LRP1 Regulates Architecture of the Vascular Wall by
Controlling PDGFRb-Dependent Phosphatidylinositol
Li Zhou1, Yoshiharu Takayama1, Philippe Boucher3, Michelle D. Tallquist2, Joachim Herz1*
1Department of Molecular Genetics, UT Southwestern Medical Center, Dallas, Texas, United States of America, 2Molecular Biology, UT Southwestern Medical Center,
Dallas, Texas, United States of America, 3Department of Pharmacology, University of Strasbourg, Strasbourg, France
Background: Low density lipoprotein receptor-related protein 1 (LRP1) protects against atherosclerosis by regulating the
activation of platelet-derived growth factor receptor b (PDGFRb) in vascular smooth muscle cells (SMCs). Activated PDGFRb
undergoes tyrosine phosphorylation and subsequently interacts with various signaling molecules, including phosphati-
dylinositol 3-kinase (PI3K), which binds to the phosphorylated tyrosine 739/750 residues in mice, and thus regulates actin
polymerization and cell movement.
Methods and Principal Findings: In this study, we found disorganized actin in the form of membrane ruffling and
enhanced cell migration in LRP1-deficient (LRP12/2) SMCs. Marfan syndrome-like phenotypes such as tortuous aortas,
disrupted elastic layers and abnormally activated transforming growth factor b (TGFb) signaling are present in smooth
muscle-specific LRP1 knockout (smLRP12/2) mice. To investigate the role of LRP1-regulated PI3K activation by PDGFRb in
atherogenesis, we generated a strain of smLRP12/2 mice in which tyrosine 739/750 of the PDGFRb had been mutated to
phenylalanines (PDGFRb F2/F2). Spontaneous atherosclerosis was significantly reduced in the absence of hypercholester-
olemia in these mice compared to smLRP12/2 animals that express wild type PDGFR. Normal actin organization was
restored and spontaneous SMC migration as well as PDGF-BB-induced chemotaxis was dramatically reduced, despite
continued overactivation of TGFb signaling, as indicated by high levels of nuclear phospho-Smad2.
Conclusions and Significance: Our data suggest that LRP1 regulates actin organization and cell migration by controlling
PDGFRb-dependent activation of PI3K. TGFb activation alone is not sufficient for the expression of the Marfan-like vascular
phenotype. Thus, regulation of PI3 Kinase by PDGFRb is essential for maintaining vascular integrity, and for the prevention
of atherosclerosis as well as Marfan syndrome.
Citation: Zhou L, Takayama Y, Boucher P, Tallquist MD, Herz J (2009) LRP1 Regulates Architecture of the Vascular Wall by Controlling PDGFRb-Dependent
Phosphatidylinositol 3-Kinase Activation. PLoS ONE 4(9): e6922. doi:10.1371/journal.pone.0006922
Editor: Harald HHW Schmidt, Monash University, Australia
Received May 19, 2009; Accepted August 7, 2009; Published September 9, 2009
Copyright: ? 2009 Zhou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by grants from NIH (HL20948, HL63762, and NS43408), the Alzheimer’s Association, the Humboldt Foundation and the Perot
Family Foundation for J.H., by NIH (HL074257) and American Heart Association (AHA) Scientific Development Grant (0330351) to M.D.T., and by a Postdoctoral
Fellowship (Grant 0825316F) from the South Central Affiliate of the AHA to L.Z.. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Low density lipoprotein receptor related protein 1 (LRP1) is a
multifunctional member of the LDL receptor (LDLR) gene family
with a unique capacity of binding over 40 distinct ligands . It
plays diverse roles in a variety of biological processes including
lipoprotein metabolism, protease degradation, activation of lyso-
Binding of apolipoprotein E (apoE) to the extracellular domain of
LRP1 removes apoE-containing lipoprotein remnants from the
circulation into the liver by endocytosis [3,4,5]. By contrast, in the
smooth muscle cells (SMCs) of the arterial wall, apoE-lipoprotein
binding inhibits platelet-derived growth factor (PDGF)-directed
SMC migration . Studies from our laboratory have shown that
LRP1 suppresses PDGF receptor b (PDGFRb) activation and
protects against atherosclerosis .
Activated PDGFRb undergoes tyrosine phosphorylation and
subsequently interacts with a variety of SH2 domain-containing
signaling molecules including phosphatidylinositol 3-kinase (PI3K),
phospholipase Cc (PLCc), Src family kinase, and phosphotyrosine
phosphatase SHP-2 . Among these interacting proteins, PI3K
which binds to the phosphorylated tyrosine 740/751 residues
(739/750 in the mouse) of PDGFRb through its p85 regulatory
subunit , is particularly important for regulating actin
organization [10,11], cell growth and migration .
LRP1 is also known as transforming growth factor b (TGFb)
receptor V (TbR-V) and appears to be required for mediating the
growth inhibitory response of TGFb, in conjunction with Smad
signaling through TbR-II and I [13,14]. TGFb signaling is
abnormally elevated in the absence of LRP1 in vivo, where analysis
of SMC-specific LRP1 knockout (smLRP12/2) mice revealed a
Marfan syndrome-like phenotype with nuclear accumulation of
PLoS ONE | www.plosone.org1September 2009 | Volume 4 | Issue 9 | e6922
phosphorylated Smad2 (p-Smad2) and disruption of elastic layers
in the vessel wall .
For the present study we have generated a new, genetically
complex strain of compound mutant mice that are LDL receptor-
deficient (LDLR2/2), lack LRP1 only in their vascular smooth
muscle cells, and express an endogenous, crippled form of the
PDGFRb that is incapable of activating PI3K. Our goal was to
test, whether increased PDGFRb signaling through PI3K is the
primary cause for the increased susceptibility to atherosclerotic
lesion development in LDLR 2/2 mice lacking LRP1 in their
SMCs, and whether PDGFRb-dependent PI3K signaling is
required for the expression of the Marfan syndrome-like
phenotype in smLRP1-deficient mice.
Lack of LRP1 expression in the SMCs results in cell
hypertrophy and vessel elongation
Earlier data from our laboratory showed that smLRP12/2;
LDLR2/2 mice are highly susceptible to atherosclerosis when
fed a high-cholesterol diet . To determine if this increased
susceptibility to atherosclerosis is preserved in smLRP12/2
mice in the absence of hypercholesterolemia, smLRP12/2 mice
either expressing or lacking LDLR were maintained on a
standard low-fat rodent chow. Determination of plasma total
cholesterol confirmed that smLRP12/2 mice did not develop
hypercholesterolemia (129.965.3 mg/dl, Table 1). Whereas,
smLRP12/2; LDLR2/2 mice had high total cholesterol levels
of 246.5661.0 mg/dl with a major increase in LDL (Figure S1).
Elongated aortas were present in the absence of hypercho-
lesterolemia in smLRP12/2 mice (Figure 1c, h). However,
atherosclerotic lesions were only visible in smLRP12/2;
LDLR2/2 mice (Figure 1e, j). To study structural changes in
the vascular wall of these elongated aortas, H&E, trichrome and
elastin staining were performed. Thickened aortic walls with
intima thickening, disarranged and hypertrophic SMCs, in-
creased extracellular collagen accumulation, and elastic lamina
disruption were observed in 11-month old smLRP12/2 mice
(Figure 1B). Vascular wall thickening was also present in young
mice at 7 weeks of age (Figure S2). Compared with wild type,
the aortic wall of smLRP12/2 mice was significantly thicker
(66.1463.32 mm vs. 39.3562.16 mm, Figure 1C). Our findings
indicate that LRP1 expression in SMCs controls the architec-
ture of the vascular wall in a plasma cholesterol-independent
LRP1 controls PI3K binding and activation by PDGFRb in
PDGFRb signaling is crucial for regulating SMC responses and
is an important contributing factor to atherogenesis. Previous work
from our laboratory showed dramatically increased expression and
activation of PDGFRb in aortas from smLRP12/2; LDLR2/2
mice on a high-cholesterol diet . To investigate the basal
expression and activation level of PDGFRb in mice maintained on
a low-cholesterol diet, aortic extracts were prepared from mice
expressing or lacking LRP1 and LDLR and analyzed by Western
blotting. Because PDGFRb activation, through transphosphory-
lation of tyrosine residues in its cytoplasmic domain, triggers a
cascade of phosphorylation events which eventually lead to the
activation of extracellular regulated-protein kinases (Erks), phos-
phorylated-Erk1/2 was used as an indicator of PDGFRb
activation . About a two-fold increase of PDGFRb expression
was detected in smLRP12/2 mice regardless of LDLR genotype
(Figure 2A, C). Increased Erk1/2 phosphorylation was also
observed in these aortas (Figure 2A). These data suggest that the
expression and activation of PDGFRb is only regulated by LRP1,
PI3K binding sites on PDGFRb are crucial for the receptor-
mediated cell responses [16,17]. To explore the functional and
biochemical interaction between LRP1 and the PDGFRb-PI3K
signaling pathway, we performed a co-immunoprecipitation assay.
Compared to wild type animals, smLRP12/2 mice showed
increased tyrosine-phosphorylation of and PI3K binding to
PDGFRb (Figure 2B), indicating that LRP1 regulates PDGFRb-
dependent activation of PI3K by controlling PDGFRb phosphor-
Disruption of PDGFRb-PI3K signaling in mice reduces
To investigate whether LRP1 regulates atherosclerosis through
the PDGFRb-dependent PI3K pathway in vivo, we generated a
compound mutant mouse model by crossing smLRP12/2;
LDLR2/2 mice to PI3K binding-deficient PDGFRb F2/F2
mutant mice, in which tyrosine residues at position 739 and 750
are mutated to phenylalanines .
We found significantly decreased atherosclerotic lesions in the
aortic arch and abdominal aorta of smLRP12/2; LDLR2/2;
PDGFRb F2/F2 mice (Figure 3A, C). H&E, trichrome and elastin
staining revealed well-arranged spindle-shaped SMCs, reduced
extracellular matrix, and virtually normal elastic layers in
smLRP12/2; LDLR2/2; PDGFRb F2/F2 aortas (Figure 3B).
Vascular wall thickness, hypercellularity and length of the aortas in
smLRP12/2; LDLR2/2; PDGFRb F2/F2 mutants were mark-
edly reduced to approximately normal levels (Figure 3B, D, E, F).
However, the prominent aneurysms of the mesenteric arteries,
which are a hallmark of smLRP2/2 mice, were notably not
abolished in smLRP12/2; LDLR2/2; PDGFRb F2/F2 mice
(Figure 3A), suggesting that atherogenesis and aneurysm formation
employ at least partially different molecular or regionally distinct
Ablation of PI3K binding to PDGFRb reverses actin
disorganization in LRP1-deficient SMCs
To characterize SMCs lacking either LRP1 or LDLR and with
crippled PI3K binding to PDGFRb in vitro, we generated primary
cells from the aortas of these mice. To eliminate fibroblast
contamination, LRP1-deficient SMCs were selected with Pseudo-
monas exotoxin A (PEA) according to previous publications
[19,20]. Primary SMCs were identified by their typical spindle
Table 1. Total cholesterol and triglyceride levels of mouse
Total Cholesterol131.3616.8 129.965.3a
Total Triglycerides 61.769.851.1620.0a
Total cholesterol and triglyceride levels were significantly higher in mice lacking
LDLR, even though they were fed a standard rodent chow diet. LRP-deficient in
smooth muscle cells did not change the lipid levels significantly.
ap.0.05 (smLRP12/2 vs WT).
bp,0.01 (LDLR2/2 vs WT).
cp,0.05 (smLRP12/2; LDLR2/2 vs smLRP12/2).
dp.0.05 (smLRP12/2; LDLR2/2 vs LDLR2/2).
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shape (Figure 4a) and the expression of smooth muscle actin
(Figure 4b). LRP1 expression was verified by both immunocyto-
chemical staining (Figure 4c, d) and Western blotting (Figure 4e).
Interestingly, the morphology of SMCs of different genotypes
looked quite distinct from each other. Actin organization was
disrupted in the SMCs lacking LRP1 expression and an ‘‘actin
ring’’ was present below the plasma membrane (Figure 4B).
However, in smLRP12/2; PDGFRb F2/F2 SMCs actin organi-
zation was restored (Figure 4B), suggesting that LRP1 mediates
actin remodeling through PI3K activation upon PDGFRb
Increased migration of LRP1-deficient SMCs is diminished
by the PDGFRb F2 mutation
Our in vivo experiments showed disarranged SMCs in the
medial layer of the aorta (Figure 1m, 3h, 3k) and disrupted elastic
laminas when LRP1 was deficient in the SMCs (Figure 1s, 3t, 3w).
To investigate if these phenomena are caused by abnormal
migration due to the absence of LRP1, we performed two different
kinds of in vitro migration assays. Compared with wild type cells,
SMCs lacking LRP1 showed markedly increased migratory
activity, both in a Boyden chamber transmigration assay
(Figure 5A, C) and in the commonly used scratch assay, in which
the migration of the cells into a denuded area of a tissue culture
dish is quantified (Figure 5B, D). Cell migration was significantly
reduced in those cells containing the PDGFRb F2/F2 mutation
(Figure 5A–D), and this correlated with the improved architecture
of the elastic layers in the aortic wall of smLRP2/2; LDLR2/2;
PDGFRb F2/F2 mice (Figure 3u, x). These findings thus confirm
that SMC migration is regulated by LRP1 through the PDGFRb-
dependent PI3K pathway.
PDGF-BB-induced chemotaxis is inhibited by blocking
PI3K activation through PDGFRb
To investigate whether PDGF-BB-induced SMC migration
involves the PDGFRb-PI3K pathway, 10 ng/ml PDGF-BB was
administrated as a chemo-attractant in the bottom well of a
Boyden chamber. As shown in Figure 5E, PDGF-BB induced the
transmigration of SMCs independent of LRP1 expression.
However, when the Y739/750 phosphorylation sites of PDGFRb
were mutated, this PDGF-BB-induced chemotaxis was completely
abolished. This finding suggests that the increased migratory
propensity of LRP1-deficient SMCs, which is further enhanced by
PDGF-BB activation of PDGFRb, is in its entirety dependent
upon the activation of PI3K by PDGFRb.
Abnormal activation of TGFb signaling is present in the
absence of LRP1
Our in vivo findings indicate that lack of LRP1 in SMCs results
in elongation of the aorta, thickening of the vascular wall, and
disruption of the elastic layers. Tortuous aorta and elastic
laminar disruption are also two key cardiovascular manifestations
Figure 1. Hypertrophic and hyperplastic SMCs and elongated aortas in smLRP12/2 mice. (A) Unopened (a–e) and Oil Red O stained (f–j)
aortas from 11-month old mice of the indicated genotypes. Mice were maintained on standard rodent chow diet. Arrows indicate lipid-laden
atherosclerotic lesions. (B) Histological analysis of thoracic aortas from 11-month old mice of the indicated genotypes. k–m: hematoxylin & eosin (HE);
Data represent mean6SD from 3 mice per group. *** p,0.001.
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of Marfan syndrome. As a connective tissue disorder with
autosomal dominant inheritance, Marfan syndrome is caused by
loss-of-function mutations in fibrillin-1, a matrix component of
extracellular microfibrils . Fibrillin-1 regulates activation of
the cytokine TGFb, and its deficiency results in enhanced TGFb
signaling [22,23]. Recent studies have shown that abnormal
activation of TGFb contributes to the pathogenesis of Marfan
syndrome . Paradoxically, loss of function mutations in
TGFb receptor I or II also result in increased TGFb signaling
and give rise to Marfan syndrome [24,25]. LRP1 is identical to
the type V TGFb receptor (TbR-V), which co-expresses with
other TGFb receptors (TbR-I, TbR-II and TbR-III) .
LRP1/TbR-V mediates TGFb induced growth inhibition in
concert with TbR-II/TbR-I/Smad2/3/4 signaling [13,14] and
TGFb signaling is increased in LRP1 deficient mouse aortas .
To evaluate the TGFb activation state in the aortas of the
different genotypes, immunofluorescent staining and Western
blotting of p-Smad2 were performed. Significantly increased
LDLR2/2 aortas. Importantly, blockade of PI3K binding to
PDGFRb did not suppress over-activation of TGFb signaling
(Figure 6A). An approximately 2.5-fold increase of Smad2
phosphorylation at Ser 465/467 was also detected in LDLR-
expressing, LRP1-deficient SMCs (Figure 6B, C). These data
indicate that in the absence of LRP1, TGFb signaling is
abnormally activated independent of the absence or presence of
the LDL receptor, and this over-activation of TGFb is not
suppressed by the PDGFRb F2 mutation.
Taken together, our findings suggest that aorta elongation and
elastic layer disruption, both hallmarks of Marfan syndrome,
require the PDGFRb dependent activation of PI3K. Activation of
the TGFb signaling pathway alone is not sufficient. Thus, LRP1
acts as a molecular switch that integrates TGFb and PDGFRb/
PI3K signals and this is essential for maintaining the integrity of
the vascular wall architecture.
In this study we have investigated the role of LRP1 for PI3K
activation by PDGFRb in SMCs, and the impact this LRP1
‘checkpoint’ has for preventing atherosclerotic lesion formation
and progression, as well as for the maintenance of vascular wall
integrity. We found that the selective genetic blockade of PI3K
activation by PDGFRb substantially suppressed spontaneous
atherosclerotic lesion development, which is prominent in
smLRP12/2; LDLR2/2 mice. Furthermore, vascular wall
elongation and medial thickening, due to SMC hyperproliferation,
increased SMC migration and disruption of elastic layers are
normalized throughout the entire aorta. Our findings suggest that
PI3K is the main driving force that promotes SMC proliferation
and migration, elastolysis, spontaneous atherosclerosis and lesion
progression in the absence of LRP1.
Prominent atherosclerotic lesions preexisted in smLRP12/2;
LDLR2/2 mice maintained on standard, low-fat and cholesterol-
free rodent chow, but not in smLRP12/2 animals of comparable
age. These data suggest that in the presence of an intact
endothelium and low plasma cholesterol levels, proliferative
signals alone are not sufficient to initiate the pathogenic
mechanisms that culminate in classic atherosclerotic plaques. By
contrast, the aorta of LRP1+/+; LDLR2/2 mice appears
Figure2. Increased expression andactivation ofPDGFRb andPI3K binding by PDGFRb in aortic extracts ofsmLRP12/2 mice. (A)Protein
extracts (10 mg/lane) from mouse aortas of the indicated genotypes were analyzed by immunoblotting for PDGFRb, p-Erk1/2, Erk1/2, and LRP1.
(B) 200 mg of aortic extracts of the indicated genotypes were immunoprecipitated with the designated antibodies (anti-PDGFRb and anti-
phosphotyrosine) to semi-quantitatively determine the interaction between PI3K and PDGFRb. Precipitated proteins were analyzed by immunoblotting
using the indicated antibodies (anti-phosphotyrosine, anti-PI3K-p85 and anti-PDGFRb). Actin served as a loading control. WB: Western blot;
IP: immunoprecipitation. (C) Expression of PDGFRb relative to the loading control was quantified using Image J software (NIH). Data are expressed as
mean6SD. ** p,0.01.
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Figure 3. Reduced atherosclerotic lesions in smLRP12/2; LDLR2/2; PDGFRb F2/F2 mutant mice. (A) Unopened (a–c) & Oil Red O stained (d–
f) aortas from 10-month old mice of the indicated genotypes. Mice were maintained on standard rodent chow diet. Arrows indicate lipid-laden
atherosclerotic lesions. (B) Histological analysis of aortas from 8-month old mice of the indicated genotypes. g–l: HE stain, m–r: trichrome stain, s–x:
elastin stain. g,h,i,m,n,o,s,t u: aortic arch; j,k,l,p,q,r,v,w,x: thoracic aorta. Scale bar, 20 mm. (C, D, E, F) Atherosclerotic lesions (C), length (D), thickness (E)
and cell number per cm2(F) of the indicated genotypes were quantified using Image J software (NIH). Results from 3 mice per group are presented as
mean6SD. * p,0.05, ** p,0.01, *** p,0.001.
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Figure 4. Disorganization of the actin cytoskeleton in LRP12/2 SMCs is prevented by blocking PI3K activation by PDGFRb. (A)
Primary SMCs generated from mouse aortas. (a) Phase contrast image. Scale bar, 80 mm. (b) Immunofluorescence using anti-smooth muscle actin
monoclonal antibody (red). Blue: DAPI staining of nuclear DNA. Scale bar, 40 mm. (c, d) Detection of LRP1 (green) by immunofluorescence by a rabbit
anti-LRP1 polyclonal antibody. Blue: DAPI staining. WT, wild type. Scale bars, 40 mm. (e) Immunoblotting was performed to verify the presence or
absence of LRP1 protein in the wild type and LRP12/2 SMCs using the same polyclonal anti-LRP1 antibody. (B) Immunofluorescence of smooth
muscle actin (red) in primary SMCs. Actin disorganization in LRP12/2 and LRP12/2; LDLR2/2 SMCs. Normal organization of the actin cytoskeleton is
restored in primary PDGFRb F2/F2 SMCs. Blue: DAPI. Scale bar: 20 mm.
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Figure 5. Primary SMC Migration. (A) Transwell migration assay. 30,000 SMCs of the indicated genotypes were added to the top compartment of
a Boyden chamber. After 6 hours of incubation, the transwell membrane was fixed and stained with hematoxylin. Cells that had migrated through
the holes on the membrane to the bottom face were counted. Scale bar, 50 mm. (B) Scratch assay. 300,000 SMCs of the indicated genotypes were
seeded into 60 mm culture dishes in a medium containing 10 mg/ml mitomicin C to prevent cell proliferation and allowed to adhere overnight. The
next day, part of the dish was denuded by scratching along a straight line (indicated by a black line behind). Cells were then allowed to migrate into
the denuded area for 24 hours prior to fixation and quantification. Scale bar, 50 mm. (C, D) Statistical analyses of the Transwell and Scratch migration
assays. Results are represented as mean6SD. *** p,0.001. (E) PDGF-BB chemotaxis assay. 10,000 SMCs of the indicated genotypes were added to the
top compartment of the Boyden chamber. 10 ng/ml PDGF-BB was added to the lower chamber of the well. After 6 hours of incubation, the transwell
membrane was fixed and stained with hematoxylin. The cells that had migrated through the holes on the membrane to the bottom face were
counted. Scale bar, 50 mm. (F) Statistical analysis of the PDGF-BB chemotaxis assay. Results are represented as mean6SD. ** p,0.01, *** p,0.001.
n=5 for all assays.
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histoanatomically normal despite increased plasma cholesterol
levels on the same chow, and extensive atherosclerotic lesions
develop only after feeding of a high-cholesterol diet for several
months . Thus, LRP1 in SMCs functions cell autonomously in
the maintenance of vascular wall integrity and protection from
In the absence of smLRP1, the mouse aorta undergoes
hyperplastic and hypertrophic changes that were apparent in
young (7 weeks) as well as older (11 months old) mice indicating
that they are not the result of aging, but the manifestation of an
intrinsic change of smooth muscle phenotype. This is most likely
caused by the increased expression and activation of PDGFRb in
smLRP12/2 mice and an accompanying increase in PI3K
association with PDGFRb. Disruption of an obligatory proathero-
genic proliferative pathway, involving PI3K and PDGFRb,
prevents or greatly reduces lesion development at sites of high
shear stress, such as the aortic arch and the abdominal aorta,
where endothelial integrity is easily compromised. Thus, by
selectively controlling SMC proliferation and migration indepen-
dent of endothelial integrity and plasma cholesterol levels in a
novel genetically complex animal model, we have been able to
isolate and demonstrate the pivotal and interdependent roles of
two central mechanisms of atherosclerotic lesion development.
Activation of the PDGFRb results in actin reorganization in the
forms of membrane ruffling and chemotaxis [11,27,28,29,30] and
thus provides an excellent functional assay for the physiological
activation of PDGFRb through other genetic manipulations, such
as the disruption of LRP1. PI3K binding to the cytoplasmic
domain of activated PDGFRb receptors requires phosphorylation
at residues 739 and 750 and this interaction in turn activates the
kinase [18,31]. Replacement of these tyrosines by non-phosphor-
ylatable phenylalanines prevents binding of PI3K and fails to
mediate membrane ruffling and cell migration [28,29]. As a result,
the pronounced edge ruffling and circular membrane ruffling as
well as greatly enhanced SMC migration that were observed in the
absence of LRP1 were virtually normalized in mice in which
Figure 6. Increased Smad2 phosphorylation at Ser 465/467 in smLRP12/2; LDLR2/2 and smLRP12/2; LDLR2/2; PDGFRb F2/F2
aortas. (A) Immunofluorescent staining of p-Smad2 (Ser 465/467, green) in aortas of atherosclerosis-free 1-month old mice. Scale bar, 20 mm.
(B) Immunoblotting of aortic extracts for Smad2 phosphorylation at Ser 465/467. b-actin served as a loading control. (C) The density of p-Smad2
signals was normalized to the corresponding b-actin signals from the same blot and quantified using Image J software (NIH). Densitometric
scanning from three independent experiments revealed a statistically significant average of 250%613% in smLRP12/2 mice and 260%629% in
smLRP12/2; PDGFRb F2/F2 mice, compared with wild type controls (92%617%). Relative ratio of p-Smad2 in mouse aortas from the indicated
genotypes was plotted. Data are presented as mean6SD. * p,0.001, n=3.
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PDGFRb-dependent PI3K activation had been genetically
disrupted. These findings show that the membrane ruffling and
increased smooth muscle migration in smLRP2/2 mice is
critically dependent upon PI3K activation, which is mediated by
PDGFRb. Nevertheless, a caveat to this interpretation is that,
although PI3K is the only known cellular signal transducer that
interacts with pY739 and 750 of PDGFRb, this do not exclude the
possibility that another unknown signal modulator also interacts
with this site and contributes to the pathogenic mechanism.
Marfan syndrome, a disorder of connective tissue architecture
with prominent manifestations in the skeletal, ocular and
cardiovascular systems, is caused by mutations in the fibrillin-1
gene [21,32] or by loss of function mutations in TGFb receptor I
or II [24,25]. TGFb signaling is abnormally elevated in fibrillin-1-
deficient mice [22,23,33] and human aortas  as well as TGFb
receptor I and II deficiency . Previous data from our
laboratory have shown nuclear accumulation of phosphorylated
Smad2, an indicator of activation of TGFb signaling, in the
LRP1(TbR-V)-deficient vascular wall . In the present study,
we have reconfirmed these Marfan syndrome-like phenotypes,
including elastic layer disruption, aorta elongation, and aneurysm
formation in the presence of increased Smad2 phosphorylation
when LRP1 is deficient in the SMCs. These phenotypic
manifestations in the vascular wall were essentially abolished in
smLRP12/2; PDGFRb F2/F2 mice, however, the increased
phosphorylation and nuclear translocation of Smad2 was not
affected by the PDGFRb mutations. These findings indicate that
TGFb activation through LRP1 precedes PDGFRb-dependent
PI3K signaling, and that activation of TGFb signaling by itself is
not sufficient to disrupt the vascular wall architecture. PDGFRb-
dependent PI3K activation appears to be necessary for the
expression of the Marfan-like phenotypes. Suppression of PI3K
activation by PDGFRb prevents the Marfan-like phenotypic
changes in the vascular wall in the presence of unabated TGFb
signaling, suggesting a pivotal role of LRP1-controlled and
PDGFRb-dependent PI3K activation in the pathogenesis of
Marfan syndrome. Selective elimination of PDGFRb-dependent
PI3K activation thus could be a potential therapeutic target for
both atherosclerosis and Marfan syndrome.
In conclusion, the current study reveals a novel PI3K-
dependent mechanism by which LRP1 is essential for controlling
the integrity of the vascular wall, and by which this multifunctional
receptor potently protects against atherosclerosis and Marfan
syndrome. The findings we have presented here shed new light on
the molecular mechanisms that control cellular growth and
migration, and which are thereby essential to the remodeling
and repair of the vascular wall and for slowing or preventing
degenerative disorders of the vascular wall.
Materials and Methods
Generation of the mouse strains
All experimental mice were maintained on a mixed C57BL/6/
129 background. Transgenic mice expressing Cre recombinase
specifically in smooth muscle cells (SM22 Cre) mated with LRP1
PDGFRb F2/F2 mouse strains were established. Paired littermates
were utilized throughout the study.
Animal & aorta preparation
Experiments were performed according to protocols approved
by the Institutional Committee for Use and Care of Laboratory
Animals. All animals were maintained on standard rodent chow
(Teklad 6% fat) with water ad libitum. Mice were sacrificed and
blood samples were collected for lipid analysis following a six-hour
fasting period. Aortas were removed intact from the root of the
aortic arch to the iliac bifurcation and preserved in 4%
paraformaldehyde (PFA) for conventional morphological study.
Oil Red O staining
Aortas were opened longitudinally under a dissecting micro-
scope (Model Z30L, Cambridge Instruments). After fixation in 4%
PFA, tissues were stained with 0.05% Oil Red O (s1848, Poly
Scientific) at 60uC for 30 min. The aortas were then rinsed twice
with 85% propylene glycol to develop the color.
Hematoxylin-eosin (H&E), Masson’s Trichrome and
Hart’s Elastin stainings were performed according to
established textbook methods [35,36].
Aortic extract preparation and Western blotting (WB)
The fat and connective tissue of the aorta were removed
carefully. The aorta was homogenized in RIPA buffer with
Proteinase Inhibitor Cocktail (P8430, Sigma) and Phosphatase
Inhibitor Cocktail II (P5726, Sigma) to inhibit tyrosine protein
phosphatases. After centrifugation at 20,000 xg for 30 minutes at
4uC, the supernatant was applied for Western blotting and the
pellet was discarded.
Briefly, aortic extract was resolved on SDS-PAGE gel and
transferred to nitrocellulose membranes (HybondTM-C Extra,
RPN303 E, Amersham Biosciences). Blots were blocked with 5%
skim milk, probed with the appropriate primary antibodies (a-
PDGFRb: #06-498, Upstate; a-p-Erk1/2: #9101, Cell Signaling;
a-LRP1: 377, Herz Lab; a-TGFbRII: #06-227, Upstate; a-p-
Smad2, #3108, Cell signaling) and then incubated with
horseradish peroxidase-conjugated anti-rabbit secondary antibody
(NA934V, Amersham Biosciences). Immunoreactive bands were
visualized using an enhanced chemiluminescence Western blotting
detection kit (RPN 2132, Amersham Biosciences).
Aorta extracts were prepared as described above but using
immunoprecipitation (IP) lysis buffer (50 mM Tris pH 7.4,
150 mM NaCl, 0.5% NP-40) instead of RIPA buffer.
Protein extract was pre-cleared with irrelevant non-immune
serum. Nonspecific binding was precipitated with pre-swollen
protein A-Agarose beads (P3476, Sigma). Supernatant was
harvested by short spin and the appropriate specific primary
antibodies were applied (a-PDGFRb: #06-498, Upstate; a-p-
Tyrosine: #05-321, Upstate). The suspension was probed at 4uC
for 2 hours and then precipitated again with protein A-Agarose
beads. The antigen-antibody-protein A Agarose complex was
collected by a brief centrifugation, washed three times with a
solution containing 50 mM Tris pH 7.4, 150 mM NaCl, 0.1%
NP-40 and then three times with the same buffer without NP-40.
The washed antigen-antibody-protein A Agarose complex was
resolved on SDS-PAGE gel and immunoblotted with the relevant
antibodies (a-p-Tyrosine: #05-321, Upstate; a-PI3K-p85: #06-
496/#05-217, Upstate; a-PDGFRb: #06-498, Upstate; a-LRP1:
377, Herz Lab; a-actin: A4700, Sigma).
Primary SMC culture
Mouse primary SMC culture was established using the explant
technique as previously described [37,38]. All aortas were
obtained from 8-week old male mice. Briefly, the aorta was
dissected under sterile conditions, rinsed with PBS containing
antibiotics, and the connective tissue and adventitia were removed
LRP1 Controls Vascular PI3K
PLoS ONE | www.plosone.org9 September 2009 | Volume 4 | Issue 9 | e6922
carefully. The aorta was opened longitudinally and the intima was
scraped on luminal surface. Then the aorta was minced into small
pieces and placed into a T25 flask with high glucose (4.5 g/L)
DMEM containing 15% FCS, 100 U/ml penicillin, 100 mg/ml
streptomycin, 20 mM L-glutamine. Both explants and cells were
cultured at 37uC in 5% carbon dioxide (CO2). Cells were detached
by incubation with 0.25% trypsin-EDTA solution. Passages 5–15
were used in this study.
Subcultured SMCs from the mouse aortic explants were
allowed to grow on glass coverslips for 24 hours after trypsiniza-
tion. The cells were fixed in situ with 95% ethanol, blocked with
5% non-immune goat serum, and probed with anti-a-smooth
muscle actin (A2547, Sigma) and LRP1 (377, Herz Lab)
antibodies. After three washes in PBS, the cells were incubated
with Alexa Fluor 594 goat anti-mouse (A11032, Molecular Probes)
and Alexa Fluor 488 goat anti-rabbit IgG antibodies (A11034,
Molecular Probes). After three more washes in PBS, coverslips
were mounted on glass slides using a DAPI-containing mounting
Hard SetTM, H-1500, Vector) and
analyzed using a fluorescence microscope (Axioplan 2 Imaging,
Carl Zeiss MicroImaging Inc.).
Boyden chamber transmigration assay
SMC migration was measured using a 12-well modified Boyden
chamber (AA12, Neuro Probe) hosting a polycarbonate filter with
8-mm pores (PFB8, Neuro Probe) as described . 36104cells in
100 ml were loaded into the top chamber of each well while the
lower chambers were filled with SMC medium. After incubating at
37uC in 5% CO2for 6 hours, non-migrated cells were scraped
from the upper surface of the filter. Cells on the lower surface were
fixed with 95% ethanol and stained with Harris Modified
Hematoxylin (HHS-16, Sigma). The number of SMCs on the
lower surface of the filter was determined by counting five
continuous high-power (2006) fields of constant area per well.
Experiments were performed three times in duplicate wells.
Two-dimensional migration (scratch) assay
Subcultured 36105SMCs were seeded into 60 mm Petri dishes.
10 mg/ml mitomycin C was applied to inhibit cell proliferation.
Cells were incubated at 37uC in 5% CO2overnight. The next day,
part of the dishwasdenudedby scratchingalong a straight line. The
dishes were put back to the incubator for 24 hours. The cells which
appeared on the denuded area were treated as migrated cells. To
quantify the migrated cells, the cells in five continuous high-power
(2006) fields were counted and statistical analysis was performed.
PDGF-BB chemotaxis assay
This experiment was performed as described above in the
Boyden chamber transmigration assay with the exception that only
16104cells were seeded. In addition, 10 ng/ml PDGF-BB was
administrated to the lower chambers as an attractant.
Aortas were isolated and fixed in 4% PFA for 1 hour. The tissues
were sliced into 8 mm cross sections after embedded in OCT. The
mounted sections were treated with 0.1% Triton X-100 for 5
minutes, blocked with 5% non-immune goat serum, and probed
with anti-p-Smad2 (#3108, Cell signaling) rabbit antibody. After
three washes in PBS, the sections were incubated with Alexa Fluor
488 goat anti-rabbit IgG antibodies (A11034, Molecular Probes).
After three more washes in PBS, coverslips were mounted on glass
slides with a DAPI-containing mounting medium (ProLong Gold
antifade reagent with DAPI, P36935, Invitrogen).
Statistical analyses were performed using two-tail Students’ t-test.
Results aregiven as mean6SD. A p,0.05 was considered significant.
LDL-cholesterol was observed in LDLR2/2 and smLRP12/2;
LDLR2/2 mice. LRP1-deficiency in smooth muscle cells had no
effect on the plasma lipoprotein profile.
Found at: doi:10.1371/journal.pone.0006922.s001 (1.21 MB TIF)
Plasma Lipoprotein Profile. A significant increase of
old mice were dissected out. 8 mm-thick cryostat sections were
stained with DAPI. Aortic wall thickening was observed in
Found at: doi:10.1371/journal.pone.0006922.s002 (4.56 MB TIF)
Light microscopy of cryostat sections. Aortas of 7-week
We thank John Shelton for excellent assistance in photography. We also
thank Wenling Niu, Isaac Rocha and Elida Priscilla Rodriguez for their
helpful technical support.
Conceived and designed the experiments: LZ YT JH. Performed the
experiments: LZ YT. Analyzed the data: LZ PB MDT JH. Contributed
reagents/materials/analysis tools: LZ YT PB MDT JH. Wrote the paper:
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