Original Research
ATG16L1 Expression in Carotid Atherosclerotic Plaques Is
Associated With Plaque Vulnerability
Joelle Magné, Peter Gustafsson, Hong Jin, Lars Maegdefessel, Kjell Hultenby, Annika Wernerson,
Per Eriksson, Anders Franco-Cereceda, Petri T. Kovanen, Isabel Gonçalves,* Ewa Ehrenborg*
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Objective—Autophagy has emerged as a cell survival mechanism critical for cellular homeostasis, which may play a
protective role in atherosclerosis. ATG16L1, a protein essential for early stages of autophagy, has been implicated in the
pathogenesis of Crohn’s disease. However, it is unknown whether ATG16L1 is involved in atherosclerosis. Our aim was
to analyze ATG16L1 expression in carotid atherosclerotic plaques in relation to markers of plaque vulnerability.
Approach and Results—Histological analysis of 143 endarterectomized human carotid atherosclerotic plaques revealed that
ATG16L1 was expressed in areas surrounding the necrotic core and the shoulder regions. Double immunofluorescence
labeling revealed that ATG16L1 was abundantly expressed in phagocytic cells (CD68), endothelial cells (CD31), and
mast cells (tryptase) in human advanced plaques. ATG16L1 immunogold labeling was predominantly observed in
endothelial cells and foamy smooth muscle cells of the plaques. ATG16L1 protein expression correlated with plaque
content of proinflammatory cytokines and matrix metalloproteinases. Analysis of Atg16L1 at 2 distinct stages of the
atherothrombotic process in a murine model of plaque vulnerability by incomplete ligation and cuff placement in carotid
arteries of apolipoprotein-E-deficient mice revealed a strong colocalization of Atg16L1 and smooth muscle cells only
in early atherosclerotic lesions. An increase in ATG16L1 expression and autophagy flux was observed during foam cell
formation in human macrophages using oxidized-LDL.
Conclusions—Taken together, this study shows that ATG16L1 protein expression is associated with foam cell formation
and inflamed plaque phenotype and could contribute to the development of plaque vulnerability at earlier stages of the
atherogenic process. (Arterioscler Thromb Vasc Biol. 2015;35:00-00. DOI: 10.1161/ATVBAHA.114.304840.)
Key Words: ATG16L1 ◼ atherosclerosis ◼ autophagy ◼ carotid plaque
A
utophagy is a ubiquitous catabolic process by which
cells degrade protein aggregates and damaged organelles
in lysosomes.1 In basal conditions, autophagy is involved in
the maintenance of normal cellular homeostasis. In response
to environmental stress, such as starvation, hypoxia, oxidative
stress, or exposure to xenobiotics, autophagy is upregulated
and provides an internal source of nutrients for energy production, promoting cell survival.2
Genetic studies in yeasts allowed the discovery of a complex molecular machinery of autophagy in mammals. The
proteins of autophagy-related genes (Atg) are organized into
functional complexes that orchestrate the major steps of the
autophagy process, including the formation of an isolation
membrane known as a phagophore, the elongation, cargo
sequestration, and assembly of a double-membrane vesicle
named the autophagosome and its fusion to the endosome and
lysosome.3
Atg16L1 plays an essential role in the autophagosome formation through its binding to the Atg12-Atg5 complex, which
is localized on the phagophore, and, once the autophagosome
is assembled, Atg16L1 dissociates from it.4,5 In humans, the
discovery of a functional ATG16L1 polymorphism associated with Crohn’s disease has shed light on autophagy as a
key mechanism involved in the regulation of immunity and
the development of inflammatory bowel diseases.6 However,
it is unknown whether ATG16L1 could contribute in humans
to the regulation of other inflammatory-driven diseases, such
as atherosclerosis. Although atherosclerosis is a common
disease, leading to the formation of plaques in the arteries,
not all plaques lead to the development of symptoms. Human
Received on: October 30, 2014; final version accepted on: February 27, 2015.
From the Atherosclerosis Research Unit, Department of Medicine, Center for Molecular Medicine, Karolinska University Hospital (J.M., P.G., H.J.,
L.M., P.E., E.E.), Division of Clinical Research Center, Department of Laboratory Medicine (K.H.), Division of Renal Medicine, Department of Clinical
Science, Technology and Intervention (A.W.), Cardiothoracic Surgery Unit, Department of Molecular Medicine and Surgery (A.F.-C.), Karolinska Institutet,
Stockholm, Sweden; Wihuri Research Institute, Helsinki, Finland (P.T.K.); and Experimental Cardiovascular Research Group and Cardiology Department,
Skåne University Hospital, Clinical Research Center, Clinical Sciences Malmö, Lund University, Sweden (I.G.).
*These authors shared senior authorship.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.114.304840/-/DC1.
Correspondence to Joelle Magné, PhD, Atherosclerosis Research Unit, Center for Molecular Medicine, L8:03, Karolinska University Hospital SE-171
76 Stockholm, Sweden. E-mail [email protected]
© 2015 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
1
DOI: 10.1161/ATVBAHA.114.304840
2 Arterioscler Thromb Vasc Biol May 2015
Nonstandard Abbreviations and Acronyms
EC
MMP
oxLDL
SMC
endothelial cell
matrix metalloproteinases
oxidized-LDL
smooth muscle cell
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atherosclerotic plaques with high risk for causing events are
considered to be vulnerable or rupture-prone. These vulnerable plaques are rich in lipids and inflammatory infiltrates and
have thin fibrous caps poor in smooth muscle cells (SMC).7
The high-risk thin-cap fibroatheromas easily rupture and suddenly precipitate local thrombosis, which is the root cause of
the clinically significant atherothrombotic complications of
the disease, notably myocardial infarction and stroke.
Analyses of autophagy-defective animal models have
revealed that autophagy plays a protective role during early
atherosclerosis.8 In Ldlr knockout mice, a macrophage-specific depletion of Atg5 has shown to increase atherosclerotic
lesion size with an expansion of the necrotic core.9 In apolipoprotein-E knockout mice, proatherogenic inflammasome
activity has been associated with defective autophagy in macrophages.10 It has recently been demonstrated that autophagy
is also involved in the clearance of lipid droplets in macrophages, thus contributing to cholesterol efflux in macrophage
foam cells.11 Indeed, studies conducted by De Meyer and
colleagues showed that autophagy can be specifically stimulated in murine macrophages by drugs, such as everolimus,
an inhibitor of mammalian target of rapamycin or imiquimod,
a toll-like receptor 7 ligand.12,13 Interestingly, implementation of second-generation eluting stents coated with mammalian target of rapamycin inhibitors has shown to reduce the
incidence of restenosis and thrombosis after angioplasty.14
Unfortunately, these drugs also enhance cytokine production
and have recently been associated with adverse effects, such
as hyperlipidemia and hyperglycemia.15,16 Therefore, deeper
understanding of the interactions between autophagy and the
pathways involved in the development of atherosclerosis is
still warranted to improve new therapeutic strategies.
In the present work, we studied whether a protein involved
in the early steps of the autophagy process is expressed in
advanced human atherosclerotic plaques by characterizing
the expression of the autophagy protein ATG16L1 in carotid
atherosclerotic plaques. Additionally, we analyzed whether
ATG16L1 is related to plaque vulnerability by assessing
plaque inflammation, as well as histological and biochemical markers characteristic of vulnerable plaque phenotype. To
further define whether ATG16L1 could be involved in plaque
vulnerability at different stages of the athero-thrombotic process, analyses of ATG16L1 expression were performed at 2
distinct stages of the plaque progression in a murine model of
plaque vulnerability induction by incomplete ligation and subsequent cuff placement in carotid arteries from apolipoproteinE-deficient mice. Because apoptosis via caspase-3 activation
recently has been shown to directly induce ATG16L1 degradation and autophagy impairment in Crohn’s disease,17,18 we
sought to determine whether ATG16L1 protein expression as
well as autophagy flux could be modulated in human primary
macrophages during foam cell formation using oxidized lowdensity lipoprotein (oxLDL) as an early atherogenic stimulus
known to trigger lipid storage, inflammatory response, and
apoptosis.
Materials and Methods
Materials and Methods are available in the online-only Data
Supplement.
Results
ATG16L1 in Human Advanced
Carotid Artery Plaques
To determine the extent of ATG16L1 expression in carotid
atherosclerotic plaques, 143 human carotid plaques were analyzed first by immunohistochemistry. As controls, 5 human
mammary arteries free of atherosclerosis were also analyzed.
The clinical characteristics of the patients are summarized
in Table 1. In carotid atherosclerotic plaques, ATG16L1 was
expressed abundantly in the shoulder regions, areas surrounding the necrotic core, and healed ruptured regions, whereas in
healthy mammary arteries free of atherosclerosis, ATG16L1
showed a homogenous expression across the vessel wall
(Figure 1). There were no significant differences in the area
stained for ATG16L1 between plaques from symptomatic and
asymptomatic patients. No significant differences were found
in the areas stained for ATG16L1 when comparing patients
with or without clinical risk factors, such as diabetes mellitus, dyslipidemia, hypertension, obesity, or statin treatment.
The area stained for ATG16L1 correlated positively with the
area stained for lipids (Oil Red O staining; r=0.341, P<0.005)
and for phagocytic cells, such as macrophages (CD68 staining; r=0.455; P<0.005; Table 2). A significant negative association was found between ATG16L1 and the SMC marker,
α-actin (r=−0.261, P<0.01; Table 2). The above data show
that in advanced atherosclerotic plaques, ATG16L1 is associated with plaques rich in lipids (necrotic core) and with
Table 1. Clinical Characteristics of the Patients
N
Age, y
Body mass index
Male/Female
143
70 (SD, 9.5)
26.8 (SD, 3.9)
96/47
Degree of stenosis,%
90 (IQR 75–95)
Diabetes mellitus, %
55
Hypertension,%
75
Smoking (in the past or currently), %
80
Dyslipidemia, %
97
Total cholesterol, mmol/L
4.3 (IQR 3.5–5.1)
LDL cholesterol, mmol/L
2.3 (IQR 1.8–3.4)
HDL cholesterol, mmol/L
1.1 (IQR 0.9–1.4)
Triglycerides, mmol/L
1.3 (IQR 0.9–1.9)
High-sensitive CRP, mg/L
3.7 (IQR 2.2–7.4)
Statins, %
88
Values are mean±SD or median±interquartile range or count as indicated.
HDL indicates high-density lipoprotein; and LDL, low-density lipoprotein.
Magné et al ATG16L1 in Carotid Atherosclerotic Plaques 3
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Figure 1. Representative micrographs of ATG16L1 staining (brown) in frozen sections of carotid atherosclerotic plaques and in frozen
section of healthy mammary artery are shown at low magnification (scale bar=500 μm). Boxed areas in carotid atherosclerotic plaques
depict fibrous cap, shoulder, necrotic core, and ruptured plaque regions at higher magnification (scale bar=50 μm). L indicates lumen; NC,
necrotic core; and Thr, thrombus.
intracellular lipids (macrophage foam cells), but poor in contractile SMCs, all characteristics of a vulnerable plaque.
ATG16L1 Expression, Inflammation,
and Extracellular Matrix Proteins
To estimate whether ATG16L1 could be related to inflammatory pathways and matrix degrading proteins involved in
Table 2. Significant Spearman Correlations Between
Plaque ATG16L1, Plaque Histology, Plaque Cytokines and
Chemokines, and Plaque MMPs
the destabilization of the atherosclerotic plaques, correlation
analyzes were performed. ATG16L1 expression, measured
histologically, was analyzed looking for possible associations
to plaque contents of cytokines and matrix metalloproteinases
(MMPs), measured by ELISA. ATG16L1 expression area
correlated positively with plaque contents of cytokines and
chemokines, namely interleukin-6, monocyte chemoattractant
protein-1, macrophage inflammatory protein-1β, regulated
on activation T-cells expressed and secreted and chemokine receptor 7 (Table 2). Additionally, plaque expression of
ATG16L1 correlated with plaque content of MMP1, MMP2,
MMP9, and MMP10 (Table 2).
Plaque Histology
α-Actin
r=−0.261*
CD68
r=0.341†
Oil red O
r=0.455†
Plaque cytokines and chemokines
IL-6
r=0.280†
MCP-1
r=0.296†
MIP-1β
r=0.241*
RANTES
r=0.249†
CCR7
r=0.458*
Plaque MMPs
MMP1
r=0.306†
MMP2
r=0.219‡
MMP9
r=0.299†
MMP10
r=0.259†
CCR7 indicates cell chemokine receptor 7; IL, interleukin; MCP-1, monocyte
chemoattractant protein-1; MIP-1β, macrophage inflammatory protein-1β;
MMP: matrix metalloproteinases; and RANTES, regulated on activation T-cells
expressed and secreted.
*P<0.01.
†P<0.005.
‡P<0.05.
Colocalization of ATG16L1 and Cell Type
Markers in Human Carotid Plaques
Transmission electron microscopy micrographs of human
carotid atherosclerotic plaques have revealed structures identified as autophagosomes in disintegrating SMCs in the fibrous
cap.8 Previous animal studies have suggested a contribution of macrophage autophagy to the atherogenic process.19
Therefore, in the present study, we sought to investigate which
cell types are the sources of ATG16L1 in human plaques.
Double immunofluorescence staining for ATG16L1 and
cell type markers showed a relative colocalization between
ATG16L1 and SMC-α-actin. ATG16L1 was abundantly
expressed in phagocytic cells, endothelial cells (EC), and
mast cells in areas surrounding intraplaque microvessels as
revealed by colocalization analysis with CD68, CD31, and
tryptase, respectively (Figure 2).
To further identify the cell types expressing ATG16L1 in
human atherosclerotic plaques, immuno-electron microscopy
studies were performed. In parallel to ATG16L1 immunogold labeling, staining of the ultrathin sections for CD68,
α-SMC-actin, and CD31 was performed to facilitate the location of intraplaque macrophages, SMCs, and ECs. Occasional
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Figure 2. Detection of cellular sources of ATG16L1 expression in the fibrous cap area (ECs and SMCs) or in the shoulder area close intraplaque microvessels (macrophages and mast cells) of carotid atherosclerotic plaque. Double immunofluorescence labeling for ATG16L1
(red, Alexa-594-tagged) and cell type markers (green, Alexa-488-tagged); CD31 (EC marker), α-SMC-actin (SMC marker), CD68 (macrophage marker) and tryptase (mast cell marker). Nuclei were stained with DAPI (blue) and differential interface contrast microscopy or
nomarski is represented (gray). Scale bar=50 μm. EC indicates endothelial cells; L, lumen; and SMC, smooth muscle cells.
cytoplasmic ATG16L1 labeling of macrophages in the carotid
plaques was observed (Figure 3A). Abundant labeling of
ATG16L1 was observed in the cytoplasm and in areas close
to membrane-bound organelles in foamy SMCs and ECs
(Figure 3B and 3C).
Atg16L1 in Vulnerable Murine Carotid Plaques
Analyses of Atg16L1 expression were performed in an
atherosclerotic carotid plaque vulnerability model in
apolipoprotein-E-deficient mice and contralateral control carotid arteries of the same mice. Approximately 50%
of the 16-week-old male mice display features of vulnerable plaques, such as intraluminal thrombus formation as
observed by hematoxilin-eosin staining (Figure 4A). To better understand whether Atg16L1 could be involved in earlier
phases of the atherogenic process, carotid arteries obtained
from this murine model of vulnerable plaques were analyzed
by immunofluorescence at 2 distinct stages of the plaque
progression. Atg16L1 expression was augmented in carotid
arteries developing intimal hyperplasia (stable fibrous cap) as
compared with uninjured healthy contralateral carotid arteries or to stable, vulnerable carotid arteries (Figure 4B). The
EC marker, CD31, was not detected in mouse carotid artery
lesions, indicating a physical disruption of the endothelial
layer induced both by the cuff placement for the injured
arteries and the sectioning of the control arteries, as previously described.20 Double immunofluorescence examination
of Atg16L1 and the macrophage marker F4/80 showed no
colocalization in either hyperplastic or vulnerable carotid
arteries (Figure I in the online-only Data Supplement). In
sharp contrast, Atg16L1 and SMC-α-actin showed a strong
colocalization in early atherosclerotic carotid plaques developing intimal hyperplasia 4 weeks after ligation (Figure 4B),
but such colocalization was not observed in advanced atherosclerotic carotid plaques showing plaque vulnerability
features (Figure 4B).
These data suggest that Atg16L1 expression might be
induced at an early initial stage of the atherogenic process.
Magné et al ATG16L1 in Carotid Atherosclerotic Plaques 5
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Figure 3. Immunogold electron micrographs of
ATG16L1 staining in frozen section of carotid
atherosclerotic plaque. Ultrathin sections of
fixed carotid plaques were stained with cell
type markers CD68 (macrophage marker),
α-SMC-actin (SMC marker), or CD31 (EC
marker) to better locate macrophages and EC
on the electron micrographs. ATG16L1 staining
shown at low magnification (scale bar=1 μm)
in a macrophage (A), an SMC (B), and endothelial cells (C). Higher magnifications (scale
bar=100 nm) of the boxed areas are shown
in the right-hand panels, in which the arrows
show selective labeling of ATG16L1 by 10 nm
gold particles. EC indicates endothelial cell;
ECM, extracellular matrix; L, lumen; MФ, macrophage; N, nucleus; and SMC, smooth
muscle cell.
ATG16L1 Expression and Apoptosis Activation
Apoptosis through caspase-3 activation has recently been
described as an important regulator of ATG16L1 stability or
degradation in Crohn’s disease.17,18 Therefore, we analyzed
whether caspase-3 cleavage could be related to ATG16L1
expression in human carotid atherosclerotic plaques.
ATG16L1 expression area correlated positively with human
carotid plaque contents of cleaved caspase-3 (r=0.186;
P=0.026). ATG16L1 and cleaved caspase-3 showed a relative
colocalization in the fibrous cap and the shoulder region of
human carotid plaques as observed by double immunofluorescence examination (Figure 5A).
We further determined whether ATG16L1 stability, as well
as autophagy flux, could be modulated during human macrophage foam cell formation and apoptosis. Human macrophages
were differentiated from monocytes isolated from buffy coats
of healthy donors and treated with oxLDL, an atherogenic
stimulus known to induce lipid accumulation, inflammatory
response, autophagy, and apoptosis (Figure 5B–5D).
As expected, a physiological dose of oxLDL (25 μM)
increased LC3-II expression, the gold standard autophagy
marker,21 in primary macrophages, in a time-dependent manner
as compared with native-LDL. The autophagy flux also increased
over time as assessed by LC3-II after pharmacological inhibition
of autophagy by Bafilomycin A1, which prevents the fusion of
the autophagosome to the lysosome (Figure 5B). Expression of
ATG16L1 uncleaved and cleaved β isoforms also increased over
time after exposure to physiological dose of oxLDL (Figure 5B).
No modulation in caspase-3 expression and no detection of the
cleaved caspase-3 isoform were observed by immunoblotting in
the lysates of these human macrophages.
Macrophages treated with a cytotoxic oxLDL dose (100
μM) for 24 h showed significantly decreased ATG16L1
expression, as well as decreased autophagy flux compared
with the cells treated with lower oxLDL doses (Figure 5C).
When the human primary macrophages were treated with a
cytotoxic oxLDL dose (100 μM) during 48 h, the expression
of cleaved caspase-3 was detected by immunofluorescence
(Figure 5D). Colocalization between ATG16L1 and cleaved
caspase-3 was not observed in primary macrophage foam
cells.
Discussion
Recent studies have provided new insights into the importance of autophagy to maintain cardiovascular functions that
are potentially relevant during atherogenesis. In this study,
ATG16L1, an essential protein involved in the first steps of
the autophagy process, was analyzed in carotid atherosclerotic
plaques. This characterization of ATG16L1 protein expression in advanced human carotid atherosclerotic plaques and
in mouse carotid plaques during the atherothrombotic process
demonstrates that ATG16L1 is associated with characteristics
of atherosclerotic lesion destabilization at early and advanced
stages of atherosclerotic plaque progression.
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Figure 4. Detection of Atg16L1 expression in frozen sections of carotid artery from a model of vulnerable carotid atherosclerotic plaques
development in apolipoprotein E-deficient mice. A, Scheme of ligation and cuff placement in the right carotid artery and representative
cross-sections stained with hematoxilin-eosin of the control left carotid artery without injury, stable plaque in the right carotid artery after
4 weeks of incomplete ligation+4 days of cuff placement and vulnerable plaque in right carotid artery after 4 weeks+4 days ligation/cuff
treatment. B, Double immunofluorescence labeling for Atg16L1 (red; Alexa-594-tagged) and α-smooth muscle cell-actin (green; Alexa488-tagged). Nuclei were stained with DAPI (blue) and differential interface contrast microscopy or nomarski is represented (gray). Scale
bar=50 μm. L indicates lumen; and Thr, thrombus.
These findings provide evidence that the essential autophagy ATG16L1 protein is abundantly expressed in advanced
human atherosclerotic plaques in the necrotic core areas
and the areas surrounding them, particularly in the shoulder regions. During the atherosclerotic process, retention
of subendothelial lipoproteins attracts highly inflammatory
cells, which engulf the lipid pools and secrete cytokines,
chemokines, and proteolytic enzymes until they die, leaving behind cell debris and cholesterol crystals, forming a
necrotic core.22 Over time, a complex feature of the atherosclerotic lesion in humans is characterized by a large
necrotic core covered by a fibrous cap of variable thickness
and shoulder regions with local infiltration of immune cells
that actively produce inflammatory cytokines and enzymes.
The presence of ATG16L1 in these active regions of human
atherosclerotic lesions strongly suggests that this protein,
which is involved in the early stage of autophagy, plays a
significant role in atherosclerosis, at least in its late stages
in humans.
Because autophagy is a ubiquitous catabolic process
induced by a plethora of physiological and atherogenic stimuli, such as cholesterol crystals, hypoxia, reactive oxygen species, or oxLDL, ATG16L1 expression was anticipated to show
a diffuse and homogenous distribution in advanced atherosclerotic plaques.8 Instead, high levels of ATG16L1 correlate
with plaque areas rich in lipid and phagocytic cells, whereas
poor in contractile SMCs, which is the classical phenotype of
a vulnerable plaque. Although descriptive, to our knowledge,
the present work is the first one to show ATG16L1 expression in human atherosclerotic plaques, thereby strengthening
the link between autophagy pathways and an inflamed plaque
phenotype.
Indeed, ATG16L1 expression was significantly positively
correlated with proinflammatory cytokines, chemokines,
Magné et al ATG16L1 in Carotid Atherosclerotic Plaques 7
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Figure 5. ATG16L1 expression and apoptosis activation in human carotid atherosclerotic plaques and human primary macrophages.
A, Double immunofluorescence labeling for ATG16L1 (red; Alexa-594-tagged) and cleaved caspase-3 (green; Alexa-488-tagged) in the
fibrous cap area or in the shoulder area of carotid atherosclerotic plaque. Nuclei were stained with DAPI (blue) and Nomarski (gray). Scale
bar=25 μm. Protein expression of ATG16L1, LC3, caspase-3, and b-actin in whole lysates of human primary macrophages treated with
native-LDL (nLDL) or oxidized-LDL (OxLDL; 25 μg/mL) during different time points (B) or during 24 h (C) at different doses of OxLDL. The
cells were pretreated with Bafilomycin A1 (Bafilo; 100 nM) during 2 h as indicated. The uncleaved and cleaved isoforms of ATG16L1 were
detected through a short or long exposure time, respectively, of the immunoblots. D, Double immunofluorescence labeling for ATG16L1
(red; Alexa-594-tagged) and cleaved caspase-3 (green; Alexa-488-tagged) in human primary macrophages treated with oxLDL (100 μg/
mL) during 48 h. Nomarski (gray). Scale bar=10 μm. L indicates lumen; and LDL, low-density lipoprotein.
and MMPs in the plaque, suggesting a close connection
between ATG16L1 and inflammatory pathways in atherosclerotic lesions. Among the 34 conserved Atg genes and
their corresponding proteins involved in the molecular
machinery of autophagy in mammals, ATG16L1 is the first
autophagy marker which has been directly related to immunity and inflammation.23 Indeed, defective autophagy has
repeatedly shown to lead to general immune disorders. In
Crohn’s disease, a polymorphism in the ATG16L1 gene is
associated with an excessive production of cytokines, such
as interleukin 1-β and interleukin-6 in peripheral blood
mononuclear cells.24 Further, studies in macrophages from
Atg16L1-deficient mice demonstrated that direct interaction
between Nod2 and Atg16L1 is a key mechanism by which
autophagy controls the endotoxin-induced inflammatory
immune response through the translocation of procytokines
into the lysosome.25 Additional investigations are needed to
understand how ATG16L1 could regulate innate immunity
in atherosclerosis.
In the present study, mast cells, highly potent immune cells,
were sources of ATG16L1 in human carotid atherosclerotic
lesions, as shown by colocalization with the mast cell–specific
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protease tryptase. Importantly, in a 3-year follow up study, it
was found that patients with high content of mast cells in endarterectomized carotid atherosclerotic plaques experienced
significantly more cardiovascular events than the patients
with lesser quantities of mast cells in the plaques.26 Indeed,
it has repeatedly been observed that the number of mast cells
in atherosclerotic plaques and their state of activation contribute to plaque vulnerability. On activation, mast cells produce
and release granules which contain different mediators, such
as heparin, histamine, proteases, growth factors, proinflammatory cytokines, and chemokines, including interleukin-6,
TNF-α, monocyte chemoattractant protein-1, and regulated
on activation T-cells expressed and secreted.27 Interestingly,
in bone marrow–derived mast cells from Atg7-deficient
mice, it has recently been demonstrated that autophagy plays
a crucial role in the release of secretory granules from mast
cells,28 thus potentially rendering autophagy of mast cells in
advanced human atherosclerotic plaques a potential target for
suppression of their plaque-weakening effects. However, further mechanistic investigations are needed to understand how
ATG16L1 could affect mast cell functions during the atherogenic process.
We also provide evidence that phagocytic cells are important cellular sources of ATG16L1 in human carotid atherosclerotic lesions as observed by colocalization with CD68, a
marker of macrophages in atherosclerotic lesions. A positive
correlation between the areas of ATG16L1 staining and Oil
Red O lipid staining suggests an expression of this autophagy
protein in foam cells. Although autophagy has been involved
in the clearance of macrophage lipid droplets and shown to
contribute to cholesterol efflux from macrophage foam cells
in mice, emerging data suggests that autophagy proteins
could also be recruited into the phagosome, accelerating the
degradation of cell corpses by the phagocytes through the
noncanonical autophagy pathway named LC3-associated
phagocytosis.11,29 The full molecular machinery involved in
the cross-talk between autophagy and phagocytosis remains
to be elucidated.
The immunogold labeling of ATG16L1 in foamy α-actinpositive SMCs demonstrates that SMCs are a cellular source
of ATG16L1 in advanced atherosclerotic plaques. In agreement with this finding, recent data have shown a large
contribution of intimal SMCs in foam cell formation in
advanced atherosclerotic lesions where 50% of the CD68positive cells expressed SMCs markers.30 Further, studies
on human primary aortic SMCs revealed that exposure of
SMCs to oxidized LDL induced not only the expression of
autophagy markers but also the expression of phagocytic
markers.31 SMCs transition from a contractile to a migratory
and synthetic phenotype, reflected by a typical loss of SMCs
markers, such as SMC-α-actin, myosin SMC heavy chains,
and vimentin, has been reported in later stages of atherosclerosis32 and could be supported by the negative correlation
between ATG16L1 and SMC-α-actin staining observed here.
When considering this negative correlation with the fact that
lipid-phagocytosing α-actin-positive SMC which are gaining CD68-positivity, that is, converted to macrophage foam
cells, will retain their α-actin positivity,30 we can infer that
our finding of a positive correlation between the areas of
ATG16L1-positivity and the areas of Oil Red O positivity (neutral lipid content) and CD68-positive macrophages
reflects rather the presence of ATG16L1 in monocytederived macrophages than in SMC-derived macrophages.
Based on this characterization of ATG16L1 protein expression in human atherosclerotic plaques, we hypothesize that
ATG16L1 might be involved in earlier stages of the atherogenic process during plaque development when macrophage
foam cells are formed, and so ultimately contribute to destabilization of the plaque.
Atg16L1 expression was also analyzed in atherosclerosisprone mice. For this purpose, we obtained carotid samples at
different stages of plaque progression in a specific model of
apolipoprotein-E-deficient mice, in which ultimately plaque
vulnerability can be induced by incomplete ligation and cuff
placement. Importantly, this validated and well-characterized
mouse model of vulnerable atherosclerotic plaques induced
by blood flow alteration display similar features as the
human vulnerable carotid plaque, for example, a large lipid
core, accumulation of macrophages in the shoulder region,
a fibrous cap, the integrity of which can be disrupted with
superimposed thrombosis.20,33 Interestingly, we observed that
contractile SMCs expressing α-actin were cellular sources of
Atg16L1 only in stable carotid atherosclerotic lesions developing intimal hyperplasia. Similar to the findings observed in
the advanced human carotid atherosclerotic plaques, no colocalization between SMC-α-actin and Atg16L1 was observed
in the carotid arteries of mice with vulnerable atherosclerotic
lesions, suggesting that ATG16L1 might be involved in SMC
function and survival particularly at an early stage of the atherosclerotic plaque progression.
Indeed, this study provides evidence of an increased
ATG16L1 expression during macrophage foam cell formation, a crucial step in the initiation of atherosclerosis. At cytotoxic oxLDL levels, an impairment of autophagy flux was
associated with a decrease in ATG16L1 expression in primary
human macrophages. Furthermore, it has recently been demonstrated that ATG16L1 contains caspase cleavage motifs and
that caspase-3 is directly involved in the degradation of the
protein. Furthermore, a concomittant decrease in ATG16L1
stability and an increase in caspase-3 cleavage were observed
in primary macrophages when treated with high doses of
TNF-α and cycloheximide, an inhibitor of protein synthesis.18
In the present study, at a cellular level, our data suggest that
apoptosis and caspase-3 activation might not be involved in
the ATG16L1 degradation when foam cell formation is triggered by oxLDL.
At the tissue level, in the human carotid atherosclerotic
plaque, the colocalization between ATG16L1 and cleaved caspase-3 indicate that other or a combination of stimuli might
trigger ATG16L1 degradation by caspase-3. Of note, recent
data have suggested that ischemic stress, such as moderate
hypoxia, could potentiate inflammatory response in human
primary macrophages.34 Therefore, further analysis of different atherogenic stimuli in different cell types will be needed
to better describe the pathways involved in the regulation of
ATG16L1 expression.
Magné et al ATG16L1 in Carotid Atherosclerotic Plaques 9
Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
Finally, recent data from autophagy-deficient mice suggest
that macrophage autophagy could be a potential therapeutic
target for the treatment of atherosclerosis.19 In the present
study of human carotid atherosclerotic plaques, we emphasize the need to better delineate the role that autophagy plays,
besides the macrophages, in other cells of the vessel wall,
such as ECs, SMCs, and mast cells, during the development
of atherosclerosis.
Several limitations of the present study need to be considered. First, examination of atherosclerotic lesions in the longitudinal axial direction has demonstrated that upstream and
downstream segments of the plaque exhibit different pattern
of blood flow, significantly influencing the morphology and
the composition of the lesion. In this study, only 1 mm fragment was used for histology because the rest of the material
was used for quantitative biochemical analysis, so examination of these transversal sections only allows partial observations of ATG16L1 expression in human atherosclerotic
plaques. To the best of our knowledge, the present investigation is the first to report endothelial cells as a major source of
autophagy activity in human atherosclerotic plaques, but we
did not investigate whether and how ATG16L1 could influence endothelial function during atherogenesis. Further studies will be needed to understand the putative link between
ATG16L1, blood flow pattern, and endothelial dysfunction.
Furthermore, the mechanistic studies of ATG16L1 expression in macrophage foam cell formation were performed on
macrophages differentiated from monocytes isolated from
healthy individuals, which may not reflect the phenotype of
resident macrophages present in human advanced atherosclerotic plaques. Further ex-vivo studies on cells extracted
and isolated from the human atherosclerotic plaques will be
needed to better understand the contribution of ATG16L1 in
inflammatory responses in resident macrophages and other
immune cells.
Taken together, these findings support a role of the autophagy protein ATG16L1 in atherogenesis and development of
plaque vulnerability, thus suggesting a new member in this
pathophysiological pathway.
Acknowledgments
We thank Olivera Werngren and Ewa Idsund Jonsson, Karolinska
Institutet, Ana Persson, Michaela Nitulescu and Lena Sundius,
Experimental Cardiovascular Research Group, Lund University,
for excellent technical support and Knut and Alice Wallenberg
Foundation (CLICK facility).
Sources of Funding
This work was supported by the Swedish Research Council,
the Swedish Heart-Lung Foundation, the Marianne and Marcus
Wallenberg’s Foundation, the Swedish Medical Society, the
Swedish Foundation for Strategic Research, Karolinska Institutet,
the Fredrik and Ingrid Thuring Foundation, the Lars Hiertas Minne
Foundation, and Skåne University Hospital Funds. Wihuri Research
Institute is maintained by the Jenny and Antti Wihuri Foundation
(Helsinki).
Disclosures
None.
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Significance
Autophagy is a cell survival mechanism which can be upregulated in response to several atherogenic signals. Human genetic approaches
have demonstrated that ATG16L1, an essential protein during early stages of the autophagy process, is involved in the pathogenesis of
inflammatory disease, such as Crohn’s disease. However, it is still unknown whether ATG16L1 plays an important role in atherosclerosis
development. This study provides the first evidence that the essential autophagy protein ATG16L1 is abundantly expressed in active regions
of human carotid atherosclerotic plaques, particularly rich in lipids, phagocytic, and inflammatory cells, which corresponds to characteristics
of vulnerable plaques. Interestingly, characterization of ATG16L1 expression during human primary macrophages foam cell formation and in
carotid atherosclerotic plaques from a murine model of plaque vulnerability revealed that ATG16L1 is also involved in earlier stages of plaque
formation. This study is pioneer in showing that ATG16L1 is linked to atherosclerotic plaque vulnerability.
Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
ATG16L1 Expression in Carotid Atherosclerotic Plaques Is Associated With Plaque
Vulnerability
Joelle Magné, Peter Gustafsson, Hong Jin, Lars Maegdefessel, Kjell Hultenby, Annika
Wernerson, Per Eriksson, Anders Franco-Cereceda, Petri T. Kovanen, Isabel Gonçalves, and
Ewa Ehrenborg
Arterioscler Thromb Vasc Biol. published online March 12, 2015;
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
Greenville Avenue, Dallas, TX 75231
Copyright © 2015 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://atvb.ahajournals.org/content/early/2015/03/12/ATVBAHA.114.304840
Data Supplement (unedited) at:
http://atvb.ahajournals.org/content/suppl/2015/03/12/ATVBAHA.114.304840.DC1
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Materials and methods
Human atherosclerotic tissues
One-hundred and forty three human carotid atherosclerotic plaques from the Carotid
Plaque Imaging Project (CPIP) were studied. This biobank includes carotid plaques
from patients undergoing carotid endarterectomies at Skåne University Hospital, Malmö.
The indications for surgery were plaques associated with ipsilateral symptoms (transient
ischemic attack, stroke or amaurosis fugax) and stenosis higher than 70%, or plaques
not associated with symptoms but with stenosis larger than 80%. Eighty-two of the
patients had suffered symptoms. Informed consent was given by each patient. The
study was approved by the local Ethics review board.
Assessment of cytokines, matrix metalloproteinases (MMPs) and cleaved caspase-3
was performed by ELISA in plaque homogenates as previously described.1 As controls,
5 human mammary arteries free of atherosclerosis were obtained from the Advanced
Study of Aortic Pathology (ASAP). This biobank includes tissue biopsies from patients
undergoing elective open heart surgery for aortic valve disease and/or ascending aortic
disease as described previously.
Histology
After surgical removal, carotid plaques were snap-frozen, and fragments of 1mm from
the most stenotic region were obtained for histology. Transversal cryosections from the
fragments were stained for plaque histology markers α-SMC-actin, CD68, Oil red O and
Masson trichrome, as described previously.2 When staining for ATG16L1, primary
antibody polyclonal rabbit anti-ATG16L1 (PM040, MBL international Corporation) and
secondary antibody polyclonal goat anti-rabbit (DakoCytomation, Glostrup, Denmark)
were used. Areas of the different stainings in the plaque (% area) were quantified blindly
using Biopix iQ 2.1.8 (Gothenburg, Sweden) after scanning with ScanScope Console
Version 8.2 (LRI imaging AB, Vista California, USA) and photographed with Aperio
image scope v.8.0 (Aperio, Vista California, USA).
Immuno-electron microscopy
After surgical removal, another 1mm fragment of the carotid plaque, consecutive to the
one used for histology, in the most stenotic region was fixed in 3 % paraformaldehyde in
0.1 M phosphate buffer, washed and then infiltrated into 2.3 M of sucrose and frozen in
liquid nitrogen. Ultrathin sectioning was performed at -95°C and placed on carbonreinforced formvar-coated, 50 mesh nickel grids. Immunogold labelling procedure was
performed as follows: grids were placed directly on drops of 2% BSA (Sigma fraction V)
and 2% Fish gelatin (GE Healthcare, Buckinghampshire, UK) in phosphate buffer to
block non-specific binding. Sections were then incubated with the rabbit anti-ATG16L1
antibody in phosphate buffer containing 0.1% BSA + 0.1% Gelatin over night in a
humidified chamber at room temperature. The sections were thoroughly washed in the
same buffer and bound antibodies were detected with protein A coated with 10 nm gold
(Biocell, BBInternational, Cardiff, England). Sections were rinsed in buffer and fixed in
2% glutaraldehyde and contrasted with 0.05% uranyl acetate and embedded in 1%
methylcellulose and examined in a Tecnai 10 (FEI company, Eindhoven, The
Netherlands) at 100 kV. Digital images were taken by a Veleta camera (Soft Imaging
System GmbH, Műnster, Germany).
Mouse model of atherosclerotic plaque vulnerability
Analyses of Atg16L1 expression were performed in an atherosclerotic carotid plaque
rupture model in ApoE-deficient mice and contralateral control carotid arteries of the
same mice.3 The model in brief consists of an incomplete ligation (Vicryl 5-0 suture,
Ethicon Endo-Surgery Inc, Blue Ash, USA) of the common right carotid artery (just
below the bifurcation) for 4 weeks, which triggers intimal hyperplasia and non-ruptured
carotid atherosclerotic lesions. To provoke rupture of the developed plaque, a conical
polyethylene cuff is placed proximal to the ligation site for 4 days.4 Approximately 50%
of the 16 week old male mice display features of ruptured plaques, such as endothelial
cracks or ulcers, and intraluminal thrombus formation. After sacrifice, injured and control
carotid arteries are embedded (Cryomount, Histolab AB, Gothenburg, Sweden) and
snap frozen. All experiments have been approved by the Stockholm Regional Board for
Experimental Animal Ethics.
Immunofluorescence
To identify in which cell types the autophagy marker ATG16L1 is expressed in human
and mouse atherosclerotic plaques, we used fluorescent double-staining. Acetone-fixed
cryosections were first incubated with rabbit anti-ATG16L1 antibody (PM040, MBL
International Corporation) at 4°C overnight, followed with AlexaFluor 594 labeled goat
anti-rabbit IgG (Life technologies, Stockholm, Sweden) for 1h. Subsequently, the
sections were incubated overnight with mouse anti-CD68 antibody (Clone KP1, DAKO,
Stockholm, Sweden), mouse anti-SMC-α-actin antibody (Clone1A4, DAKO, Stockholm,
Sweden), mouse anti-CD31 antibody (Clone JC70A, DAKO, Stockholm, Sweden) or
mouse anti-human mast cell tryptase antibody (Clone AA1, DAKO, Stockholm, Sweden)
followed by AlexaFluor 488 labeled goat anti-mouse IgG (Life technologies, Stockholm,
Sweden), with subsequent staining of the nuclei using DAPI. To better identify
macrophages in the mice carotid atherosclerotic plaques, sections were incubated with
rat anti-F4/80 (Clone BM8, Abcam, Stockholm, Sweden) followed with AlexaFluor488
labeled goat anti-rat IgG (Life technologies, Stockholm, Sweden). For the fluorescent
double staining between cleaved caspase-3 and ATG16L1, the sections were incubated
with mouse anti-ATG16L1 antibody (M150-5, MBL International Corporation, Woburn,
USA) then with rabbit anti-cleaved caspase-3 antibody (9961, Cell signaling, Danvers,
USA). The specificity of the ATG16L1 antibody was confirmed by incubation with
isotype-matched control IgG. Images were obtained using a Zeiss LSM700 confocal
laser microscope using x20, 0.8NA objective lens (for the atherosclerotic lesions) or
using a Leica SP5 confocal laser microscope using x100 oil, 1.4NA objective lens, (for
the macrophages). Each image consisted of a Z-stack of 15 to 20 optical slices taken at
0.3µm intervals.
Primary human macrophage studies
To determine whether oxidized-LDL could stimulate ATG16L1 expression, human
monocytes were isolated from fresh buffy coats of healthy donors (Blood Transfusion
Center, Karolinska University Hospital, Stockholm, Sweden) as previously described.5
In brief, human peripheral blood mononuclear cells were isolated from buffy coats by
endotoxin-free Ficoll density gradient centrifugation. Monocytes were then separated
from lymphocytes by high density hyper-osmotic Percoll density gradient centrifugation
and separated from platelets and dead cells on a low-density iso-osomotic Percoll
density gradient. Monocytes were cultured in RPMI-1640 medium (Invitrogen,
Stockholm, Sweden) supplemented with penicillin-streptomycin, L-glutamine (2mM) and
5% FBS (Invitrogen, Stockholm, Sweden). The cells were seeded in 6-well plates at a
density of 0.8×106cells/mL and differentiated in the presence of recombinant mouse MCSF (100 ng/ml; PeproTech, Stockholm, Sweden) over seven days. The primary human
macrophages were then treated with home-made copper-oxidized LDL (OxLDL) at
different time points (2, 6, 24 or 48 hours) or at different dose (25, 50 or 100µg/mL).
Native-LDL (nLDL, 25µg/mL) was used for controls.
For the autophagy flux assessment, the cells were pretreated during 2h with Bafilomycin
A1 (100nM; B1793, Sigma-Aldrich, Stockholm, Sweden).
Protein expression of ATG16L1, LC3, Caspase-3 and β-Actin were analyzed in the
whole cell lysate by immunoblotting using rabbit anti-ATG16L1 antibody (PM040, MBL
International Corporation, Woburn, USA), rabbit anti-LC3 antibody (NB100-2220, Novus
biological, Cambridge, UK), anti-rabbit caspase-3 antibody (9962, Cell signaling,
Danvers, USA) and anti-mouse β-actin antibody (Sigma-Aldrich, Stockholm, Sweden)
as previously described.6
Statistics
Variables are presented as percentages or mean (standard deviation, SD), median
(inter-quartile range, IQR), depending on their distribution (normal or not, respectively).
Spearman rho was used to determine correlations. Significance was considered at
P<0.05. SPSS 20.0 was used for the statistical analysis.
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