Inhibition of epidermal growth factor receptor attenuates atherosclerosis via decreasing inflammation and oxidative stress

Abstract

Atherosclerosis is a progressive disease leading to loss of vascular homeostasis and entails fibrosis, macrophage foam cell formation, and smooth muscle cell proliferation. Recent studies have reported that epidermal growth factor receptor (EGFR) is involved vascular pathophysiology and in the regulation of oxidative stress in macrophages. Although, oxidative stress and inflammation play a critical role in the development of atherosclerosis, the underlying mechanisms are complex and not completely understood. In the present study, we have elucidated the role of EGFR in high-fat diet-induced atherosclerosis in apolipoprotein E null mice. We show increased EGFR phosphorylation and activity in atherosclerotic lesion development. EGFR inhibition prevented oxidative stress, macrophage infiltration, induction of pro-inflammatory cytokines, and SMC proliferation within the lesions. We further show that EGFR is activated through toll-like receptor 4. Disruption of toll-like receptor 4 or the EGFR pathway led to reduced inflammatory activity and foam cell formation. These studies provide evidence that EGFR plays a key role on the pathogenesis of atherosclerosis, and suggests that EGFR may be a potential therapeutic target in the prevention of atherosclerosis development.

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Scientific RepoRts | 7:45917 | DOI: 10.1038/srep45917
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Inhibition of epidermal growth
factor receptor attenuates
atherosclerosis via decreasing
inammation and oxidative stress
Lintao Wang1,*, Zhouqing Huang2,*, Weijian Huang2,*, Xuemei Chen1, Peiren Shan2,
Peng Zhong1, Zia Khan3, Jingying Wang1, Qilu Fang1, Guang Liang1 & Yi Wang1
Atherosclerosis is a progressive disease leading to loss of vascular homeostasis and entails brosis,
macrophage foam cell formation, and smooth muscle cell proliferation. Recent studies have reported
that epidermal growth factor receptor (EGFR) is involved vascular pathophysiology and in the
regulation of oxidative stress in macrophages. Although, oxidative stress and inammation play a
critical role in the development of atherosclerosis, the underlying mechanisms are complex and not
completely understood. In the present study, we have elucidated the role of EGFR in high-fat diet-
induced atherosclerosis in apolipoprotein E null mice. We show increased EGFR phosphorylation and
activity in atherosclerotic lesion development. EGFR inhibition prevented oxidative stress, macrophage
inltration, induction of pro-inammatory cytokines, and SMC proliferation within the lesions. We
further show that EGFR is activated through toll-like receptor 4. Disruption of toll-like receptor 4 or the
EGFR pathway led to reduced inammatory activity and foam cell formation. These studies provide
evidence that EGFR plays a key role on the pathogenesis of atherosclerosis, and suggests that EGFR
may be a potential therapeutic target in the prevention of atherosclerosis development.
Coronary atherosclerosis is the principal cause of coronary artery disease and, therefore, a major cause of mortal-
ity and morbidity globally1,2. Atherosclerosis is now recognized as a systemic, lipid-driven inammatory disease
of medium-sized and large arteries leading to multifocal plaque development3–5. e formation and progression
of atherosclerotic plaques involves aberrant inammatory cell recruitment, foam cell formation, smooth muscle
cell (SMC) proliferation and increased matrix synthesis, production of reactive oxygen species (ROS), and arterial
remodeling6,7. Among these changes, chronic inammation8 and ROS9,10 appear to play dominant roles. During
the inammatory stage of atherosclerosis, low-density lipoprotein (LDL) is taken up in the arterial wall and is oxi-
dized by excessive ROS. Macrophages scavenge oxidized-LDL (ox-LDL) forming lipid-laden foam cells11. Studies
have shown that ox-LDL also induces ROS production and release of inammatory factors, which attribute for
the progression of atherosclerosis12,13. e mechanisms driving ox-LDL-induced inammation, increased oxida-
tive stress, and atherosclerotic lesion progression are not fully dened.
Epidermal growth factor receptor (EGFR; also known as ErbB1) has recently been implicated in vascular
pathophysiological processes associated with excessive remodeling. Activation of EGFR occurs either by bind-
ing of ligands such as epidermal growth factor (EGF) and heparin bound-EGF, or by transactivation. EGFR is
expressed in macrophages, vascular smooth muscle cells, endothelial cells, and cardiomyocytes, and these cells
also secrete EGFR ligands. It has been reported that EGFR plays a role in foam cell transformation, and cellu-
lar dysfunction and proliferation of vascular SMCs14. EGFR activation by metalloproteinase meprin-α medi-
ates ox-LDL-induced oxidative stress in macrophages15. Furthermore, EGFR leads to downstream activation of
transcription factors such as nuclear factor-κ B (NF-κ B) and stimulates pro-inammatory gene transcription in
macrophages16–18. Recent ndings have also suggested that EGF-like ligands may serve as biomarkers for active
1Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou,
Zhejiang, 325035, China. 2Department of Cardiology, the First Aliated Hospital, Wenzhou Medical University,
Wenzhou, Zhejiang, 325035, China. 3Department of Pathology and Laboratory Medicine, Western University,
London, ON N6A5C1, Canada. *These authors contributed equally to this work. Correspondence and requests for
materials should be addressed to G.L. (email: wzmcliangguang@163.com) or Y.W. (email: yi.wang1122@gmail.com)
Received: 07 December 2016
Accepted: 06 March 2017
Published: 04 April 2017
OPEN

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Scientific RepoRts | 7:45917 | DOI: 10.1038/srep45917
inammatory atherosclerosis in a primate model of atherosclerosis19. We have recently shown that inhibition
of EGFR eectively protects cardiac damage and remodeling by attenuating oxidative stress in a type 1 diabetic
mice model20. Taken together, these ndings suggest an important role of EGFR in atherosclerosis. Uncovering
this role and the mechanism of EGFR activation may lead to the development of new therapeutic modalities for
patients with coronary artery disease.
In the present study, we have deciphered the role of EGFR in atherosclerotic lesion formation by utilizing the
apolipoprotein E (ApoE) null mice. We inhibited EGFR in these mice by two specic small-molecule EGFR inhib-
itors AG1478 and 542 (Fig.1a). We have recently shown that these inhibitors eectively block EGFR activation
and attenuate angiotensin II-induced cardiac hypertrophy and dysfunction21. We have further delineated a novel
mode of EGFR activation in atherosclerosis. Our studies show that EGFR is phosphorylated and activated in ath-
erosclerotic lesion formation. Inhibition of EGFR prevents oxidative stress, induction of inammatory cytokines,
and foam cell formation. We further show that EGFR activation in macrophages involves toll-like receptor 4.
Results
Increased EGFR phosphorylation in aortas of HFD-fed ApoE−/− mice. We rst wanted to know if
inhibiting EGFR alters serum lipid levels since elevated low-density lipoproteins (LDL) have been shown to be
strongly related to the development of atherosclerosis. ApoE−/− mice fed a high fat diet (HFD) exhibited increased
serum levels of LDL and triglycerides (TG) as compared to mice fed a control/low fat diet (LFD) (Fig.1b). We
also tested the serum insulin level and found that HFD induced the increase in serum insulin while AG or 542 did
not aect the insulin level (SupplementaryFig.S1). Inhibiting EGFR through 542 or AG1478 for 8 weeks showed
no signicant dierences in the levels of serum lipids between the HFD mice and the treated groups (Fig.1b,c).
Examination of aorta tissues by immunohistochemistry showed increased levels of EGFR expression and phos-
phorylation in HFD-fed mice compared to LFD-fed mice (Fig.1d and SupplementaryFig.S2). Interestingly,
treatment of mice with 542 and AG1478 reduced the levels of p-EGFR immunoreactivity but not total EGFR. We
also noted activation of predominant signaling proteins downstream of EGFR, namely extracellular signal-reg-
ulated kinase (ERK) and Akt. Immunouorescent staining analysis for p-ERK and p-AKT in aorta tissues found
that administration with EGFR inhibitors signicantly blocked HFD-induced ERK and Akt phosphorylation in
aortas of ApoE−/− mice (SupplementaryFig.S3a–d). Proteins isolated from aorta tissues conrmed these results
(Fig.1e and SupplementaryFig.S4a–d). ese results show increased EGFR phosphorylation and activity in
atherosclerotic lesions in mice.
Atherogenesis is characterized by developing atheromas driven by progressive uptake of LDL cholesterol by
macrophages, becoming lipid-laden foam cells accumulated in the subendothelial space. Additionally, the aber-
rant growth of SMCs and endothelial cells (ECs) create intimal thickening, and together with foam cells, produce
a local environment containing a wide range of secreted mediators such as growth factors and pro-inammatory
molecules. erefore, all three cell types (macrophages, SMCs, and ECs) contribute to the development of ath-
erosclerosis. We performed the evaluation of p-EGFR localization at the atherosclerotic plaques in aortas of the
ApoE− /−
mice by colocalization immunouorescence staining. e results indicated that p-EGFR were increased
in these three cell types in HFD mice relative to control mice (SupplementaryFig.S5). However, statistical anal-
ysis shows that the ratio of p-EGFR-positive macrophages in total macrophages (41.85%) is higher than the ratio
of p-EGFR-positive SMCs in total SMCs (28.84%) and the ratio of p-EGFR-positive ECs in total ECs (7.13%).
us, our ndings suggest that macrophages are mainly associated aberrant EGFR phosphorylation in the lesion.
Despite the critical role of SMCs and ECs, we selected macrophages for in vitro stu dy.
AG1478 and 452 treatment prevented atherosclerotic plaque development in HFD-fed ApoE−/−
mice. Although we did not nd a dierence in serum lipid levels upon EGFR inhibition, reduced EGFR acti-
vation in aortas prompted us to examine dierences in the degree of atherosclerotic lesions. We performed Oil
Red O staining of the entire aorta to measure the severity of these lesions. Our results show signicantly increased
lesion area in ApoE−/− mice fed a HFD compared to LFD as expected (Fig.2a,b). Treatment of mice with AG1478
and 542 decreased the atherosclerotic lesion area to approximately half of that observed in untreated HFD-fed
mice (Fig.2a,b). Additional assessment through H&E and Oil Red O staining showed that the plaque areas in the
aortic sinus of EGFR inhibitor-treated mice were signicantly smaller than in untreated HFD-fed mice (Fig.2c,d,
and SupplementaryFig.S6a). Increased plaque area accompanied increased macrophage inltration as assessed
through CD68 staining (Fig.2e and SupplementaryFig.S6b). Likewise, smooth muscle proliferation in the aor-
tic sinus of untreated mice were signicantly higher than in mice treated with EGFR inhibitors (Fig.2f, and
SupplementaryFig.S6c). All these pathological changes were attenuated by administration with either AG1478
or 542.
A hallmark of a variety of brotic diseases, including atherosclerosis, is extensive deposition of extracellular
matrix. We tested the eect of EGFR inhibition on brosis in aorta tissues of HFD-fed ApoE−/− mice. Treatment
of mice with AG and 542 prevented HFD-induced collagen deposition as highlighted by Masson Trichrome and
Sirus Red staining (SupplementaryFig.S7a,b). ese results were conrmed by determining mRNA levels of
collagen 1, and brogenic factors connective tissue growth factor (C-TGF) and transforming growth factor-β 1
(TGF-β ). In addition, we assessed TGF-β protein levels and show that both AG and 542 prevented HFD-induced
expression of TGF-β (SupplementaryFig.S7c–f). ese results suggest that EGFR inhibition renders ApoE−/−
mice resistant to atherosclerosis.
AG1478 and 542 inhibited HFD-induced inflammation and oxidative stress in aortas. We
sought to clarify whether inammation and oxidative stress were involved in the attenuation of atherosclerotic
plaque development by EGFR inhibition. e levels of inammatory factors and adhesion molecules includ-
ing tumor necrosis factor-α (TNF-α ), interleukin-6 (IL-6), vascular cell adhesion molecule-1 (VCAM-1), and

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Figure 1. Administration of EGFR inhibitors blocked EGFR signaling activation in HFD-fed ApoE−/−
mouse artery. (a) e structures of AG1478 and compound 542. ApoE−/− mice were fed with HFD for 8
weeks, and treated with AG1478 (AG, 10 mg/kg/day) or 542 (10 mg/kg/day) for 8 weeks by oral gavage. (b,c)
Serum levels of LDL and TG. (d) Representative microscopic images of EGFR and p-EGFR immunochemical
staining in artery tissues. (e) Western blot analysis of p-EGFR, p-AKT and p-ERK in artery tissues, with the
densitometric quantications shown in SupplementaryFig.S3. e gels were run under the same experimental
conditions. Shown are cropped gels/blots (e gels/blots with indicated cropping lines are shown in the
SupplementaryFig.20). (LFD = low fat diet, HFD = high fat diet; n = 7 in each group; ##P < 0.01, vs LFD; ns, not
signicant vs HFD). e quantication results for all staining images were shown in the Supplementary File.

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Figure 2. EGFR inhibitors prevented atherosclerotic plaque development in HFD-fed ApoE−/− mice.
ApoE−/− mice were fed with HFD for 8 weeks, and treated with AG1478 (AG, 10 mg/kg/day) or 542 (10 mg/
kg/day) for 8 weeks by oral gavage. (a,b) Atherosclerosis plaque staining in the artery using Oil Red staining
(a), with the quantication of atherosclerotic plaque lesion area (b) (n = 7; #P < 0.05, vs LFD; *P < 0.05, vs
HFD). (c) H&E staining in the aortic valve. (d) Oil Red O staining in aortic valve (lower panels show higher
magnication). (e) Immunouorescence staining with anti-CD68 in the artery tissues. (f) Histochemical
staining with anti-α -smooth muscle actin in the artery tissues. All images are representative from 7 mice per
group, and the quantication results for all staining images were shown in the Supplementary File.

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intracellular adhesion molecule-1 (ICAM-1) in atherosclerotic aortas were markedly higher in HFD-fed mice
compared to LFD-fed control mice and EGFR inhibitor-treated mice (Fig.3a–d). e fact that EGFR inhibition
prevented induction of inammatory cytokines and adhesion molecules point to an important role of EGFR early
in the disease course.
We next examined parameters of tissue remodeling as excessive inammatory cytokines may increase the
expression and activity of matrix metalloproteinases (MMPs). As shown in Fig.3e,f, MMP2 expression and
MMP9 activity in aortas of HFD-fed mice were signicantly increased compared to the LFD-fed mice. Both
MMP2 and MMP9 have been shown to be involved in atherosclerosis22 and serve as markers of tissue remodeling
and progression of atherosclerotic lesions. Treatment of mice with 452 and AG1478 reduced MMP2 and MMP9
to levels comparable to LFD-fed mice. Mirroring the pattern of inammatory markers, dihydroethidium (DHE)
uorescence staining for reactive oxygen species (ROS) and nitrotyrosine (3-NT) immunohistochemistry showed
increased oxidative stress in aortas of HFD-fed mice (Fig.3g,h, and SupplementaryFig.S8a,b). Both measures of
Figure 3. EGFR inhibitors prevented inammation and oxidative stress in the atherosclerotic plaques of
HFD-fed ApoE−/− mice. ApoE−/− mice were fed with HFD for 8 weeks, and treated with AG1478 (AG, 10 mg/
kg/day) or 542 (10 mg/kg/day) for 8 weeks by oral gavage. (a–e) Real time qPCR analysis of TNF-α (a), IL-6 (b),
VCAM-1 (c), ICAM-1 (d), MMP2 (e). (f) MMP-9 activity in the atherosclerotic plaques as measured by gelatin
zymography. e gels were run under the same experimental conditions. Shown are cropped gels/blots (e
gels/blots with indicated cropping lines are shown in the SupplementaryFig.20). (n = 7 per group, #P < 0.05,
vs LFD; *P < 0.05, **P < 0.01, vs HFD). (g,h) Representative images of Dihydroethidium (DHE) and anti-3-
Nitrotyrosine (3-NT) staining in aortic valve tissues. e quantication results for all staining images were
shown in the Supplementary File.

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Scientific RepoRts | 7:45917 | DOI: 10.1038/srep45917
oxidative stress were decreased by AG1478 and 542, indicating that EGFR inhibition reduces inammation and
ROS in the development of atherosclerosis.
EGFR inhibitors suppress inflammation in ox-LDL-stimulated macrophages and vascular
smooth muscle cells (SMCs). EGFR signaling has been shown to mediate lipopolysaccharide-induced
inammation via regulating the activation of nuclear factor-κ B (NF-κ B) in macrophages18. Given that EGFR
inhibitors 452 and AG1478 are able to attenuate atherosclerosis through reducing inammation in mice, we
investigated the anti-inammatory eects of EGFR inhibitors in oxidized-LDL (ox-LDL)-stimulated primary
macrophages. Brief exposure of macrophages to ox-LDL induced EGFR phosphorylation, but not EGFR expres-
sion, as detected by western bott method (Fig.4a and SupplementaryFig.S9) and immunouorescence staining
(Fig.4b and SupplementaryFig.S10). e levels of p-EGFR were greatly reduced when cells were pre-treatment
with 542 or AG1478 (Fig.4b and SupplementaryFig.S10). Downstream signaling proteins Akt and ERK were
also phosphorylated by ox-LDL and inhibited with 542 and AG1478 (Fig.4c and SupplementaryFig.S11a).
As macrophage NF-κ B18 has been shown to be critical in inammation, we assessed its activation by western
blotting and cell staining. Our results show increased NF-κ B p65 subunit in the nuclear protein fraction and
increased nuclear staining of macrophages stimulated by ox-LDL, as detected by western bott method (Fig.4d
and SupplementaryFig.S11b) and immunouorescence staining (Fig.4e and SupplementaryFig.S12). In both
assays, 542 and AG1478 markedly inhibited ox-LDL-induced NF-κ B activation.
Activation of EGFR and downstream signaling proteins by ox-LDL was also associated with induction of
pro-inammatory cytokines TNF-α and IL-6 at both protein and mRNA levels in cultured macrophages. As
expected, AG1478 or 542 prevented this induction (Fig.4f,g, SupplementaryFig.S13a,b). mRNA analysis also
showed that AG1478 and 542 suppressed the expression of adhesion molecules ICAM-1 and VCAM-1 induced
by ox-LDL (Fig.4h,i). We then examined MMPs as our studies in aorta tissues showed dysregulated expres-
sion and activity in atherosclerotic lesions. In cultured macrophages, ox-LDL increased MMP2 expression and
MMP9 activity and both of these changes were prevented by AG and 542 pretreatment (Fig.4j,k). We also tested
the anti-inammatory eects of EGFR inhibitors in cultured SMCs and show responses similar to macrophages
(SupplementaryFig.S14a,b).
EGFR inhibition prevented ox-LDL-induced ROS production, mitochondrial damage, and
foam cell formation. Increased EGFR phosphorylation was found to play a vital role in the production of
ROS by ox-LDL in macrophages15. Here, we determined the eects of EGFR inhibitors on ox-LDL-stimulated
ROS generation in macrophages. Exposure of macrophages to ox-LDL for 6 h signicantly increased ROS gen-
eration as indicated by DCFH-DA/DHE uorescence staining (Fig.5a and SupplementaryFig.S15) and ow
cytometry (Fig.5b). Pretreatment with 452 or AG1478 was able to block increased ROS generation. Similar
results were obtained in SMCs (SupplementaryFig.S16). To understand how EGFR induces ROS produc-
tion following ox-LDL stimulation, we tested the eects of EGFR inhibitors on the expression and activity of
NADPH oxidase (NOX) in macrophages. NOX1 has recently been shown to activate infiltrating immune
cells, increasing ROS levels in aortic sinus of diabetic mice23. Our results showed that both AG1478 and 451
signicantly reversed ox-LDL-induced NADP/NADPH ratio and inhibited ox-LDL-induced NOX-1 expres-
sion (SupplementaryFig.S17). In addition, we tested the determination of NO level and iNOS expression in
oxLDL-stimulated macrophages. It was observed that pre-treatment with EGFR inhibitors signicantly blocked
oxLDL-induced overproduction of NO and overexpression of iNOS (SupplementaryFig.S18).
We next examined mitochondrial membrane potential as it is well known that increased ROS levels result in
the mitochondrial dysfunction. Loss of mitochondrial membrane potential (Dψ m) is catastrophic for cells and
leads to the release of cytochrome C into the cytosol. We tested mitochondrial membrane potential loss by using
potential-sensitive ratiometric uorescence dye JC-1. As shown in Fig.5c, ox-LDL caused a pronounced decrease
in mitochondrial Dψ m indicating a reduction of highly energized mitochondria. In contrast, pretreatment with
EGFR inhibitors (AG1478 or 542) for 1 h attenuated the ox-LDL-induced decrease in mitochondrial Dψ m.
Once lipids are taken up in the arterial wall and oxidized by ROS, macrophages scavenge these modied lipids
and become foam cells. We, therefore, investigated the role of EGFR in ox-LDL uptake by macrophages. Cells
were incubated with DiI-labled ox-LDL (DiI-ox-LDL) with or without pretreatment with EGFR inhibitors and
analyzed by uorescence microscopy and ow cytometry. Here, we report that inhibition of EGFR prevented
ox-LDL update in macrophages (Fig.5d,e, and SupplementaryFig.S19a). We conrmed these results by staining
macrophages exposed to ox-LDL with Oil Red O (Fig.5f and SupplementaryFig.S19b). ese studies show that
EGFR inhibition reduced formation of foam cells.
ox-LDL induces EGFR activation through toll-like receptor/Src in macrophages. Our studies
have shown that EGFR inhibition prevented atherosclerotic lesion formation, inammation, ROS generation,
and foam cell formation. However, it remains unclear as to how ox-LDL activates EGFR signaling. EGFR lig-
ands including heparin binding-EGF (HB-EGF)24,25, epiregulin (EREG)26, TGF-α
25,27, and β -cellulin28 are associ-
ated with human atherosclerosis and potentially may contribute to the EGFR activation. Our studies show rapid
phosphorylation of EGFR suggesting direct activation rather than through elaboration of typical EGF ligands.
Recent studies have suggested that EGFR can also be activated without the typical ligands29, and it can function
in intracellular membranes30. Toll-like receptor 4 (TLR4) has been reported to be directly activated by ox-LDL
and mediate pathological pathways and phenotypes31–33. In addition, expression of TLR4-induced genes in
lipopolysaccharide-stimulated myeloid cells requires EGFR kinase activity18. We have also found that TLR4 and
c-Src mediate palmitic acid-induced EGFR activation in cardiomyocyte-like H9c2 cells34. us, we tested whether
TLR4/c-Src mediates ox-LDL-induced EGFR activation in macrophages. We collected primary macrophages
from TLR4−/− mice and wildtype (WT) mice. Protein analysis of cultured primary macrophages showed that

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Scientific RepoRts | 7:45917 | DOI: 10.1038/srep45917
Figure 4. Inhibiting EGFR blocks ox-LDL-induced inammation in macrophages. (a) ox-LDL activates
EGFR in macrophages. MPMs were stimulated with ox-LDL (50 μ g/mL) for dierent time points. Cell lysates
were analyzed for p-EGFR and EGFR. (b,c) AG and 542 suppressed ox-LDL-induced activation of EGFR.
MPMs were pretreated with 542 (10 μ M or indicated concentrations), AG1478 (10 μ M), or vehicle (DMSO, 1 μ L)
for 1 h and then stimulated with ox-LDL (50 μ g/mL) for 15 min. Immunouorescence staining for p-EGFR and
DAPI was performed (b) and the levels of p-EGFR, p-ERK, and p-AKT in cell lysates were detected by western
blot (c). (d,e) AG and 542 suppressed ox-LDL-induced activation of NF-κ B. MPMs were pretreated with 542
(10 μ M), AG1478 (10 μ M), or vehicle (DMSO, 1 μ L) for 1 h and then stimulated with ox-LDL (50 μ g/mL) for 1 h.
Levels of nuclear NF-κ B p65 were assessed by western blotting with Lamin B as a loading control (d), or were
detected by anti-p65 immunouorescence staining (e). (f–j) AG and 542 inhibited ox-LDL-induced release of
cytokines. MPMs were pretreated with 542 (2.5, 5 or 10 μ M) and AG1478 (10 μ M) for 1 h and then stimulated

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ox-LDL increased c-Src and EGFR/ERK/AKT phosphorylation in WT macrophages but not in macrophages
derived from TLR4−/− mice (Fig.6a). In addition, pretreatment of primary macrophages with AG1478 and c-Src
inhibitor (PP2) reduced EGFR phosphorylation (Fig.6b). Interestingly, AG1478 pretreatment only blocked EGFR
phosphorylation but did not alter c-Src possibly indicating that c-Src is upstream of EGFR activation.
We reasoned that if TLR4 mediated ox-LDL-induced EGFR phosphorylation then inammatory activity
downstream of EGFR would not be evident in cells from TLR−/− mice. Indeed, ox-LDL failed to induce IL-1β ,
IL-6, and TNF-α release from TLR4−/− macrophages (Fig.6c). Similarly, inhibition of TLR4 through TAK242
prevented ox-LDL update and foam cell formation (Fig.5d–f). ese results suggest that TLR4/c-Src signaling
mediates EGFR activation downstream of ox-LDL and leads to foam cell formation.
Discussion
e development of atherosclerosis is tightly associated with chronic inammation and oxidative stress in the
arterial plaque3,4,35. Fibro-fatty plaque formation and SMC proliferation are also hallmarks of atherosclerosis. In
the present study, we evaluated whether EGFR-dependent pathways play a role in the development of atheroscle-
rosis in ApoE−/− mice. Mice fed a HFD for 8 weeks showed accelerated atherosclerotic lesions characterized by
accumulation of SMCs and macrophages. In addition, formation of foam cells, induction of inammatory factors
including IL-6, ICAM-1 and TNF-α , accompanied increased EGFR phosphorylation and activity. Inhibition of
EGFR using AG1478 or compound 452 signicantly ameliorated these abnormalities without altering serum LDL
levels. Our results indicated that p-EGFR were increased in all three cell types (macrophages, SMCs, and ECs),
which contribute mainly to atherosclerosis, in HFD-fed ApoE−/− mice. We conrmed our ndings in cultured
macrophages and SMCs challenged with ox-LDL. Finally, we identied a novel mechanism of oxLDL-induced
EGFR activation involving TLR4 in macrophages. ese ndings indicate a detrimental eect of activated EGFR
in the pathogenesis of atherosclerosis, and that exacerbated EGFR phosphorylation contributes to the progression
of atherosclerotic plaque formation, likely through increased inammation and oxidative stress.
Oxidative stress plays a key role in the progression of cardiovascular disease. In particular, ROS very com-
monly accompanies the development of typical characteristics of atherosclerosis10,36. Excessive ROS generation
can directly damage the cell membrane, proteins and DNA. Mitochondrial DNA has also been proposed to be
susceptible to oxidative damage37,38. Recent studies show that increasing ROS production participates in inam-
mation, disturbed blood blow and abnormal shear stress, and arterial wall remodeling39,40. In addition, Park and
colleagues7 reported that oxidative stress contributes to structural remodeling through SMC proliferation and
enhanced inammation. In the present work, we found oxidative stress markers in the arteries of ApoE−/− mice
were increased. Increased oxidative damage was associated with artery remodeling and enhanced inammation
in vivo (Figs3 and 4). Interestingly, oxidative stress as well as SMC proliferation was signicantly attenuated by
AG1478 and 452 treatments. ese results conrm that ROS is involved in the development of atherosclerosis and
clearly show the involvement of EGFR in ROS production and SMCs proliferation. e mechanisms leading to
enhanced ROS generation through EGFR are just recently being claried and may involve EGFR/AKT41,42. MAPK
pathways are also reported to be involved in the ROS production in macrophages43. In addition, several reports
conrmed EGFR-PI3K-AKT/ERK signaling pathway responsible for ROS generation44–46. In a related system,
we have shown that EGFR inhibitors signicantly blocked NOX expression and activity in high glucose-induced
H9c2 cell20. Here, we show that the same EGFR/AKT-ERK activation pathway enhances ROS production in ath-
erosclerotic lesion of ApoE−/− mice, which were markedly reversed by EGFR inhibitors AG1478 or 452. We also
show that ox-LDL-stimulated macrophages utilize the NOX and iNOS pathways for ROS generation.
In addition to ROS (and likely downstream of ROS), inammation plays an important role in the initiation
and progression of atherosclerosis8,47. Multiple cell types including monocytes/macrophages, T-lymphocytes,
SMCs and mast cells8 are present in atherosclerotic plaques from the earliest lesions to ruptured plaques. ese
cells accompany various inammatory and tissue remodeling factors including TNF-α , IL-6, ICAM-1, VCAM-1
and MMPs48. We established that increased EGFR signaling activation is associated with artery inammation and
lipid accumulation in macrophages. Recently, we have found that administration of EGFR inhibitors (AG1478
and 542) signicantly prevented HFD-induced inammation in ApoE−/− mouse hearts34 and both ApoE− /−
and C57B/L6 mouse kidneys49. at is to say, EGFR inhibition may prevents systemic inammatory changes in
HFD-fed mice. EGFR inhibition using AG 1478 or 452 alleviated atherosclerotic lesions in ApoE−/− mice through
decreasing macrophages inltration, foam cell formation and possibly matrix metalloproteinase secretion. ese
ndings suggest EGFR activation is responsible for the pathophysiological development of atherosclerosis.
Consistent with our observations, a recent study by Liang et al. showed that meprin-α activated EGFR activity
to induce oxidative stress in ox-LDL-stimulated macrophage15. e authors showed that meprin-α promotes the
formation of atherosclerotic plaques and ROS production, and both are reversed with AG1478 treatment. Herein,
with ox-LDL (50 μ g/mL) for 24 h (in panels f and g) or 6 h (in panels h–j). e levels of IL-6 (d) and TNF-α (e)
in the cultural medium were detected by ELISA. e mRNA levels of ICAM-1 (h), VCAM-1 (i), and MMP-2 (j)
were detected by real-time qPCR assay. (k) AG and 542 inhibited ox-LDL-induced MMP9 activity. MPMs were
pretreated with 542 or AG1478 at indicated concentrations for 1 h and then stimulated with ox-LDL (50 μ g/mL)
for 48 h. MMP-9 activity in the medium was measured by gelatin zymography. (n = 4 independent experiments,
##P < 0.01, vs control; *P < 0.05, **P < 0.01, ***P < 0.001, vs ox-LDL). For panels a, c, d, and k, the gels were run
under the same experimental conditions. Shown are cropped gels/blots (e gels/blots with indicated cropping
lines are shown in the SupplementaryFig.20). e quantication results for all staining images were shown in
the Supplementary File.

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Figure 5. AG and 542 inhibit ox-LDL-induced ROS production and foam cell formation in primary
macrophages. (a,b) AG and 542 inhibited the production of O2- or H2O2 by ox-LDL. Primary macrophages
were pretreated with 542 and AG at 10 μ M for 1 h, followed by the incubation with ox-LDL (50 μ g/mL) for
30 min. DHE and DCFH-DA probes were loaded and cells were detected using uorescence microscope (a).
DCFH-DA probes were loaded and cells were analyzed by ow cytometry for H2O2 level (b). (c) AG and 542
attenuates ox-LDL-induced mitochondrial injury. Primary macrophages pretreated with AG or 542 at 10 μ M
for 1 h were incubated with oxLDL (50 μ g/mL) for 24 h. Cells were subjected to JC-1 staining for mitochondrial
membrane potential analysis. (d,e) Primary macrophages were pretreated with 542, AG1478, or TAK242 at 10 μ
M for 1 h, followed by the incubation with Dil-ox-LDL (100 μ g/mL) for 30 min. Cells were then processed by
ow cytometry (d) or uorescence imaging (e). (f) Primary macrophages were pretreated with 542, AG1478,
or TAK242 at 10 μ M for 1 h and then stimulated with ox-LDL (100 μ g/mL) for 30 min and then stained with Oil
Red O. (Data are representative from n = 4 independent experiments; the quantications were shown in the
Supplementary le). e quantication results for all staining images were shown in the Supplementary File.

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we observed that all these abnormalities appeared to be reduced by EGFR inhibitor AG1478 or 452, indicating
EGFR activation play a critical role.
Our studies have shown that AG1478 and 452 inhibit the phosphorylation of ERK and p65 nuclear transloca-
tion in ox-LDL-induced macrophages. is indicates that EGFR functions upstream of ERK and NF-κ B in mac-
rophages. Rapid activation of EGFR in cultured cells points to a mode of action rather than elaboration of typical
EGF ligands. Rapid activation of EGFR by ox-LDL has been reported in vascular cells50–53, though the mecha-
nisms are unknown. We identied TLR4 as a potential activator of EGFR in macrophages. TLR has been shown
to be important in activated macrophages, regulating nucleotide-binding domain and leucine-rich repeat con-
taining (NLR) family, pyrin domain containing 3 (NLRP3) inammasomes15,54–56. We showed phosphorylation
of EGFR/AKT/ERK to be decient in macrophages derived from TLR4−/− mice. Moreover, downstream eects
of EGFR activation including induction of inammatory factors (IL-6, IL-1β and TNF-α ) and MCP-1 secretion
was lacking in TLR4−/− macrophages challenged with ox-LDL. Furthermore, inhibition of TLR4 prevents foam
Figure 6. oxLDL-induced EGFR activation requires TLR4/c-Src. (a) EGFR is not activated by ox-LDL
in the TLR4 knockout-derived macrophages. Primary macrophages isolated from TLR4 knockout mice
and C57/B6 WT mice were stimulated with ox-LDL (50 μ g/mL) for 15 min. p-EGFR/EGFR, p-c-Src/c-Src,
p-AKT/AKT, and p-ERK/ERK levels were determined by western blotting. (b) c-Src inhibitor PP2 prevents
ox-LDL-induced EGFR activation. Primary macrophages were pretreated with AG1478 or PP2 at 10 μ M for
1 h, followed by the incubation with ox-LDL (50 μ g/mL) for 15 min. Total proteins were extracted to detect
the levels of p-EGFR/EGFR and p-c-Src/c-Src using western blot analysis. (c) Primary macrophages isolated
from TLR4 knockout mice and C57/B6 and stimulated with ox-LDL (50 μ g/mL) for 24 h. Culture medium was
used to detect the levels of TNF-α , IL-6 and IL-1β by ELISA. (n = 4 independent experiments, #P < 0.05, vs
control WT; **P < 0.01, vs ox-LDL-WT). For panels a and b, the gels were run under the same experimental
conditions. Shown are cropped gels/blots (e gels/blots with indicated cropping lines are shown in the
SupplementaryFig.20).

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cell formation. ese observations suggest that TLR4 plays its pro- activity through regulating the activation of
EGFR/AKT/ERK signal pathway.
It is worth noting that many pharmacological interventions, including statins, angiotensin-converting enzyme
inhibitors, niacin and calcium channel blockers, target ROS and inammation to abrogate the development of
atherosclerosis10. In the present study, the newly synthesized EGFR inhibitor 452 showed eective prevention of
atherosclerosis development. e eect produced by 452 was comparable to AG1478 in improving inammation
and ROS production both in vitro and in vivo. EGFR inhibitors already constitute the rst-line therapy for a
number of cancers and our studies suggest another clinically signicant indication where EGFR inhibitors may
be of therapeutic benet.
Material and Methods
Reagents and cell culture. AG1478 were purchased from Sigma-Aldrich (St. Louis, MO). Compound 542
(Fig.1a) was prepared with a purity of 99.2% as described in our previous study34. AG1478 and compound
542 were dissolved in dimethyl sulfoxide (DMSO) for in vitro experiments and in 1% sodium carboxyl methyl
cellulose (CMC-Na) for in vivo experiments. Antibodies against GAPDH, p-EGFR and p-AKT were purchased
from Cell Signaling (Danvers, MA, USA). Antibodies against p-ERK, TGF-β , Collagen4, cleaved caspase 3, Bax,
Bcl-2, and TLR4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against CD68 was
purchased from Abcam (Cambridge, MA). Human vascular smooth muscle cell line was purchased from R&S
Biotech. Co., LTD (Shanghai, China).
Preparation of mouse peritoneal macrophages. Mouse primary peritoneal macrophages (MPMs)
were isolated from C57BL/6 mice and cultured as shown by us previously57. Briey, C57BL/6 mice were sim-
ulated by intraperitoneal injection of 6% thioglycollate solution (0.3 g beef extract, 1 g tryptone, 0.5 g sodium
chloride dissolved in 100 ml ddH2O, and ltrated through 0.22-μ m lter membrane, 3 ml per mouse) and kept
in a pathogen-free condition for 3 days before mouse peritoneal macrophages (MPMs) isolation. Mice were
euthanized by rising CO2 inhalation, in accordance with Schedule 1 of the Animals (Scientic Procedures) Act
(1986). Total MPMs were harvested by washing the peritoneal cavity with PBS containing 30 mM of EDTA (8 ml
per mouse), centrifuged, and suspended in RPMI-1640 medium (Gibco/BRL life Technologies, Eggenstein,
Germany) with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 100 U/ml penicillin, and 100 mg/ml strep-
tomycin. Nonadherent cells were removed by washing with medium 3 h aer seeding. Experiments were under-
taken aer the cells were rmly adhered to the culture plates.
Real-time quantitative PCR. Total RNA was isolated from cells and artery tissues using TRIZOL (ermo
Fisher, Carlsbad, CA). Both reverse transcription and quantitative PCR were carried out using a two-step M-MLV
Platinum SYBR Green qPCR SuperMix-UDG kit (ermo Fisher) in Eppendorf Mastercycler ep realplex detec-
tion system (Eppendorf, Hamburg, Germany). Primers were obtained from ermo Fisher (Shanghai, China).
Primer sequences are listed in SupplementaryTableS1. mRNA levels of target genes was normalized to β -actin.
Western immunoblot analysis. Lysates from cells or homogenized artery tissues were separated by 10%
SDS-PAGE and electro-transferred onto a nitrocellulose membrane. Each membrane was pre-incubated for
1.5 h at room temperature in Tris-buered saline (pH 7.6, containing 0.05% Tween 20 and 5% non-fat milk).
Membranes were then incubated with specic antibodies. Immunoreactive bands were detected by incubating
with secondary antibody conjugated to horseradish peroxidase and visualizing using enhanced chemilumines-
cence reagent (Bio-Rad, Hercules, CA). e amounts were analyzed using Image J analysis soware version 1.38e
(NIH) and normalized to their respective controls.
Oil red staining. Macrophages were incubated with 100 μ g/mL ox-LDL (Biomedical Technologies) in RPMI 1640
media for 24 h. At the time of analysis, cells were xed in 4% paraformaldehyde for 15 min, washed with PBS, and
incubated with a 0.5% working solution of Oil Red O (Jiancheng Bioengineering Institute, Nanjing, China) for 15 min.
MMP-9 gelatinase activity. Following treatment of cell, 25 μ L cell-free condition media was collected by
centrifugation. Media was mixed with 25 μ L of Laemmli buer without β -mercaptoethanol and separated using
10% SDS-PAGE containing 1 mg/mL gelatin. e gels were incubated in Zymogram renaturing Buer (0.25%
Triton X 100 solution) for 1 h at room temperature followed by incubation overnight in Zymogram developing
buer (50 mmol/L Tris base, 50 mmol/L Tris-HCl, 0.2 mmol/L NaCl, 5 mmol/L CaCl2 and 0.02% Brij 35). Gels
were stained with Coomassie Blue R-250 solution to get clear bands against a dark blue background where the
proteases had digested the substrate.
Dil-ox-LDL uptake and binding assays. ox-LDL lipoproteins were labeled with the uorescent probe
DiI. For uptake assays, mouse peritoneal macrophages were incubated in fresh media containing 50 μ g/mL
DiI-Ox-LDL for 3 h at 37 °C. For the binding assays, cells were incubated for 15 min at 4 °C to stop membrane
internalization. Cells were visualized under a Nikon epi-uorescence microscope equipped with a digital cam-
era (Tokyo, Japan). Finally, cells were analyzed by ow cytometry (FACScalibur; Becton Dickinson, San Diego,
CA, USA). The results are expressed in terms of specific median intensity of fluorescence after subtracting
auto-uorescence of cells (absence of DiI-Ox-LDL).
Enzyme-linked immunosorbent assay. Mouse macrophages were pretreated with the compounds for
2 h, then treated with 50 mg/mL ox-LDL for 24 h. Aer treatment, the culture media and cells were collected

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Scientific RepoRts | 7:45917 | DOI: 10.1038/srep45917
separately. e levels of tumor necrosis factor alpha (TNF-α ) and interleukin-6 (IL-6) in the media were deter-
mined by enzyme-linked immunosorbent assay (ELISA) (eBioScience, San Diego, CA). e total quantity of the
inammatory factor in the media was standardized to the total protein amount of the viable cell pellets.
Mitochondrial Membrane Potential (Δ ψ) analysis. Cells were seeded onto glass slides (Orange
Scientic. E.U). JC-1 assay reagent (Beyotime BioTech., Nanjing, China) was diluted in culture media and cells
were incubated for 20 min to stain the mitochondria. Aer 2 to 3 rinses, cells were inspected using an Axiovert
200 uorescent inverted microscope (Zeiss, Germany). Both monomeric (excitation at 488 nm, emission 500–
550 nm) as well as aggregation (excitation 488 nm, emission at 575–620 nm) were registered using the microscope.
Measurements of the level of serum lipid and biochemical indicators. e components of serum
lipid including the total triglycerides (TG), low-density lipoprotein (LDL), Total cholesterol (TCH). (Nanjing
Jiancheng, Jiangsu, China).
Determination of ROS generation by uorescent microscope and ow cytometry. In order to
analyze ROS generation, we used Dichloro-dihydro-uorescein diacetate (DCFH-DA) which measures H2O2 and
allows for ROS determination in live cells. e uorescence intensity for 10,000 events was acquired using FACS,
and cellular images were captured under the Nikon uorescence microscope.
Determination of NADPH oxidase activity. Aer treatments, NADPH oxidase activity in cells was meas-
ured using NADP/NADPH Quantication colorimetric Kit (BioVision Inc., Milpitas, CA) as previously described45.
Animal experiments. Male ApoE−/− mice (18–20 g, 8 weeks) on C57BL/6 background were purchased
from HFK Bioscience Co. Ltd (Beijing, China). Mice were housed at a constant room temperature with a 12:12 h
light–dark cycle and fed with a standard rodent diet. Mice were acclimatized to the laboratory for at least 3 days
before initiating studies. All animal care and experimental procedures were approved by the Wenzhou Medical
University Animal Policy and Welfare Committee (wydw2014-0058). All animal experiments were performed
conform the NIH guidelines (Guide for the care and use of laboratory animals).
ApoE−/− mice were randomly divided into four weight-matched groups (n = 7, total 28 mice). 7 mice were
fed with standard animal low-fat diet containing 10 kcal.% fat, 20 kcal.% protein and 70 kcal.% carbohydrate
(MediScience Diets Co. LTD, Yangzhou, China, Cat. #MD12031) served as the normal control group (LFD),
while the remaining 21 mice were fed with high-fat diet containing 60 kcal.% fat, 20 kcal.% protein and 20 kcal.%
carbohydrate (HFD, MediScience Diets Co. LTD, Yangzhou, China, Cat. #MD12033) for 16 weeks. Since 9th week
HFD-fed mice were then divided into three groups: HFD (n = 7), AG1478-treated HFD (HFD + AG, n = 7) and
542-treated HFD (HFD+542, n = 7). AG and 542 compounds were administered orally at 10 mg/kg/day for the
last 8 weeks. e HFD and LFD groups received 1% CMC-Na solution alone. Bodyweight was recorded weekly
aer AG/542 administration. Mice were euthanized by rising CO2 inhalation, in accordance with Schedule 1 of
the Animals (Scientic Procedures) Act (1986), and blood was collected by cardiac puncture into a syringe con-
taining 4% trisodium citrate (1:10, v/v). Artery tissues were embedded in 4% paraformaldehyde for microscopic
analysis and/or snap-frozen in liquid nitrogen for gene and protein expression analysis.
Histology and analysis of atherosclerotic lesions. For analysis of plaque lesion in aortic sinus, the
heart and proximal aorta were removed and embedded in optimum cutting temperature compound. Serial 10
μ m-thick cryosections from the middle portion of the ventricle to the aortic arch were collected. Sections were
stained with oil red O and hematoxylin. For en face analyses of lesions in the entire aorta, whole aorta was dis-
sected out, opened longitudinally from heart to the iliac arteries, and stained with Oil Red O.
Five μ m frozen sections were stained with hematoxylin and eosin (H&E) for histopathological observation.
Paran sections (5 μ m) were stained with 0.1% Sirius Red and Masson trichrome for collagen deposition and
brosis.
Immunohistochemistry. Paran sections were deparanization and rehydration. Slides were incubated
with 3% H2O2 for 10 min to block endogenous peroxidase activity. Slides were blocked with 1% bovine serum
albumin in for 30 min and then incubated overnight at 4 °C with p-EGFR and smooth muscle α -actin antibody
(1:200). Horseradish peroxidase-conjugated secondary antibody (Santa Cruz; 1:500) and DAB were used for
detection.
Frozen sections were used for immunouorescence. Slides were blocked using 1% bovine serum albumin for
30 min and incubated overnight at 4 °C with CD68 antibody (1:200). FITC-conjugated secondary antibody (Santa
Cruz; 1:500) was used for detection. Slides were counterstained with DAPI.
Statistical analysis. Data are presented as means ± SEM. Dierences between groups were determined by
student’s t test or ANOVA multiple comparisons as appropriate using in GraphPad Pro (GraphPad, San Diego,
CA). Dierences were considered to be signicant at P < 0.05.
References
1. Heidenreich, P. A. et al. Forecasting the future of cardiovascular disease in the United States: a policy statement from the American
Heart Association. Circulation 123, 933–944 (2011).
2. Murray, C. J. & Lopez, A. D. Measuring the global burden of disease. N Engl J Med 369, 448–457 (2013).
3. Moore, . J. & Tabas, I. Macrophages in the pathogenesis of atherosclerosis. Cell 145, 341–355, doi: 10.1016/j.cell.2011.04.005
(2011).
4. Libby, P., ider, P. M. & Hansson, G. . Progress and challenges in translating the biology of atherosclerosis. Nature 473, 317–325
(2011).

www.nature.com/scientificreports/
13
Scientific RepoRts | 7:45917 | DOI: 10.1038/srep45917
5. Tabas, I., Williams, . J. & Boren, J. Subendothelial lipoprotein retention as the initiating process in atherosclerosis: update and
therapeutic implications. Circulation 116, 1832–1844 (2007).
6. Bentzon, J. F., Otsua, F., Virmani, . & Fal, E. Mechanisms of plaque formation and rupture. Circ es 114, 1852–1866 (2014).
7. Par, S. & Laatta, E. G. ole of inammation in the pathogenesis of arterial stiness. Yonsei medical journal 53, 258–261 (2012).
8. Conti, P. & Shai-Dasthagirisaeb, Y. Atherosclerosis: a chronic inammatory disease mediated by mast cells. Central-European
journal of immunology/Polish Society for Immunology and eleven other Central-European immunological societies 40, 380–386 (2015).
9. Assinger, A. et al. Hypochlorite-oxidized LDL induces intraplatelet OS formation and surface exposure of CD40L–a prominent
role of CD36. Atherosclerosis 213, 129–134 (2010).
10. He, F. & Zuo, L. edox oles of eactive Oxygen Species in Cardiovascular Diseases. Int J Mol Sci 16, 27770–27780 (2015).
11. Steinberg, D., Parthasarathy, S., Carew, T. E., hoo, J. C. & Witztum, J. L. Beyond cholesterol. Modifications of low-density
lipoprotein that increase its atherogenicity. N Engl J Med 320, 915–924 (1989).
12. Moore, . J. & Freeman, M. W. Scavenger receptors in atherosclerosis: beyond lipid uptae. Arterioscler romb Vasc Biol 26,
1702–1711 (2006).
13. Shi, Y. et al. Oxidized low-density lipoprotein activates p66Shc via lectin-lie oxidized low-density lipoprotein receptor-1, protein
inase C-beta, and c-Jun N-terminal inase inase in human endothelial cells. Arterioscler romb Vasc Biol 31, 2090–2097 (2011).
14. Schreier, B., Gele, M. & Grossmann, C. ole of epidermal growth factor receptor in vascular structure and function. Curr Opin
Nephrol Hypertens 23, 113–121, doi: 10.1097/01.mnh.0000441152.62943.29 (2014).
15. Gao, P. et al. Induction of oxidative stress by oxidized LDL via meprinalpha-activated epidermal growth factor receptor in
macrophages. Cardiovasc es 97, 533–543 (2013).
16. Troib, A. & Azab, A. N. Eects of psychotropic drugs on Nuclear Factor appa B. European review for medical and pharmacological
sciences 19, 1198–1208 (2015).
17. Zhang, H., Chalothorn, D., Jacson, L. F., Lee, D. C. & Faber, J. E. Transactivation of epidermal growth factor receptor mediates
catecholamine-induced growth of vascular smooth muscle. Circ es 95, 989–997 (2004).
18. Chattopadhyay, S. et al. EGF inase activity is required for TL4 signaling and the septic shoc response. EMBO ep 16,
1535–1547, doi: 10.15252/embr.201540337 (2015).
19. Stanic, B., Pandey, D., Fulton, D. J. & Miller, F. J., Jr. Increased epidermal growth factor-lie ligands are associated with elevated
vascular nicotinamide adenine dinucleotide phosphate oxidase in a primate model of atherosclerosis. Arterioscler romb Vasc Biol
32, 2452–2460, doi: 10.1161/ATVBAHA.112.256107 (2012).
20. Liang, D. et al. EGF inhibition protects cardiac damage and remodeling through attenuating oxidative stress in STZ-induced
diabetic mouse model. Journal of molecular and cellular cardiology 82, 63–74, doi: 10.1016/j.yjmcc.2015.02.029 (2015).
21. Qian, Y. et al. Novel Epidermal Growth Factor eceptor Inhibitor Attenuates Angiotensin II-Induced idney Fibrosis. J Pharmacol
Exp er 356, 32–42, doi: 10.1124/jpet.115.228080 (2016).
22. Wagsater, D., Zhu, C., Bjoregren, J., Sogsberg, J. & Erisson, P. MMP-2 and MMP-9 are prominent matrix metalloproteinases
during atherosclerosis development in the Ldlr(− /− )Apob(100/100) mouse. Int J Mol Med 28, 247–253, doi: 10.3892/ijmm.2011.693
(2011).
23. Di Marco, E. et al. Eects of NOX4 and NOX1 on immune cell-mediated inammation in the aortic sinus of diabetic mice. Clin Sci
(Lond), doi: 10.1042/CS20160249 (2016).
24. Naata, A. et al. Localization of heparin-binding epidermal growth factor-lie growth factor in human coronary arteries. Possible
roles of HB-EGF in the formation of coronary atherosclerosis. Circulation 94, 2778–2786 (1996).
25. Dreux, A. C., Lamb, D. J., Modjtahedi, H. & Ferns, G. A. e epidermal growth factor receptors and their family of ligands: their
putative role in atherogenesis. Atherosclerosis 186, 38–53, doi: 10.1016/j.atherosclerosis.2005.06.038 (2006).
26. Taahashi, M. et al. Epiregulin as a major autocrine/paracrine factor released from E- and p38MAP-activated vascular smooth
muscle cells. Circulation 108, 2524–2529, doi: 10.1161/01.CI.0000096482.02567.8C (2003).
27. Mueller, S. G., Paterson, A. J. & udlow, J. E. Transforming growth factor alpha in arterioles: cell surface processing of its precursor
by elastases. Mol Cell Biol 10, 4596–4602 (1990).
28. Tamura, . et al. Immunohistochemical localization of Betacellulin, a member of epidermal growth factor family, in atherosclerotic
plaques of human aorta. Atherosclerosis 155, 413–423 (2001).
29. Charaborty, S. et al. Constitutive and ligand-induced EGF signalling triggers distinct and mutually exclusive downstream
signalling networs. Nat Commun 5, 5811, doi: 10.1038/ncomms6811 (2014).
30. Yamashita, M. et al. Epidermal growth factor receptor is essential for Toll-lie receptor 3 signaling. Sci Signal 5, ra50, doi: 10.1126/
scisignal.2002581 (2012).
31. Wang, X., Sun, Y., Yang, H., Lu, Y. & Li, L. Oxidized Low-Density Lipoprotein Induces Apoptosis in Cultured Neonatal at
Cardiomyocytes by Modulating the TL4/NF-appaB Pathway. Scientic reports 6, 27866, doi: 10.1038/srep27866 (2016).
32. Yang, . et al. Oxidized low-density lipoprotein promotes macrophage lipid accumulation via the toll-lie receptor 4-Src pathway.
Circulation journal: ocial journal of the Japanese Circulation Society 79, 2509–2516, doi: 10.1253/circj.CJ-15-0345 (2015).
33. Zhang, X. et al. Involvement of TL4 in oxidized LDL/beta2GPI/anti-beta2GPI-induced transformation of macrophages to foam
cells. J Atheroscler romb 21, 1140–1151 (2014).
34. Li, W. et al. EGF Inhibition Blocs Palmitic Acid-induced inammation in cardiomyocytes and Prevents Hyperlipidemia-induced
Cardiac Injury in Mice. Scientic reports 6, 24580, doi: 10.1038/srep24580 (2016).
35. Tabas, I. & Bornfeldt, . E. Macrophage Phenotype and Function in Dierent Stages of Atherosclerosis. Circ es 118, 653–667, doi:
10.1161/CICESAHA.115.306256 (2016).
36. Cai, H. & Harrison, D. G. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ es 87, 840–844 (2000).
37. Jacson, M. J. et al. Antioxidants, reactive oxygen and nitrogen species, gene induction and mitochondrial function. Molecular
aspects of medicine 23, 209–285 (2002).
38. Wallace, D. C. Mitochondrial genetics: a paradigm for aging and degenerative diseases? Science New Yor, N.Y. 256, 628–632 (1992).
39. Griendling, . . & Ushio-Fuai, M. edox control of vascular smooth muscle proliferation. J Lab Clin Med 132, 9–15 (1998).
40. Alexander, . W. e Jeremiah Metzger Lecture. Pathogenesis of atherosclerosis: redox as a unifying mechanism. Transactions of the
American Clinical and Climatological Association 114, 273–304 (2003).
41. rieg, T., Cui, L., Qin, Q., Cohen, M. V. & Downey, J. M. Mitochondrial OS generation following acetylcholine-induced EGF
receptor transactivation requires metalloproteinase cleavage of proHB-EGF. Journal of molecular and cellular cardiology 36, 435–443
(2004).
42. Chen, X. et al. Integrin alpha1beta1 controls reactive oxygen species synthesis by negatively regulating epidermal growth factor
receptor-mediated ac activation. Mol Cell Biol 27, 3313–3326 (2007).
43. Yu, J. Y. et al. Mycotoxin zearalenone induces AIF- and OS-mediated cell death through p53- and MAP-dependent signaling
pathways in AW264.7 macrophages. Toxicology in vitro: an international journal published in association with BIBA 25,
1654–1663 (2011).
44. Nolly, M. B. et al. e signaling pathway for aldosterone-induced mitochondrial production of superoxide anion in the myocardium.
J Mol Cell Cardiol 67, 60–68 (2011).
45. Lien, G. S. et al. Epidermal growth factor stimulates nuclear factor-appaB activation and heme oxygenase-1 expression via c-Src,
NADPH oxidase, PI3, and At in human colon cancer cells. PLoS One 9, e104891, doi: 10.1371/journal.pone.0104891 (2014).

www.nature.com/scientificreports/
14
Scientific RepoRts | 7:45917 | DOI: 10.1038/srep45917
46. Yang, J., Li, Q., Zhou, X. D., olosov, V. P. & Perelman, J. M. Naringenin attenuates mucous hypersecretion by modulating reactive
oxygen species production and inhibiting NF-appaB activity via EGF-PI3-At/E MAPinase signaling in human airway
epithelial cells. Mol Cell Biochem 351, 29–40 (2014).
47. osenfeld, M. E. Inammation and atherosclerosis: direct versus indirect mechanisms. Curr Opin Pharmacol 13, 154–160 (2013).
48. Braganza, D. M. & Bennett, M. . New insights into atherosclerotic plaque rupture. Postgrad Med J 77, 94–98 (2001).
49. Fang, Q. et al. EGF mediates hyperlipidemia-induced renal injury via regulating inammation and oxidative stress: the detrimental
role and mechanism of EGF activation. Oncotarget 7, 24361–24373, doi: 10.18632/oncotarget.8222 (2016).
50. Cantley, L. C. et al. Oncogenes and signal transduction. Cell 64, 281–302 (1991).
51. Carpenter, G. eceptors for epidermal growth factor and other polypeptide mitogens. Annual review of biochemistry 56, 881–914,
doi: 10.1146/annurev.bi.56.070187.004313 (1987).
52. Carraway, . L. & Carraway, C. A. Signaling, mitogenesis and the cytoseleton: where the action is. Bioessays 17, 171–175, doi:
10.1002/bies.950170212 (1995).
53. Suc, I. et al. Activation of EGF receptor by oxidized LDL. FASEB J 12, 665–671 (1998).
54. Cole, J. E., assiteridi, C. & Monaco, C. Toll-lie receptors in atherosclerosis: a ‘Pandora’s box’ of advances and controversies. Tren ds
Pharmacol Sci 34, 629–636 (2013).
55. Hovland, A. et al. e complement system and toll-lie receptors as integrated players in the pathophysiology of atherosclerosis.
Atherosclerosis 241, 480–494 (2015).
56. Palova-Jelinova, L. et al. Pepsin digest of wheat gliadin fraction increases production of IL-1beta via TL4/MyD88/TIF/MAP/
NF-appaB signaling pathway and an NLP3 inammasome activation. PLoS One 8, e62426 (2013).
57. Pan, Y. et al. Attenuation of high-glucose-induced inammatory response by a novel curcumin derivative B06 contributes to its
protection from diabetic pathogenic changes in rat idney and heart. J Nutr Biochem 24, 146–155, doi: 10.1016/j.
jnutbio.2012.03.012S0955-2863(12)00096-4 [pii] (2013).
Acknowledgements
Financial support was provided by the National Natural Science Foundation of China (81470565, 81600341,
81570347, and 81500657), and Natural Science Funding of Zhejiang Province (LY16H310013). Guang Liang is
the guarantor of this work and had full access to all the data in the study and takes responsibility for the integrity
of the data and the accuracy of the data analysis.
Author Contributions
L.W., X.C., P.S., and Q.F. performed the research G.L., Z.H., and Y.W. designed the research study W.H., P.Z., and
J.W. contributed essential reagents or tools G.L., Y.W., and Z.H. analysed the data G.L. and Z.K. wrote the paper.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing Interests: e authors declare no competing nancial interests.
How to cite this article: Wang, L. et al. Inhibition of epidermal growth factor receptor attenuates atherosclerosis
via decreasing inammation and oxidative stress. Sci. Rep. 7, 45917; doi: 10.1038/srep45917 (2017).
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