 10 min under rotating conditions. Data are shown as means ± standard errors of the means (SEM). p < 0.05 by a
one-way analysis of variance followed by Tukey's test. (C) HUVECs were pretreated with 50 μM of cholesterol or
7-ketocholesterol for 18 h, incubated with 5 μM p38 MAPK inhibitor (SB203580) for 30 min and stimulated with TNF
α for an additional 4 h. Western blotting was used to evaluate the expression of E-selectin, ICAM-1, and VCAM-1
proteins as described in the Materials and Methods. Representative blots from three independent experiments are
significantly reduced in the presence of a p38 MAPK inhibitor (SB203580). Treatment with
SB203580 also inhibited the dramatic upregulation of E-selectin induced by 7-ketocholesterol
and TNFα. These data suggest that the p38 MAPK activation pathway plays a significant role
in 7-ketocholesterol-enhanced inflammatory responses.
To improve our understanding of how 7-ketocholesterol-dependent mechanisms
contribute to the regulation of E-selectin expression, we undertook a detailed analysis of
transcriptional responses in HUVECs. E-selectin expression is mediated by activation of the
transcription factor activation transcription factor 2 (ATF-2) and principally involves
p38MAPK . A western blotting analysis of nuclear extracts from HUVECs revealed that
7-ketocholesterol enhanced the phosphorylation of ATF-2; additionally, this activation was
significantly inhibited by p38MAPK inhibitor treatment (Fig 5).
8 / 13
Fig 5. Effects of 7-ketocholesterol on ATF-2 activity in human umbilical vascular endothelial cells (HUVECs).
HUVECs were pretreated with 50 μM of cholesterol or 7-ketocholesterol for 18 h, incubated with 5 μM p38MAPK
inhibitor (SB203580) for 30 min and stimulated with tumor necrosis factor (TNF)-α for an additional 4 h. Western
blotting was used to evaluate the nuclear levels of phospho-ATF-2 and ATF-2 as described in the Materials and
Methods. Representative blots from three independent experiments are shown.
Leukocyte±endothelial interactions have been recognized as crucial factors in the development
of atherosclerosis and subsequent cardiovascular disease. 7-Ketocholesterol is a major dietary
oxysterol and component of the non-enzymatic cholesterol oxidation pathway, along with
singlet oxygen and free radical oxidation [16±17]. The present study demonstrates that
7-ketocholesterol significantly enhances leukocyte±endothelial interactions. The adhesion of monocytes
to endothelial cells is responsible for monocyte infiltration and subsequent differentiation into
macrophages, which contribute to the formation of atherosclerotic lesions. These results are
consistent with the observed accumulation of 7-ketocholesterol in plaque areas, which leads to
plaque destabilization and rupture [8,18,19].
The initial adhesive interactions between leukocytes and the endothelium, known as
capturing and rolling, are subsequently enhanced by leukocyte activation. Consequently, leukocytes
attach to the endothelium and remain stationary. First, we confirmed that 7-ketocholesterol
enhances the adhesion of THP-1 cells to stimulated HUVECs in a non-static adhesion assay.
Interestingly, we found that 7-ketocholesterol significantly increased not only the numbers of
THP-1 cells that firmly adhered to the HUVECs, but also the numbers of ªrollingº THP-1 cells
under more physiological conditions. These data suggest that 7-ketocholesterol causes
inflammation at a very early stage of atherosclerosis. Lectin-like adhesion glycoproteins, such as
Eselectin, mediate leukocyte rolling, while the firm adhesion and subsequent transendothelial
migration of leukocytes are mediated by the interactions of integrins (CD11/CD18, VLA-4) on
leukocytes with immunoglobulin-like adhesion molecules on endothelial cells (e.g., ICAM-1,
VCAM-1) . Previous studies have shown that 7-ketocholesterol induced the expression of
ICAM-1 and VCAM-1 on human endothelial cells [
]. In this study, 7-ketocholesterol
dramatically induced the expression of E-selectin on HUVECs, which is responsible for the
increase in THP-1 cell rolling. Interestingly the bands of adhesion molecules in
7-ketocholesterol-treated HUVEC were reproducibly not in line with other bands under this condition. The
9 / 13
Fig 6. Schematic representation of the signaling pathways involved in the 7-ketocholesterol-induced
leukocyteendothelial interactions. 7-ketocholesterol induces E-selectin expression mediated by ATF-2 and involves the
p38MAPK activation pathway, which together increase the number of leukocyte interaction to endothelial cells.
possible reason is that 7-ketocholesterol may induce post-transcriptional modification of those
adhesion molecules or other structural changes, which may imply an important biological
consequence of 7-ketocholesterol treatment.
Additionally, we demonstrated the effects of 7-ketocholesterol on inflammatory chemokine
expression in endothelial cells. Notably, this oxysterol enhanced the expression of IL-8 mRNA
in HUVECs. IL-8 can rapidly cause rolling monocytes to adhere firmly to monolayers
expressing E-selectin [
]. By contrast, we did not observe significant changes in the mRNA
expression of MCP-1, which is involved in monocyte recruitment .
Previous studies have described inflammatory pathways involving 7-ketocholesterol in
various cell types and have implicated different inflammatory pathways [25±27]. The expression of
genes encoding adhesion molecules is regulated by various signaling molecules, including
] and MAPK. Several studies revealed that 7-ketocholesterol-induced inflammatory
signals involve the TLR4-mediated NF-kB signaling pathway [
]. First, we speculated that
the NF-κB pathway would mediate the upregulation of adhesion molecules in response to
7-ketochlesterol in cytokine-activated endothelial cells. We found that 7-ketocholesterol
partially enhanced the activation of NF-κB in HUVECs by suppressing the degradation of IκB
and the nuclear translocation of NF-κB. Dixon et al. suggested that several potent inducers of
10 / 13
these transcriptional factors increase E-selectin transcription and expression without requiring
significant NF-κB involvement . Taken together, these data suggest that NF-kB activation
may influence but does not fully control the 7-ketocholesterol-induced inflammatory pathway.
To further our understanding of the molecular targets of 7-ketocholesterol in inflammatory
signaling pathways, we examined the effects of 7-ketocholesterol on the activity of MAPKs
such as c-JNK and p38 MAPK, which regulate the induction of several genes encoding
inflammatory factors. The stimulation of HUVECs with 7-ketocholesterol induced the
phosphorylation of p38 MAPK but not c-JNK. Similar results have been observed in
7-ketocholesterolinduced intestinal inflammation [
]. Huang et al. reported that a JNK inhibitor (SP600125)
did not suppress any of the inflammatory markers activated by 7-ketocholesterol [
Therefore, we focused on the activation of p38 MAPK, rather than JNK, to verify the mechanism by
which 7-ketocholesterol affects inflammatory signaling. The increase in TNFα-induced THP-1
cell adhesion to HUVECs as mediated by 7-ketocholesterol was significantly decreased by the
inhibition of p38 MAPK. The dramatic expression of E-selectin on response to
7-ketocholesterol was also blocked by p38 MAPK inhibition. These data indicate that the p38 MAPK
activation pathway plays a significant role in the inflammatory responses enhanced by
Recent studies have clarified a regulatory role for ATF-2 in inflammation [
]. ATF-2 is
known as the transcriptional regulator of the E-selectin promoter . We found that
7-ketocholesterol enhanced the phosphorylation of ATF-2 in nuclear extracts from HUVECs, and
that this phenomenon was significantly inhibited by treatment with a p38 MAPK inhibitor.
Taken together, these results suggest that the process by which 7-ketocholesterol induces
Eselectin expression is mediated by ATF-2 and involves the p38 MAPK activation pathway,
which together increased the number of THP-1 cells rolling on HUVECs (Fig 6).
In conclusion, 7-ketocholesterol enhances leukocyte±endothelial interactions by
upregulating the expression of adhesion molecules, presumably via a p38 MAPK-dependent pathway.
This finding suggests that a large dietary oxysterol intake or oxysterol accumulation may be a
risk factor for atherosclerosis development.
Conceptualization: Mariko Tani, Masayuki Yoshida.
Formal analysis: Mariko Tani.
Funding acquisition: Mariko Tani, Mizuko Osaka, Masayuki Yoshida.
Investigation: Mariko Tani, Yuko Kamata, Michiyo Deushi.
Project administration: Mariko Tani, Masayuki Yoshida.
Supervision: Masayuki Yoshida.
Visualization: Mariko Tani.
Writing ± original draft: Mariko Tani.
Writing ± review & editing: Mariko Tani, Mizuko Osaka, Masayuki Yoshida.
11 / 13
Lemaire S, Lizard G, Monier S, Miguet C, Gueldry S, Volot F, et al. Different patterns of IL-1beta
secretion, adhesion molecule expression and apoptosis induction in human endothelial cells treated with
7alpha-, 7beta-hydroxycholesterol, or 7-ketocholesterol. FEBS Lett. 1998; 440(3):434±9., PMID:
Deckert V, Duverneuil L, Poupon S, Monier S, Le Guern N, Lizard G, et al. The impairment of
endothelium-dependent arterial relaxation by 7-ketocholesterol is associated with an early activation of protein
kinase C. Br J Pharmacol. 2002; 137(5):655±62. https://doi.org/10.1038/sj.bjp.0704920 PMID:
Sung SC, Kim K, Lee KA, Choi KH, Kim SM, Son YH, et al. 7-Ketocholesterol upregulates interleukin-6
via mechanisms that are distinct from those of tumor necrosis factor-alpha, in vascular smooth muscle
cells. J Vasc Res. 2009; 46(1):36±44. https://doi.org/10.1159/000135663 PMID: 18515973
Lemaire-Ewing S, Berthier A, Royer MC, Logette E, Corcos L, Bouchot A, et al.
7beta-Hydroxycholesterol and 25-hydroxycholesterol-induced interleukin-8 secretion involves a calcium-dependent
activation of c-fos via the ERK1/2 signaling pathway in THP-1 cells: oxysterols-induced IL-8 secretion is
calcium-dependent. Cell Biol Toxicol. 2009; 25(2):127±39. https://doi.org/10.1007/s10565-008-9063-0
Nishio E, Watanabe Y. Oxysterols Induced Apoptosis in Cultured Smooth Muscle Cells through CPP32
Protease Activation and bcl-2 Protein Downregulation. Biochem. Biophys. Res. Commun., 1996; 226
(3): 928±34. https://doi.org/10.1006/bbrc.1996.1452 PMID: 8831713
Garcia-Cruset S, Carpenter KL, Guardiola F, Stein BK, Mitchinson MJ. Oxysterol profiles of normal
human arteries, fatty streaks and advanced lesions. Free Radic Res. 2001; 35: 31±41. PMID:
Sato K, Nakano K, Katsuki S, Matoba T, Osada K, Sawamura T, et al. Dietary Cholesterol Oxidation
Products Accelerate Plaque Destabilization and Rupture Associated with Monocyte
Infiltration/Activation via the MCP-1-CCR2 Pathway in Mouse Brachiocephalic Arteries: Therapeutic Effects of
Ezetimibe. J Atheroscler Thromb. 2012; 19(11):986±98 PMID: 22785139
Hitsumoto T, Takahashi M, Iizuka T, Shirai K. Clinical significance of serum 7-ketocholesterol
concentrations in the progression of coronary atherosclerosis. J Atheroscler Thromb. 2009; 16(4):363±70.
Ross R. AtherosclerosisÐan inflammatory disease. N Engl J Med. 1999; 14; 340(2):115±26. https://
doi.org/10.1056/NEJM199901143400207 PMID: 9887164
Libby P. Current concepts of the pathogenesis of the acute coronary syndromes. Circulation. 2001; 17;
104(3):365±72. PMID: 11457759
Cybulsky MI, Iiyama K, Li H, Zhu S, Chen M, Iiyama M, et al. A major role for VCAM-1, but not ICAM-1,
in early atherosclerosis. J Clin Invest. 2001; 107(10):1255±62. https://doi.org/10.1172/JCI11871 PMID:
van Reyk DM, Brown AJ, Hult'en LM, Dean RT, Jessup W. Oxysterols in biological systems: sources,
metabolism and pathophysiological relevance. Redox Rep. 2006; 11:255±262 https://doi.org/10.1179/
135100006X155003 PMID: 17207307
Yoshida M, Sawada T, Ishii H, Gerszten RE, Rosenzweig A, Gimbrone MA Jr, et al. Hmg-CoA
reductase inhibitor modulates monocyte-endothelial cell interaction under physiological flow conditions in
vitro: involvement of Rho GTPase-dependent mechanism. Arterioscler Thromb Vasc Biol. 2001; 21
(7):1165±71. PMID: 11451746
Jacobsen MC, Dusart PJ, Kotowicz K, Bajaj-Elliott M, Hart SL, Klein NJ, et al. A critical role for ATF-2
transcription factor in the regulation of E-selectin expression in response to non-endotoxin components
of Neisseria meningitidis. Cell Microbiol. 2016; 18(1):66±79. https://doi.org/10.1111/cmi.12483 PMID:
Rodriguez IR, Fliesler SJ. Photodamage generates 7-keto- and 7-hydroxycholesterol in the rat retina
via a free radical-mediated mechanism. Photochem Photobiol. 2009; 85:1116±1125. https://doi.org/10.
1111/j.1751-1097.2009.00568.x PMID: 19500292
Brown AJ, Dean RT, Jessup W. Free and esterified oxysterol: formation during copper-oxidation of low
density lipoprotein and uptake by macrophages. J Lipid Res. 1996; 37:320±335. PMID: 9026530
Brown A.J., Leong S.-L., Dean R., Jessup J W. 7-Hydroperoxycholesterol and its products in oxidized
low density lipoprotein and human atherosclerotic plaque. J. Lipid Res. 1997; 38; 1730±45. PMID:
12 / 13
1. Dulak J , JoÂzkowicz A , Dichtl W , Alber H , Schwarzacher SP , Pachinger O , et al. Vascular endothelial growth factor synthesis in vascular smooth muscle cells is enhanced by 7-ketocholesterol and lysophosphatidylcholine independently of their effect on nitric oxide generation . Atherosclerosis . 2001 ; 159 ( 2 ): 325 ± 32 . PMID: 11730812
Crisby M. , Nilsson J. , Kostulas V. , BjoÈrkhem I. , Diczfalusy U. Localization of sterol 27-hydroxylase
immuno-reactivity in human atherosclerotic plaques . Biochim. Biophys. Acta . 1997 ; 1344 ( 3 ): 278 ± 85 .
pool Life Sciences ; 2010 .
21. Shimozawa M , Naito Y , Manabe H , Uchiyama K , Kuroda M , et al. 7 -Ketocholesterol enhances the expression of adhesion molecules on human aortic endothelial cells by increasing the production of reactive oxygen species . Redox Rep . 2004 ; 9(6):370±5 . https://doi.org/10.1179/135100004225006902 PMID: 15720835
22. Yamagata K , Tanaka N , Suzuki K. Epigallocatechin 3 -gallate inhibits 7-ketocholesterol-induced monocyte-endothelial cell adhesion . Microvasc Res . 2013 ; 88 : 25 ±31 https://doi.org/10.1016/j.mvr. 2013 . 03 . 006 PMID: 23567873
23. Gerszten RE , Garcia-Zepeda EA , Lim YC , Yoshida M , Ding HA , et al. MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions . Nature . 1999 ; 398 : 718 ±23 https:// doi.org/10.1038/19546 PMID: 10227295
pathology: enhanced biological insights . Atherosclerosis 2002 ; 160 ( 1 ): 91 ±102 PMID: 11755926
25. Lordan S , Mackrill JJ , O'Brien NM . Oxysterols and mechanisms of apoptotic signaling: implications in the pathology of degenerative diseases . J Nutr Biochem . 2009 ; 20 : 321 ±336 https://doi.org/10.1016/j. jnutbio. 2009 . 01 .001 PMID: 19345313
26. Torocsik D , Szanto A , Nagy L . Oxysterol signaling links cholesterol metabolism and inflammation via the liver X receptor in macrophages . Mol Aspects Med . 2009 ; 30 : 134 ±152 https://doi.org/10.1016/j. mam. 2009 . 02 .002 PMID: 19248804
27. Moreira EF , Larrayoz IM , Lee JW , Rodriguez IR . 7 -Ketocholesterol is present in lipid deposits in the primate retina: potential implication in the induction of VEGF and CNV formation . Invest Ophthalmol Vis Sci . 2009 ; 50 : 523 ±532 https://doi.org/10.1167/iovs.08-2373 PMID: 18936140
28. Lenardo M.J.; Baltimore D. NF-κB: A pleiotropic mediator of inducible and tissue-specific gene control . Cell 1989 , 58 , 227±229 PMID: 2665943
29. Aye IL , Waddell BJ , Mark PJ , Keelan JA . Oxysterols exert proinflammatory effects in placental trophoblasts via TLR4-dependent, cholesterol-sensitive activation of NF-κB . Mol Hum Reprod . 2012 ; 18 : 341 ±353 https://doi.org/10.1093/molehr/gas001 PMID: 22238372
30. Huang JD , Amaral J , Lee JW , Rodriguez IR . 7 -Ketocholesterol-Induced Inflammation Signals Mostly through the TLR4 Receptor Both In Vitro and In Vivo. PLoS One . 2014 ; 18 ; 9 ( 7 ):e100985 https://doi. org/10.1371/journal.pone. 0100985 PMID: 25036103
31. Dixon G.L. , Heyderman R .S., van der Ley P., and Klein N.J. High-level endothelial E-selectin (CD62E) cell adhesion molecule expression by a lipopolysaccharide-deficient strain of Neisseria meningitidis despite poor activation of NF-kappaB transcription factor . Clin Exp Immunol . 2004 ; 135 : 85 ±93 https:// doi.org/10.1111/j.1365- 2249 . 2004 . 02335 . x PMID : 14678268
32. Guina T , Deiana M , Calfapietra S , Cabboi B , Maina M , et al. The role of p38 MAPK in the induction of intestinal inflammation by dietary oxysterols: modulation by wine phenolics . Food Funct . 2015 ; 6 ( 4 ): 1218 ±28 https://doi.org/10.1039/c4fo01116c PMID: 25736858
33. Yu T , Li YJ , Bian AH , Zuo HB , Zhu TW , et al. The regulatory role of activating transcription factor 2 in inflammation . Mediators Inflamm . 2014 ; 2014 : 950472. https://doi.org/10.1155/ 2014 /950472 PMID: 25049453
34. Miyata Y , Fukuhara A , Otsuki M , Shimomura I. Expression of activating transcription factor 2 in inflammatory macrophages in obese adipose tissue . Obesity (Silver Spring) . 2013 ; 21 ( 4 ): 731 ± 6
35. Jacobsen MC , Dusart PJ , Kotowicz K , Bajaj-Elliott M , Hart SL . A critical role for ATF2 transcription factor in the regulation of E-selectin expression in response to non-endotoxin components of Neisseria meningitidis . Cell Microbiol . 2016 ; 18 ( 1 ): 66 ±79 https://doi.org/10.1111/cmi.12483 PMID: 26153406