A neutralizing IL-11 antibody reduces vessel hyperplasia in a mouse carotid artery wire injury model

Abstract

Vascular restenosis remains a major problem in patients with coronary artery disease (CAD) and
peripheral artery disease (PAD). Neointimal hyperplasia, defined by post-procedure proliferation
and migration of vascular smooth muscle cells (VSMCs) is a key underlying pathology. Here we
investigated the role of Interleukin 11 (IL-11) in a mouse model of injury-related plaque development.
Apoe−/− mice were fed a hyperlipidaemic diet and subjected to carotid wire injury of the right
carotid. Mice were injected with an anti-IL11 antibody (X203), IgG control antibody or buffer. We
performed ultrasound analysis to assess vessel wall thickness and blood velocity. Using histology
and immunofluorescence approaches, we determined the effects of IL-11 inhibition on VSMC
and macrophages phenotypes and fibrosis. Treatment of mice with carotid wire injury using X203
significantly reduced post-endothelial injury vessel wall thickness, and injury-related plaque, when
compared to control. Immunofluorescence staining of the injury-related plaque showed that X203
treatment did not reduce macrophage numbers, but reduced the number of VSMCs and lowered
matrix metalloproteinase 2 (MMP2) levels and collagen content in comparison to control. X203
treatment was associated with a significant increase in smooth muscle protein 22α (SM22α) positive
cells in injury-related plaque compared to control, suggesting preservation of the contractile VSMC
phenotype. Interestingly, X203 also reduced the collagen content of uninjured carotid arteries as
compared to IgG, showing an additional effect on hyperlipidemia-induced arterial remodeling in the
absence of mechanical injury. Therapeutic inhibition of IL-11 reduced vessel wall thickness, attenuated
neointimal hyperplasia, and has favorable effects on vascular remodeling following wire-induced
endothelial injury. This suggests IL-11 inhibition as a potential novel therapeutic approach to reduce
arterial stenosis following revascularization in CAD and PAD patients.

Full text

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A neutralizing IL‑11 antibody
reduces vessel hyperplasia
in a mouse carotid artery wire
injury model
David Schumacher1,2,15, Elisa A. Liehn3,4,5,6,15, Pakhwan Nilcham2, David Castaño Mayan7,8,9,
Chutima Rattanasopa7, Kaviya Anand7, Gustavo E. Crespo‑Avilan6,10,11,
Sauri Hernandez‑Resendiz6,10, Roshni R. Singaraja7,8,9, Stuart A. Cook6,10,12,16 &
Derek J. Hausenloy6,8,10,13,14,16*
Vascular restenosis remains a major problem in patients with coronary artery disease (CAD) and
peripheral artery disease (PAD). Neointimal hyperplasia, dened by post‑procedure proliferation
and migration of vascular smooth muscle cells (VSMCs) is a key underlying pathology. Here we
investigated the role of Interleukin 11 (IL‑11) in a mouse model of injury‑related plaque development.
Apoe−/− mice were fed a hyperlipidaemic diet and subjected to carotid wire injury of the right
carotid. Mice were injected with an anti‑IL11 antibody (X203), IgG control antibody or buer. We
performed ultrasound analysis to assess vessel wall thickness and blood velocity. Using histology
and immunouorescence approaches, we determined the eects of IL‑11 inhibition on VSMC
and macrophages phenotypes and brosis. Treatment of mice with carotid wire injury using X203
signicantly reduced post‑endothelial injury vessel wall thickness, and injury‑related plaque, when
compared to control. Immunouorescence staining of the injury‑related plaque showed that X203
treatment did not reduce macrophage numbers, but reduced the number of VSMCs and lowered
matrix metalloproteinase 2 (MMP2) levels and collagen content in comparison to control. X203
treatment was associated with a signicant increase in smooth muscle protein 22α (SM22α) positive
cells in injury‑related plaque compared to control, suggesting preservation of the contractile VSMC
phenotype. Interestingly, X203 also reduced the collagen content of uninjured carotid arteries as
compared to IgG, showing an additional eect on hyperlipidemia‑induced arterial remodeling in the
absence of mechanical injury. Therapeutic inhibition of IL‑11 reduced vessel wall thickness, attenuated
neointimal hyperplasia, and has favorable eects on vascular remodeling following wire‑induced
endothelial injury. This suggests IL‑11 inhibition as a potential novel therapeutic approach to reduce
arterial stenosis following revascularization in CAD and PAD patients.
OPEN
1Institute of Experimental Medicine and Systems Biology, University Hospital, RWTH Aachen University,
Aachen, Germany. 2Department of Anesthesiology, University Hospital, RWTH Aachen University, Aachen,
Germany. 3Department of Cardiology, Angiology and Intensive Medicine, University Hospital Aachen, Aachen,
Germany. 4Victor Babes National Institute of Pathology, Bucharest, Romania. 5Department of Intensive Care
and Intermediate Care, University Hospital, RWTH Aachen University, Aachen, Germany. 6National Heart
Research Institute Singapore, National Heart Centre, Singapore 169609, Singapore. 7Translational Laboratories
in Genetic Medicine, Agency for Science, Research and Technology, Singapore 138648, Singapore. 8Yong Loo
Lin School of Medicine, National University Singapore, Singapore 169857, Singapore. 9Cardiovascular Research
Institute, National University Health System, Singapore 119228, Singapore. 10Cardiovascular and Metabolic
Disorders Program, Duke-National University of Singapore Medical School, 8 College Road, Singapore 169857,
Singapore. 11Department of Biochemistry, Medical Faculty, Justus Liebig-University, Giessen, Germany. 12MRC
LMS, London W12 0NN, UK. 13The Hatter Cardiovascular Institute, University College London, London WC1E
6BT, UK. 14Cardiovascular Research Center, College of Medical and Health Sciences, Asia University, Taichung,
Taiwan. 15These authors contributed equally: David Schumacher and Elisa A. Liehn. 16These authors jointly
supervised this work: Stuart A. Cook and Derek J. Hausenloy. *email: derek.hausenloy@duke-nus.edu.sg
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Abbreviations
IL-11 Interleukin-11
VSMC Vascular smooth muscle cells
TGF-β Transforming growth factor-beta
ANGII Angiotensin-II
SMA Smooth muscle actin
MMP2 Matrix metalloproteinase 2
SM22 α Smooth muscle protein 22α
Despite advances in stent design and revascularization therapies, vascular restenosis remains a major problem in
patients with coronary artery disease (CAD) and peripheral artery disease (PAD)1–4. In-stent restenosis can lead
to severe complications such as cardiac ischemia and chronic limb threatening ischemia, and new therapeutic
strategies are needed to prevent these complications. Vascular smooth muscle cells (VSMCs) switching from its
contractile phenotype to a synthetic phenotype is a major contributor to neointimal hyperplasia, the key pathol-
ogy underlying vascular restenosis2,3.
VSMCs are specialized cells found within the medial layer of the vasculature where their primary function
is to regulate vessel tone and blood pressure. In response to vascular injury, VSMCs proliferate, migrate into the
tunica intima and assume a synthetic phenotype which is an adaptive response but results in vessel wall thicken-
ing. e synthetic VSMC phenotype is characterized by secretion of extracellular matrix, leading to brosis and
inammation. e cellular transition to a synthetic phenotype is termed phenotypic switching and plays a key
role in arterial restenosis, aortic remodelling, and the development of atherosclerosis5–11.
Two key factors associated with VSMC phenotypic switching and vascular pathologies such as atherosclerosis
and arterial restenosis are transforming growth factor-beta (TGFβ) and angiotensin-II (ANGII)12–14. Fibroblast-
to-myobroblast dierentiation and VSMC phenotypic switching share many similarities, including the secretion
of extracellular matrix, cell proliferation and migration, and both transitions can be triggered by the same stimuli.
We have recently discovered that IL-11, a little studied cytokine of the IL-6 family, is important for broblast
activation downstream of both TGFβ1 and ANGII as well as for VSMC phenotypic switching, in response to
the same stimuli15,16. We hypothesized that IL-11 might play a role in vessel hyperplasia and investigated the
eects of the neutralizing IL-11 antibody (X203) or an isotype control IgG antibody in a carotid wire-induced
endothelial injury mouse model.
Material and methods
All experiments and methods were performed in accordance with relevant guidelines and regulations. All animal
experiments were performed in accordance with ARRIVE (Animal Research: Reporting of InVivo Experiments)
guidelines and approved by the Biomedical Sciences Institute Singapore Institutional Animal Care Committee
at A*STAR (161165). All methods are reported in accordance with ARRIVE guidelines.
Mouse husbandry. All experiments were approved by the Biomedical Sciences Institute Singapore Insti-
tutional Animal Care Committee at A*STAR (161165). Mice were maintained on a 12h dark–light cycle, with
adlibitum access to water and were fed with lipid-rich Western-Type Diet (D12079B, Research Diets, NJ), as
indicated. Plasma for all experiments was isolated from blood withdrawn from the orbital sinus in EDTA coated
capillary tubes. Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), low-density lipopro-
tein (LDL-C), high-density lipoprotein (HDL-C), triglycerides (TG) and total cholesterol were measured using
Cobas c111 (Roche Diagnostics, Switzerland).
Carotid artery wire injury model of vascular restenosis. Male, 10 to 12week Apoe−/− mice (C57BL/6J
background, Charles River Laboratory, Italy) were fed lipid-rich Western-Type Diet17 for a total of 3weeks:
1week before and 2weeks aer wire injury. Only male mice were used for this study to avoid the interference
of estrogen eects on injury-related plaque with our target of interest IL-11. For the wire injury procedure, mice
were anesthetized (100mg/kg ketamine hydrochloride, 10mg/kg xylazine i.p.) and subjected to endothelial
denudation of the le common carotid artery using a 1cm insertion of a exible 0.36mm guide wire through
a transverse arteriotomy of the external carotid artery, as previously described17. Prior to surgery and for up to
2days post-surgery, we performed analgesia with subcutaneous injection of Buprenorphine (0.05–0.1mg/kg).
Mice were randomly divided in three treatment groups: (1) PBS, (2) IgG, and (3) anti-IL-11 antibody treatment.
Twenty mg/kg anti-IL-11 mouse monoclonal antibody (Clone 3C6; X203) or control IgG (Clone 11E10) were
administered via intra-peritoneal injections16, twice per week beginning on the day of wire injury and continu-
ing for 2weeks (Fig.1A).
X203 was generated in mice using a cDNA encoding amino acid 22–199 of human IL-11 cloned into expres-
sion plasmids (Aldevron Freiburg GmbH, Freiburg, Germany), as described previously18. Its ecacy has already
been demonstrated in arterial remodeling16, myocardial infarction15, liver brosis19 and pulmonary brosis18.
Ultrasound measurements of the carotid arteries and heart. Mice were anesthetized with 2% iso-
urane and monitored to maintain heart rate above 500beats/min during measurements. Measurements were
performed in B-Mode and M-Mode. Velocities were recorded and measured in B-Mode (2D-realtime) using
angle correction and vessel diameters (wall thickness) were recorded and analyzed in M-Mode using a 40MHz
transducer and a small-animal ultrasound imager (Vevo 3100, FUJIFILM Visualsonics, Toronto, Canada) as well
as the VevoLab Soware (FUJIFILM Visualsonics, Toronto, Canada).
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Histology and immunohistochemistry. Two weeks following wire injury, mice were anesthetized
(100mg/kg ketamine, 10mg/kg xylazine, i.p.) and carotid arteries were excised, xed in formalin and embed-
ded in paran. e carotid arteries were then cut in 5µm serial sections starting from the bifurcation until
500µm, for all the collected sections. Verhoe Van Gieson Elastic stain was performed as recommended by the
manufacturer (ab150667, Abcam, Cambridge, UK) in 10 serial sections (50µm apart, starting from the bifurca-
tion) for the le side and 4 serial sections (50µm apart, starting from the bifurcation for the right side). Injury-
related plaque areas were determined for all sections using Diskus soware (Hilgers, Königswinter, Germany),
as previously described17. e average of all 10 sections for the le side (4 for right side) was considered as nal
restenosis area for each vessel.
For further measurements and to minimize variability of arterial layers aer mechanical injury, we performed
the measurements in whole vessel wall and have referred to it as injury-related plaque. Serial sections (3 sections
per mouse, 100µm apart) were stained to analyze the injury-related plaque and vessel for early dierentiation
of VSMCs (SM22α ab14106, Abcam, Cambridge, UK) and mature VSMCs (smooth muscle actin, M 0851 clone
1A4, DAKO, Germany), macrophages (Mac2, CL8942AP, Cedarlane, Germany) and MMP2 (ab110186, Abcam,
Cambridge, UK). e sections were counterstained with DAPI for quantication of total cells. Positive-stained
cells were counted in the injury-related plaque in each section and expressed as cells per injury-related plaque,
percentage of all cells or percentage of positive area from the total injury-related plaque area. e results are
represented as average of the measurements of all 3 slides.
ree serial sections, 100µm apart, were stained with Gomori’s 1-step trichrome stain (ab150686, Abcam,
Cambridge, UK). Blue-stained collagen content was analyzed with Cell P Soware (Olympus, Hamburg, Ger-
many) and expressed as a percentage of the injury-related plaque area. Final results were represented as average
of the measurements of all 3 slides.
Figure1. e eect of anti-IL-11 antibody (X203) treatment on plasma lipids. (A) A schematic of the
experimental design. (B) Plasma concentration of AST, ALT, triglycerides, total cholesterol, LDL cholesterol and
HDL cholesterol (N = 12–14/group, One-way ANOVA, Tukey’s multiple comparison test, Values: mean ± SD).
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Statistical analysis. Data are presented as mean ± SD. Statistical analysis were performed with Prism 6.1
soware (GraphPad). For analyses between more than 2 groups we used 1-way ANOVA followed by Tukey’s
multiple comparison test. P values of < 0.05 were considered signicant.
Results
X203 treatment for 2 weeks did not aect blood lipids. To investigate the eect of IL-11 inhibi-
tion on neointimal hyperplasia, we performed wire injury in mice randomly assigned to receive either control
IgG, the anti-IL-11 antibody X203, or no treatment (Fig.1A). Since circulating lipids play an important role in
arteriosclerosis and neointimal hyperplasia, we rst assessed plasma lipids. With short term treatment, there
were no dierences in triglyceride, total cholesterol, LDL cholesterol or HDL cholesterol levels in mice across
experimental groups (Fig.1B). Furthermore, liver transaminases were similar in all treatment groups (Fig.1B)
suggesting that systemic anti-IL-11 antibody administration did not aect plasma lipids or liver function over
the experimental time course.
X203 treatment reduced post‑wire injury neointimal hyperplasia. To determine the eect of IL-11
inhibition on neointimal hyperplasia, we performed ultrasound analyses of injured carotid arteries. Wall thick-
ness was signicantly reduced in the X203 treated group compared to controls, whilst there were no dierences
in blood ow velocity (Fig.2A,B). Interestingly, the ultrasound measurements of right-side, uninjured carotid
artery show signicant thinning aer X203 treatment, while velocity showed no dierences (Fig.2C,D), dem-
onstrating the eect of the X203 treatment on arterial remodeling. Original acquired ultrasound images are now
presented in a supplementary gure (Suppl. Fig.1).
In addition, injury-related plaque (Fig.3A), neointimal areas (Fig.3B) were signicantly reduced in X203-
treated mice compared to controls. ere were no dierences in tunica media area between the treatment groups
(Fig.3C) Representative images are shown in Fig.3D. Analyzing the right, uninjured carotid arteries, we found
no dierences in total vessel area (Fig.3E), intima (Fig.3F) or media (Fig.3G). Representative images are shown
in Fig.3H. One out of 12 control carotid arteries (Suppl. Fig.2A) and one out of 13 IgG-treated carotid arteries
developed native atherosclerotic plaques, whereas none of the 14 X203-treated carotid arteries developed native
atherosclerotic plaque.
X203 treatment had no eect on post‑endothelial injury macrophage injury‑related plaque
inltration. Inammation and macrophages play an important role in arteriosclerosis and vascular
restenosis20. erefore, we assessed the eect of IL-11 inhibition on the number of macrophages inltrating the
Figure2. e eect of anti-IL-11 antibody (X203) treatment on vessel wall thickness. (A) Velocity and wall
thickness of injured le carotid artery (N = 10/group, One-way ANOVA, Tukey’s multiple comparison test,
Values ± SD). (B) Representative M-Mode images of injured le carotid artery. Brackets show carotid artery size,
and arrows point out the measured wall thickness. (C) Velocity and wall thickness of uninjured right carotid
artery (N = 10/group, One-way ANOVA, Tukey’s multiple comparison test, Values ± SD). (D) Representative
M-Mode images of uninjured right carotid artery. Brackets show carotid artery size, and arrows point out the
measured wall thickness.
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injury-related plaque area. Neither the proportion of macrophages nor the absolute number of macrophages
were dierent across the treatment groups (Fig.4A–C). e right carotid arteries showed no staining or isolated
subendothelial staining for macrophage marker Mac2 (Fig.4D), except the one carotid artery from the control
groups presenting with native atherosclerotic plaque, which showed predominately macrophages inltration
(Suppl. Fig.2B).
X203 treatment reduced post‑endothelial injury VSMC accumulation. Given the central role
of VSMC switching to a synthetic phenotype characterized by proliferation and migration in post-endothelial
injury neointimal hyperplasia, we investigated the eect of IL-11 inhibition on VSMC accumulation and pheno-
type. X203 treatment reduced the numbers of VSMCs in the injury-related plaque (Fig.5A,B,D), and increased
the numbers of VSMCs expressing SM22α, a contractile marker (Fig.5C,E), when compared to controls, sug-
Figure3. e eect of anti-IL-11 antibody (X203) treatment on neointimal hyperplasia. (A) Injury-related
plaque area (N = 11/group, One-way ANOVA, Tukey’s multiple comparison test, Values ± SD). (B) Intima area
(N = 11/group, One-way ANOVA, Tukey’s multiple comparison test, Values ± SD). (C) Media area (N = 11/group,
One-way ANOVA, Tukey’s multiple comparison test, Values ± SD). (D) Representative images of Verhoe Van
Gieson Elastic stain (scale bar 200μm). (E) Vessel area of control uninjured right carotid arteries (N = 12–14/
group, One-way ANOVA, Tukey’s multiple comparison test, Values ± SD). (F) Intima area (N = 12–14/group,
One-way ANOVA, Tukey’s multiple comparison test, Values ± SD). (G) Media area (N = 12–14/group, One-way
ANOVA, Tukey’s multiple comparison test, Values ± SD). (H) Representative images of Verhoe Van Gieson
Elastic stain (scale bar 200μm).
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gesting that most VSMCs in the injury-related plaques of the X203-treated mice were contractile in phenotype,
suggesting atheroprotection. e right uninjured carotid arteries showed mainly vascular smooth muscle cells
in the vessel wall (Fig.5F).
X203 treatment reduced post‑endothelial injury injury‑related plaque brosis. Several recent
studies have reported the pro-brotic properties of IL-1115,16. Given the critical role of VSMC switching to a
synthetic phenotype in post-endothelial injury neointimal hyperplasia, which is characterized by the secretion
of extracellular matrix, we investigated the eect of IL-11 inhibition on MMP2 expression and collagen content.
X203 treatment decreased MMP2 expression (Fig.6A,B) and reduced collagen content (Fig.6C,D) in the injury-
related plaque, compared to controls, again suggesting that inhibition of IL-11 is atheroprotective via reducing
VSMC phenotype switching. Interestingly, the uninjured right carotid arteries showed a signicant reduction of
collagen content aer X203 treatment (Fig.6E,F), demonstrating the anti-brotic role of X203 treatment and
protection against arterial remodeling. is may account for the right carotid arteries appearing thinner during
the ultrasound measurements.
Figure4. e eect of anti-IL-11 antibody (X203) treatment on macrophage inltration. (A) Macrophages
per injury-related plaque (N = 11/group, One-way ANOVA, Tukey’s multiple comparison test, Values ± SD).
(B) Macrophage proportion of all cells (N = 11/group, One-way ANOVA, Tukey’s multiple comparison test,
Values ± SD). (C) Representative images of Mac2 immunouorescence staining and corresponding DAPI
staining (insets) (scale bar 100µm). Transparent red-lines were traced to delineate the injury-related plaque area
used for quantications. (D) Representative images of Mac2 staining in the right uninjured carotid arteries and
corresponding DAPI staining (insets) (scale bar 100µm).
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Discussion
We demonstrate for the rst time that inhibition of IL-11 reduces vessel wall thickness and neointimal hyper-
plasia in a carotid wire-induced endothelial injury mouse model. ese ndings wereassociated with benecial
eects on post-endothelial injury injury-related plaque remodelling as evidenced by decreased accumulation
of VSMCs, increased proportion of contractile VSMCs, lower MMP2 expression, and reduced collagen content
in the injury-related plaque. Interestingly, inhibition of IL-11 reduced the collagen content of uninjured carotid
arteries as compared to either IgG or buer control mice, showing an additional eect on hyperlipidemia-induced
arterial remodeling in the absence of mechanical injury.
Figure5. e eect of anti-IL-11 antibody (X203) treatment on injury-related plaque VSMC accumulation.
(A) SMA+ VSMCs per injury-related plaque (N = 11/group, One-way ANOVA, Tukey’s multiple comparison
test, Values ± SD). (B) SMA+ VSMCs percent of all cells (N = 11/group, One-way ANOVA, Tukey’s multiple
comparison test, Values ± SD). (C) SM22α expression in VSMCs in the injury-related plaque (N = 11/group,
One-way ANOVA, Tukey’s multiple comparison test, Values ± SD). (D) Representative images of SMA
immunouorescence staining (red) and corresponding DAPI staining (blue, insets) in le, injured carotid
arteries (scale bar 100µm). Transparent yellow-lines were traced to delineate the injury-related plaque area
used for quantications. (E) Representative images of SM22α (green) and SMA (red) immunouorescence
co-staining (yellow, scale bar 50μm). Transparent blue-lines were traced to delineate the injury-related plaque
area used for quantications. (F) Representative images of SMA immunouorescence staining (red) and
corresponding DAPI staining (blue, insets) in right, uninjured carotid arteries (scale bar 100µm).
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Autocrine IL-11 signaling is a key downstream eector of TGFβ1 and ANGII in dierent cell types involved
in extracellular matrix (ECM) production15,16,18,19. In cardiac broblasts15, lung broblasts18 and hepatic stellate
cells19, IL-11 is required for ERK-dependent myobroblast activation. Interestingly, TGFβ1 induces IL-11 secre-
tion from aortic and coronary artery VSMCs21,22. Recently, we showed a role of IL-11 in phenotypic switching
of VSMCs and discovered the existence of an autocrine loop of IL-11 activity in VSMCs, which is required
downstream of both TGFβ1 and ANGII for phenotypic switching to occur in the context of aortic modelling16.
However, the role of IL-11 in phenotypic switching and function of VSMCs in the setting of neointimal hyper-
plasia following endothelial injury has not been investigated. It is known that X203 treatment has a positive
eect on arterial remodeling in the context of hypertension16, on cardiac brosis and healing aer myocardial
infarction15, on liver brosis19 and in idiopathic pulmonary brosis18.
In this study, we showed that inhibiting IL-11 reduced neointimal proliferation and had favorable eects on
injury-related plaque remodelling following wire-induced endothelial injury in the mouse carotid artery. As
expected, carotid wire injury induced neointimal hyperplasia in control mice as evidenced by an increase in vessel
wall thickness and tunica intima area, and treatment with IgG had no eect on these parameters. Treatment with
the anti-IL-11 antibody X203 signicantly reduced vessel wall thickness (with no eects on carotid artery veloc-
ity) and decreased total injury-related plaque area (with a reduction in tunica intima area but no eect on media
area). e reduction in injury-related plaque area with X203 treatment was associated with decreased numbers of
VSMCs, with an increased expression of SM22α, a marker for VSMCs with a preserved contractile phenotype23.
ese ndings are consistent with our prior study showing that genetic or antibody-mediated inhibition of
IL-11 attenuated VSMC phenotypic switching16. Several studies have identied macrophages to be important
contributors to vascular restenosis24,25. However, we found no dierences in macrophage injury-related plaque
Figure6. e eect of anti-IL-11 antibody (X203) treatment on post-endothelial injury injury-related plaque
brosis. (A) MMP2 positive staining per injury-related plaque (N = 11/group, One-way ANOVA, Tukey’s
multiple comparison test, Values ± SD). (B) Representative images of MMP2 immunouorescence staining
(green, scale bar 100μm). Insets represent higher magnication of positive cells inside the restenosis plaque
(scale bar 50μm). Transparent red-lines were traced to delineate the injury-related plaque area used for
quantications. (C) Collagen content (blue) of the injury-related plaque (N = 11/group, One-way ANOVA,
Tukey’s multiple comparison test Values ± SD). (D) Representative images of Gomori stain (collagen in blue,
muscle in red, scale bar 50μm). (E) Collagen content (blue) of right, uninjured carotid arteries (N = 12–14/
group, One-way ANOVA, Tukey’s multiple comparison test Values ± SD). (F) Representative images of Gomori
stain (collagen in blue, muscle in red, scale bar 50μm).
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inltration with X203 treatment, suggesting that the benecial eects of inhibiting IL-11 on reducing neointimal
hyperplasia were independent of macrophage accumulation into the injury-related plaque at this timepoint.
In our previous study we showed that IL-11 induces phenotypic switching of VSMCs to a synthetic phenotype
characterized by secretion of collagen and extracellular matrix proteins, including MMP216, which is involved
with phenotype switching and migration26,27. Furthermore, expression of the VSMC contractile marker SMA22α
was also decreased in response to IL-11 antibodytreatment in the same study16. Consistent with a pathological
role for IL-11 on VSMC function, we show here that treatment with X203 decreased injury-related plaque MMP2
levels and increased injury-related plaque SMA22α levels, suggesting a favorable eect of IL-11 inhibition on
vascular remodelling following wire-induced endothelial injury.
We highlight that while IL-11 was discovered three decades ago, there is very little known of its eects in the
vasculature. Limited earlier studies have suggested IL-11 as anti-inammatory, anti-brotic and pro-regenera-
tive28 and in the vasculature, IL-11 has been thought to inhibit VSMC proliferation and plaque formation29, the
opposite of what we demonstrate here. One reason for the general misunderstanding of IL-11 function relates to
the repeated use of recombinant human IL-11 in mouse models of disease. Paradoxically, it was recently shown
that rhIL-11 is a competitive inhibitor of mouse IL-11 in mouse cells and thus much of the earlier literature may
need to be reconsidered30.
In conclusion, we show for the rst time that inhibition of IL-11 reduced neointimal hyperplasia following
endothelial injury and had favorable eects on vascular remodelling. ese ndings position the IL-11 antibody
(X203) as a novel therapeutic strategy for preventing post-angioplasty/stent restenosis and improving outcomes
in CAD and PAD patients undergoing revascularization.
Received: 11 May 2021; Accepted: 24 September 2021
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Acknowledgements
We thank Roya Soltan and Melanie Garbe for excellent technical assistance.
Author contributions
Conceptualization: D.J.H., S.A.C.; Methodology: E.A.L., D.S., R.R.S., S.A.C., D.J.H.; Formal analysis and inves-
tigation: D.S., E.A.L., P.N., D.C.M., C.R., K.A., G.C., S.H.R., R.R.S.; Writing—original dra preparation: D.S.,
E.A.L.; Writing—review and editing: R.R.S., D.J.H., S.A.C.; Funding acquisition: D.J.H.; Resources: D.J.H., E.A.L.,
S.A.C.; Supervision: D.J.H., S.A.C. All authors commented on previous versions of the manuscript and all authors
read and approved the nal manuscript.
Funding
is study was supported by the Interdisciplinary Centre for Clinical Research IZKF Aachen (junior research
group to E.A.L.). DS is supported by the Clinician Scientist program of the Faculty of Medicine of the RWTH
Aachen University. RRS was supported by the Agency for Science, Research and Technology (A*STAR) and
the National University of Singapore (NUS). SHR is supported by the Singapore Ministry of Health’s National
Medical Research Council under its Open Fund-Young Individual Research Grant (OF-YIRG)–(NMRC/
OFYIRG/0078/2018). SAC is supported by NMRC STaR award and the Tanoto Foundation. DJH is supported
by the Duke-NUS Signature Research Programme funded by the Ministry of Health, Singapore Ministry of
Health’s National Medical Research Council under its Clinician Scientist-Senior Investigator scheme (NMRC/
CSA-SI/0011/2017), Centre Grant (CGAug16M006), and Collaborative Centre Grant scheme (NMRC/
CGAug16C006). is article is based upon work from COST Action EU-CARDIOPROTECTION CA16225
supported by COST (European Cooperation in Science and Technology).
Competing interests
SAC is an inventor on the patent applications: WO/2017/103108 (TREATMENT OF FIBROSIS),
WO/2018/109174 (IL-11 ANTIBODIES), WO/2018/109170 (IL-11RA ANTIBODIES). S.A.C. is a shareholder
of Enleofen Bio PTE LTD. e remaining authors (DS, EAL PN, DCM, CR, KA, GEC, SHR, RRS and DJH)
declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 021- 99880-y.
Correspondence and requests for materials should be addressed to D.J.H.
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