Integrin ␤1 Subunit Controls Mural Cell Adhesion,
Spreading, and Blood Vessel Wall Stability
Sabu Abraham, Naoko Kogata, Reinhard Fässler, Ralf H. Adams
Abstract—Growth, maturation, and integrity of the blood vessel network require extensive communication between the
endothelial cells, which line the vascular lumen, and associated mural cells, namely vascular smooth muscle cells and
pericytes. Pericytes extend long processes, make direct contact with the capillary endothelium, and promote vascular
quiescence by suppressing angiogenic sprouting. Vascular smooth muscle cells are highly contractile, extracellular
matrix–secreting cells that cover arteries and veins and provide them with mechanical stability and elasticity. In the
damaged blood vessel wall, for example in atherosclerotic lesions, vascular smooth muscle cells lose their differentiated
state and acquire a highly mitotic, so-called “synthetic” phenotype, which is thought to promote pathogenesis. Among
other factors, extracellular matrix molecules and integrin family cell–matrix receptors may regulate this phenotypic
transition. Here we show that the inactivation of the gene encoding the integrin ␤1 subunit (Itgb1) with a Cre-loxP
approach in mice leads to mural cell defects and postnatal lethality. Integrin ␤1– deficient vascular smooth muscle cells
display several hallmarks of the synthetic phenotype: Cell proliferation is enhanced, whereas differentiation and their
ability to support blood vessels are compromised. Similarly, mutant pericytes are poorly spread but present in larger
numbers. Our analysis of this mutant model shows that integrin ␤1–mediated cell–matrix adhesion is a major
determinant of the mural cell phenotype. (Circ Res. 2008;102:562-570.)
Key Words: integrin 䡲 adhesion 䡲 blood vessel 䡲 vascular smooth muscle cell 䡲 pericyte
Targeted inactivation of the Lama4 gene (laminin ␣4) is
compatible with embryonic angiogenesis and cardiac development, but mutant capillaries have basement membrane
defects and are dilated and fragile.10 Binding to these matrix
substrates is mediated by integrin receptors, which, in turn,
control cellular responses such as adhesion, spreading, motility, proliferation, and survival. Integrins function as heterodimers consisting of ␣ and ␤ subunits. Gene targeting
experiments have uncovered that complexes containing the
integrin molecules ␣4, ␣5, ␣7, ␣V, and ␤8 are essential for
vascular morphogenesis.11,12 Because these integrins play
important roles in many different tissues and cell types, the
mutant phenotypes may reflect primary defects in ECs,
PCs/VSMCs, or other cell populations. Alterations in the
local matrix and/or integrin expression are also thought to
promote a phenotypic switch of VSMCs in response to vessel
wall injury or in atherosclerosis. Affected SMCs acquire a
mitotic, poorly differentiated (“synthetic”) phenotype and
share features with immature embryonic VSMCs.4,13–15
To gain better insight into integrin function in the vessel
wall, we inactivated the Itgb1 gene encoding integrin ␤1 in
mural cells. Integrin ␤1 is a particularly promiscuous subunit
that can partner with 11 distinct ␣ chains and thereby mediate
T
he function and integrity of the vascular system requires
reciprocal interactions between the endothelial layer of
the vessel wall and the more peripherally located vascular
smooth muscle cells (VSMCs) and pericytes (PCs). PCs
associate tightly with the endothelial cells (ECs) of capillaries, small venules, and immature blood vessels so that both
cell types are in direct contact, coupled by junctions, and
enclosed by a single basement membrane layer.1–3 By contrast, VSMCs are found on more mature and larger caliber
blood vessels, lack direct EC contact, and surround the outer
basement membrane surface in one or several sheets.1,3,4
Fully differentiated VSMCs are highly contractile and help to
provide the vasculature with mechanical stability and elasticity. Smooth muscle cells (SMCs) are also a major source of
the matrix in the vessel wall.4,5
The close relationship between matrix proteins and blood
vessel morphogenesis is highlighted by the phenotypes of
knockout mice lacking extracellular matrix (ECM) components. Loss of fibronectin or collagen IV results in lethality
around midgestation because of defects in the embryonic
heart and vasculature.6 – 8 The alternatively spliced EIIIA and
EIIIB regions of fibronectin are essential for normal vascular
remodeling, VSMC association, and embryonic survival.9
Original received July 9, 2007; resubmission received November 14, 2007; revised resubmission received December 12, 2007; accepted January 3,
2008.
From the Cancer Research UK London Research Institute (S.A., N.K., R.H.A.), Vascular Development Laboratory, London, UK; Max-Planck-Institute
of Biochemistry (R.F.), Department of Molecular Medicine, Martinsried, Germany; Max-Planck-Institute for Molecular Biomedicine (R.H.A.),
Department of Tissue Morphogenesis, and University of Münster, Faculty of Medicine, Münster, Germany.
Correspondence to Ralf H. Adams, Vascular Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields,
London WC2A 3PX, United Kingdom. E-mail ralf.adams@cancer.org.uk
© 2008 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.107.167908
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binding to a diversity of matrix substrates including collagen
I and IV, laminins, vitronectin, and fibronectin. This versatility may explain why the global inactivation of the Itgb1
gene in mice is incompatible with survival beyond embryonic
day (E)5.5.11 To circumvent this early developmental block,
conditional Itgb1 mutants permitting tissue-specific gene
targeting with the Cre-loxP approach have been generated
previously.16 We combined these mice with Pdgfrb-Cre
transgenics,17 a strain of mice expressing Cre recombinase
under the control of a Pdgfrb (the gene for platelet-derived
growth factor receptor ␤) genomic DNA fragment. Because
of Pdgfrb-Cre expression in PCs and VSMCs of the skin and
other tissues,17 this gene-targeting strategy allowed us to
investigate the function of the integrin ␤1 subunit in mural
cells without disrupting its expression in the endothelium.
Materials and Methods
Animal Models
Mice carrying a loxP-flanked Itgb1 gene (Itgb1lox/lox) and Pdgfrb-Cre
have been reported previously.16,17 For the generation of mutants,
Pdgfrb-Cre Itgb1lox/⫹ double heterozygotes were bred to Itgb1lox/lox
mice in a mixed 129⫻C57BL/6 genetic background. Cre-negative
littermates were used as controls. H-2KbtsA58 immorto18 or Rosa26 –
enhanced yellow fluorescent protein (EYFP) Cre reporter transgenes19 were introduced for the isolation of immortalized VSMCs or
flow cytometric experiments, respectively. All animal experiments
complied with the relevant laws and were approved by the Cancer
Research UK Animal Ethics Committee.
Analysis of Tissues
For immunofluorescence on sections, tissue samples were fixed
overnight in 4% paraformaldehyde at 4°C and embedded in paraffin
for sectioning. Microtome sections were blocked with 0.1% BSA and
1.5% goat serum in PBS (30 minutes) before overnight incubation
with primary antibody diluted in blocking solution. Primary antibodies were rat monoclonal anti–integrin ␤1 (Chemicon, 1:100), rabbit
polyclonal anti-fibronectin (Sigma, 1:200), anti– collagen IV
(Chemicon, 1:200), anti–phospho-histone H3 (Upstate, 1:100), and
goat polyclonal anti–smoothelin B (Santa Cruz Biotechnology,
1:100). After washing, samples were incubated in secondary antibody (anti-rabbit Alexa Fluor-488 or Fluor-546, anti-rat Alexa
Fluor-488, Molecular Probes, 1:500) and counterstained with 4⬘,6diamidino-2-phenylindole (DAPI) (Sigma, 1:1000). Fluorescence
was visualized using a Leica DM IRBE light microscope.
For whole-mount staining, skin samples were fixed overnight in
4% paraformaldehyde, washed with PBS, blocked in 1% goat serum
in PBS containing 0.1% Tween 20 (3 hours at room temperature),
followed by overnight incubation with primary antibodies (diluted in
blocking solution) at 4°C. Samples were washed 3 times (1 hour
each) in PBS, incubated for 3 hours at room temperature with
secondary antibodies diluted in blocking solution, and washed as
before. Primary antibodies were rat anti-mouse platelet endothelial
cell adhesion molecule-1 MEC13.3 (Pharmingen, 1:100), mouse
anti-human ␣-smooth muscle actin (SMA) (Clone1A4, Sigma,
1:400), polyclonal rabbit anti-desmin (Abcam, 1:200), rat monoclonal anti-endomucin (gift from Dietmar Vestweber, Max-PlanckInstitute for Molecular Biomedicine, Münster, Germany), and rabbit
anti-fibronectin (Sigma, 1:400). A Zeiss LSM510 Meta was used for
confocal microscopy. Sample analysis by electron microscopy has
been described previously.17
Flow Cytometry
Mesenteric tissue from E17.5 or postnatal day (P) 2 Pdgfrb-Cre
Itgb1lox/lox Rosa26-EYFP or control mice was incubated for 2 hours at
37°C in 2 mL of PBS containing 400 U/mL collagenase (GIBCO).
After the addition of 10 mL of medium (10% FCS in DMEM), cells
Integrin ␤1 Function in Mural Cells
563
were dispersed with a Pasteur pipette, sieved (pore size, 100␮m),
collected by centrifugation (1000 rpm, 5 minutes) and washed with
5 mL of medium. Cells were resuspended in 500 ␮L of DMEM
containing 2% FCS, incubated with integrin ␤1 antibody for 45
minutes and anti-rat secondary antibody conjugated to Alexa Fluor647 (Molecular Probes). Propidium iodide (Invitrogen) staining
selected live cells, which were analyzed with a FACSCalibur system
(BD Biosciences).
Smooth Muscle Cell Isolation, Culture,
and Analysis
The isolation of SMCs from adult aortas, as well as the culture,
verification, transfection, and staining of these cells; the analysis of
fibronectin fibrillogenesis; and video microscopic and automatic cell
shape analysis are described in the online data supplement, available
at http://circres.ahajournals.org.
Results
Targeting of Itgb1 in Mural Cells
Itgb1Pdgfrb-Cre mutants, generated by breeding Pdgfrb-Cre
transgenic mice17 into a background of animals carrying a
loxP-flanked version of the Itgb1 gene (Itgb1lox/lox),16 were
obtained at E17.5 almost at the expected Mendelian ratio
(18.8% instead of 25%) predicted for our breeding scheme
(see Materials and Methods; Figure 1B). Even though these
embryos were of normal size and appeared healthy, their skin
contained visibly dilated blood vessels (Figure 1A). After
birth, a gradually increasing fraction of mutants died and only
3 survivors were obtained beyond P10 (Figure 1B). Immunofluorescence with anti–integrin ␤1 antibodies on skin
sections from P2 animals validated our genetic approach.
Whereas the anti–integrin ␤1 signal labels both endothelial
cells and VSMCs in control littermates, specific staining is
only visible in the Itgb1Pdgfrb-Cre endothelium but absent from
␣-SMA–positive cells (Figure 1C). Residual mural integrin
␤1 expression in mutant survivors at later postnatal stages
suggests incomplete gene inactivation, and we therefore
excluded these animals from our study. Flow cytometric
analysis showed that integrin ␤1 is gradually depleted during
development and that the majority of Pdgfrb-Cre–positive
cells in postnatal mutants (74.2% at P1) has lost the protein
(Figure 1D).
Vascular Smooth Muscle Cell Defects in
Itgb1 Mutants
Analysis of the Itgb1Pdgfrb-Cre dermal vasculature by whole-mount
immunofluorescence revealed the presence of vascular aneurysms, local distensions of blood vessels, affecting both arteries
and veins already in embryos (Figure 2A and 2B). These
aneurysms correspond with regions with poor VSMC coverage,
suggesting that this phenotype is linked to mural cell defects. At
P2, mutant VSMCs show a highly rounded, button-like morphology. Because of poor spreading, individual cells are often
separated by gaps so that Itgb1Pdgfrb-Cre arteries and veins lack
continuous SMC coverage (Figure 2C and 2D). Staining with
anti–␣-SMA antibodies shows that the cytoskeleton of control
VSMCs is aligned into a sheet of parallel fibers that surrounds
the endothelium tightly. By contrast, Itgb1 mutant mural cells
are highly disorganized and fail to align with neighboring cells.
Moreover, ␣-SMA is no longer polarized to one surface of the
VSMCs in the absence of integrin ␤1 (Figure 2E).
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Figure 1. Gene targeting of Itgb1 in
mural cells. A, Phenotype of freshly isolated E17.5 embryos. The bottom images
show higher magnifications of insets.
Note dilated blood vessels in the
Itgb1Pdgfrb-Cre skin (right). B, Number of
Itgb1Pdgfrb-Cre mice (mutants) among the
total of embryos/pups analyzed at indicated stages. Percentage (%) of mutant
vs total mice relative to the expected
ratio (25% for this breeding scheme)
indicates lethality of Itgb1 mice. C, Double immunofluorescence with antibodies
against integrin ␤1 (green) and ␣-SMA
(red) on skin sections from P2 animals,
confirming the absence of integrin ␤1 in
VSMCs but not the endothelium of
Itgb1Pdgfrb-Cre mutants (right). Bottom
images show individual channels of
insets at higher magnification. Nuclei
were stained with DAPI. Arrows indicate
mutant or control VSMCs. D, Flow cytometric analyses of integrin ␤1 expression
cells isolated from Pdgfrb-Cre Itgb1lox/lox
ROSA26-YFP Cre reporter mice at E17.5
or P1. Cells from ROSA26-YFP (without
anti–integrin ␤1 antibody) and PdgfrbCre ROSA26-YFP double transgenic
embryos at E17.5 were used as controls,
as indicated. Numbers in quadrants (purple boxes) indicate fractions of integrin
␤1– expressing (top) and YFP-positive
(Cre-expressing) integrin ␤1–negative
(bottom right quadrant) cell populations
relative to total cells. Scale bars: 1 mm
(A) and 20 ␮m (C).
On the ultrastructural level, mutant mural cells fail to
associate with the subendothelial basement membrane, appear rounded and poorly spread, and are frequently located in
some distance from the endothelium. As a consequence, Itgb1
vessel walls are not covered by a layer of tightly packed
VSMCs and instead appear loosely organized (Figure 2F).
Integrin ␤1 Controls the Spreading of PCs
Because PCs play critical roles in the stabilization of capillary
beds2,3 and are targeted by the Pdgfrb-Cre transgene,17 we
analyzed the morphology PCs in Itgb1 mutants. Wholemount staining with antibodies directed against desmin, an
intermediate filament protein and PC marker, allows the
visualization of the PCs that cover the dermal vasculature
with an extensive lattice of fine processes (Figure 3A).
Desmin-positive PCs are also present in the Itgb1Pdgfrb-Cre skin,
and their number is even significantly increased compared
with control littermates (Figure 3A and 3E). However, mutant
cells lack the characteristic slender and stretched morphology
of normal PCs, and their processes appear short (Figure 3B).
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565
Figure 2. VSMC defects in Itgb1 mutants.
A through E, Confocal images of E17.5 (A
and B) or P2 skins (C through E) after
whole-mount immunostaining with antibodies detecting ECs (platelet endothelial cell adhesion molecule-1, green)
and/or VSMCs (␣-SMA, red). B and D,
Higher-magnification images of insets in
A and C, respectively. Arteries (a) and
veins (v) are labeled. Arrows indicate aneurysm in A and B and rounded VSMCs
in D and E. F, Electron micrographs of
dermal blood vessels. Black arrows
point at loosely associated Itgb1Pdgfrb-Cre
VSMCs. Endothelial (ec) and blood cells
(bc) are labeled. Scale bars: 50 ␮m (A
and C), 20 ␮m (E), and 2 ␮m (F).
Several lines of evidence suggest that loss of integrin ␤1
affects the interaction of PCs with ECs. In electron micrographs, mutant PCs have a round morphology, fail to wrap
around dermal capillaries, and areas of endothelial-PC contact are small (Figure 3D). Whereas control PCs show no
appreciable ␣-SMA immunofluorescence, mutant PCs are
SMA-positive, similar to what has been reported previously
for the poorly attached PCs covering the tumor vasculature.20
Furthermore, distended capillary diameters in the Itgb1Pdgfrb-Cre
skin suggest that mutant PCs, despite their presence in greater
Figure 3. Defective morphology and
association of Itgb1 PCs. A through C,
Whole-mount staining of control and
Itgb1Pdgfrb-Cre dermal vasculature with
anti-desmin (green), anti–␣-SMA (red or
blue), and/or anti-endomucin (red) antibodies as indicated. Arteries (a) and
veins (v) are indicated in A. Desminpositive PCs lack long cellular processes
(arrows in B) and are more abundant
(asterisks label cell bodies in B). C,
Channel with anti–␣-SMA signal of
images shown in B. D, Electron micrographs of dermal capillaries. Itgb1Pdgfrb-Cre
mutants lack PC processes seen around
the endothelium in controls (black
arrows). PCs (pc), endothelial (ec), and
blood cells (bc) are indicated. E, Quantitative analysis showing larger capillary
diameters (5.0⫾0.4 vs 6.7⫾0.7 ␮m;
*P⫽0.011 by 2-tailed t test assuming
unequal variances) and increased PC
numbers (48.5⫾6.2 vs 61.8⫾2.6 per
0.04 mm2 area; *P⫽0.017 by 2-tailed t
test assuming unequal variances) in P2
Itgb1Pdgfrb-Cre head skin. Values are ⫾SD
and based on 4 mutant and control
samples, respectively. Scale bars: 50 ␮m
(A), 20 ␮m (B), and 2 ␮m (D).
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numbers, are not supporting the endothelium sufficiently
(Figure 3E). In contrast to other mutant models in which the
loss or defective association of mural cells leads to edema
formation and hemorrhaging,17,21 we did not find extravasated red blood cells in Itgb1 mutants. The angiogenic growth
of the vascular network, as judged by the size of the
vascularized area (Figure IA in online data supplement), the
number of blood vessel branch points, and endothelial proliferation (data not shown), was also not significantly altered
when mural cells had lost integrin ␤1 expression.
Integrin ␤1 Controls the Shape, Adhesion, and
Motility of Cultured Mural Cells
To investigate the role of integrin ␤1 in cultured mural cells,
we isolated aortic smooth cell cells from adult Itgb1lox/lox
homozygous mice, which also carried the H-2Kb-tsA58
immorto transgene, allowing the inducible expression of a
temperature-sensitive SV40 T antigen.18 Transient expression
of Cre recombinase in these cells yielded integrin ␤1–
deficient (Itgb1KO) SMCs. Deletion of the loxP-flanked region
in the Itgb1 gene of these cells was verified by genotyping
PCR (data not shown), and immunofluorescence confirmed
the absence of integrin ␤1 protein (Figure 4A and 4B).
Mock-transfected cells were used as controls (Figure 4A and
4B). Visualization of the focal adhesions by anti-paxillin
antibody staining and of the actin cytoskeleton shows that
loss of Itgb1 in cultured VSMCs phenocopies morphological
changes observed in vivo. Itgb1KO SMCs are round, poorly
spread, and lack cellular protrusions seen in control cells
(Figure 4A and 4C through 4E). Absence of integrin ␤1
expression does not prevent the formation of focal adhesions,
even though they are short and disorganized in comparison
with control cells (Figure 4A and 4B). As expected, reexpression of green fluorescent protein–tagged integrin ␤1 in
Itgb1KO SMCs restores their morphology, so that they resemble control cells (supplemental Figure II).
Consistent with the binding affinities of ␤1-containing
integrin heterodimers, attachment and spreading on collagen
I and fibronectin matrix substrates is delayed in Itgb1KO
SMCs (Figure 5A and 5B). Similarly, cell motility and the
persistence of migration are significantly reduced in the
absence of integrin ␤1 (Figure 5C and 5D and supplemental
Figure I).
ECM Protein Expression in Itgb1 Blood Vessels
Previous work has indicated that integrins are not only
necessary for cell–matrix adhesion but, at least in some
tissues, for the proper expression and deposition of ECM
proteins.22 Evaluation of laminin, fibronectin, and collagen
IV by immunofluorescence shows that all these matrix
proteins are expressed in Itgb1Pdgfrb-Cre dermal blood vessels
(Figure 6A through 6C and supplemental Figure III). However, we noted that Itgb1 ␣-SMA–positive cells protrude
through the matrix layer into the surrounding dermis and are
no longer fully ensheathed by collagen IV, as is the case for
control blood VSMCs (Figure 6A and 6B). Whereas fibronectin is still located within the basement membrane that
separates the endothelium from the VSMC layer, its assembly
into long parallel fibrils is defective in Itgb1Pdgfrb-Cre mutants
Figure 4. Characterization of cultured integrin ␤1– deficient
VSMCs. A, Anti–integrin ␤1 (red) and anti-paxillin immunofluorescence of control and integrin ␤1– deficient (Itgb1KO) aortic
SMCs, as indicated. B, Details of individual channels of insets in
A. Arrows indicate integrin ␤1–positive focal adhesions (upper
left). Note focal adhesion morphology is abnormal in Itgb1KO
cells (bottom right). C, Organization of the actin cytoskeleton
(phalloidin, green) in control and mutant cells. Focal adhesions
are labeled by anti-paxillin staining (red). Arrows indicate cortical
actin in Itgb1KO VSMCs. D, Example images of phalloidinstained control (left) and Itgb1KO (right) cells from automatic cell
shape analysis (see Materials and Methods). Automatically identified and analyzed cells are in green and rejected cells are in
orange. E, Histogram of shape factor data. Height of bars indicates cell numbers. Larger shape factor of integrin ␤1– deficient
cells indicates decreased complexity of cell outlines, that is,
more rounded cell shapes. Scale bars: 10 ␮m (A, C, and E).
Abraham et al
Integrin ␤1 Function in Mural Cells
567
Figure 5. Regulation of cell adhesion
and motility by integrin ␤1. A, Diagram
showing the spreading of control and
Itgb1KO cells on collagen I or fibronectin substrates. Fraction of spread cells
at indicated times were determined by
video microscopy (see Materials and
Methods). B, Still images of video
microscopy showing areas containing
spread (black arrowheads) and
unspread (white arrowheads) control
and mutant cells 90 minutes after plating. C, Diagrams showing tracks from
control (n⫽16) and Itgb1KO (n⫽13) cells
with starting points shifted to the origin. Distances are indicated (in ␮m).
Similar tracks were recorded in 2 further repeat experiments but are not
shown in the figure. D, Graphic representation of migration speeds (␮m/h) of
control (left, n⫽16) and Itgb1KO cells
(right, n⫽13). Circles correspond to
median speeds, boxes to 50%, and
whiskers to 80% of values. Migration
of mutant cells (14.0⫾1.4 ␮m/h) is significantly reduced compared with control cells (24.6⫾2.2 ␮m/h). ***P⬍0.001
by ANOVA.
(Figure 6C and 6D). Confocal analysis reveals that mutant
blood vessels lack long fibronectin fibrils and the ECM
protein is instead predominantly accumulated in a circular
fashion underneath VSMCs (Figure 6D). Similarly, cultured
Itgb1KO SMCs are only capable of limited fibronectin fibrillogenesis (Figure 6E).
Proliferation and Differentiation of Itgb1 VSMCs
To gain a better understanding of the defects in mutant vessel
walls, we evaluated whether the loss of integrin ␤1 has any
effect on the survival, proliferation, or differentiation of
mural cells. TUNEL staining shows no overlap between
apoptotic (TUNEL-positive) nuclei and anti–␣-SMA antibody signal in both control and mutant skin sections (data not
shown), arguing against an essential role of integrin ␤1 in the
protection of mural cells against cell death. By contrast,
VSMC proliferation, assayed with antibodies against the mitotic
marker phospho-histone H3, is increased in Itgb1Pdgfrb-Cre
mutants (Figure 7A through 7C). Thus, changes in the
morphology of Itgb1 VSMCs are accompanied by upregulated proliferation reminiscent of the SMC behavior seen in
response to vascular injury.
The loss of late differentiation markers is another feature of
the synthetic VSMC phenotype.4,15 We therefore analyzed the
expression of smoothelin B, an actin-binding protein that is a
late VSMC differentiation marker linked to SMC contractility.23 Whereas smoothelin B is abundantly present in
␣-SMA–positive cells of control vessels, such staining is
missing in the Itgb1 dermal vasculature (Figure 7D and 7E).
The expression of various transcriptional factors with established roles in the regulation of the smooth muscle differentiation program is also reduced and/or delayed in cultured
integrin ␤1– deficient SMCs (supplemental Figure IV). All
these findings together are consistent with a failure of
VSMCs to acquire a fully differentiated and functional
phenotype in the absence of integrin ␤1.
Discussion
Regulation of Mural Cell Function by Integrin ␤1
Our findings demonstrate that integrin ␤1 is a critical regulator of mural cell morphology and function in vivo and in
vitro. In the skin of Itgb1Pdgfrb-Cre mutants, VSMCs are poorly
spread, lack the normal alignment with neighboring cells, and
support the vasculature insufficiently. This leads to poorly
organized blood vessel walls and the formation of aneurysms.
Similarly, mutant PCs, albeit present in increased numbers,
fail to extend the long processes that are characteristic for this
cell type and lack proper interactions with the endothelium.
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March 14, 2008
Figure 6. Matrix protein expression in Itgb1Pdgfrb-Cre blood vessels. A through E, Immunofluorescence with antibodies directed
against fibronectin or collagen IV (col IV) and ␣-SMA (red), as
indicated. Sectioned P2 skins (A through C), confocal images of
whole-mount samples (D) or immunostaining of cultured Itgb1KO
and control SMCs (E) are shown. B, Higher-magnification
images of insets in A. Arrows point to rounded VSMCs protruding through the collagen IV layer. D, Long fibronectin fibers
(arrows) in the proximity of ␣-SMA–positive VSMCs are present
in control but not in Itgb1Pdgfrb-Cre dermal blood vessels. E, Itgb1KO
cells display limited fibrillogenesis of plated fibronectin compared
with control SMCs, which are capable of assembling long fibers
(arrows). Nuclei in A through C and E were stained with DAPI
(blue). Scale bars: 20 ␮m (A, C, and D) and 10 ␮m (E).
Ectopic expression of ␣-SMA by integrin ␤1– deficient PCs
is highly reminiscent of PC defects in the deregulated
vasculature of tumors20 and most likely reflects changed gene
expression in response to the loose association of these cells
Figure 7. Loss of integrin ␤1 affects VSMC proliferation and differentiation. A, B, D, and E, Immunofluorescence on P2 skin
sections with antibodies against phosphorylated histone H3
(p-histone H3, green), smoothelin B (green), and ␣-SMA (red) as
indicated. Nuclei were stained with DAPI (gray). Staining of red
blood cells (*) is unspecific. B, The isolated green channel of
mutant image in A. Arrows point at proliferating VSMCs in (A
and B) and at control or Itgb1 VSMCs in E. C, Quantitation of
p-histone H3–stained mutant and control blood vessel section.
Bars represent proportion of control or Itgb1 vessels with a certain VSMC proliferation index (mitotic VSMCs/total VSMCs).
Ninety-five percent of control sections contain no phosphorylated histone H3–stained VSMCs. E, Green channel (smoothelin B)
of images shown in D. Scale bars: 20 ␮m (A through E).
with the capillary network. The dilation of capillaries in the
Itgb1Pdgfrb-Cre skin is also consistent with insufficient support
by mural cells. Even though we were unable to establish that
these vascular defects were directly responsible for the
lethality of the mutant mice, some form of cardiovascular
insufficiency or the rupture of dilated and fragile blood
vessels are the most likely cause of death. Embryonic and
perinatal growth and development is accompanied by a
Abraham et al
significant increase in blood pressure,24,25 and the severity of
the mural cell defects suggests that the Itgb1Pdgfrb-Cre mutant
vasculature may be too weak to cope with these stronger
hemodynamic forces.
Because many of the known molecular players controlling
mural cell biology are linked to integrins, it is worthwhile to
compare the different mutant phenotypes. For example,
signaling by platelet-derived growth factor (PDGF) B and its
receptor PDGFR␤ is essential for the proliferation, chemotactic guidance, and association of PCs/VSMCs. Studies in
cultured cells have established that this pathway synergistically cooperates with integrins.26,27 However, this crosstalk
involves integrin ␣v␤3 rather than complexes with ␤1, and
we found that Itgb1Pdgfrb-Cre mutants do not recapitulate the
dramatic reduction of PCs/VSMCs caused by the inactivation
of the Pdgfb or Pdgfrb genes.3
We have shown previously that that mural cells require
ephrin-B2, a small transmembrane protein and ligand for Eph
family receptor tyrosine kinases, for their correct association
with capillaries and small caliber arteries and veins.17 Even
though cultured ephrin-B2– deficient aortic SMCs display
prominent focal adhesion defects, the accelerated but random
motility of these cells appears very distinct from the comparably static Itgb1 KO cells. Thus, mural cell-specific integrin
␤1 mutants fall into a separate phenotypic category characterized by compromised PC and VSMCs spreading, migration, and differentiation.
Redundancy Versus Functional Specificity of
Integrin Receptors
Several mutant mice lacking individual integrin ␣ subunits
develop vascular defects, suggesting that these molecules
may partner with ␤1 in PCs and VSMCs. Integrin ␣5␤1 is
strongly expressed in SMCs, and the knockout of the Itga5
gene encoding the ␣5 subunit reproduces the very severe
vascular defects seen in embryos lacking fibronectin, the
major ligand of ␣5␤1.11,28 The midgestation lethality of Itga5
embryos is likely to reflect a combination of problems
affecting the heart, the endothelium, and, possibly, mural
cells. Tissue-specific loss-of-function studies will be required
to identify the roles of integrin ␣5 in individual cell types.
Similar to ␣5, the majority of Itga4 (integrin ␣4) mutants die
at midgestation, which has been attributed to failed chorioallantoic fusion.29 A fraction of mutants surviving up to
E14.5 displays defective distribution of PCs/VSMCs in the
cranial vasculature.30 Adhesion and motility of cultured Itga4
cells was also reduced, similar to Itgb1KO SMCs.30,31 It also
has been shown that the laminin receptor integrin ␣7␤1 is
expressed in SMCs, and cerebrovascular hemorrhaging in the
knockout mice has been attributed to VSMC defects.32,33
Given that integrin ␤1 forms functional complexes with all
the subunits mentioned above, mediates adhesion to fibronectin, laminin, and collagen substrates, and also controls fibronectin fibrillogenesis in the vessel wall, it is surprising that VSMC
survival or proliferation are not disrupted in Itgb1Pdgfrb-Cre
mutants. Similarly, mutant PCs are compromised in their
ability to spread and support ECs but are actually present in
increased numbers. These data suggest that other integrins,
such as ␣v␤3 or ␣v␤5, provide sufficient adhesion for
Integrin ␤1 Function in Mural Cells
569
necessary promitotic and antiapoptotic signals. Indeed, the
integrins ␣v␤3 or ␣v␤5 are expressed by SMCs and can bind
a set of ECM molecules that partially overlaps with ␤1containing receptor complexes.34
Integrin ␤1 and the Synthetic VSMC Phenotype
Changes in the matrix environment or the expression, cell
surface presentation, binding, or signaling properties of integrins may cause or contribute to pathological changes in the
vasculature. For example, it has been shown that laminin and
type I or type IV collagens help to maintain a differentiated,
contractile phenotype of cultured arterial SMCs, whereas the
cell attachment (RGD) motif of fibronectin has the opposite
effect.35–38 Experimentally induced vascular injury reduces
the local expression and activity of ␤1 integrins in vascular
cells, whereas ␣v␤3 or ␣v␤5 and the corresponding matrix
substrates are upregulated.5,39 – 41 Moreover, ␤3 integrins favor a poorly differentiated and highly motile SMC phenotype,
and knockout mice are protected against pathological VSMC
migration and neointima formation.5,40 – 44
The sum of these findings suggests that different integrin
complexes may have opposite roles in the regulation of the
SMC phenotype. The defects in Itgb1Pdgfrb-Cre mutant mice
directly confirms that integrin ␤1 is essential for VSMC
morphology, differentiation, and function and may provide
useful leads for future research investigating tissue repair and
vascular regeneration processes.
Acknowledgments
We thank A. Compagni and P. Lindblom for preliminary characterization of the Itgb1 mutant phenotype, D. Vestweber for reagents, D.
Zicha for help with the analysis of Itgb1 cells, and N. Hogg for
critically reading the manuscript.
Sources of Funding
This work was funded by Cancer Research UK and the European
Molecular Biology Organization Young Investigator Programme
(R.H.A.).
Disclosures
None.
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