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Atvbaha 120 314459
Atvbaha 120 314459
Atvbaha 120 314459
ORIGINAL RESEARCH
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.
A
therosclerotic lesion progression is a chronic inflam- atherosclerosis.1 In human and murine models, unlike
matory process characterized by fatty streaks, healthy arteries, the ECM of atherosclerotic arteries con-
leukocytes, smooth muscle cells (SMCs), calcifica- tains abundant deposits of Fn (fibronectin), suggesting a
tion, and extracellular matrix (ECM) remodeling. During functional role for Fn in the pathophysiology of athero-
chronic inflammation, ECM proteins or their fragments sclerosis.2,3 Fn is alternatively spliced that results in vari-
can modulate the migration of several cell types, includ- able inclusion of extra domain A (EDA), extra domain B
ing endothelial cells (ECs), SMCs, and monocyte/mac- (EDB), and the type III homologies connecting segment.
rophages, all known to contribute to different stages of 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
2 July 2020 Arterioscler Thromb Vasc Biol. 2020;40:00–00. DOI: 10.1161/ATVBAHA.120.314459
Doddapattar et al Role of SMC vs Endothelial Fn-EDA in Atherogenesis
alcohol and stained with Oil Red O. Hearts containing aortic µg/mL, Invitrogen, no. A11081), goat anti-rat 488 (4 µg/
Original Research - VB
roots were dissected and fixed overnight in 4% paraformalde- mL, Invitrogen, no. A-11006), goat anti-rabbit IgG 546 (4
hyde before embedding in paraffin. 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-
Extent and Composition of Atherosclerotic ries no. H:1200). For the chromogenic method, slides were
Lesions incubated with streptavidin-HRP for 40 minutes room tem-
The whole aortae were isolated and stained with Oil Red O, perature and washed thrice with PBS for 5 minutes and incu-
and the en face lesion area was measured by morphometry bated with DAB substrate (Vector laboratories no. SK400)
using NIH ImageJ software to measure atherosclerosis lesion for <2 minutes until color develops. Hematoxylin counter-
progression. Lesion areas in the aortic sinus were quantified by stained slides were dehydrated, mounted using permount,
using 5 µm thick serial cross-sections that were cut through and examined using an Olympus fluorescent microscope
the aorta beginning at the origin of the aortic valve leaflets and (BX51). Mean fluorescence intensity was quantified using
stained by VerHoeffs/Van Gieson method. The cross-sectional the ImageJ software as previously described (Jensen, 2013).
lesion area from each mouse was calculated by taking the Protein colocalization was analyzed using the Pearson colo-
mean value of 4 sections (each 80 µm apart, beginning at the calization coefficients (http://rsb.info.nih.gov/ij/plugins/
aortic valve leaflets and spanning 320 µm) as described previ- track/jacop.html). Colocalization was considered positive for
ously.18 For quantification, NIH ImageJ software was used. values ranging from 0.5 to 1. Measurements were obtained
from 2 different fields per murine section, 4 to 6 different
Bone Marrow Transplantation fields for human samples. For negative control, incubation
without primary antibodies and with isotype-matched immu-
Bone marrow transplantation (BMT) of either Fn-EDAfl/
noglobulins was used. A mean for each mouse was calcu-
Tie2Cre+ Apoe−/− or Fn-EDAfl/flApoe−/− mice were performed
fl
lated using the mean value of 4 sections (each 80 µm apart,
on 6-weeks-old mice. Mice were irradiated with 2 doses of
beginning at the aortic valve leaflets and spanning 320 µm).
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 Determination of Plasma Total Cholesterol and
excised femurs and tibias of euthanized mice, suspended in Lipid Levels
sterile PBS and injected into the retro-orbital venous plexus Overnight fasted blood from each mouse was collected in hep-
of lethally irradiated recipient mice. Post-transplantation, mice arinized tubes by retro-orbital venous plexus puncture. Plasma
were maintained in sterile cages and fed autoclaved food and was separated and analyzed for total cholesterol and triglyceride
water ad libitum. Following BMT experiments were performed:
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Results are reported as the mean±SEM. For statistical analysis, ure 2D) within aortic lesions were comparable between
the Prism Graph software package was used. Shapiro-Wilk test Fn-EDAEC-KO and control mice. Inflammatory cytokines
was used to check normality, and the Bartlett test was used TNF-α and IL-1β in plasma (Figure VI in the Data Supple-
to check equal variance. The statistical significance of the dif- ment), total cholesterol, and triglyceride levels (Table I in
ference between means was assessed using the unpaired the Data Supplement) were comparable among groups.
Student t test (for normally distributed data) or Mann Whitney Together, these results suggest that EC-derived Fn-EDA
test (for not normally distributed data) and 1-way ANOVA fol- contributes to early atherosclerosis, but not advanced
lowed by Tukey multiple comparisons test. P<0.05 was consid- atherosclerosis, most likely by promoting neutrophil and
ered significant. macrophage recruitment via VCAM-1.
4 July 2020 Arterioscler Thromb Vasc Biol. 2020;40:00–00. DOI: 10.1161/ATVBAHA.120.314459
Doddapattar et al Role of SMC vs Endothelial Fn-EDA in Atherogenesis
Original Research - VB
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that SMC-derived Fn-EDA contributes to advanced ath- previous evidence suggesting that Fn-EDA is proath-
erosclerosis, but not early atherosclerosis. 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
DISCUSSION that EC-derived Fn-EDA contributes to early atheroscle-
Understanding the mechanisms that contribute to the rosis, whereas SMC-derived Fn-EDA contributes to late
initiation and progression of atherosclerosis is vital in atherosclerosis, suggesting cell and stage-specific role
developing novel strategies to limit the lesion progres- of Fn-EDA during atherosclerosis.
sion before reaching the clinical manifestations. Abun- Previous studies have shown that Fn-EDA was
dant deposits of Fn-EDA have been found in the ECM of associated with ECs during early lesion progression
both human and murine atherosclerotic plaques. Despite but absent from adjacent uninvolved endothelium.12 A
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- in plasma total cholesterol and triglyceride levels. Despite
phages or early foam cells during early lesion progres- the fact that the atherogenic process initiates with the
sion.12 Another study found that cFn deposition by α5β1 deposition of LDL, several studies suggest that chronic
integrin promotes early atherosclerosis.14 Although these inflammation contributes to atherosclerotic lesion pro-
studies suggest that EC-derived Fn-EDA may contrib- gression, and lesion size does not always correlate with
ute to early atherosclerosis,12 there was no definitive evi- an increase in plasma cholesterol and LDL levels.20–22 It
dence that has examined how the lack of Fn-EDA from is known that ECM within the lesions can trap and retain
different specific cell type affects early atherogenesis. lipids.23 There could be several possibilities by which
Using Fn-EDAEC-KO and Fn-EDASMC-KO mice, we found EC-derived Fn-EDA may contribute to early atheroscle-
that Fn-EDA derived from ECs, but not from SMCs, con- rosis. First, by promoting early atherogenic inflammation
tributes to early atherosclerosis, independent of changes via integrin α5β1 as suggested previously.14 Second,
6 July 2020 Arterioscler Thromb Vasc Biol. 2020;40:00–00. DOI: 10.1161/ATVBAHA.120.314459
Doddapattar et al Role of SMC vs Endothelial Fn-EDA in Atherogenesis
Original Research - VB
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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 neutrophils and known to contribute to adhesion, and
factor-κB) p65 activation and VCAM-1 expression.14,24 transmigration.25 Neutrophil depletion has been shown to
In agreement with these studies, we observed signifi- improve early atherosclerosis in mice.26 Studies suggest
cantly decreased VCAM-1 expression in Fn-EDAEC-KO that neutrophils contribute to early atherosclerosis via
mice. Third, by potentiating neutrophil interactions with multiple mechanisms including neutrophil extracellular
the vessel wall. We found that reduced lesion size in Fn- trap formation and interacting with monocytes to prime
EDAEC-KO mice was associated with decreased neutrophil them for inflammatory response.27,28 Fourth, by promoting
and macrophage content within lesions. The EDA of Fn NF-κB p65-mediated inflammation in both neutrophils
contains additional binding sites for integrin α9β1 and and monocytes/macrophages via innate immune recep-
α4β1. Integrin α9β1 is highly expressed on activated tor TLR-4 (toll-like receptor 4). Fn-EDA is an endogenous
ligand for TLR-4. Previously, we have shown that cFn lesions.12 Consistent with these studies, we provide evi-
potentiates NF-κB p65-mediated inflammation in bone- dence that SMC-derived Fn-EDA, but not EC-derived
marrow-derived neutrophils and macrophages via TLR- Fn-EDA, contributes to late atherosclerosis by recruit-
4.11 In line with our observations, a recent study found ment of monocytes/macrophages without altering SMCs
that EC-derived Fn-EDA was increased by disturbed flow and collagen content. It is possible that the leak of circu-
and interacted with TLR-4 to promote inflammation.29 lating Fn-EDA into the vessel wall over time may mask
Herein, we found that in human diseased coronary the effectiveness of Fn-EDA deletion in Fn-EDAEC-KO
lesions, a majority of Fn-EDA staining was colocalized mice on late atherogenesis. Such a possibility is mini-
with SMCs, whereas Fn-EDA staining colocalized with mal because lack of Fn-EDA, specifically, in the plasma
ECs was less when compared with SMCs. Our results does not affect atherogenesis, suggesting that the Fn-
are in agreement with the previous study, where less Fn- EDA produced by cells present in atherosclerotic plaque
EDA staining was associated with ECs in the advanced 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 Role of SMC vs Endothelial Fn-EDA in Atherogenesis
possibilities by which SMC-derived Fn-EDA may con- 6. Glukhova MA, Frid MG, Shekhonin BV, Vasilevskaya TD, Grunwald J,
Original Research - VB
Saginati M, Koteliansky VE. Expression of extra domain A fibronectin
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levels of cellular fibronectin in diabetes. Diabetes Care. 2001;24:323–327.
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ARTICLE INFORMATION 14. Al-Yafeai Z, Yurdagul A Jr, Peretik JM, Alfaidi M, Murphy PA, Orr AW.
Endothelial FN (Fibronectin) deposition by α5β1 integrins drives athero-
Received September 20, 2019; accepted May 11, 2020. genic inflammation. Arterioscler Thromb Vasc Biol. 2018;38:2601–2614.
doi: 10.1161/ATVBAHA.118.311705
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Original Research - VB
10 July 2020 Arterioscler Thromb Vasc Biol. 2020;40:00–00. DOI: 10.1161/ATVBAHA.120.314459