Arteriosclerosis, Thrombosis, and Vascular Biology

ORIGINAL RESEARCH

Differential Roles of Endothelial Cell-Derived

and Smooth Muscle Cell-Derived Fibronectin
Containing Extra Domain A in Early and Late
Atherosclerosis
Prakash Doddapattar, Rishabh Dev, Manish Jain, Nirav Dhanesha, Anil K. Chauhan

OBJECTIVE: The extracellular matrix of atherosclerotic arteries contains abundant deposits of cellular Fn-EDA (fibronectin
containing extra domain A), suggesting a functional role in the pathophysiology of atherosclerosis. Fn-EDA is synthesized
by several cell types, including endothelial cells (ECs) and smooth muscle cells (SMCs), which are known to contribute
to different stages of atherosclerosis. Although previous studies using global Fn-EDA-deficient mice have demonstrated
that Fn-EDA is proatherogenic, the cell-specific role of EC versus SMC-derived-Fn-EDA in atherosclerosis has not been
investigated yet.

APPROACH AND RESULTS: To determine the relative contribution of different pools of Fn-EDA in atherosclerosis, we generated
mutant strains lacking Fn-EDA in the ECs (Fn-EDAEC-KO) or smooth muscle cells (Fn-EDASMC-KO) on apolipoprotein E-deficient
(Apoe−/−) background. The extent of atherosclerotic lesion progression was evaluated in whole aortae, and cross-sections
of the aortic sinus in male and female mice fed a high-fat Western diet for either 4 weeks (early atherosclerosis) or 14
weeks (late atherosclerosis). Irrespective of sex, Fn-EDAEC-KO, but not Fn-EDASMC-KO mice, exhibited significantly reduced
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early atherogenesis concomitant with decrease in inflammatory cells (neutrophil and macrophage) and VCAM (vascular cell
adhesion molecule)-1 expression levels within the plaques. In late atherosclerosis model, irrespective of sex, Fn-EDASMC-
KO
mice exhibited significantly reduced atherogenesis, but not Fn-EDAEC-KO mice, that was concomitant with decreased
macrophage content within plaques. Lesional SMCs, collagen content, and plasma inflammatory cytokines (TNF-α [tumor
necrosis factor α] and IL (interleukin)-1β), total cholesterol, and triglyceride levels were comparable among groups.

CONCLUSIONS: EC-derived Fn-EDA contributes to early atherosclerosis, whereas smooth muscle cell-derived Fn-EDA
contributes to late atherosclerosis.

Key Words: atherosclerosis ◼ cytokines ◼ endothelial cells ◼ fibronectin ◼ macrophages

A
therosclerotic lesion progression is a chronic inflam-
matory process characterized by fatty streaks,
leukocytes, smooth muscle cells (SMCs), calcifica-
tion, and extracellular matrix (ECM) remodeling. During
chronic inflammation, ECM proteins or their fragments
can modulate the migration of several cell types, includ-
ing endothelial cells (ECs), SMCs, and monocyte/mac-
rophages, all known to contribute to different stages of
atherosclerosis.1 In human and murine models, unlike
healthy arteries, the ECM of atherosclerotic arteries con-
tains abundant deposits of Fn (fibronectin), suggesting a
functional role for Fn in the pathophysiology of athero-
sclerosis.2,3 Fn is alternatively spliced that results in vari-
able inclusion of extra domain A (EDA), extra domain B
(EDB), and the type III homologies connecting segment.
The amino acid sequence and splicing patterns of the

 
Correspondence to: Prakash Doddapattar, Department of Internal Medicine, University of Iowa, 3120 Medical Labs, Iowa City, IA 52242, Email prakash-doddapattar@
uiowa.edu; or Anil K. Chauhan, Department of Internal Medicine, University of Iowa, 3120 Medical Labs, Iowa City, IA 52242, Email anil-chauhan@uiowa.edu
The Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/ATVBAHA.120.314459.
For Sources of Funding and Disclosures, see page xxx.
© 2020 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at www.ahajournals.org/journal/atvb

Arterioscler Thromb Vasc Biol. 2020;40:00–00. DOI: 10.1161/ATVBAHA.120.314459 July 2020   1

 Doddapattar et al
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Nonstandard Abbreviations and Acronyms
α-SMA alpha smooth muscle cell actin
BMT bone marrow transplantation
EC endothelial cell
ECM extracellular matrix
EDB extra domain B
Fn-EDA fibronectin containing extra domain A
IFN interferon
IL interleukin
LDL-R low-density lipoprotein receptor
MCP-1 monocyte chemoattractant protein 1
mTOR mammalian target of rapamycin
NF-κB nuclear factor-κB
PCR polymerase chain reaction
SMC smooth muscle cell
TGF-β transforming growth factor β
TLR-4 toll-like receptor 4
TNF-α tumor necrosis factor α
VCAM-1 vascular cell adhesion molecule 1
EDA and EDB are highly conserved (>90%) in the ver-
tebrates, with either total inclusion or exclusion, includ-
ing human, mouse, rat, chicken, and zebrafish.4 Although
the predominant form of Fn in plasma of healthy humans
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contains negligible amounts of EDA or EDB, inclusion of
these alternatively spliced domains has been observed in
animal models of vascular injuries,5,6 and patients at risk
for diabetes mellitus,7 and atherosclerosis.8,9
Several studies have demonstrated that global dele-
tion of Fn-EDA in apolipoprotein E-deficient (Apoe−/−)
or LDL-R (low-density lipoprotein receptor)-deficient
mice reduces atherosclerosis suggesting that Fn-EDA
is proatherogenic.10–12 Fn-EDB accumulation has also
been found in human atherosclerotic plaques13; however,
whether it is proatherogenic remains unclear. The Fn
present in the atherosclerotic lesions could arise from
several sources, including the leak of plasma Fn (pFn)
from the circulation, Fn-derived from ECs, and Fn-derived
from SMCs. Studies using human aortic ECs showed
that EC-derived Fn regulates integrin α5β1-mediated
adhesion formation, Fn fibrillogenesis, and inflammation
in response to oxidized LDL, suggesting EC-derived Fn
may contribute to early atherogenesis.14 Other studies
showed that Fn-EDA is expressed in the vicinity of SMCs
and was a characteristic feature of synthetic SMCs that
are known to contribute to different stages of athero-
sclerosis.6,15 Despite these observations, there are no
studies that provide conclusive evidence on the role of
EC-derived versus SMC-derived Fn-EDA in atherogen-
esis. Also unclear is whether Fn-EDA derived from these
cell types contributes to the early or late atherogenesis.
Herein, using mutant mice, we provide evidence that
2   July 2020 Arterioscler Thromb Vasc Biol. 2020;40:00–00. DOI: 10.1161/ATVBAHA.120.314459
Role of SMC vs Endothelial Fn-EDA in Atherogenesis
Highlights
• The extracellular matrix of atherosclerotic arteries
contains abundant deposits of Fn-EDA (fibronectin
containing extra domain A), suggesting a functional
role in the pathophysiology of atherosclerosis.
• Endothelial cell-derived Fn-EDA, but not smooth
muscle cell-derived Fn-EDA, contributes to early
atherosclerosis in mice.
• Smooth muscle cell-derived Fn-EDA, but not endo-
thelial cell-derived Fn-EDA, is the primary determi-
nant that contributes to late atherosclerosis.
EC-derived Fn-EDA contributes to early atherosclerosis,
whereas SMC-derived Fn-EDA contributes to advanced
atherosclerosis.
MATERIALS AND METHODS
The data that support the findings of this study are available
from the corresponding author upon reasonable request. For
details of reagents and murine strains, please see the Major
Resources Table in the Data Supplement.
Mice
Six-weeks-old male and female Fn-EDAfl/flApoe−/− mice on
the C57BL/6J background were used in the present study.11
Fn-EDAfl/flApoe−/− mice were crossed with the Apoe−/− Tie2Cre+
mice to generate Fn-EDAfl/flTie2Cre+Apoe−/− (Fn-EDAEC-KO, EC
KO of Fn-EDA) as described.16 SM22αCre+ mice (004746, Tg
[Tagln-cre]1 Her/J; The Jackson Laboratory) were crossed with
Apoe−/− mice to generate SM22αCre+Apoe−/− mice. Fn-EDAfl/
fl
Apoe−/− mice were crossed to SM22αCre+Apoe−/− mice to
generate Fn-EDAfl/flSM22αCre+Apoe−/− mice (Fn-EDASMC-KO,
smooth muscle KO of Fn-EDA) as described.15 Mice were gen-
otyped by polymerase chain reaction (PCR) according to proto-
cols from The Jackson Laboratory and as described before.15–17
The University of Iowa Animal Care and Use Committee
approved all protocols.
Mice Diet Feeding and Preparation of Tissues
Both male and female mice (Fn-EDAfl/flTie2Cre+Apoe−/−,
Fn-EDAfl/fl Apoe−/−, EDAfl/flSM22αCre+Apoe−/−) were fed a high-
fat Western diet (20% milk fat and 0.2% cholesterol, Harlan
Teklad) beginning at 6 weeks of age until they were euthanized
at 5 months of age (ie, 14 weeks on high-fat Western diet to
induce late atherosclerosis). Mice were fed a normal labora-
tory diet (NIH-31 modified mouse diet: 7913) before high-fat
Western diet. All mice were fed 4 weeks of high-fat Western
diet beginning at 6 weeks of age to study early atherosclerosis.
For plasma isolation, blood samples were collected in heparin-
ized tubes by retro-orbital plexus puncture after overnight fast-
ing. Before tissue isolation, mice were anesthetized with 100
mg/kg ketamine/10 mg/kg xylazine and perfused via the left
ventricle with 10 mL PBS, followed by 10 mL of 4% parafor-
maldehyde under physiological pressure. After perfusion, aor-
tae were isolated, dehydrated for 5 minutes in 60% isopropyl
 Doddapattar et al
alcohol and stained with Oil Red O. Hearts containing aortic
roots were dissected and fixed overnight in 4% paraformalde-
hyde before embedding in paraffin.
Extent and Composition of Atherosclerotic
Lesions
The whole aortae were isolated and stained with Oil Red O,
and the en face lesion area was measured by morphometry
using NIH ImageJ software to measure atherosclerosis lesion
progression. Lesion areas in the aortic sinus were quantified by
using 5 µm thick serial cross-sections that were cut through
the aorta beginning at the origin of the aortic valve leaflets and
stained by VerHoeffs/Van Gieson method. The cross-sectional
lesion area from each mouse was calculated by taking the
mean value of 4 sections (each 80 µm apart, beginning at the
aortic valve leaflets and spanning 320 µm) as described previ-
ously.18 For quantification, NIH ImageJ software was used.
Bone Marrow Transplantation
Bone marrow transplantation (BMT) of either Fn-EDAfl/
Tie2Cre+ Apoe−/− or Fn-EDAfl/flApoe−/− mice were performed
fl
on 6-weeks-old mice. Mice were irradiated with 2 doses of
6.5-Gy at an interval of 4 hours between the first and sec-
ond irradiations before BMT as described.18 Under sterile
conditions, bone marrow cells (1×107) were extracted from
excised femurs and tibias of euthanized mice, suspended in
sterile PBS and injected into the retro-orbital venous plexus
of lethally irradiated recipient mice. Post-transplantation, mice
were maintained in sterile cages and fed autoclaved food and
water ad libitum. Following BMT experiments were performed:
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(1) irradiated Fn-EDAfl/flApoe−/− mice reconstituted with BM
from Fn-EDAfl/fl Apoe−/−, (2) irradiated Fn-EDAfl/flApoe−/− mice
reconstituted with BM from Fn-EDAfl/flTie2Cre+Apoe−/−, (3)
irradiated Fn-EDAfl/flTie2Cre+Apoe−/− mice reconstituted with
BM from Fn-EDAfl/fl Apoe−/− donors. Successful BMT was con-
firmed after 4 weeks by PCR to check the presence of the
genomic DNA of the respective donor mice in peripheral blood
cells. Total blood cell counts were obtained using an automated
veterinary hematology analyzer (ADVIA) to ascertain that BMT
did not affect the number of BM-derived blood cells.
Immunohistochemistry of Murine Samples
Tissue preparation and histochemical staining were per-
formed as described.18 Briefly, antigen retrieval was per-
formed before immunohistochemical staining. Slides were
rehydrated, and sections were blocked with 5% serum in
Tris-buffered saline at room temperature from the species in
which the secondary antibody was raised. For chromogenic
detection, endogenous peroxidase activity was quenched
with 0.1% hydrogen peroxide (Fisher Scientific, no. H325) in
methanol for 15 minutes. Sections were stained for Lys6G.2
(clone, 1A8, Biorad no. MCA6077GA), Mac3 (1.5 µg/
mL BD Pharmingen no. 550292) and smooth muscle cell
actin (α-SMA, Sigma, no. A5228), fibronectin EDA (Clone
Fn-3E2, Sigma, no. F6140), CD31 (ab28364), VCAM-1
(no. SC-13160). After overnight incubation at 4°C, slides
were washed thrice with PBS for 5 minutes and incubated
with biotinylated secondary antibody for 1 hour at room
temperature. For immunofluorescence, goat anti-rat 546 (4
Arterioscler Thromb Vasc Biol. 2020;40:00–00. DOI: 10.1161/ATVBAHA.120.314459
Role of SMC vs Endothelial Fn-EDA in Atherogenesis
µg/mL, Invitrogen, no. A11081), goat anti-rat 488 (4 µg/
Original Research - VB
mL, Invitrogen, no. A-11006), goat anti-rabbit IgG 546 (4
µg/mL, Invitrogen no. A11035), and goat anti-mouse 488
(Catalog no. A32723) were used. Nuclei were stained using
the Antifade mounting medium with DAPI (Vector laborato-
ries no. H:1200). For the chromogenic method, slides were
incubated with streptavidin-HRP for 40 minutes room tem-
perature and washed thrice with PBS for 5 minutes and incu-
bated with DAB substrate (Vector laboratories no. SK400)
for <2 minutes until color develops. Hematoxylin counter-
stained slides were dehydrated, mounted using permount,
and examined using an Olympus fluorescent microscope
(BX51). Mean fluorescence intensity was quantified using
the ImageJ software as previously described (Jensen, 2013).
Protein colocalization was analyzed using the Pearson colo-
calization coefficients (http://rsb.info.nih.gov/ij/plugins/
track/jacop.html). Colocalization was considered positive for
values ranging from 0.5 to 1. Measurements were obtained
from 2 different fields per murine section, 4 to 6 different
fields for human samples. For negative control, incubation
without primary antibodies and with isotype-matched immu-
noglobulins was used. A mean for each mouse was calcu-
lated using the mean value of 4 sections (each 80 µm apart,
beginning at the aortic valve leaflets and spanning 320 µm).
Determination of Plasma Total Cholesterol and
Lipid Levels
Overnight fasted blood from each mouse was collected in hep-
arinized tubes by retro-orbital venous plexus puncture. Plasma
was separated and analyzed for total cholesterol and triglyceride
(both from DiaSys no. 113009911923, no. 157109911923)
levels by using enzymatic colorimetric assays according to the
manufacturer’s protocol.
Picrosirius Red Staining for Collagen
Quantification of interstitial collagen content of the aortic
sinus was performed as described.11 Briefly, serial cross-
sections of 5 µm were stained with Weigert’s hematoxylin
and washed in running tap water for 10 minutes followed by
incubation for 4 hours in a freshly prepared 0.1% solution
of Sirius Red F3B (Sigma-Aldrich, no. 365548) in saturated
aqueous picric acid (Sigma-Aldrich, no. P-6744). Sections
were rinsed twice in acidified (0.01 N HCl) distilled water,
dehydrated, and mounted in Permount (Fisher Scientific, no.
SP15-500). Polarization microscopy was used to analyze
picrosirius red staining. Total collagen, thick mature orange-
red fibers, and thin immature green fibers were quantified
as described19 using NIH ImageJ software with a defined
threshold (minimum 100 and maximum 200). A mean for
each mouse was calculated using the mean value of 4 sec-
tions (each 80 µm apart, beginning at the aortic valve leaf-
lets and spanning 320 µm).
ELISA Assay for TNF-α and IL-1β
Overnight fasted plasma samples were used for determination
of TNF (tumor necrosis factor)-α and IL (interleukin)-1β with
commercially available mouse ELISA kits (TNF-α, no. MTA00B;
IL-1β, no. MLB00C; R&D Systems) according to the kit manu-
facturer’s instructions.
July 2020   3
 Doddapattar et al
Statistical Analysis
Original Research - VB
Results are reported as the mean±SEM. For statistical analysis,
the Prism Graph software package was used. Shapiro-Wilk test
was used to check normality, and the Bartlett test was used
to check equal variance. The statistical significance of the dif-
ference between means was assessed using the unpaired
Student t test (for normally distributed data) or Mann Whitney
test (for not normally distributed data) and 1-way ANOVA fol-
lowed by Tukey multiple comparisons test. P<0.05 was consid-
ered significant.
RESULTS
EC-Derived Fn-EDA Contributes to Early
Atherosclerosis But Not Late Atherosclerosis
We generated Fn-EDAfl/flTie2Cre+Apoe−/− mice to deter-
mine the role of EC-derived Fn-EDA in atherosclerosis.
Because Tie2 is also expressed by hematopoietic cells
in addition to ECs, Fn-EDAfl/flTie2Cre+Apoe−/− mice were
transplanted with bone marrow from Fn-EDAfl/flApoe−/−
mice, which constitutively express Fn-EDA in all cells
and tissues. This strategy resulted in mutant mice that
are deficient for Fn-EDA in ECs (Fn-EDAEC-KO). Controls
were irradiated littermates Fn-EDAfl/flApoe−/− mice that
received bone marrow from Fn-EDAfl/flApoe−/− mice or
Fn-EDAfl/flTie2Cre+Apoe−/− mice. Male and female mice
were examined separately to determine sex-based dif-
ferences. Mice were fed a normal laboratory diet for
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10 weeks until the complete reconstitution of newly
transplanted bone marrow and then a high-fat Western
diet for 4 or 14 weeks. Genomic PCR confirmed the
presence of the Tie2Cre gene in Fn-EDAfl/flTie2Cre+A
poe−/− mice (Figure IA in the Data Supplement). RT-PCR
confirmed the absence of Fn-EDA mRNA in the ECs but
not in other cells of Fn-EDAfl/flTie2Cre+Apoe−/− mutant
mice (Figure IB in the Data Supplement).
Next, we compared the extent of atherosclerosis in
whole aortae (by staining with Oil Red O and quantify-
ing en face lesion area) and aortic sinus (by staining
with VerHoeffs/Van Gieson and quantifying the cross-
sectional area). In the early atherosclerosis model, irre-
spective of sex, Fn-EDAEC-KO mice exhibited reduced
atherosclerosis lesion progression in aortae and aortic
sinus when compared with controls (P<0.05, Figure 1A
and 1B, and Figure IIA and IIB in the Data Supplement).
The decrease in lesion size in the Fn-EDAEC-KO mice
was associated with reduced neutrophil (Figure 1C) and
macrophage content (Figure III in the Data Supplement)
and decreased VCAM-1 expression (Figure 1D) within
lesions suggesting reduced endothelial activation. Rep-
resentative negative controls are shown (Figure IV in the
Data Supplement). In late atherosclerosis model, lesion
area in the aortae and aortic sinus (Figure 2A and 2B),
macrophage content (Figure 2C), total collagen content
including both thick mature and thin immature fibers
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Role of SMC vs Endothelial Fn-EDA in Atherogenesis
(Figure V in the Data Supplement), and α-SMA (Fig-
ure 2D) within aortic lesions were comparable between
Fn-EDAEC-KO and control mice. Inflammatory cytokines
TNF-α and IL-1β in plasma (Figure VI in the Data Supple-
ment), total cholesterol, and triglyceride levels (Table I in
the Data Supplement) were comparable among groups.
Together, these results suggest that EC-derived Fn-EDA
contributes to early atherosclerosis, but not advanced
atherosclerosis, most likely by promoting neutrophil and
macrophage recruitment via VCAM-1.
SMC-Derived Fn-EDA Contributes to Late
Atherosclerosis But Not Early Atherosclerosis
We generated Fn-EDAfl/flSM22αCre+Apoe−/− to deter-
mine the role of SMC-derived Fn-EDA in atherosclerosis.
Genomic PCR confirmed the presence of the SM22αCre
gene in Fn-EDAfl/flSM22αCre+Apoe−/− (Figure VIIA in the
Data Supplement). RT-PCR confirmed the absence of
Fn-EDA mRNA in SMCs but not in other cells of Fn-
EDAfl/flSM22αCre+Apoe−/− mutant mice (Figure VIIB
in the Data Supplement). Male and female mice were
first fed a normal laboratory diet for 6 weeks, and then
a high-fat Western diet for 4 or 14 weeks. In the early
atherosclerosis model, irrespective of sex, we observed
that atherosclerosis lesion progression in the aorta and
aortic sinus was comparable between Fn-EDASMC-KO and
controls (Figure 3A and 3B and Figure VIIIA and VIIIB
in the Data Supplement). Lesion neutrophil (Figure 3C),
macrophage influx (Figure IX in the Data Supplement),
and VCAM-1 expression (Figure 3D) were comparable
among groups. In late atherosclerosis model, irrespec-
tive of sex, we found that the mean lesion areas in the
aortae and aortic sinus were significantly smaller in Fn-
EDASMC-KO mice when compared with controls (P<0.05,
Figure 4A and 4B and Figure VIIIC and VIIID in the
Data Supplement). The decrease in lesion size in the
Fn-EDASMC-KO mice was associated with reduced mac-
rophage infiltration (Figure 4C) with unaltered collagen
(Figure X in the Data Supplement and α-SMA content
(Figure 4D). Inflammatory cytokines TNF-α and IL-1β,
total cholesterol, and triglycerides levels in the plasma
and leukocyte counts were comparable among groups
(Figure XI and Tables II and III in the Data Supplement).
Next, using immunostaining, we determined the per-
centage of Fn-EDA associated with SMCs (α-SMA-
positive) and ECs (CD31-positive) in autopsy samples
from patients with coronary artery disease. We found a
marked expression of Fn-EDA within advanced lesions
from diseased coronary arteries, whereas staining was
virtually absent in arteries from healthy controls (Figure
XII in the Data Supplement). Double-label immunos-
taining revealed a 3-fold increase in Fn-EDA staining
associated with SMCs (Fn-EDA/α-SMA-positive) when
compared with ECs (Fn-EDA/CD31-positive; Figure XII
in the Data Supplement). Together, these results suggest
 Doddapattar et al Role of SMC vs Endothelial Fn-EDA in Atherogenesis

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Figure 1. Fn-EDAEC-KO mice exhibit reduced early atherosclerosis.

All the mice were females and fed a high-fat Western diet for 4 wk. A, Representative photomicrographs and quantification of Oil red O-stained
en face lesion area in the whole aortae. B, Representative photomicrographs of VerHoeffs/Van Geison-staining and quantification of cross-
sectional lesion area in aortic sinuses. C, Representative photomicrographs and quantification of neutrophil-stained (Lys6G.2-positive cells)
in aortic sinuses. Scale bar=100 μm. D, Representative photomicrographs and quantification of VCAM-1 (vascular cell adhesion molecule
1-stained positive cells) area in aortic sinuses. Scale bar=100 μm. Values are expressed as mean±SEM. Each dot represents a single mouse
(n=10–13/group). Statistical analysis: 1-way ANOVA followed by Tukey multiple comparisons test.

that SMC-derived Fn-EDA contributes to advanced ath-
erosclerosis, but not early atherosclerosis.
DISCUSSION
Understanding the mechanisms that contribute to the
initiation and progression of atherosclerosis is vital in
developing novel strategies to limit the lesion progres-
sion before reaching the clinical manifestations. Abun-
dant deposits of Fn-EDA have been found in the ECM of
both human and murine atherosclerotic plaques. Despite
previous evidence suggesting that Fn-EDA is proath-
erogenic, the role of EC-derived versus SMC-derived
Fn-EDA in atherogenesis remains unclear. Using Fn-
EDAEC-KO and Fn-EDASMC-KO mice, we provide evidence
that EC-derived Fn-EDA contributes to early atheroscle-
rosis, whereas SMC-derived Fn-EDA contributes to late
atherosclerosis, suggesting cell and stage-specific role
of Fn-EDA during atherosclerosis.
Previous studies have shown that Fn-EDA was
associated with ECs during early lesion progression
but absent from adjacent uninvolved endothelium.12 A

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Figure 2. Endothelial-derived Fn-EDA (fibronectin containing extra domain A) does not contribute to late atherosclerosis.
All the mice were females and fed a high-fat Western diet for 14 wk. A, Representative photomicrographs and quantification of Oil red O-
stained en face lesion area in the whole aortae. B, Representative photomicrographs of VerHoeffs/Van Geison-staining and quantification
of cross-sectional lesion area in aortic sinuses. Scale bar=500 μm. C, Representative photomicrographs and quantification of macrophage
(mac3-positive cells) in aortic sinuses. Scale bar=500 μm. D, Representative photomicrographs and quantification of smooth muscle cell-
stained (⍺-SMA-positive) area in aortic sinuses. Scale bar=100 μm Values are expressed as mean±SEM. Each dot represents a single mouse
(n=10–13/group). Statistical analysis: 1-way ANOVA followed by Tukey multiple comparisons test.

minimal Fn-EDA staining was also found around macro-
phages or early foam cells during early lesion progres-
sion.12 Another study found that cFn deposition by α5β1
integrin promotes early atherosclerosis.14 Although these
studies suggest that EC-derived Fn-EDA may contrib-
ute to early atherosclerosis,12 there was no definitive evi-
dence that has examined how the lack of Fn-EDA from
different specific cell type affects early atherogenesis.
Using Fn-EDAEC-KO and Fn-EDASMC-KO mice, we found
that Fn-EDA derived from ECs, but not from SMCs, con-
tributes to early atherosclerosis, independent of changes
in plasma total cholesterol and triglyceride levels. Despite
the fact that the atherogenic process initiates with the
deposition of LDL, several studies suggest that chronic
inflammation contributes to atherosclerotic lesion pro-
gression, and lesion size does not always correlate with
an increase in plasma cholesterol and LDL levels.20–22 It
is known that ECM within the lesions can trap and retain
lipids.23 There could be several possibilities by which
EC-derived Fn-EDA may contribute to early atheroscle-
rosis. First, by promoting early atherogenic inflammation
via integrin α5β1 as suggested previously.14 Second,

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Figure 3. Smooth muscle cell-derived Fn-EDA (fibronectin containing extra domain A) does not contribute to early
atherosclerosis.
All the mice were females and fed a high-fat Western diet for 4 wk. A, Representative photomicrographs and quantification of Oil red O-stained
en face lesion area in the whole aortae. B, Representative photomicrographs of VerHoeffs/Van Geison-staining and quantification of cross-
sectional lesion area in aortic sinuses. Scale bar=500 μm. Lower: C, Representative photomicrographs and quantification of neutrophil-stained
(Lys6G.2-positive cells) in aortic sinuses. Scale bar=100 μm. D, Representative photomicrographs and quantification of VCAM-1 (vascular
cell adhesion molecule 1-stained positive cells) in aortic sinuses. Scale bar=100 μm. Each dot represents a single mouse (n=8–14/group).
Statistical analysis: unpaired Student t test.

by enhancing oxidized LDL-mediated NF-κB (nuclear
factor-κB) p65 activation and VCAM-1 expression.14,24
In agreement with these studies, we observed signifi-
cantly decreased VCAM-1 expression in Fn-EDAEC-KO
mice. Third, by potentiating neutrophil interactions with
the vessel wall. We found that reduced lesion size in Fn-
EDAEC-KO mice was associated with decreased neutrophil
and macrophage content within lesions. The EDA of Fn
contains additional binding sites for integrin α9β1 and
α4β1. Integrin α9β1 is highly expressed on activated
neutrophils and known to contribute to adhesion, and
transmigration.25 Neutrophil depletion has been shown to
improve early atherosclerosis in mice.26 Studies suggest
that neutrophils contribute to early atherosclerosis via
multiple mechanisms including neutrophil extracellular
trap formation and interacting with monocytes to prime
them for inflammatory response.27,28 Fourth, by promoting
NF-κB p65-mediated inflammation in both neutrophils
and monocytes/macrophages via innate immune recep-
tor TLR-4 (toll-like receptor 4). Fn-EDA is an endogenous

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Figure 4. Fn-EDASMC-KO mice exhibit reduced late atherosclerosis.

All the mice were females and fed a high-fat Western diet for 14 wk. A, Representative photomicrographs and quantification of Oil red
O-stained en face lesion area in the whole aortae. B, Representative photomicrographs of VerHoeffs/Van Geison-staining and quantification of
cross-sectional lesion area in aortic sinuses. Scale bar=500 μm. C, Representative photomicrographs and quantification of macrophage (mac3-
positive cells) area in aortic sinuses. Scale bar=500 μm. D, Representative photomicrographs and quantification of smooth muscle cell-stained
(⍺-SMA-positive) area in aortic sinuses. Scale bar=100 μm. Each dot represents a single mouse (n=8–14/group). Statistical analysis: unpaired
Student t test.

ligand for TLR-4. Previously, we have shown that cFn
potentiates NF-κB p65-mediated inflammation in bone-
marrow-derived neutrophils and macrophages via TLR-
4.11 In line with our observations, a recent study found
that EC-derived Fn-EDA was increased by disturbed flow
and interacted with TLR-4 to promote inflammation.29
Herein, we found that in human diseased coronary
lesions, a majority of Fn-EDA staining was colocalized
with SMCs, whereas Fn-EDA staining colocalized with
ECs was less when compared with SMCs. Our results
are in agreement with the previous study, where less Fn-
EDA staining was associated with ECs in the advanced
lesions.12 Consistent with these studies, we provide evi-
dence that SMC-derived Fn-EDA, but not EC-derived
Fn-EDA, contributes to late atherosclerosis by recruit-
ment of monocytes/macrophages without altering SMCs
and collagen content. It is possible that the leak of circu-
lating Fn-EDA into the vessel wall over time may mask
the effectiveness of Fn-EDA deletion in Fn-EDAEC-KO
mice on late atherogenesis. Such a possibility is mini-
mal because lack of Fn-EDA, specifically, in the plasma
does not affect atherogenesis, suggesting that the Fn-
EDA produced by cells present in atherosclerotic plaque
contributes to atherogenesis.10 There could be several

8   July 2020 Arterioscler Thromb Vasc Biol. 2020;40:00–00. DOI: 10.1161/ATVBAHA.120.314459
 Doddapattar et al
possibilities by which SMC-derived Fn-EDA may con-
tribute to late atherosclerosis. First, by modulating SMCs
from contractile to synthetic phenotype via integrins.6,15
SMC-derived Fn-EDA contributes to synthetic pheno-
type via Akt1/mTOR (mammalian target of rapamycin)
signaling.15 In addition to ECM components, synthetic
SMC produces many cytokines such as PDGF, TGF
(transforming growth factor)-β, IFN (interferon)-γ, and
MCP-1 (monocyte chemoattractant protein 1) that may
promote monocyte/macrophage recruitment.30 Second,
by potentiating TLR-4 signaling in monocytes/macro-
phages. In addition to integrins including α5β1, α3β1,
αvβ1, αvβ3, Fn-EDA is an endogenous ligand for TLR-4.
Previously, using double-deficient mice, we have shown
that TLR-4 partially contributes to Fn-EDA-mediated
advanced atherosclerosis, most likely by potentiating
NF-κB p65-mediated inflammation.11
In conclusion, our results provide novel insights
into the role of different pools of Fn-EDA at different
stages of atherogenesis. This study further supports
the notion that therapeutically targeting Fn-EDA may
have the potential for the prevention and treatment
of atherosclerosis in patients at high risk for coronary
heart disease.
ARTICLE INFORMATION
Received September 20, 2019; accepted May 11, 2020.
Downloaded from http://ahajournals.org by on May 31, 2020
Affiliation
From the Division of Hematology/Oncology, Department of Internal Medicine,
University of Iowa, Iowa City.
Sources of Funding
The AKC lab is supported by grants from the National Institutes of Health grants
(R35HL139926, R01NS109910, and U01NS113388) and by Established In-
vestigator Award 18EIA33900009 from American Heart Association. P. Dodda-
pattar is supported by a postdoctoral grant from the American Heart Association
(18POST33960179), and N. Dhanesha is supported by the American Society of
Hematology Scholar award.
Disclosures
None.
REFERENCES
1. Raines EW. The extracellular matrix can regulate vascular cell migration,
proliferation, and survival: relationships to vascular disease. Int J Exp Pathol.
2000;81:173–182. doi: 10.1046/j.1365-2613.2000.00155.x
2. Shekhonin BV, Domogatsky SP, Idelson GL, Koteliansky VE, Rukosuev VS.
Relative distribution of fibronectin and type I, III, IV, V collagens in normal
and atherosclerotic intima of human arteries. Atherosclerosis. 1987;67:9–
16. doi: 10.1016/0021-9150(87)90259-0
3. Pedretti M, Rancic Z, Soltermann A, Herzog BA, Schliemann C,
Lachat M, Neri D, Kaufmann PA. Comparative immunohistochemical stain-
ing of atherosclerotic plaques using F16, F8 and L19: three clinical-
grade fully human antibodies. Atherosclerosis. 2010;208:382–389. doi:
10.1016/j.atherosclerosis.2009.07.043
4. Hynes RO. The evolution of metazoan extracellular matrix. J Cell Biol.
2012;196:671–679. doi: 10.1083/jcb.201109041
5. Dubin D, Peters JH, Brown LF, Logan B, Kent KC, Berse B,
Berven S, Cercek B, Sharifi BG, Pratt RE. Balloon catheterization induced
arterial expression of embryonic fibronectins. Arterioscler Thromb Vasc Biol.
1995;15:1958–1967. doi: 10.1161/01.atv.15.11.1958
Arterioscler Thromb Vasc Biol. 2020;40:00–00. DOI: 10.1161/ATVBAHA.120.314459
Role of SMC vs Endothelial Fn-EDA in Atherogenesis
6. Glukhova MA, Frid MG, Shekhonin BV, Vasilevskaya TD, Grunwald J,
Original Research - VB
Saginati M, Koteliansky VE. Expression of extra domain A fibronectin
sequence in vascular smooth muscle cells is phenotype dependent. J Cell
Biol. 1989;109:357–366. doi: 10.1083/jcb.109.1.357
7. Kanters SD, Banga JD, Algra A, Frijns RC, Beutler JJ, Fijnheer R. Plasma
levels of cellular fibronectin in diabetes. Diabetes Care. 2001;24:323–327.
doi: 10.2337/diacare.24.2.323
8. Lemańska-Perek A, Krzyżanowska-Gołąb D, Pupek M, Klimeczek P,
Witkiewicz W, Kątnik-Prastowska I. Analysis of soluble molecular fibro-
nectin-fibrin complexes and EDA-fibronectin concentration in plasma of
patients with atherosclerosis. Inflammation. 2016;39:1059–1068. doi:
10.1007/s10753-016-0336-0
9. Fiechter M, Frey K, Fugmann T, Kaufmann PA, Neri D. Comparative in
vivo analysis of the atherosclerotic plaque targeting properties of eight
human monoclonal antibodies. Atherosclerosis. 2011;214:325–330. doi:
10.1016/j.atherosclerosis.2010.11.027
10. Pulakazhi Venu VK, Uboldi P, Dhyani A, Patrini A, Baetta R, Ferri N,
Corsini A, Muro AF, Catapano AL, Norata GD. Fibronectin extra domain
A stabilises atherosclerotic plaques in apolipoprotein E and in LDL-
receptor-deficient mice. Thromb Haemost. 2015;114:186–197. doi:
10.1160/TH14-09-0790
11. Doddapattar P, Gandhi C, Prakash P, Dhanesha N, Grumbach IM,
Dailey ME, Lentz SR, Chauhan AK. Fibronectin splicing variants con-
taining extra domain A promote atherosclerosis in mice through toll-like
receptor 4. Arterioscler Thromb Vasc Biol. 2015;35:2391–2400. doi:
10.1161/ATVBAHA.115.306474
12. Tan MH, Sun Z, Opitz SL, Schmidt TE, Peters JH, George EL. Deletion of
the alternatively spliced fibronectin EIIIA domain in mice reduces athero-
sclerosis. Blood. 2004;104:11–18. doi: 10.1182/blood-2003-09-3363
13. Yu M, Ortega CA, Si K, Molinaro R, Schoen FJ, Leitao RFC, Xu X,
Mahmoudi M, Ahn S, Liu J, et al. Nanoparticles targeting extra domain B of
fibronectin-specific to the atherosclerotic lesion types III, IV, and V-enhance
plaque detection and cargo delivery. Theranostics. 2018;8:6008–6024. doi:
10.7150/thno.24365
14. Al-Yafeai Z, Yurdagul A Jr, Peretik JM, Alfaidi M, Murphy PA, Orr AW.
Endothelial FN (Fibronectin) deposition by α5β1 integrins drives athero-
genic inflammation. Arterioscler Thromb Vasc Biol. 2018;38:2601–2614.
doi: 10.1161/ATVBAHA.118.311705
15. Jain M, Dhanesha N, Doddapattar P, Chorawala MR, Nayak MK,
Cornelissen A, Guo L, Finn AV, Lentz SR, Chauhan AK. Smooth mus-
cle cell-specific fibronectin-EDA mediates phenotypic switching
and neointimal hyperplasia. J Clin Invest. 2020;130:295–314. doi:
10.1172/JCI124708
16. Dhanesha N, Chorawala MR, Jain M, Bhalla A, Thedens D, Nayak M,
Doddapattar P, Chauhan AK. Fn-EDA (Fibronectin Containing Extra Domain
A) in the plasma, but not endothelial cells, exacerbates stroke outcome
by promoting thrombo-inflammation. Stroke. 2019;50:1201–1209. doi:
10.1161/STROKEAHA.118.023697
17. Muro AF, Chauhan AK, Gajovic S, Iaconcig A, Porro F, Stanta G, Baralle FE.
Regulated splicing of the fibronectin EDA exon is essential for proper skin
wound healing and normal lifespan. J Cell Biol. 2003;162:149–160. doi:
10.1083/jcb.200212079
18. Doddapattar P, Dhanesha N, Chorawala MR, Tinsman C, Jain M, Nayak MK,
Staber JM, Chauhan AK. Endothelial cell-derived von willebrand factor, but
not platelet-derived, promotes atherosclerosis in apolipoprotein E-defi-
cient mice. Arterioscler Thromb Vasc Biol. 2018;38:520–528. doi:
10.1161/ATVBAHA.117.309918
19. Ovchinnikova O, Robertson AK, Wågsäter D, Folco EJ, Hyry M, Myllyharju J,
Eriksson P, Libby P, Hansson GK. T-cell activation leads to reduced col-
lagen maturation in atherosclerotic plaques of apoe(-/-) mice. Am J Pathol.
2009;174:693–700. doi: 10.2353/ajpath.2009.080561
20. Gandhi C, Khan MM, Lentz SR, Chauhan AK. ADAMTS13 reduces vascular
inflammation and the development of early atherosclerosis in mice. Blood.
2012;119:2385–2391. doi: 10.1182/blood-2011-09-376202
21. Boring L, Gosling J, Cleary M, Charo IF. Decreased lesion formation in
CCR2-/- mice reveals a role for chemokines in the initiation of atheroscle-
rosis. Nature. 1998;394:894–897. doi: 10.1038/29788
22. Sharrett AR. Serum cholesterol levels and atherosclerosis. Coron Artery Dis.
1993;4:867–870. doi: 10.1097/00019501-199310000-00005
23. Chait A, Wight TN. Interaction of native and modified low-density lipopro-
teins with extracellular matrix. Curr Opin Lipidol. 2000;11:457–463. doi:
10.1097/00041433-200010000-00003
24. Yurdagul A Jr, Sulzmaier FJ, Chen XL, Pattillo CB, Schlaepfer DD, Orr AW.
Oxidized LDL induces FAK-dependent RSK signaling to drive NF-κB
July 2020   9
 Doddapattar et al
activation and VCAM-1 expression. J Cell Sci. 2016;129:1580–1591. doi:
Original Research - VB
10.1242/jcs.182097
25. Mambole A, Bigot S, Baruch D, Lesavre P, Halbwachs-Mecarelli L. Human
neutrophil integrin alpha9beta1: up-regulation by cell activation and synergy
with beta2 integrins during adhesion to endothelium under flow. J Leukoc
Biol. 2010;88:321–327. doi: 10.1189/jlb.1009704
26. Drechsler M, Megens RT, van Zandvoort M, Weber C, Soehnlein O. Hyper-
lipidemia-triggered neutrophilia promotes early atherosclerosis. Circulation.
2010;122:1837–1845. doi: 10.1161/CIRCULATIONAHA.110.961714
27. Soehnlein O. Multiple roles for neutrophils in atherosclerosis. Circ Res.
2012;110:875–888. doi: 10.1161/CIRCRESAHA.111.257535
Downloaded from http://ahajournals.org by on May 31, 2020
10   July 2020 Arterioscler Thromb Vasc Biol. 2020;40:00–00. DOI: 10.1161/ATVBAHA.120.314459
Role of SMC vs Endothelial Fn-EDA in Atherogenesis
28. Warnatsch A, Ioannou M, Wang Q, Papayannopoulos V. Inflamma-
tion. Neutrophil extracellular traps license macrophages for cyto-
kine production in atherosclerosis. Science. 2015;349:316–320. doi:
10.1126/science.aaa8064
29. Qu D, Wang L, Huo M, Song W, Lau CW, Xu J, Xu A, Yao X, Chiu JJ,
Tian XY, et al. Focal tlr4 activation mediates disturbed flow-induced endo-
thelial inflammation. Cardiovasc Res. 2019;
30. Raines EW, Ferri N. Thematic review series: The immune system and
atherogenesis. Cytokines affecting endothelial and smooth mus-
cle cells in vascular disease. J Lipid Res. 2005;46:1081–1092. doi:
10.1194/jlr.R500004-JLR200