Deficiency of IL12p40 (Interleukin 12 p40) Promotes Ang II

(Angiotensin II)–Induced Abdominal Aortic Aneurysm

Neekun Sharma, Rishabh Dev, Anthony M. Belenchia, Annayya R. Aroor,
Adam Whaley-Connell, Lakshmi Pulakat, Chetan P. Hans

Objective—Abdominal aortic aneurysm is caused by the accumulation of inflammatory cells in the aortic wall. Our recent
studies demonstrated that inhibition of Notch signaling attenuates abdominal aortic aneurysm formation by shifting
the macrophage balance towards anti-inflammatory (M2) phenotype. Using IL12p40−/− (interleukin 12 p40) mice, we
investigated the effects of M2-predominant macrophages on the development of abdominal aortic aneurysm.
Approach and Results—Male (8–10 week-old) wild-type and IL12p40−/− mice (n=15) on C57BL/6 background were infused
with Ang II (angiotensin II, 1000 ng/kg per minute) by implanting osmotic pumps subcutaneously for 28 days. In the
IL12p40−/− mice, Ang II significantly increased the maximal intraluminal diameter (9/15) as determined by transabdominal
ultrasound imaging. In addition, IL12p40-deletion significantly increased aortic stiffness in response to Ang II as measured
by pulse wave velocity and atomic force microscopy. Histologically, IL12p40−/− mice exhibited increased maximal external
diameter of aorta and aortic lesions associated with collagen deposition and increased elastin fragmentation compared with
wild-type mice infused with Ang II. Mechanistically, IL12p40 deficiency by siRNA (small interfering RNA) augmented
the Tgfβ2-mediated Mmp2 expression in wild-type bone marrow–derived macrophages without affecting the expression
of Mmp9. No such effects of IL12p40 deficiency on MMP2/MMP9 was observed in human aortic smooth muscle cells or
fibroblasts. Depletion of macrophages in IL12p40−/− mice by clodronate liposomes significantly decreased the maximal
external diameter of aorta and aortic stiffness in response to Ang II as determined by imaging and atomic force microscopy.
Conclusions—IL12p40 depletion promotes the development of abdominal aortic aneurysm, in part, by facilitating
recruitment of M2-like macrophages and potentiating aortic stiffness and fibrosis mediated by Tgfβ2.
Visual Overview—An online visual overview is available for this article.   (Arterioscler Thromb Vasc Biol. 2019;39:212-223.
DOI: 10.1161/ATVBAHA.118.311969.)
Downloaded from http://ahajournals.org by on June 21, 2023

Key Words: aneurysm ◼ fibrosis ◼ macrophage ◼ mice ◼ transforming growth factor

A bdominal aortic aneurysm (AAA) is a common aortic

disorder and 10th leading cause of death among men
over 65 years of age.1,2 AAAs tend to expand asymptomati-
cally until aortic rupture or dissection occurs, and once these
rupture, the overall mortality rate is >80%.3 The treatment of
AAA is confined to surgery and endovascular repair because
there are no effective pharmacological therapies to date.2,4
Thus, there is a need to define the molecular and cellular
mechanism of the disease to develop effective pharmacolog-
ical therapies.
The major factors contributing to AAA pathogenesis in-
clude infiltration of activated inflammatory cells and deg-
radation of extracellular matrix (ECM) along with loss of
vascular smooth muscle cells (SMCs).5–7 It is now well es-
tablished that innate and adaptive immune cells, including
macrophages, neutrophils, B cells, T cells, natural killer cells,
and dendritic cells contribute significantly to aortic aneurysm
development.8–10 Recent data from human AAA patients and
mouse models have reported the presence of macrophages in
the aneurysmal aorta wall, accumulating predominantly in the
adventitia.11–14
In response to different stimuli, macrophages differentiate
towards a spectrum of polarization with distinct functions.
The extreme ends of this polarized spectrum are called as pro-
inflammatory (M1) or anti-inflammatory (M2) macrophages
and serve distinct functions in the progression of AAA devel-
opment. Although heterogeneity of these macrophages have
been reported in various vascular diseases, recent studies in-
cluding ours have identified predominance of proinflamma-
tory macrophages within the inflamed aortic tissues.15–17 By
contrast, few studies have also reported the presence of anti-in-
flammatory macrophages in the aneurysmal tissues, which are
implicated in ECM remodeling and tissue repair.18,19 Existence
of these diverse macrophage phenotypes with pathogenic and
reparative roles has provided a complexity in understanding
the pathogenesis of AAA. Although inflammatory process

Received on: July 20, 2018; final version accepted on: November 26, 2018.
From the Department of Cardiovascular Medicine (N.S., R.D., A.M.B., L.P., C.P.H.), Department of Medical Pharmacology and Physiology (A.R.A.,
C.P.H.), Harry S. Truman Memorial Veterans’ Hospital (A.W.-C.), and Dalton Cardiovascular Research Center (N.S., R.D., A.M.B., L.P., C.P.H.), University
of Missouri, Columbia.
The online-only Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/ATVBAHA.118.311969.
Correspondence to Chetan P. Hans, PhD, School of Medicine-Cardiovascular Medicine and Medical Pharmacology and Physiology, University of
Missouri, Room 324B,134 Research Park Dr, Columbia, MO 65211. Email hanscp@health.missouri.edu
© 2018 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at https://www.ahajournals.org/journal/atvb DOI: 10.1161/ATVBAHA.118.311969

212
 Sharma et al   M2-Polarized Macrophage and AAA   213
Nonstandard Abbreviations and Acronyms
AAA abdominal aortic aneurysm
AFM atomic force microscopy
Ang II angiotensin II
BMDM bone marrow–derived macrophage
ECM extracellular matrix
IFN-γ interferon-γ
IL interleukin
MAPK mitogen-activated protein kinase
Mmp matrix metalloproteinase
PCR polymerase chain reaction
PWV pulse wave velocity
Smad mothers against decapentaplegic homolog 3
SMC smooth muscle cell
Tgfβ transforming growth factor β
Timp tissue inhibitor of metalloproteinase
WT wild type
seems to contribute to AAA, approaches to limit progression
of AAA by use of anti-inflammatory drugs for treating the
AAA patients have not been successful.20–22 These observa-
tions warrant in-depth studies about the role of inflammatory
factors in the development of AAA by examining specific
roles of M1 or M2 macrophages.
Among the various key players known to regulate macro-
phage phenotype is IL12 (interleukin 12), a proinflammatory
heterodimeric cytokine composed of p35 and p40 subunits.23,24
Downloaded from http://ahajournals.org by on June 21, 2023
Principally expressed by monocytes/macrophages and den-
dritic cells, it has been shown that macrophages from IL12p40
deficient mice are biased toward M2 activation profile.25,26 It
is also reported that IL12p40 deficiency enhances bleomycin-
induced fibrosis; however, it protects against silica-induced
fibrosis.27 Similarly, deficiency of IL12p35 enhances cardiac
fibrosis by promoting M2 macrophages and TGFβ (transform-
ing growth factor-β) production.26 Additionally, IL12p35 defi-
ciency presents robust silica-induced pulmonary inflammation
and fibrosis.28 Interestingly, studies on the independent role of
M2 macrophages leading to fibrosis and vascular diseases have
emerged.29,30 In the recent studies, low dose of Ang II (angi-
otensin II) infusion differentiated Ly6Chi monocytes into M2
macrophages in the aortic wall.30 Chemokine (C-C motif) re-
ceptor 2 antagonist prevented the accumulation of these mac-
rophages and was associated with reduction in fibrosis, elastin
loss, and blood pressure in these studies.30 Our lab has pre-
viously published that inhibition of Notch signaling protects
against the development of AAA associated with decreased
M1 polarization and increased M2 polarization of macro-
phages.15,31 However, direct roles of M2-predominant macro-
phages in the development of AAA are not yet determined.
In the present study, we sought to determine the impact of
M2-like macrophages on AAA development. We hypothesized
that deletion of IL12p40 and subsequent enhanced M2 macro-
phages aortic infiltration would accelerate AAA formation in
the setting of Ang II infusion through development of perivas-
cular fibrosis and elastin fragmentation. Our results showed
that deficiency of IL12p40 leads to enhanced AAA formation
associated with increased Tgfβ2 mediated proteolytic activity.
Materials and Methods
The authors declare that all supporting data are available within the
article and in the online-only Data Supplement. The authors also de-
clare that we will make analytic methods and study materials avail-
able to other researchers on request from the corresponding author
(C.P. Hans).
Animals, Design, and Aneurysmal Model
Eight-week old, male wild-type (WT; C57BL/6J; 000664) and
IL12p40−/− mice (B6.129S1; 002693) were purchased from The Jackson
Laboratory. In addition to 10 generations crossing with C57BL/6 mice
by The Jackson Laboratory, IL12p40−/− mice were further crossed to
C57BL/6 mice for 7 to 8 generations in our laboratory to generate on
homogenous C57BL/6J background. The WT and IL12p40−/− mice
were first bred to generate IL12p40+/− mice, and the male and female
IL12p40+/− mice were further interbred to generate WT and IL12p40−/−
littermates, which were used for the studies. Genotyping was per-
formed according to The Jackson Laboratory’s protocol.
Only male mice were studied because of low incidence of Ang
II–induced AAA in female mice as detailed in an Arteriosclerosis,
Thrombosis, and Vascular Biology Council statement.32 Mice were
kept on a 12 hours/12 hours light/dark cycle with standard chow.
Aneurysmal studies were performed on these mice by infusing Ang
II for 28 days using published protocols.13,15 Animals were randomly
allocated to Ang II infusion or control. For the end point data anal-
ysis, samples and data were blinded wherever possible to reduce bias.
Briefly, mice were anesthetized in a closed chamber with 1% to 2%
isoflurane in oxygen for 2 to 5 minutes until immobile. Each mouse
was then removed and taped on a heated (37±2°C) procedure board
with 1.0±1.5% isoflurane administered via nosecone during minor
surgery. Mini osmotic pumps (Model 2004; Alzet, Cupertino, CA)
containing Ang II (1000 ng/min per kg; Sigma) were implanted sub-
cutaneously in the neck region of anesthetized mice. The mice were
observed daily for normal behavior or sudden death. The mice that
died in response to Ang II were immediately necropsied to determine
the cause of death. At the end of the study, mice were terminated
with an overdose of anesthetics ketamine (100 mg/kg) and xylazine
(20 mg/kg). The aortas were dissected, fixed in 10% formalin, and
processed for macroscopic and histological studies. All the animal-
related experiments were approved by the Animal Care and Use
Committee at the University of Missouri (Columbia, MO). All the
animal experiments conform the National Institutes of Health guide-
lines (Guide for the care and use of laboratory animals).
Sample Size Calculation and Outcome Variables
Our primary outcome variable in the current study was an increase
in the external aortic diameter by ≥50% to define the presence or
absence of AAA. Based on our previous work and reports from other
labs using this definition, we powered the study with our main out-
come variable of maximal external aortic diameter using (α=0.05).
We estimated our sample size for this variable to n=10 per group to
determine if the data is statistically significant assuming a 5% sig-
nificance level using t test. For the pathological outcome measures,
we included cellular infiltration, elastin fragmentation, abundance of
collagen, and increased Mmp2 (matrix metalloproteinase) expres-
sion. For the functional outcome, aortic stiffness as determined by 2
independent methods (pulse wave velocity [PWV] and atomic force
microscopy [AFM]) was used.33,34
Transabdominal Ultrasound Imaging and
Quantification of Aortic Aneurysms
For ultrasonic imaging, mice were restrained for <15 s to put into the
anesthesia chamber, followed by anesthetization with oxygen and
vaporized isoflurane (≈2%). Loss of spinal reflexes was confirmed via
toe pinching, and the loss of corneal reflex was assessed by gentle
touch of the eye with a soft tissue paper technique. The animals were
placed on a heated (41°C) imaging stage in supine position while
under anesthesia. The body temperature, heartbeat, and respiration
 214   Arterioscler Thromb Vasc Biol   February 2019
rates were continuously monitored during the imaging procedure. For
abdominal aorta measurements, the abdominal hairs were removed by
applying hair removal cream followed by cleaning with wet gauze.
Warmed ultrasound gel was applied to the abdominal surface and ul-
trasound MS550D probe (40 MHz) applied to the gelled surface to
collect B-mode, M-Mode, ECG-based Kilohertz Visualization mode
images, as well as Power Doppler measurements, by the imaging
system (Vevo 2100, VisualSonics). Short and long axis scans of aortas
were performed on the abdominal aorta from the level of the left renal
arterial branch through to the suprarenal region. Cine loops of 100
frames were acquired throughout the renal region on the abdominal
aorta and used to determine the maximal diameters of the abdominal
aorta in the suprarenal region. To define consistency, all the ultrasound
data were collected in a blinded fashion by an experienced faculty
member in the core facility at Dalton Cardiovascular Research Center.
The Ang II–induced AAA were defined as having at least 50%
increase in the maximal intraluminal and external diameters of the
abdominal aorta compared with the control mice.15,35 The maximal
intraluminal diameters of the suprarenal abdominal aorta were quan-
tified in vivo by ultrasound imaging. For quantification of the max-
imal external diameters, suprarenal abdominal aortic diameters were
measured using ZEN lite software (Zen 2.3 blue edition; Zeiss, NY)
by an independent researcher ex vivo under a microscope. The av-
erage suprarenal aortic width was 0.87 mm in control mice, and con-
sequently, we defined AAA as >1.31 mm. For aortic rupture, mice
were closely monitored for acute rupture incidences for first 10 days
of Ang II infusion. The mice which died post-Ang II infusion im-
mediately underwent autopsy to determine the cause of death. The
aortic rupture was defined by the presence of blood clot in the chest
cavity and hemorrhage of abdominal aorta between the celiac artery
and the left renal artery.36 These aortas were isolated and examined
histologically for the presence of disrupted elastic laminae at the site
of rupture, with extravasation of blood.
Aortic Stiffness Measurement
Downloaded from http://ahajournals.org by on June 21, 2023
In vivo aortic stiffness was measured locally in the abdominal aorta by
PWV technique by analyzing ECG-based Kilohertz Visualization data
collected at day 14 and 28 of Ang II infusion using VevoVasc software
as described previously.34 Briefly, PWV was determined by simulta-
neous tracking of R-wave of the ECG and the pulse wave along the 2
locations of suprarenal abdominal aorta. VevoVasc software was used to
calculate PWV as a ratio of the distance (d) between 2 locations along
the aorta and time delay (∆t) of the pulse wave between both locations
and is expressed in m/s. The ex vivo aortic stiffness was determined in
the abdominal aortic sections by AFM.33 To evaluate the stiffness, a 2
mm segment of the abdominal aorta was obtained from mice. The aorta
was opened longitudinally, and the adventitial surface of each explant
was fastened to a glass coverslip using cell-tak (BD Biosciences).
The aortic stiffness within intact aortic explants was measured using
a nano-indentation protocol with AFM according to previously
described procedures.33 AFM measurements were conducted at room
temperature. The measurements for both PWV and AFM were con-
ducted following the 2-man principle, who were blinded to the study.
Blood Pressure Measurement
Blood pressure was measured noninvasively on conscious mice using
a CODA volume pressure recording tailcuff system (Kent Scientific
Corporation, Torrington, CT) as described previously.15 The CODA
system analyzes 6 measurements: systolic, diastolic pressure, mean
pressure, rate, blood flow, and blood volume. Briefly, mice were
acclimated for 2 days to restraint tubes and trial measurements. On
the third day, after 5 acclimation cycles, 25 individual blood pressure
measurements (technical replicates) were taken; all false readings (as
determined by the diagnostic software) were excluded, and any an-
imal failing to register at least 20 (80%) true readings were excluded
from analysis. Data was trimmed to exclude the lowest and highest
5% of measured values, and the mean was used to represent each an-
imal. The measurement of blood pressure was performed blindly with
respect to experimental groups.
Histology and Immunohistochemistry
After fixation, the abdominal aortas from experimental mice were
rinsed with PBS and processed for paraffin embedding. Serial sections
(5 μm) were prepared by cutting abdominal aorta into 2 equal halves
and sectioned throughout the tissue. The sections of the abdominal aorta
at regular intervals (200 μm) were subjected to hematoxylin and eosin,
elastin, and Masson trichrome stain for histoarchitectural evaluation of
aneurysm as described previously.37 The serial tissue sections obtained
from these mice were further subjected to immunohistochemistry.
For immunohistochemistry, the abdominal aortas were stained with
antibodies for Moma2 (monocyte+macrophage antibody; ab33451),
Cd206 (MCA2235T, BioRad), Tgfβ2 (ab36495), Mmp2 (ab37150),
Mmp9 (ab38898), phospho-p38 MAPK (mitogen-activated protein
kinase1:800; 9211, cell signaling), and phospho-smad3 (mothers
against decapentaplegic homolog 3; 1:200; ab52903) as described.37
The specificity of all the antibodies were confirmed using appropriate
IgG controls (I-2000–1, I-4000–1, I-1000–5; Vector laboratories) in
place of primary antibodies at same concentrations. The intensity of
the immunostaining was evaluated by obtaining 6 images from random
areas of interest at ×40 or ×100 from each tissue (n=6) and quantified
using Fiji version of Image J following the software directions.38 The
images were blinded to reduce bias during quantification.
Cell Isolation, siRNA Transfection, RNA
Extraction, and Quantitative Real-Time PCR
Bone marrow–derived macrophages (BMDMs) were isolated from
6- to 8-week old WT or IL12p40−/− mice by previously established
protocol.15,31 Briefly, femur and tibia bones were flushed with culture
media under aseptic conditions, and cells were collected and treated
with ACK (ammonium-chloride-potassium) lysis buffer (Gibco) to
lyse red blood cells. The remaining bone marrow–derived cells were
cultured in ultra-low 6 well tissue culture plates. After 6 hours, cells
were treated with macrophage colony stimulating factor (10 ng/mL,
R&D systems) in complete medium (RPMI [Roswell Park Memorial
Institute] 1640+10% human serum+1% penicillin-streptomycin) with
a media change every other day. At day 7, cells were serum starved in
1% FCS-RPMI for 2 hours followed by stimulation with lipopolysac-
charide (100 ng/mL, Sigma Aldrich) and IFN-γ (interferon-γ; 20 ng/
mL, Biolegend) or IL4/IL13 (10 ng/mL each) for 24 hours to polarize
into M1 or M2 states, respectively. For naive macrophages, cells
were treated with vehicle only. For gene expression analysis, media
was removed, and the cell pellets were mixed with RLT lysis buffer
(Qiagen). Total RNA was extracted using the RNeasy kit (Qiagen)
following the manufacturers’ instructions. The concentration of
extracted RNA was measured using a Nanodrop ND1000 to verify that
the 260:280 and 260:230 ratios were ≈2. For transfection studies of
BMDMs, IL12p40 siRNA (small interfering RNA; sc-39641), Tgfβ2
siRNA (sc-39803), and control siRNA (sc-37007) were used with
Lipofectamine RNA iMAX reagent (Invitrogen) and the OptiMEM
media (31985-070, Invitrogen) using the manufacturer’s protocol.
In some wells, IL12p40−/− BMDMs were treated with SB431542,
Tgfβ receptor kinase inhibitor (15 nmol/L, Tocris Biosciences), or
human recombinant Tgfβ2 protein (10 ng/mL, Fisher) for 48 hours.
Quantitative real-time polymerase chain reaction (PCR) was per-
formed on CFX connect real-time PCR detection system (Biorad) in
triplicate. Cycle threshold for Rpl13a was used to normalize gene
expression. The primer sequences for genes are available on request.
Western Blot Analysis
The supernatants from BMDMs grown in 60 mm culture dishes were
collected, and the media were concentrated 50-fold using Amicon
ultra-4 centrifugal filter units (Millipore). In some dishes, the cells
were treated with Ang II (200 nm; Sigma) for 24 hours or IL4/IL13
(20 ng/mL each, R&D systems) for 48 hours. The concentrated pro-
teins were mixed with equal volume of 2X Laemmli sample buffer
(Biorad) containing β-mercaptoethanol and boiled for 8 minutes at
95°C. After centrifugation, 20 µL of the samples were then loaded
onto SDS-PAGE (10% wt/vol) and electrophoretically transferred
to polyvinylidene fluoride (PVDF) membranes. After blocking, the
 Sharma et al   M2-Polarized Macrophage and AAA   215
membranes were probed with antibodies against Tgfβ1 (ab92486;
1:2000) and Tgfβ2 (ab36495; 1:2000). Membranes were then incu-
bated with appropriate HRP (horseradish peroxidase)-conjugated
secondary antibodies. Proteins on the membranes were visualized by
chemiluminescence detection kit (Super signal west femto maximum
sensitivity substrate; Thermo Scientific). The proteins in the media
were normalized to total protein contents of the cell extracts.
Aortic Ring Assay and Gelatin Zymography
Aorta from WT and IL12p40−/− mice were isolated, and aortic rings
were obtained for transfection or treatment as described previously.37
The aortic rings were treated with nonspecific siRNA or Tgfβ2 siRNA
using Lipofectamine as described above. The aortic rings were also
treated with SB431542 or human recombinant Tgfβ2 for 48 hours as
described above. For gelatin zymography, the aortic tissue lysates and
culture supernatants from aortic rings and IL12p40−/− BMDMs with
various treatments were used. The supernatants were concentrated
to 50-fold using Amicon ultra-10 centrifugal filter units (Millipore).
Aortic tissue lysates were prepared in RIPA (radioimmunoprecipi-
tation assay) buffer containing protease and phosphatase inhibitor.
Samples were briefly sonicated and centrifuged at 4°C at 10 000 rpm
for 20 minutes. After analysis of protein content (BCA Protein assay
kit, Pierce) in supernatants, gelatinase activity was detected by load-
ing equal amount of protein (20 µg) onto 7.5% SDS-PAGE gels con-
taining 1 mg/mL gelatin. Bands of lysis in Coomassie Blue–stained
gels were derived for bands corresponding to the pro and activated
forms of MMP2 and MMP9.
Macrophages Depletion, Flow Cytometry, and
Ang II Infusion
The macrophages in IL12p40 mice were depleted using liposomes
−/−
containing clodronate (dichloromethylene diphosphonate; clodronate-
Liposomes.org). Animals received 150 μL intraperitoneal injections
Downloaded from http://ahajournals.org by on June 21, 2023
of control or clodronate liposome 2× at 3-day interval. One day after
second injection, aneurysmal studies were conducted in these mice for
14 days by methods described above. Some mice were sacrificed after
2 clodronate injections to verify the absence of macrophages in spleens
by flow cytometry (Figure IX in the online-only Data Supplement).
Flow cytometry cell surface staining with standard procedures was
done using BV605 anti-F4/80 antibody as described previously.39 The
cells were analyzed by BD LSRFortessa X-20 instrument.
Statistical Analysis
Statistical analyses were performed using GraphPad Prism version
7.0 (GraphPad Software, Inc, CA) and SAS Proc GLM (SAS 9.4).
All the data were assessed for normality and equal variance using
GraphPad Prism and SAS Proc GLM. Continuous data were exam-
ined by Shapiro-Wilk test for normality. Unpaired 2-tailed Student t
test was used to determine statistical difference between 2 groups for
normally distributed continuous variables. For comparison of multiple
groups, ANOVA followed by Tukey multiple comparison analysis or
2-way ANOVA followed by Bonferroni post hoc tests were used. For
data without normal distribution, nonparametric Mann-Whitney U
test or Kruskal-Wallis test were applied. In data that revealed unequal
variance, Kolmogorov-Smirnov test was applied. For incidence of
AAA, Fisher exact test was performed. In the case of blood pressure,
repeated measure 2-way ANOVA was used to determine between and
within group differences, with time as the repeating factor. All data
are presented as mean±SEM. P<0.05 was considered statistically sig-
nificant for all tests.
Results
IL12p40 Deficiency Induces BMDM Polarization
Toward M2 Macrophage Phenotype
We initially characterized the BMDMs from C57BL6 (WT)
and IL12p40−/− mice for a panel of M1 and M2 genes by
quantitative PCR. In accordance with the published reports,25
BMDMs from IL12p40−/− mice showed significantly
decreased expression of proinflammatory (M1) macrophage
markers including iNos, Tnfα, and Notch1 (Figure IA in the
online-only Data Supplement). Concomitantly, the BMDMs
from IL12p40−/− mice showed modest increase in the ex-
pression of anti-inflammatory (M2) macrophage markers,
including Il10 and Tgfβ1 (Figure IB in the online-only Data
Supplement). In addition, the Western blot measurements
showed that IL12p40−/− macrophages spontaneously secrete
higher amount of Tgfβ2 in the supernatant compared with
that of WT (Figure IC in the online-only Data Supplement).
This increase in Tgfβ2 was consistent with Ang II or IL4/IL13
stimulation of macrophages. However, the Tgfβ1 expression
by IL12p40−/− macrophages remain unchanged, although it
was increased at basal level (Figure IC in the online-only Data
Supplement). In addition, the BMDMs were confirmed to be
>90% double positive for CD11b and F4/80 (Figure II in the
online-only Data Supplement). These results indicate that the
BMDMs from the IL12p40−/− mice represent a macrophage
profiling with predominance of anti-inflammatory genes (M2)
and decreased expression of proinflammatory genes (M1).
IL12p40 Deficiency Exacerbates Ang II–Induced
AAAs
To examine the direct role of M2-like macrophages in AAA
pathogenesis, WT and IL12p40−/− mice (n=15) were infused
with Ang II subcutaneously for 28 days. Transabdominal ul-
trasound imaging showed increased maximal intraluminal
diameter in the suprarenal region of the abdominal aorta of
IL12p40−/− mice at day 14 and 28 of Ang II, whereas no such
increase in maximal intraluminal diameter in this region was
observed in the WT mice infused with Ang II (Figure 1A and
1C). Aortic dissection-mediated mortality was observed in
13% of the IL12p40−/− mice (2 out of 15) in response to Ang II,
whereas no mortality was observed in WT mice with same treat-
ment (Figure 1D). The macroscopic examination of the aortas at
day 28 demonstrated that only 13% of the WT mice (2/15) de-
veloped AAA formation in response to Ang II as determined by
the maximal external aortic diameter in the suprarenal aortic re-
gion (Figure 1B, 1D, and 1E). The extent of AAA development
significantly increased to 60% (9/15) in IL12p40−/− mice at day
28 and was significantly higher compared with WT mice in re-
sponse to Ang II (Figure 1E). No change in the maximal aortic
diameter was observed in the saline-treated WT or IL12p40−/−
mice (Figure 1D). Ang II infusion increased systolic, diastolic,
and mean blood pressure in all mice, and no significant differ-
ences were observed between these groups at day 28 (Figure
III in the online-only Data Supplement). Similar increase in
the total cholesterol, low-density lipids, and high-density lip-
ids contents in the serum of both WT and IL12p40−/− mice was
observed in response to Ang II infusion at day 28 (Figure IV in
the online-only Data Supplement).
Next, we investigated whether increase in maximal intralu-
minal diameter in abdominal aorta of IL12p40−/− mice is corre-
lated with the aortic wall functions. Aortic stiffness as measured
by PWV using VevoVasc software was significantly higher in
IL12p40−/− compared with WT at day 28 of Ang II infusion
(1.76±0.40 versus 1.15±0.23 m/s; Figure 1F). Furthermore,
 216   Arterioscler Thromb Vasc Biol   February 2019
Downloaded from http://ahajournals.org by on June 21, 2023

Figure 1. IL12p40 (interleukin 12 p40) deficiency increases Ang II (angiotensin II)–induced abdominal aortic aneurysm (AAA) development and aortic stiffness.
A, Representative transabdominal ultrasound images obtained at day 0, 14, and 28 using a VisualSonics Vevo2100 showing increase in the luminal expansion
of abdominal aorta in the wild-type (WT) mice (top) and IL12p40−/− mice (bottom) in response to Ang II. Dashed yellow lines outline the lumen. B, Represen-
tative aortas from the experimental mice showing maximal aortic width in the suprarenal region of the aorta. Images were taken using Zeiss Stemi 2000-C
microscope. Scale bar, 1 mm. C, Quantification of maximal intraluminal diameters of abdominal aorta by ultrasound. D, Quantification of ex vivo maximal
external diameters of abdominal aortas by microscopy (n=6; control mice and n=15; Ang II mice). E, Incidence of AAA and aortic aneurysm rupture in WT and
IL12p40−/− mice in response to Ang II. F, Pulse wave velocity calculated from measurements of abdominal aortic pulse pressure as determined by ECG-based
Kilohertz Visualization in response to Ang II at day 28 (n=12). G, Bar graph showing increased aortic stiffness in IL12p40−/− mice compared with WT-Ang II
obtained by atomic force microscopy (n=8 per group). Two-way repeated measures ANOVA followed by Bonferroni post hoc analysis was used for statistics
in C. Kruskal-Wallis test was applied for statistics in D. Fisher exact test was used for comparing AAA incidence and mortality because of rupture in E. Mann-
Whitney test was applied for statistics in F and Kolmogorov-Smirnov test was applied in G.*P<0.05, **P<0.01, ***P<0.001. ns indicates nonsignificant.

AFM confirmed the increase in the aortic stiffness of the ab-
dominal aorta in IL12p40−/− mice than WT mice in response
to Ang II (8.72±3.21 versus 3.70±1.94 kpa; Figure 1G). These
results demonstrate that IL12p40 deficiency is associated with
aortic dysfunctions and increased AAA incidence.
IL12p40 Deficiency Increases Structural
Damages of Aorta in Response to Ang II
The tissue sections from different groups were subjected to
histological analysis to characterize the abdominal aortic
lesions using hematoxylin and eosin, Verhoeff-Van Gieson
(elastin), and Masson trichrome (collagen) staining of aorta.
Hematoxylin and eosin, elastin staining, and quantitative
data demonstrated that infusion of Ang II in WT mice in-
duced marginal adventitial thickening with minimal infiltra-
tion of inflammatory cells and no visible elastin fragmentation
(Figure 2C, 2G, and 2M). IL12p40 deficiency increased struc-
tural deformity as evidenced by thickening and noticeable
cellular infiltration in adventitial layer and increased frag-
mentation of elastin layer in response to Ang II (Figure 2D,
 Sharma et al   M2-Polarized Macrophage and AAA   217
Downloaded from http://ahajournals.org by on June 21, 2023

Figure 2. IL12p40 (interleukin 12 p40) deficiency increases structural damage of aorta in response to Ang II (angiotensin II). A–D, Transverse sections of ab-
dominal aorta stained with H&E (hematoxylin and eosin) illustrating the extent of abdominal aortic aneurysm progression at day 28 in response to saline (A–B)
or Ang II (C–D). E–H, Representative images of Verhoeff-Van Gieson staining demonstrating the extent of elastin fragmentation in IL12p40−/− mice infused with
Ang II at day 28. I–L, Representative collagen staining in the abdominal aorta of experimental groups visualized in blue using trichrome staining. M–N, Quanti-
fication of medial elastin degradation (M) and collagen deposition (N) in experimental groups (n=6 for each group). Scale bar=50 µm. **P<0.01, ***P<0.001. ns
denotes nonsignificant in Tukey multiple comparisons test. WT indicates wild-type.

2H, and 2M). In addition, the degree of collagen deposition
was significantly higher in the adventitial region of IL12p40−/−
compared with that of WT mice infused with Ang II indicating
that IL12p40 deficiency promotes aortic fibrosis (Figure 2L
and 2N and Figure VH in the online-only Data Supplement).
WT and IL12p40−/− control mice did not show any observable
difference in the structure of aorta in the suprarenal region
(Figure 2A, 2B, 2E, 2F, 2I, and 2J). These data demonstrate
that deficiency of IL12p40 in C57BL/6 mice induces elastin
fragmentation and increased collagen deposition in the ab-
dominal aorta exhibiting the characteristics features of AAA
in response to Ang II.
IL12p40 Deficiency Increases Infiltration
of M2 Macrophages in Aortic Lesions
Next, we characterized the immune cells infiltrated into AAA
lesions in IL12p40−/− mice using immunohistochemistry.
Aortic sections were stained with antibodies against common
macrophage marker (Moma2) and M2-macrophage specific
markers (Cd206 and Tgfβ2). Increased immunostaining of
Moma2-positive macrophages was observed within the remod-
eled adventitia of IL12p40−/− mice (arrows in Figure 3D),
whereas no such Moma2-positive cells were observed in WT
mice infused with Ang II (Figure 3C). Furthermore, the infil-
trated cells in the aorta of IL12p40−/− mice in response to Ang
II were found to be positive for both M2 macrophage mark-
ers, Cd206 and Tgfβ2 (Figure 3H and 3L). No marked in-
crease in the immunostaining for iNos (inducible nitric oxide
synthase; an M1 polarization marker) was observed in the
adventitia of IL12p40−/− mice or WT mice infused with Ang
II (data not shown). Interestingly, Tgfβ2 positive M2 macro-
phages were found to be penetrating towards the medial region
of the abdominal aortic sections in IL12p40−/− mice (arrows in
Figure 3H and 3L). WT and IL112p40−/− control mice did not
show any observable difference in cellular immunostaining
for Moma2, Cd206, or Tgfβ2 in the aortic tissue (Figure 3A,
3B, 3E, 3F, 3I, and 3J). These data demonstrated that mac-
rophages and Cd206-positive as well as Tgfβ2-positive cells
 218   Arterioscler Thromb Vasc Biol   February 2019

Figure 3. Recruitment of M2 macrophages in the aorta of IL12p40−/− (interleukin 12 p40) mice after 28 d of Ang II (angiotensin II) infusion. A–D, Immunohisto-
chemistry showing expression of Moma2 (monocyte+macrophage antibody), a macrophage marker, in the aortic tissue at day 28 of saline or Ang II treated mice.
E–H, Immunohistochemistry showing expression of Cd206, an M2-macrophage marker in the aortic tissue at day 28 of saline or Ang II mice. I–L, Immunostain-
ing of Tgfβ2 (transforming growth factor β) in the abdominal aorta of experimental mice. Some of the immunopositive cells are pointed with arrows (scale bar=50
µm). WT indicates wild-type.

(markers of M2 macrophages) seem to be infiltrating in the validate these observations, we immunostained the abdominal
Downloaded from http://ahajournals.org by on June 21, 2023

adventitial and medial regions of the aorta in IL12p40−/− mice
during pathogenesis of AAA.
IL12p40 Deficiency Induces Upregulation
of Tgfβ2 and Mmp2 Expression
TGFβ is one of the upstream signaling proteins known to alter
the structure and composition of ECM and known to play
an important role in vascular remodeling and fibrosis.40 We
examined if Tgfβ signaling could be associated with patho-
logical process of extensive matrix degradation in IL12p40−/−
mice in response to Ang II. To this end, we inhibited IL12p40
in WT BMDMs with IL12p40 siRNA and checked for expres-
sion of Tgfβ and downstream Mmps. As expected, increased
expression of Tgfβ2, not the Tgfβ1 mRNA was observed
in the WT BMDMs with IL12p40 deficiency (Figure 4A).
IL12p40 deficiency in WT BMDMs also increased the ex-
pression of Mmp2 mRNA without affecting Mmp9 expression
(Figure 4A). Deficiency of IL12p40 in fibroblasts and SMCs
using IL12p40-specific siRNA did not change the gene ex-
pression of Tgfβ2 and Mmp2 (Figure VII in the online-only
Data Supplement and data not shown). To confirm the increase
in Mmp2 is Tgfβ2-dependent, we treated IL12p40−/− BMDMs
with either Tgfβ pathway inhibitor (SB431542) or human re-
combinant TGFβ2 and checked the expression of Mmps and
Timps (tissue inhibitor of metalloproteinase). Interestingly,
we observed increased expression of Mmp2 with recombinant
TGFβ2 treatment (Figure 4B). We confirmed the increased
expression of TgfβR1 with recombinant TGFβ2 treatment
(Figure VIII in the online-only Data Supplement). To further
aortic sections from WT and IL12p40−/− mice with antibodies
specific to Mmp2 and Mmp9. As expected, significant increase
in the immunostaining of Mmp2 was observed in the adven-
titial region of IL12p40−/− mice than WT mice in response to
Ang II (Figure 4C–4F and 4K). However, there was no change
in the expression of Mmp9 in the aortic sections of WT and
IL12p40−/− mice infused with Ang II (Figure 4G–4J and 4L).
Taken together, it is conceivable that increase in Tgfβ2 may
play a role in increasing the expression of MMP2.
Furthermore, to confirm that Tgfβ2 is the mediator for
Mmp2 activity, we performed gelatin zymography studies
in aortic rings and macrophages with Tgfβ modulation. As
shown in Figure 4M, we found increased activity of Mmp2
in aortic rings as well as supernatants from IL12p40−/− mice
compared with WT both at basal level and with stimulation
of TGFβ2. The Mmp2 activity was decreased with SB431542
or Tgfβ2 siRNA (Figure 4M). Similarly, macrophages from
IL12p40−/− mice showed increased Mmp2 at basal level and
with stimulation with TGFβ2 (Figure 4N). This Mmp2 ac-
tivity was alleviated with SB431542 or Tgfβ2 siRNA treat-
ment. Finally, to confirm that Tgfβ pathway is activated in
aortic tissues, we immunostained the abdominal aortic sec-
tions with phospho-smad3 and phospho-p38 MAPK from all
4 experimental groups (Figure 5). Interestingly, we found sig-
nificantly increased number of nuclei positive for phosphory-
lated forms of both smad3 and p38 in the IL12p40−/− tissues
in response to Ang II (Figure 5D, 5H, 5I, and 5J). This data
suggested that Tgfβ pathway is activated and may play a role
in increasing the AAA in IL12p40−/− mice.
 Sharma et al   M2-Polarized Macrophage and AAA   219
Downloaded from http://ahajournals.org by on June 21, 2023

Figure 4. IL12p40 (interleukin 12 p40) deficiency increases Tgfβ2 (transforming growth factor β) dependent Mmp (matrix metalloproteinase) activity. A, mRNA
expression of IL12p40, Mmp2, Mmp9, Tgfβ1, and Tgfβ2 in wild-type (WT) bone marrow–derived macrophage (BMDMs) transfected with IL12p40-specific siRNA
(small interfering RNA) or negative control siRNA as measured by quantitative real-time polymerase chain reaction. B, mRNA expression of Mmps and Timps in
IL12p40−/− BMDMs treated with Tgfβ receptor kinase inhibitor (SB431542) or hr (human recombinant) TGFβ2. Representative immunohistochemistry images of
MMP2 (C–F) and MMP9 (G–J) in abdominal aorta of experimental mice. Quantification of MMP2 (K) and MMP9 (L) immunostaining in the adventitial region of
abdominal aorta from experimental mice. M and N, Representative gelatin zymography for pro-MMP2 (matrix metalloproteinase 2), active MMP2 and pro-MMP9
in aortic rings (M, top), supernatants from aortic rings (M, bottom), and supernatants from BMDMs of indicated groups (N). Scale bar=50 µm. *P<0.05, **P<0.01,
***P<0.001, ns denotes nonsignificant in paired 2-tailed Student t test (A and B) or in Tukey multiple comparisons test (K–L). Ang II indicates angiotensin II.

Depletion of Macrophages in IL12p40−/− Mice
Reduces Fibrosis and Aortic Stiffness
Finally, to confirm the active contributions of M2 macro-
phages in AAA pathogenesis, we depleted macrophages using
clodronate liposomes in IL12p40−/− mice and characterized
Ang II–induced aneurysm formation. Intraperitoneal injec-
tion of clodronate-containing liposomes resulted in a decrease
in F4/80+ macrophages in the mouse spleen evident by flow
cytometry for macrophage marker, F4/80 (Figure VII in the
online-only Data Supplement). At day 14 of Ang II infusion, a
significant decrease in the maximal external diameter of aorta
was observed in IL12p40−/− mice treated with clodronate than
control group. (Figure 6A and 6B). Aortic stiffness was also sig-
nificantly attenuated in the clodronate-treated IL12p40−/− mice
 220   Arterioscler Thromb Vasc Biol   February 2019

Figure 5. Tgfβ (transforming growth factor β) pathway is activated in IL12p40−/− (interleukin 12 p40) mice in response to Ang II (angiotensin II). Representative
immunohistochemistry images of phospho-smad3 (mothers against decapentaplegic homolog 3; A–D) and phospho-p38 MAPK (mitogen-activated protein
kinase) staining (E–H) in abdominal aorta of experimental mice. Quantification of phospho-smad3 (I) and phospho-p38 (J) nuclear immunostaining in the ab-
Downloaded from http://ahajournals.org by on June 21, 2023

dominal aorta from experimental mice. Scale bar=50 µm. *P<0.05, ***P<0.001, ns denotes nonsignificant in Tukey multiple comparisons test (I and J). WT
indicates wild-type.

than control IL12p40−/− mice in response to Ang II (Figure 6C).
Histological analysis of the aortic tissues revealed decreased
adventitial remodeling and reduced cellular infiltration in the
macrophages depleted group of IL12p40−/− mice (Figure 6D–
6I). Interestingly, there was less collagen deposition in the
adventitial region of clodronate injected IL12p40−/− Ang II
mice compared with IL12p40−/− Ang II alone as determined by
Trichrome staining (Figure 6H–6I). These results suggest that
increased fibrosis and arterial stiffness in the IL12p40−/− mice
may primarily be mediated by macrophages.
Discussion
The goal of the current study was to examine the direct role
of M2-like macrophages in the pathogenesis of Ang II–
induced AAA using IL12p40−/− mice as a model. We preferred
C57BL/6 mice over the well-known Apoe−/− mouse model be-
cause Apoe−/− mice have been shown to have a skew towards
increased expression of M1-associated genes even at naive
state.41 Moreover, the development of AAA in Apoe−/− mice is
critically driven by M1-polarized macrophages. However, we
aimed to determine the role of M2 macrophages in the fibrotic
events independent of inflammation observed in Apoe−/− mice
in response to Ang II. We demonstrated that predominance
of M2-like macrophages in IL12p40−/− mice increases Tgfβ2
mediated fibrosis resulting in increased Mmp2 expression.
These events lead to focal aortic enlargement along with
medial destruction thus causing AAA development. IL12,
composed of p35 and p40 subunits, is a well-known proin-
flammatory cytokine important in controlling macrophage
phenotype and T-cell effector functions.24 Previous studies
have illustrated that the macrophages from p35 or p40 defi-
cient mice have a bias towards the M2 activation profile.25,27
Although, both the p35 and p40 deficient mice are associated
with extensive cardiac fibrosis mediated by Tgfβ, herein, we
report the direct role of M2-like macrophages in the pathogen-
esis of AAA in IL12p40−/− mice.
The M2 macrophages are characterized by their involve-
ment in tissue remodeling, immune regulation, tumor pro-
motion, and phagocytic activity. These cells are generally
intended to create anti-inflammatory environment and pro-
mote healing. However, when the lesion is persistent, M2
macrophages secrete large amounts of profibrotic factor, such
as Tgfβ. The activation of Tgfβ signaling, a multifunctional
cytokine, is implicated in various cellular process, such as
proliferation, angiogenesis, and wound healing. Tgfβ sig-
naling, a modulator of the structure and composition of the
ECM, contributes to pathological tissue fibrosis of the heart,
lung, and liver in many vascular diseases.42–44 Here, we dem-
onstrated that in response to Ang II, activation of Tgfβ sig-
naling and fibrotic events in the aorta of IL12p40−/− mice are
associated with increased collagen deposition in the adventi-
tial region. Similarly, we observed infiltration of macrophages
in the aorta of IL12p40−/− mouse that was positive for Tgfβ2
immunostaining. Significant increase in Tgfβ2 expression in
 Sharma et al   M2-Polarized Macrophage and AAA   221

Figure 6. Depletion of macrophages in IL12p40−/− (interleukin 12 p40) mice attenuates structural damage of aorta. A, Representative aorta from IL12p40−/−
mice treated with PBS liposome and clodronate liposome followed by Ang II (angiotensin II) infusion for 14 d. B, Quantification of maximal aortic width from
aortas of IL12p40−/− control and clodronate-treated mice at day 14 of Ang II infusion (n=11). C, Abdominal aortic stiffness as measured by atomic force
microscopy at day 14 of Ang II infusion in IL12p40−/− control and clodronate-treated mice (n=8). Representative H&E (hematoxylin and eosin) image (D–E),
elastin staining (F–G), and Trichrome staining (H–I) from IL12p40−/− experimental mice at day 14 of Ang II infusion. **P<0.01, ***P<0.001 in Mann-Whitney test.
Scale bar=50 µm.
Downloaded from http://ahajournals.org by on June 21, 2023

the WT BMDMs transfected with IL12p40 siRNA suggested
that Tgfβ2 is the major pathway affected by M2 macrophages
associated with IL12p40 deficiency. Overall, these results
implicated that Tgfβ2 may be one of the major players during
pathogenesis of AAA in IL12p40−/− mouse.
The role of Tgfβ in AAA pathogenesis has been shown
to be variable depending on the stage of the disease or the
environment milieu of the vascular injury.45 The systemic in-
hibition of Tgfβ during Ang II infusion augmented expansion
and rupture of abdominal aorta.46 Similarly, protective role of
Tgfβ was reported in the C57BL/6 mice in response to Ang
II.47 In addition, our previous studies have shown that Notch1
haploinsufficiency stabilizes AAA by promoting macrophage
differentiation towards M2 phenotype mediated by Tgfβ2.31
However, genetic deletion of Tgfβ receptor signaling in SMCs
did not accelerate AAA in elastase model of mice.48 Moreover,
increased Tgfβ signaling is pathogenic in experimental
Marfan syndrome of aortic aneurysm and is associated with
abnormal aortic wall remodeling, dilatation, and aneurysm
expansion.49 In the present study, we observed infiltration of
M2-like macrophages is associated with increased Tgfβ2 and
fibrosis leading to augmented AAA. Potential explanation for
these variable results could be the cellular source of Tgfβ be-
cause many cells, including immune cells, vascular SMCs,
(myo)fibroblasts, and platelets, contribute to Tgfβ production.
Thus, the pathological effect of Tgfβ could be intrinsic to par-
ticular cell type. Similarly, a baseline level of Tgfβ signaling
may be necessary to preserve aortic structure, but the exces-
sive production may be pathogenic in vascular pathologies. In
our study, excessive production of M2-macrophage specific
Tgfβ2 could be a key process leading to fibrosis and enhancing
the pathogenesis of AAA in IL12p40−/− mice. Both the classic
and alternative pathways of Tgfβ signaling are known to play
a role in regulating ECM remodeling.40 We observed increased
immunostaining of both phospho-smad3 and phospho-p38 in
IL12p40−/− tissues in response to Ang II. Because alternative
Tgfβ signaling pathway is known to induce Mmp2 and Mmp9
expression,40,50 our study indicates that Tgfβ signaling leads to
proteolytic destruction of elastin fragments by the production
of Mmps. This is evident by increased activity of Mmp2 in
macrophages and aortic rings from IL12p40−/− mice. Although
Mmp9 activity was higher compared with Mmp2 in mac-
rophages, Mmp2 was present only in IL12p40−/− BMDMs,
which was eliminated by inhibition of Tgfβ2. Similarly, the
aortic rings experiment clearly showed the increased activity
of Mmp2 in IL12p40−/− aortas. The increased Mmp2 activity
in the aortic rings might also be contributed by vascular SMC–
rich medial layer. It will be interesting to determine if mac-
rophages directly influence vascular SMCs to secrete Mmps
or it is mediated through secreted factors, such as Tgfβ2.
Evidently, further studies will highlight such interactions be-
tween macrophages and SMCs. Taken together, increased ex-
pression of Mmp2 in the aortic sections of IL12p40−/− mice
and BMDMs with IL12p40 deficiency suggest that Mmp2 is
the major contributor for elastin degradation. Thus, increased
Tgfβ signaling associated Mmp2 may be one of the most
important mechanisms leading to elastin degradation during
AAA development in IL12p40−/− mice.
 222   Arterioscler Thromb Vasc Biol   February 2019
A critical balance between proinflammatory (M1) and
anti-inflammatory (M2) macrophages may be a decisive fac-
tor in the initiation and progression of AAA over time.17 In
the well-established Ang II–induced Apoe−/− mice model,
proinflammatory M1 macrophages are the major vascular in-
sult and are known to play a critical role in the initiation of
AAA and is followed by fibrotic events modulated by M2
macrophages at the later stage of the disease.14 In contrast, in
IL12p40−/− mice, fibrosis seems to be an early event and play a
causal role during the AAA development. These observations
are supported by the evidence that depletion of macrophage
in IL12p40−/− mice led to decreased collagen deposition and
reduced aortic stiffness. Aortic stiffness has been shown to
be an early event in the development of AAA51; however, di-
rect relationship between fibrosis and aortic stiffness has not
been established. It is likely that the early fibrotic event medi-
ated by Tgfβ2 and increased Mmp2 expression may be lead-
ing to elastin degradation and structural abnormality in the
IL12p40−/− mice. This might be true as we did not see a major
structural abnormality and elastin degradation in response to
macrophage depletion in IL12p40−/− mice at day 14. These
data support the role of M2 macrophages in the fibrosis of
aortic tissue and remodeling followed by the structural ab-
normality leading to AAA.
In conclusion, deficiency of IL12p40 is associated with
exacerbated AAA in C57BL/6 mice as shown by excessive
accumulation of ECM leading to pathological matrix remod-
eling, elastin degradation, and fragile vascular wall. The de-
velopment of AAA in IL12p40−/− mice in response to Ang II
Downloaded from http://ahajournals.org by on June 21, 2023
may be a useful experimental model to address the role of
M2 macrophages in AAA progression. It will be interesting
to examine the precise and direct role of M2 macrophages
and Tgfβ2 activity in chemical (elastase and CaCl2) models
of AAA. Similarly, it will be interesting to examine the down-
stream mechanisms and targets of M2 macrophage-mediated
Tgfβ activity in promoting the AAA. Overall, these novel
roles of M2 macrophages demonstrated in our study open
up new avenues to understand AAA pathogenesis. Although
there is limited information available on the direct roles of
IL12 in human AAA, our study indicate that a careful ex-
amination of the aortic aneurysm is required with regard to
the inflammatory and fibrotic component to treat AAA in the
clinical settings.
Acknowledgments
We thank Zhe Sun at imaging core facility at Dalton Cardiovascular
Research Center for capturing the ultrasound images and Cell and
Immunology Core facility, University of Missouri for flow cytom-
etry data.
Sources of Funding
This work was supported by Grants R01HL124155 (C.P. Hans),
1R01HL118376 (L. Pulakat), 1R01HL138988 (L. Pulakat),
Veterans Affairs Merit System (BX003391; A. Whaley-Connell),
and funding from the Research Institute at the University of
Missouri to C.P. Hans.
Disclosures
None.
References
1. Benjamin EJ, Virani SS, Callaway CW, et al; American Heart Association
Council on Epidemiology and Prevention Statistics Committee and Stroke
Statistics Subcommittee. Heart disease and stroke statistics-2018 update: a
report from the American Heart Association. Circulation. 2018;137:e67–
e492. doi: 10.1161/CIR.0000000000000558
2. Sakalihasan N, Limet R, Defawe OD. Abdominal aortic aneurysm. Lancet.
2005;365:1577–1589. doi: 10.1016/S0140-6736(05)66459-8
3. Thompson RW, Geraghty PJ, Lee JK. Abdominal aortic aneurysms: basic
mechanisms and clinical implications. Curr Probl Surg. 2002;39:110–230.
4. Moxon JV, Parr A, Emeto TI, Walker P, Norman PE, Golledge J.
Diagnosis and monitoring of abdominal aortic aneurysm: current
status and future prospects. Curr Probl Cardiol. 2010;35:512–548. doi:
10.1016/j.cpcardiol.2010.08.004
5. Michel JB, Martin-Ventura JL, Egido J, Sakalihasan N, Treska V,
Lindholt J, Allaire E, Thorsteinsdottir U, Cockerill G, Swedenborg J;
FAD EU Consortium. Novel aspects of the pathogenesis of aneurysms
of the abdominal aorta in humans. Cardiovasc Res. 2011;90:18–27. doi:
10.1093/cvr/cvq337
6. Kuivaniemi H, Platsoucas CD, Tilson MD III. Aortic aneurysms:
an immune disease with a strong genetic component. Circulation.
2008;117:242–252. doi: 10.1161/CIRCULATIONAHA.107.690982
7. Correction to: Heart disease and stroke statistics-2018 update: a report
from the American Heart Association. Circulation. 2018;137:e493. doi:
10.1161/CIR.0000000000000573
8. Koch AE, Haines GK, Rizzo RJ, Radosevich JA, Pope RM, Robinson PG,
Pearce WH. Human abdominal aortic aneurysms. Immunophenotypic
analysis suggesting an immune-mediated response. Am J Pathol.
1990;137:1199–1213.
9. Forester ND, Cruickshank SM, Scott DJ, Carding SR. Functional char-
acterization of T cells in abdominal aortic aneurysms. Immunology.
2005;115:262–270. doi: 10.1111/j.1365-2567.2005.02157.x
10. Dale MA, Ruhlman MK, Baxter BT. Inflammatory cell phenotypes in
AAAs: their role and potential as targets for therapy. Arterioscler Thromb
Vasc Biol. 2015;35:1746–1755. doi: 10.1161/ATVBAHA.115.305269
11. Raffort J, Lareyre F, Clément M, Hassen-Khodja R, Chinetti G, Mallat
Z. Monocytes and macrophages in abdominal aortic aneurysm. Nat Rev
Cardiol. 2017;14:457–471. doi: 10.1038/nrcardio.2017.52
12. Turner GH, Olzinski AR, Bernard RE, Aravindhan K, Boyle RJ, Newman
MJ, Gardner SD, Willette RN, Gough PJ, Jucker BM. Assessment of mac-
rophage infiltration in a murine model of abdominal aortic aneurysm. J
Magn Reson Imaging. 2009;30:455–460. doi: 10.1002/jmri.21843
13. Daugherty A, Manning MW, Cassis LA. Angiotensin II promotes athero-
sclerotic lesions and aneurysms in apolipoprotein E-deficient mice. J Clin
Invest. 2000;105:1605–1612. doi: 10.1172/JCI7818
14. Rateri DL, Howatt DA, Moorleghen JJ, Charnigo R, Cassis LA,
Daugherty A. Prolonged infusion of angiotensin II in apoE(-/-) mice
promotes macrophage recruitment with continued expansion of ab-
dominal aortic aneurysm. Am J Pathol. 2011;179:1542–1548. doi:
10.1016/j.ajpath.2011.05.049
15. Hans CP, Koenig SN, Huang N, Cheng J, Beceiro S, Guggilam A,
Kuivaniemi H, Partida-Sánchez S, Garg V. Inhibition of Notch1 signaling
reduces abdominal aortic aneurysm in mice by attenuating macrophage-
mediated inflammation. Arterioscler Thromb Vasc Biol. 2012;32:3012–
3023. doi: 10.1161/ATVBAHA.112.254219
16. Qin Z, Bagley J, Sukhova G, Baur WE, Park HJ, Beasley D, Libby P, Zhang
Y, Galper JB. Angiotensin II-induced TLR4 mediated abdominal aortic
aneurysm in apolipoprotein E knockout mice is dependent on STAT3. J
Mol Cell Cardiol. 2015;87:160–170. doi: 10.1016/j.yjmcc.2015.08.014
17. Dale MA, Xiong W, Carson JS, Suh MK, Karpisek AD, Meisinger TM,
Casale GP, Baxter BT. Elastin-derived peptides promote abdominal aortic
aneurysm formation by modulating M1/M2 macrophage polarization. J
Immunol. 2016;196:4536–4543. doi: 10.4049/jimmunol.1502454
18. Boytard L, Spear R, Chinetti-Gbaguidi G, Acosta-Martin AE, Vanhoutte J,
Lamblin N, Staels B, Amouyel P, Haulon S, Pinet F. Role of proinflamma-
tory CD68(+) mannose receptor(-) macrophages in peroxiredoxin-1 expres-
sion and in abdominal aortic aneurysms in humans. Arterioscler Thromb
Vasc Biol. 2013;33:431–438. doi: 10.1161/ATVBAHA.112.300663
19. Dutertre CA, Clement M, Morvan M, Schäkel K, Castier Y, Alsac JM,
Michel JB, Nicoletti A. Deciphering the stromal and hematopoietic cell
network of the adventitia from non-aneurysmal and aneurysmal human
aorta. PLoS One. 2014;9:e89983. doi: 10.1371/journal.pone.0089983
20. Baxter BT, Terrin MC, Dalman RL. Medical management of small ab-
dominal aortic aneurysms. Circulation. 2008;117:1883–1889. doi:
10.1161/CIRCULATIONAHA.107.735274
 Sharma et al   M2-Polarized Macrophage and AAA   223
21. Meijer CA, Stijnen T, Wasser MN, Hamming JF, van Bockel JH,
36. Saraff K, Babamusta F, Cassis LA, Daugherty A. Aortic dissection
Lindeman JH; Pharmaceutical Aneurysm Stabilisation Trial Study
Group. Doxycycline for stabilization of abdominal aortic aneu-
rysms: a randomized trial. Ann Intern Med. 2013;159:815–823. doi:
10.7326/0003-4819-159-12-201312170-00007
22. Lindeman JH, Rabelink TJ, van Bockel JH. Immunosuppression and the
abdominal aortic aneurysm: Doctor Jekyll or Mister Hyde? Circulation.
2011;124:e463–e465. doi: 10.1161/CIRCULATIONAHA.110.008573
23. Trinchieri G. Interleukin-12: a cytokine produced by antigen-presenting
cells with immunoregulatory functions in the generation of T-helper cells
type 1 and cytotoxic lymphocytes. Blood. 1994;84:4008–4027.
24. Cooper AM, Khader SA. IL-12p40: an inherently agonistic cytokine.
Trends Immunol. 2007;28:33–38. doi: 10.1016/j.it.2006.11.002
25. Bastos KR, Alvarez JM, Marinho CR, Rizzo LV, Lima MR. Macrophages
from IL-12p40-deficient mice have a bias toward the M2 activation profile.
J Leukoc Biol. 2002;71:271–278.
26. Li Y, Zhang C, Wu Y, Han Y, Cui W, Jia L, Cai L, Cheng J, Li H, Du
J. Interleukin-12p35 deletion promotes CD4 T-cell-dependent mac-
rophage differentiation and enhances angiotensin II-Induced cardiac
fibrosis. Arterioscler Thromb Vasc Biol. 2012;32:1662–1674. doi:
10.1161/ATVBAHA.112.249706
27. Huaux F, Arras M, Tomasi D, Barbarin V, Delos M, Coutelier JP, Vink A,
Phan SH, Renauld JC, Lison D. A profibrotic function of IL-12p40 in ex-
perimental pulmonary fibrosis. J Immunol. 2002;169:2653–2661.
28. Fairweather D, Frisancho-Kiss S, Yusung SA, Barrett MA, Davis SE,
Steele RA, Gatewood SJ, Rose NR. IL-12 protects against coxsackievirus
B3-induced myocarditis by increasing IFN-gamma and macrophage and
neutrophil populations in the heart. J Immunol. 2005;174:261–269.
29. Carlson S, Helterline D, Asbe L, Dupras S, Minami E, Farris S, Stempien-
Otero A. Cardiac macrophages adopt profibrotic/M2 phenotype in
infarcted hearts: role of urokinase plasminogen activator. J Mol Cell
Cardiol. 2017;108:42–49. doi: 10.1016/j.yjmcc.2016.05.016
30. Moore JP, Vinh A, Tuck KL, Sakkal S, Krishnan SM, Chan CT, Lieu M,
Samuel CS, Diep H, Kemp-Harper BK, Tare M, Ricardo SD, Guzik TJ,
Sobey CG, Drummond GR. M2 macrophage accumulation in the aortic
wall during angiotensin II infusion in mice is associated with fibrosis,
elastin loss, and elevated blood pressure. Am J Physiol Heart Circ Physiol.
Downloaded from http://ahajournals.org by on June 21, 2023
2015;309:H906–H917. doi: 10.1152/ajpheart.00821.2014
31. Cheng J, Koenig SN, Kuivaniemi HS, Garg V, Hans CP. Pharmacological
inhibitor of notch signaling stabilizes the progression of small abdominal
aortic aneurysm in a mouse model. J Am Heart Assoc. 2014;3:e001064.
doi: 10.1161/JAHA.114.001064
32. Robinet P, Milewicz DM, Cassis LA, Leeper NJ, Lu HS, Smith JD.
Consideration of sex differences in design and reporting of experimental
arterial pathology studies-statement from ATVB council. Arterioscler
Thromb Vasc Biol. 2018;38:292–303. doi: 10.1161/ATVBAHA.117.309524
33. Aroor AR, Jia G, Habibi J, Sun Z, Ramirez-Perez FI, Brady B, Chen D,
Martinez-Lemus LA, Manrique C, Nistala R, Whaley-Connell AT, Demarco
VG, Meininger GA, Sowers JR. Uric acid promotes vascular stiffness,
maladaptive inflammatory responses and proteinuria in western diet fed
mice. Metabolism. 2017;74:32–40. doi: 10.1016/j.metabol.2017.06.006
34. Sehgel NL, Zhu Y, Sun Z, Trzeciakowski JP, Hong Z, Hunter WC,
Vatner DE, Meininger GA, Vatner SF. Increased vascular smooth
muscle cell stiffness: a novel mechanism for aortic stiffness in hyperten-
sion. Am J Physiol Heart Circ Physiol. 2013;305:H1281–H1287. doi:
10.1152/ajpheart.00232.2013
35. Lutshumba J, Liu S, Zhong Y, Hou T, Daugherty A, Lu H, Guo Z, Gong
MC. Deletion of BMAL1 in smooth muscle cells protects mice from ab-
dominal aortic aneurysms. Arterioscler Thromb Vasc Biol. 2018;38:1063–
1075. doi: 10.1161/ATVBAHA.117.310153
Highlights
• IL12p40−/− (interleukin 12 p40) mice depict M2-predominant macrophage phenotype.
• Deficiency of IL12p40 augments Ang II (angiotensin II)–induced abdominal aortic aneurysm in C57BL/6 mice.
• The development of abdominal aortic aneurysm in IL12p40−/− mice is associated with enhanced fibrotic events and medial layer degeneration
via upregulation of Tgfβ2 (transforming growth factor β) and Mmp2 (matrix metalloproteinase 2).
• Our findings suggest that M1/M2 macrophage balance rather than predominance of 1 phenotype is necessary for tissue homeostasis.
precedes formation of aneurysms and atherosclerosis in angiotensin
II-infused, apolipoprotein E-deficient mice. Arterioscler Thromb Vasc
Biol. 2003;23:1621–1626. doi: 10.1161/01.ATV.0000085631.76095.64
37. Sachdeva J, Mahajan A, Cheng J, Baeten JT, Lilly B, Kuivaniemi H, Hans
CP. Smooth muscle cell-specific Notch1 haploinsufficiency restricts the
progression of abdominal aortic aneurysm by modulating CTGF expres-
sion. PLoS One. 2017;12:e0178538. doi: 10.1371/journal.pone.0178538
38. Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source plat-
form for biological-image analysis. Nat Methods. 2012;9:676–682. doi:
10.1038/nmeth.2019
39. Sharma N, Trinidad CV, Trembath AP, Markiewicz MA. NKG2D signal-
ing between human NK cells enhances TACE-mediated TNF-α release. J
Immunol. 2017;199:2865–2872. doi: 10.4049/jimmunol.1700647
40. Jones JA, Spinale FG, Ikonomidis JS. Transforming growth factor-beta
signaling in thoracic aortic aneurysm development: a paradox in patho-
genesis. J Vasc Res. 2009;46:119–137. doi: 10.1159/000151766
41. Baitsch D, Bock HH, Engel T, Telgmann R, Müller-Tidow C, Varga G, Bot
M, Herz J, Robenek H, von Eckardstein A, Nofer JR. Apolipoprotein E
induces antiinflammatory phenotype in macrophages. Arterioscler Thromb
Vasc Biol. 2011;31:1160–1168. doi: 10.1161/ATVBAHA.111.222745
42. Khalil H, Kanisicak O, Prasad V, Correll RN, Fu X, Schips T, Vagnozzi
RJ, Liu R, Huynh T, Lee SJ, Karch J, Molkentin JD. Fibroblast-specific
TGF-β-Smad2/3 signaling underlies cardiac fibrosis. J Clin Invest.
2017;127:3770–3783. doi: 10.1172/JCI94753
43. Murray LA, Chen Q, Kramer MS, Hesson DP, Argentieri RL, Peng X,
Gulati M, Homer RJ, Russell T, van Rooijen N, Elias JA, Hogaboam CM,
Herzog EL. TGF-beta driven lung fibrosis is macrophage dependent and
blocked by Serum amyloid P. Int J Biochem Cell Biol. 2011;43:154–162.
doi: 10.1016/j.biocel.2010.10.013
44. Meng XM, Nikolic-Paterson DJ, Lan HY. TGF-β: the master regulator of
fibrosis. Nat Rev Nephrol. 2016;12:325–338. doi: 10.1038/nrneph.2016.48
45. Daugherty A, Chen Z, Sawada H, Rateri DL, Sheppard MB. Transforming
growth factor‐β in thoracic aortic aneurysms: good, bad, or irrelevant? J
Am Heart Assoc. 2017;6:e005221. doi: 10.1161/JAHA.116.005221
46. Chen X, Rateri DL, Howatt DA, Balakrishnan A, Moorleghen JJ, Cassis
LA, Daugherty A. TGF-β neutralization enhances angII-induced aortic
rupture and aneurysm in both thoracic and abdominal regions. PLoS One.
2016;11:e0153811. doi: 10.1371/journal.pone.0153811
47. Wang Y, Ait-Oufella H, Herbin O, Bonnin P, Ramkhelawon B, Taleb S,
Huang J, Offenstadt G, Combadière C, Rénia L, Johnson JL, Tharaux
PL, Tedgui A, Mallat Z. TGF-beta activity protects against inflammatory
aortic aneurysm progression and complications in angiotensin II-infused
mice. J Clin Invest. 2010;120:422–432. doi: 10.1172/JCI38136
48. Gao F, Chambon P, Offermanns S, Tellides G, Kong W, Zhang X, Li W.
Disruption of TGF-β signaling in smooth muscle cell prevents elastase-
induced abdominal aortic aneurysm. Biochem Biophys Res Commun.
2014;454:137–143. doi: 10.1016/j.bbrc.2014.10.053
49. Neptune ER, Frischmeyer PA, Arking DE, Myers L, Bunton TE, Gayraud
B, Ramirez F, Sakai LY, Dietz HC. Dysregulation of TGF-beta activation
contributes to pathogenesis in Marfan syndrome. Nat Genet. 2003;33:407–
411. doi: 10.1038/ng1116
50. Gomes LR, Terra LF, Wailemann RA, Labriola L, Sogayar MC. TGF-β1
modulates the homeostasis between MMPs and MMP inhibitors through
p38 MAPK and ERK1/2 in highly invasive breast cancer cells. BMC
Cancer. 2012;12:26. doi: 10.1186/1471-2407-12-26
51. Raaz U, Zöllner AM, Schellinger IN, et al. Segmental aortic stiffening
contributes to experimental abdominal aortic aneurysm development.
Circulation. 2015;131:1783–1795. doi: 10.1161/CIRCULATIONAHA.
114.012377