Molecular Medicine
Milk Fat Globule Protein Epidermal Growth Factor-8
A Pivotal Relay Element Within the Angiotensin II and Monocyte
Chemoattractant Protein-1 Signaling Cascade Mediating Vascular Smooth
Muscle Cells Invasion
Zongming Fu,* Mingyi Wang,* Marjan Gucek, Jing Zhang, James Wu, Liqun Jiang,
Robert E. Monticone, Benjamin Khazan, Richard Telljohann, Julie Mattison, Simon Sheng,
Robert N. Cole, Gaia Spinetti, Gianfranco Pintus, Lijuan Liu, Frank D. Kolodgie, Renu Virmani,
Harold Spurgeon, Donald K. Ingram, Allen D. Everett, Edward G. Lakatta,* Jennifer E. Van Eyk*
Downloaded from http://ahajournals.org by on June 14, 2020
Abstract—Advancing age induces aortic wall thickening that results from the concerted effects of numerous signaling
proteins, many of which have yet to be identified. To search for novel proteins associated with aortic wall thickening,
we have performed a comprehensive quantitative proteomic study to analyze aortic proteins from young (8 months) and
old (30 months) rats and identified 50 proteins that significantly change in abundance with aging. One novel protein,
the milk fat globule protein epidermal growth factor 8 (MFG-E8), increases 2.3-fold in abundance in old aorta.
Transcription and translation analysis demonstrated that aortic MFG-E8 mRNA and protein levels increase with aging
in several mammalian species including humans. Dual immunolabeling shows that MFG-E8 colocalizes with both
angiotensin II and monocyte chemoattractant protein (MCP)-1 within vascular smooth muscle cells (VSMCs) of
the thickened aged aortic wall. Exposure of early passage VSMCs from young aorta to angiotensin II markedly
increases MFG-E8 and enhances invasive capacity to levels observed in VSMCs from old rats. Treatment of
VSMCs with MFG-E8 increases MCP-1 expression and VSMCs invasion that are inhibited by the MCP-1 receptor
blocker vCCI. Silencing MFG-E8 RNA substantially reduces MFG-E8 expression and VSMCs invasion capacity.
The data indicate that arterial MFG-E8 significantly increases with aging and is a pivotal relay element within the
angiotensin II/MCP-1/VSMC invasion signaling cascade. Thus, targeting of MFG-E8 within this signaling axis
pathway is a potential novel therapy for the prevention and treatment of the age-associated vascular diseases such
as atherosclerosis. (Circ Res. 2009;104:1337-1346.)
Key Words: MFG-E8 䡲 angiotensin II 䡲 monocyte chemoattractant protein-1
䡲 vascular smooth muscle cells 䡲 aging
P
roinflammatory processes and associated elevated invasion capacity of vascular smooth muscle cells (VSMCs)
are increased within the diffuse thickening of the arterial wall
that evolves with advancing age.1– 4 In humans, this ageassociated arterial remodeling is an independent risk factor
for the epidemic of quintessential human cardiovascular
diseases, ie, atherosclerosis, hypertension, and stroke.1,3,5,6
The age-associated arterial wall thickening and other aspects
of arterial remodeling are evolutionarily conserved in various
mammalian species, including rodents, nonhuman primates,
and humans.1,3,7–14 The thickened arterial intima is formed
because of VSMCs invasion and proliferation and is not
limited to secretion within the subendothelial space.2,3,7,14 A
growing body of evidence indicates that VSMCs within the
arterial media begin to express and activate proteases such as
matrix metalloprotease (MMP)2 and calpain-1, which enable
cytoskeletal remodeling and degradation of (1) basement
membranes that surround VSMCs, (2) adjacent matrix, and
(3) elastic lamina. Thus VSMCs invasion into the subendothelial space is driven, at least in part, by angiotensin (Ang)
Original received September 8, 2008; revision received April 28, 2009; accepted May 4, 2009.
From the Department of Medicine (Z.F., S.S., J.E.V.E.); Technical Implementation and Coordination Core (M.G., R.N.C., J.E.V.E.), The Johns
Hopkins Bayview Proteomics Center; Department of Neurology (L.L.); Department of Pediatrics (A.D.E.); Department of Biomedical Engineering
(J.E.V.E.); and Department of Biological Chemistry (J.E.V.E.), The Johns Hopkins University, Baltimore, Md; Laboratory of Cardiovascular Science
(M.W., L.J., R.E.M., B.K., R.T., H.S., E.G.L.) and Laboratory of Experimental Gerontology (J.M.), National Institute on Aging, NIH, Baltimore, Md;
Medstar Research Institute (J.Z., J.W.), Hyattsville, Md; IRCCS MultiMedica (G.S.), Milano, Italy; Department of Biomedical Sciences (G.P.), University
of Sassari, Italy; CVPath (F.D.K., R.V.), International Registry of Pathology, Gaithersburg, Md; and Nutritional Neuroscience and Aging Laboratory
(D.K.I.), Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge.
This manuscript was sent to Joseph Loscalzo, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
*These authors contributed equally to this work.
Correspondence to Mingyi Wang, MD, PhD, Laboratory of Cardiovascular Science, National Institute on Aging-National Institutes of Health,
Baltimore, MD 21224. E-mail: mingyiw@grc.nia.nih.gov
© 2009 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.108.187088
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Table.
June 19, 2009
Differentially Abundant Proteins Identified by 2-D DIGE and iTRAQ
Protein Class and Protein Name
Accession*
iTRAQ† O/Y
DIGE‡ O/Y
PTM§
MFGM_RAT
2.31⫾0.21
2.53⫾0.15
Glycosylation
Apoptosis/cell cycle/proliferation
MFG-E8
Serine protease HTRA1
HTRA1_MOUSE
2.03⫾0.17
ND
Integral membrane protein 2B
ITM2B_RAT
2.28⫾0.63
ND
Cysteine and glycine-rich protein 2
CSRP2_RAT
1.56⫾0.08
ND
PLF4_RAT
3.09⫾1.5
ND
IBP7_MOUSE
1.40⫾0.2
ND
Platelet factor 4 (CXCL4)
Insulin-like growth factor-binding protein 7 (IGFBP-7)
Cell Metabolism
Creatine kinase M-type
KCRM_RAT
0.58⫾0.02
ND
Glycerol 3-Pdehydrogenase 1
GPDA_RAT
0.58⫾0.10
0.52⫾0.08
Cytochrome c oxidase subunit 5B
COX5B_RAT
0.66⫾0.02
ND
Glutamine synthetase
GLNA_RAT
0.58⫾0.10
ND
2-oxoglutarate dehydrogenase E1 component
ODO1_RAT
0.67⫾0.11
ND
2,4-dienoyl-CoA reductase
DECR_RAT
0.63⫾0.06
ND
3-ketoacyl-CoA thiolase
THIM_RAT
0.66⫾0.07
0.60⫾0.07
Hydroxyacyl-coenzyme A dehydrogenase
HCDH_RAT
0.61⫾0.12
ND
Trifunctional enzyme subunit 
ECHA_RAT
0.66⫾0.07
ND
Long-chain-fatty-acid–CoA ligase 1
ACSL1_RAT
0.63⫾0.12
0.62⫾0.07
Isocitrate dehydrogenase [NADP]
IDHP_RAT
0.75⫾0.05
0.65⫾0.05
Pyruvate carboxylase
PYC_RAT
0.93⫾0.02
0.56⫾0.07
RBP1_RAT
1.49⫾0.02
ND
Actin-related protein 2/3 complex subunit 4
ARPC4_MOUSE
1.54⫾0.07
ND
STE20-like serine/threonine-protein kinase
SLK_RAT
0.59⫾0.03
ND
Calponin-1
CNN1_RAT
1.42⫾0.06
1.69⫾0.06
Calponin-3
CNN3_RAT
1.45⫾0.07
1.75⫾0.06
Cytoskeleton/invasion/migration
RalA-binding protein 1
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Phosphorylation
Extracellular matrix/cell adhesion
Transforming growth factor -3
TGFB3_RAT
2.22⫾0.21
ND
Clusterin
CLUS_RAT
1.89⫾0.26
ND
72 kDa type IV collagenase (MMP-2)
MMP2_RAT
1.40⫾0.11
ND
Biglycan
PGS1_RAT
1.56⫾0.06
ND
Collagen ␣-1(I) chain¶
CO1A1_RAT
0.39⫾0.11
0.35⫾0.05
Collagen alpha-2(I) chain¶
CO1A2_RAT
0.54⫾0.14
0.45⫾0.06
FINC_RAT
1.68⫾0.10
2.1⫾0.19
ND
Fibronectin 1
SPARC-like protein 1
SPRL1_RAT
1.47⫾0.14
VTNC_MOUSE
1.91⫾0.09
ND
Apolipoprotein A-I
APOA1_RAT
0.68⫾0.13
0.60⫾0.04
Kininogen 1
KNT1_RAT
1.67⫾0.22
1.86⫾0.19
Glycosylation
Periostin (OSF-2)
POSTN_MOUSE
2.43⫾0.13
2.46⫾0.19
Glycosylation
Apolipoprotein E
APOE _RAT
1.55⫾0.29
ND
Fetuin-A; ␣-2-HS-glycoprotein
FETUA_RAT
0.61⫾0.20
ND
Fetuin-B
FETUB_RAT
0.66⫾0.05
ND
Vitronectin
(Continued)
*Accession is from SwissProt database; †changes in abundance with P⬍0.05 and ⬎1.5-fold in 4 animals in each group using iTRAQ; ‡changes
in abundance with P⬍0.05 and ⬎1.5-fold in 8 animals in each group using 2DE (ND indicates not detected by this method); §potential PTM based
on PNGase F and phosphostaining experiments (see the online data supplement); ¶soluble form of collagen decreased, whereas the insoluble form
that is known to increase with aging will not be able to get observed by 2DE or iTRAQ.
Fu et al
Table.
MFG-E8 Mediates VSMC Invasion
1339
Continued
Protein Class and Protein Name
Accession*
iTRAQ† O/Y
DIGE‡ O/Y
Adenylate cyclase type 5
ADCY5_RAT
1.54⫾0.08
ND
␣-1-antiproteinase
A1AT_RAT
0.66⫾0.09
0.58⫾0.09
␣-1-inhibitor III
A1I3_RAT
0.67⫾0.14
0.60⫾0.08
Carbonic anhydrase 3
CAH3_RAT
0.63⫾0.23
ND
Carboxylesterase 1
EST2_RAT
0.49⫾0.03
ND
Contrapsin-like protease inhibitor 1
SPA3K_RAT
0.66⫾0.05
ND
Contrapsin-like protease inhibitor 3
SPA3L_RAT
0.62⫾0.10
ND
Ferritin light chain
FRIL1_RAT
1.56⫾0.12
1.91⫾0.09
Major urinary protein
MUP_RAT
0.46⫾0.04
ND
Solute carrier family 2; facilitated glucose transporter member 4
GTR4_RAT
2.01⫾0.02
ND
Transthyretin
TTHY_RAT
0.59⫾0.18
0.63⫾0.12
TRI47_MOUSE
1.97⫾0.09
ND
PTM§
Miscellaneous
Tripartite motif-containing protein 47
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II.7–14 VSMC invasion is also facilitated by an intimal–medial
concentration gradient of monocyte chemoattractant protein
(MCP)-1, and platelet-derived growth factor (PDGF)-BB.14
Transcription, translation, and activation of MMP2,
calpain-1, MCP-1, and PDGF-BB are linked to an ageassociated increase in local arterial Ang II signaling via the
Ang II type 1 receptor.7–14
However, numerous yet identified proteins that have crucial roles in the coordinated VSMCs invasion process likely
change in abundance or become posttranslationally modified
with aging. A complete understanding of mechanisms involved in the increased VSMCs invasive capacity with aging
requires identification of the proteome changes within functional classes and characterization of interconnections between known pathways, as well as indicating previously
unknown key regulators. To this end, we have performed a
comprehensive quantitative proteomic study using both 2D
gel electrophoresis (2DE)-based and mass spectrometry
(MS)-based iTRAQ (isobaric tag for relative and absolute
quantification) approaches to analyze aortic proteins obtained
from young (8 months) and old (30 months) rats (schematic
diagram of proteomic experimental design; Figure I in the
online data supplement, available at http://circres.ahajournals.
org). Furthermore, we used 2DE in combination with Pro-Q
Diamond Phosphoprotein Gel Stain and deglycosylation experiments to characterize selected posttranslational modifications of proteins. Using these approaches in combination with
quantitative RT-PCR, and Western blotting, immunostaining,
and VSMCs functional assays, we have discovered (1) that at
least 50 proteins change abundance in the aorta associated
with aging; (2) that milk fat globule protein epidermal growth
factor 8 (MFG-E8) (also known as lactadherin) markedly
increases with aging, not only in rat aorta but also in
nonhuman primate and human aorta; and (3) that MFG-E8 is
a novel link between proinflammatory molecules Ang II and
MCP-1 signaling and promotes the invasive capacity of
VSMCs.
Materials and Methods
For details on the materials and methods used in the present study
(including arterial specimens, isolation of aorta and preparation of
Glycosylation
aortic proteins, 2D silver-stained and DIGE, in-gel tryptic digestion
and peptide desalting, mass spectrometry analysis and protein
identification, detection of phosphoproteins and glycoproteins,
iTRAQ analysis, Ingenuity pathway analysis, real-time PCR
analysis, Western blotting analysis, immunohistochemistry and
immunofluorescence, VSMCs isolation and culture, MFG-E8
small interfering [si]RNA silencing, VSMCs invasion assay, and
statistical analysis), see the online data supplement, available at
http://circres.ahajournals.org.
Results
Quantification and Identification of the Aortic
Proteome by 2DE and iTRAQ MS-Based Analysis
Using 2DE (silver-stained and DIGE gels), we have obtained
2D gel maps of 286 identified nonredundant proteins from rat
aorta and observed 18 proteins (Table) that significantly
change abundance (⫾1.5-fold) with aging (annotated protein
gel maps for young and old aorta are shown in Online Figures
II and III; corresponding to identifications listed in Online
Table II). Using iTRAQ, 880 proteins were quantified and
between both methods, 50 proteins were shown to have
significantly different abundance associated with aging
(⫾1.5-fold) (Online Table III). The results of the 2 methods
were complimentary, increasing the confidence of the results.
Totally 923 nonredundant proteins were detected by 2 methods. The majority of the proteins detected by 2DE (243
[85%]) are also quantified by iTRAQ. Approximately 5%
(50) of the proteins identified have significantly different
abundance with aging (Table). Of the proteins found to
differ with aging, 5 had posttranslational modifications.
Acidic calponin 3 was found to be a phosphoprotein
(Online Figure IV), whereas MFG-E8 (Figure 1A and 1B),
␣-1-inhibitor III (data not shown), kininogen 1 (data not
shown), and periostin (Online Figures V and VI) were
detected as N-linked glycoproteins.
Bioinformatic Functional Analysis of Differentially
Abundant Age-Associated Arterial Proteins
To identify potential roles of differentially abundant proteins
with aging, proteins were grouped into various functional
classes based on Swiss-Prot protein function and gene ontol-
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June 19, 2009
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Figure 1. Quantification and characterization of MFG-E8 by 2DIDE and iTRAQ in aging rat aorta. A, Enlarged region of 2D DIGE gel
map showing that MFG-E8 is more abundant in aged aorta than young adult samples (2.53⫾0.15, n⫽8). Three solid white arrows point
to gel spots identified as MFG-E8. Identifications were performed using light chromatography–MS/MS. B, iTRAQ MS spectra showing
that MFG-E8 is more abundant in aged than young adult aorta. Representative MS spectra of 2 peptides VLPLSWHNR (409 – 417) and
CLVTEDTQR (79 – 87) indicate the abundance of MFG-E8 is ⬇2.5-fold increased.
ogy, and if needed, literature search (Table),15,16 with proteins
being clustered into a number of cellular pathways, including
apoptosis/cell cycle/proliferation, cytoskeleton/invasion, extracellular matrix/cell adhesion, metabolism, etc (Table).
Notably, the majority of those proteins were related to the
aging process or to the Ang II signaling cascade in other
organs, ie, prostate, skeletal muscle, skin, etc (see the expanded Discussion section in the online data supplement), but
many had not been previously linked specifically to arterial
aging.7–14 Ingenuity Pathway Analysis (IPA) (http://www.ingenuity.com) shows that the primary pathway was cellular
movement, where 14 of these proteins could be linked
together (Online Figure VII, A). The proteins include
MFG-E8, apolipoprotein E, insulin-like growth factor
binding protein 7, MMP2, biglycan, calponin 1, clusterin,
collagen type I ␣ 1 chain, collagen type 1 ␣ 2 chain,
fibronectin, periostin, serine protease HTRA1, transforming growth factor (TGF)-3, and vitronectin. The proteins
increase in abundance in the old aorta. Together with Ang
II, MCP-1, nuclear factor B, PDGF, and other molecules,
the proteins form a network of cellular movement, a salient
feature of invasion (Online Figure VII, A). Interestingly,
MFG-E8, a protein that markedly increased 2.3-fold in
abundance with aging, is shown to be directly linked to
protein kinase AKT17,18 and extracellular signal-regulated
kinase 1/218 in this network (Online Figure VII, A).
MFG-E8 is a multifunctional glycoprotein originally found
in milk and mammary epithelial cells.18 –27 It is of particular interest because its internal fragment, known as
medin, is an essential component of amyloid plaque
deposition in aged human arteries.25,26 Its involvement in
this cellular movement network indicates it may play an
important role in regulating VSMCs invasive capability.
Specifically, links between MFG-E8 and key molecules in
vascular remodeling, such as Ang II and MCP-1, have not
been established. We focused on MFG-E8 to determine
Fu et al
MFG-E8 Mediates VSMC Invasion
1341
how it specifically relates to the cell invasion signaling
axis.
Transcriptome, Immunolocalization, and
Posttranslational Modification Analysis of MFG-E8
Using quantitative RT-PCR, MFG-E8 mRNA levels were
increased 2.7-fold in older compared to younger rat aorta
(Figure 2A; P⬍0.05). Western blotting analyses of 1DE (data
not shown) and 2DE (Figure 2B) confirmed the increased
abundance of MFG-E8 in older versus younger aorta. In
addition to changes in the abundance of MFG-E8 with aging,
3 arterial MFG-E8 spots with similar molecular weight (MW)
were observed in the 2DE of the sample from old aorta
(Figure 2B). Importantly, treatment of the tissue samples with
PNGase F, which specifically removes N-linked carbohydrates, shifted the triplicate spots to a lower MW, as anticipated for posttranslational modification by deglycosylation
(Figure 2D). The newly appeared spots were identified as
MFG-E8 by mass spectrometry. Immunofluorescence
staining demonstrated that the intensity of glycosylated
MFG-E8 increases within the old compared to the young
aortic wall, and that MFG-E8 staining colocalizes with that
of ␣ smooth muscle actin (␣-SMA), a marker of VSMCs
(Figure 2C).
Age-Associated Increase in MFG-E8 Expression Is
Conserved Across Species
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To determine whether the age-associated increase in MFG-E8
expression is conserved across various mammalian species,
aortic tissues from nonhuman primates and humans were
used for Western blotting and immunostaining studies. In
nonhuman primates, Western blot analysis shows that the
abundance of aortic MFG-E8 increases ⬇9.0-fold with aging,
with the molecular weight indicating that it is glycosylated
(Figure 3A). Immunolabeling analysis indicates that MFG-E8
staining (brown color) markedly increases in both the intima
and the media aortic wall in older versus younger monkeys
(Figure 3B). Similarly, in humans, Western blotting analysis
and dual immunofluorescence staining of MFG-E8 indicate
an age-associated increase ⬇6.5-fold of the glycosylated
form of MFG-E8 (Figure 3C and 3D), predominantly located
in the inner media and intimae and also colocalized with
␣-SMA, as in the rat and monkey (Figure 3C and 3B).
Exogenous Administration of Ang II to Young
VSMCs Mimics Aging by Elevating
MFG-E8 Production
Although bioinformatic functional analysis indicates that
MFG-E8 is involved in a signaling network of cell movement
(Online Figure VII, A), it fails to place it specifically
downstream of the Ang II signaling cascade, a central feature
of arterial aging.7–14 Dual immunofluorescence demonstrates
that both Ang II and MFG-E8 expression within VSMCs
are closely associated and that both markedly increase
within the rat aortic wall, particularly in the thickened aged
intima (Figure 4A). Its negative control is provided in
Online Figure VIII.
We used early passage VSMCs to detect a potential
functional link between Ang II and MFG-E8. Both glycosy-
Figure 2. Validation and location of the MFG-E8 expression
within the rat aortic wall. A, Average MFG-E8 transcriptome
(n⫽4). B, 2DE Western blot of MFG-E8. C, Immunofluorescence
staining for MFG-E8 (green) and ␣-SMA (red). Counterstaining
nuclei with a DAPI dye (blue). Merged image shows staining
overlap (yellow) (original magnification, ⫻200). D, Enlarged
region of DIGE 2DE showing PNGase F–treated MFG-E8 compared to an untreated endogenous sample (red) from the old rat
aorta. PNGase F treatment (green) results in the removal of
N-linked carbohydrates, shifting the spot position to a lower
molecular weight.
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June 19, 2009
Figure 3. The age-associated arterial
MGF-E8 increase is conserved in mammalian species including humans. A, Representative Western blots of monkey aortic MFG-E8 (left) and average data (right)
(n⫽4). *P⬍0.05, old vs young. B, Immunohistostaining for MFG-E8 (brown color)
within the monkey thoracic aortic wall
(original magnification, ⫻100). L indicates
lumen; M, media. C, Representative
Western blots of human aortic MFG-E8
(left) and average data (right) (n⫽5).
*P⬍0.05, old vs young. D, Double immunofluorescence staining for MFG-E8
(green) and ␣-SMA (red) and merged
image (yellow) within the human thoracic
aortic wall (original magnification, ⫻100).
L indicates lumen; M, media.
Downloaded from http://ahajournals.org by on June 14, 2020
lated (upper bands) and unglycosylated forms (lower bands)
of MFG-E8 protein (Figure 4B, top) were detected, and both
increased with aging in early passage VSMCs (Figure 4B,
bottom).27 Treatment of young VSMCs with Ang II induced
MFG-E8 production, predominantly in the native form (unglycosylated) in a dose-dependent manner, up to the levels of
old untreated VSMCs (Figure 4B, bottom). Importantly,
elevated MFG-E8, including glycosylated and unglycosylated
forms, in old cells is significantly reduced (⬇40%) by
treatment with [Sar1, Gly8]-Ang II acetate hydrate, an Ang II
type 1 receptor blocker (Online Figure IX). This novel
finding indicates that MFG-E8 is a downstream molecule of
Ang II initiated signaling.
MFG-E8 Stimulates Functional MCP-1
Production in Aging VSMCs and Consequently
Enhanced Invasion
Previous studies indicate that chronic infusion of Ang II to
young rats increases MCP-1 expression and that the acute
exposure of early passage VSMCs from young rat aortae
potentiates their invasive capacity, in part, via enhanced
MCP-1 expression.10,28,29
We probed for a relationship between MFG-E8 and
MCP-1. Dual immunohistostaining of old aortae demonstrated MFG-E8 colocalization with MCP-1, preferentially in
the thickened intima (Figure 5A and in Online Figure X), and
immunocytostaining of old VSMCs indicates that MFG-E8
also colocalizes with MCP-1, preferentially in the perinuclear
region (Figure 5B). MFG-E8 treatment of early passage
VSMCs increased the functional dimer product of MCP-1
(Figure 5C).10,30 Conversely, MFG-E8 silencing (⬎70%)
substantially reduces MCP-1 expression and its dimerization
within VSMCs but does not effect MCP-1 mRNA levels
(Online Figure XI, A and B). Furthermore, the Ang II–
induced increase in MCP-1 expression is markedly inhibited
by MFG-E8 silencing (Online Figure XI, C). Notably, the
results in Figures 5 and 6 suggest that Ang II is upstream of
MFG-E8, and that MCP-1 is downstream of MFG-E8 within
Fu et al
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Figure 4. Interplay between Ang II and MFG-E8 in vivo and in
vitro. A, Immunofluorescence staining for MFG-E8 (green) and
Ang II (red) and merged image (yellow) in rat aortic wall.
Nuclei counterstaining with DAPI (blue) (original magnification,
⫻400). L indicates lumen; M, media. B, Representative 1DE
Western blots of MFG-E8 in smooth muscle cells isolated
from young or old rat aorta with or without Ang II treatment
with indicated concentration levels (upper images). The MFG-E8
immunoreactivity doublet is the N-linked glycosylated form (upper
bands) and unglycosylated (lower bands), as shown by deglycosylation of the sample with PNGase F resulting in enhancement of
the lower molecular weight band. Average data (lower graph).
*P⬍0.05 old untreated and young treated cells vs young control
cells.
the Ang II signaling cascade that potentiates VSMCs invasion. These data establish novel interactions among Ang II,
MCP-1, and MFG-E8 (Online Figure VII, B).
Next, we determined whether MFG-E8 is required for
enhanced VSMCs invasion and whether it acts in this
respect via MCP-1. Modified Boyden chamber analysis (Figure
6A, left) shows that, in control, VSMC invasive capability
significantly was increased in old compared to young cells.
MFG-E8 siRNA successfully knocked down MFG-E8 protein
expression in both young and old VSMCs (Figure 6A, right) and
reduced the invasive capability of both young and old VSMCs,
eliminating age differences in invasive capacity that were present in control (Figure 6A, left). Exposure of VSMCs to Ang II
increased the invasive capability of VSMCs in an age-dependent
fashion, and this increase was substantially reduced by MFG-E8
silencing (Figure 6B). The addition of exogenous MFG-E8 also
increased VSMCs invasive capability in an age-dependent
fashion (Figure 6C), and these effects were substantially
reduced by treatment with a specific MCP-1 receptor blocker
vCCI (Figure 6C).
Discussion
In this study, we present for the first time a proteome profile
of arterial aging in FXBN rats, a well-characterized rodent
MFG-E8 Mediates VSMC Invasion
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Figure 5. Interaction between MFG-E8 and MCP-1 in vivo and
in vitro. A, Immunofluorescence staining for MFG-E8 (green) and
MCP-1 (red) and merged image (yellow) in old human aorta.
Nuclei counterstaining with DAPI (blue) (original magnification,
⫻200). L indicates lumen; M, media. B, Immunofluorescence
photomicrographs (⫻400) for MFG-E8 (red color), MCP-1 (green
color), nuclei (blue color), and colocalization of MFG-E8 and
MCP-1 (yellow color, right images) within VSMCs. C, Representative Western blots show that the functional dimer of MCP-1
increases in VSMC treated with MFG-E8 in both young and old
cells in a dose-dependent manner.
model with similarities to the human anatomic and physiological phenotype. Using a combination of 2DE and iTRAQ,
we identified and characterized 923 proteins within the aortic
wall, of which 50 significantly change abundance with aging,
some of which also have posttranslational modifications such
as phosphorylation and glycosylation. The proteins with
significant abundance changes (Table) observed in this study
play significant roles in a number of cellular pathways,
including cellular movement, cell proliferation and apoptosis,
energy metabolism, etc, and may contribute significantly
to vascular remodeling with aging. Some of these proteins
have been related to aging in other organs, ie, prostate,
skeletal muscle, and skin, etc (for an expanded discussion
regarding the proteins, see the online data supplement).
Only a few proteins have been specifically linked to
arterial aging, such as MMP2 or TGF-.7–14 These proteins, including Ang II, MCP-1, MFG-E8, MMP2, and
TGF-, etc, are involved in a network of cellular movement (Online Figure VII, A).
Furthermore, the present results establish molecular links
between Ang II, MFG-E8, and MCP-1 in aged VSMCs. It has
been previously established that Ang II and Ang II type 1
receptor expression and signaling increase within the arterial
wall with aging.3,8,9,12,14 Exogenously infused Ang II to young
animals produces the structural molecules of the arterial wall
resembling old untreated animals,9,12 whereas chronic blockade
of Ang II signaling in animals reverses and retards these
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June 19, 2009
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Figure 6. VSMC invasion assay. A, Average invasion
analysis of VSMCs with and without siRNA of
MFG-E8 (left graph). *P⬍0.05, old vs young cells
chemoattracted by PDGF (10 ng/mL); #P⬍0.05,
young and old cells with MFG-E8 silencing vs sham
silencing, respectively. Representative Western blots
show that the majority of MFG-E8 protein is
knocked down via siRNA approach (right). B, Average invasion analysis of VSMCs with and without
siRNA of MFG-E8 and Ang II treatment. *P⬍0.05,
old vs young cells with treated with Ang II (100
nmol) and chemoattracted by PDGF (10 ng/mL);
#P⬍0.05, young and old cells with MFG-E8 silencing
vs sham silencing, respectively. C, Average invasion
analysis of VSMCs treated with and without MFG-E8
plus vCCI. *P⬍0.05 young and old cells treated with
MFG-E8 (50 ng/mL) vs untreated controls, respectively; #P⬍0.05, young and old cells treated with
MFG-E8 plus vCCI vs MFG-E8 treated, respectively.
age-associated structural molecular changes.31–33 Ang II can
promote expression of MCP-1,10,28,29 a potent chemokine that is
an element of the AKT-associated proinflammatory network and
plays an important role in chemotaxis and atherosclerosis.34
Interestingly, MFG-E8 stood out in our proteomic analyses
because it had not previously been linked directly with Ang
II/MCP-1, VSMCs, or aging, although it has been shown to
induce cell-specific apoptosis, metastasis, and angiogenesis.18 –24
Previous studies have shown that MFG-E8 is abundantly expressed in metabolically active tissues, ie, atherosclerotic
plaques25,26,35 and tumors.24 We have shown that MFG-E8 is
N-linked glycosylated and increases in abundance in aortae of
old versus young rats (Table). This age-related increase in
transcription and translation of MFG-E8 is preserved across
species (rat, nonhuman primates, human), suggesting it may
have a central role in arterial aging. As such, we investigated the
interplay between MFG-E8 and Ang II, and for the first time, we
uncovered the important finding that Ang II induces MFG-E8 in
VSMCs isolated from rat aortae. In fact, MFG-E8 is required for
the Ang II to increase MCP-1. Ang II was previously shown to
coexpress with MCP-1.10,28,29 Furthermore, we show that Ang II
or MCP-1 also colocalizes with MFG-E8 in the arterial wall,
particularly in the aged thickened intimae. These findings
suggest not only that MCP-1 receives Ang II signals but also that
this cellular information is relayed via MFG-E8. Of note,
treatment of isolated VSMCs with MFG-E8 induces the biologically active dimer of MCP-1 in a dose-dependent manner. This
action of MFG-E8 is profound, because the active MCP-1
homodimer is a potent chemoattractant of smooth muscle cells,
if produced locally.30
In addition, MFG-E8 enhanced the Ang II–induced invasion potential of the isolated VSMCs (Figure 6). We observed
that the invasiveness of old VSMCs is greater than young
cells and that this effect depends on Ang II signaling (Figure
6). However, silencing MFG-E8, a downstream molecule of
Ang II signaling, similarly reduces the invasive capacity of
both young and old cells, whereas exogenous MFG-E8
treatment of young VSMCs increases their invasiveness, up
to the levels of old untreated cells. Interestingly, a CCR2
blocker, vCCI, can interrupt MFG-E8/MCP-1 signaling, sub-
Fu et al
Downloaded from http://ahajournals.org by on June 14, 2020
stantially inhibiting this effect. Thus, MFG-E8 is a novel link
between Ang II/MCP-1 signaling and VSMCs invasive capacity within the aortic wall.
Taken together, our study demonstrates a comprehensive
nonbiased proteomic approach to better understand the biology of age-associated thickening of the aorta and has identified a novel Ang II/MFG-E8/MCP-1 signaling axis (Online
Figure VII, B) that links the cellular movement signaling
cascades with the age-associated increase in smooth muscle
cell invasive capability. The abundance changes of other
proteins observed in this proteomic study suggest that coordinated actions occur through this signaling axis. Notably,
recent findings show that MFG-E8 is expressed in advanced
human atherosclerotic plaques, and MGF-E8 deficiency results in the accumulation of apoptotic cells and accelerates
atherosclerosis in mice.36 Previously, we have performed
immunostaining for inflammatory cells, including monocyte/
macrophage and lymphocyte markers, and assessed apoptosis
by TUNEL staining in the vasculature of young and old
animals. Results have shown that these cells are not detected
in both the intima and the media, rarely observed in the
adventitia.11 Obviously, the regulation of these proteins is
complex; however, based on the evidence mentioned above
and provided in this report, it is likely that the accumulation
of MFG-E8 in the arterial wall plays a critical role in arterial
aging. Our data show MFG-E8 accumulates in the aorta with
aging and promotes VSMC invasion, an important step in
vascular thickening and arthrosclerosis. Further studies will
show whether MFG-E8 can be a potential molecular target
for the intervention to prevent/retard human arterial aging,
arthrosclerosis, and other vascular diseases that involve
inflammation and smooth muscle cell invasion.
Acknowledgments
We thank Robert O’Meally for technical support and Lesley Kane
for critical reading of the manuscript.
Sources of Funding
This research was supported by the Intramural Research Program of
the National Institute on Aging, NIH; and the National Heart, Lung
and Blood Institute Proteomic Initiative (contract no. HV28120).
Disclosures
None.
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