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First published online 14 May 2008
doi: 10.1242/dev.016378
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Pacific Vascular Research Laboratory, Division of Vascular Surgery, Department of Surgeryand Department of Anatomy, University of California, San Francisco, CA 94143, USA.
Author for correspondence (e-mail:
wangr{at}surgery.ucsf.edu)
Accepted 25 April 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Angiogenesis, Integrin, Endothelial cell, Vascular development, Extracellular matrix
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This
observation has led to intensive investigation of integrin-ECM interactions as
crucial regulators of angiogenesis. Although it is established that
integrin-ECM interactions are involved angiogenesis, it is unclear which
integrin-ligand pairs are required for the angiogenic process. For example,
the integrins 
vβ3 and vβ5 are upregulated in ECs
during wound healing and in certain tumor vasculatures, and antibody or
small-molecule inhibition of these integrins blocks angiogenesis in vivo
(Eliceiri and Cheresh, 2001).
However, the unexpected finding that
Itgb3-/-/Itgb5-/- mice are viable and fertile
has challenged the notion that v integrins are required for
angiogenesis and calls for further investigation into the precise role of
integrins in angiogenesis.
At least six β1 integrins (1β1, 2β1,
3β1, 5β1, 6β1 and vβ1) are
expressed in ECs, and several β1 heterodimers are thought to be
pro-angiogenic. The collagen receptors 1β1 and 2β1 are
upregulated by vascular endothelial growth factor, and antibody-based
inhibition of these integrins blocks tumor angiogenesis
(Senger et al., 1997). In
addition, tumor angiogenesis in Itga1-/- mice is reduced
compared with controls (Pozzi et al.,
2000). Fibronectin and its primary integrin receptors,
4β1 and 5β1, are upregulated in blood vessels of
several human tumors, and inhibition of 4β1, 5β1 or
fibronectin with antibody- or peptide-based approaches blocks angiogenesis in
vivo (Garmy-Susini et al.,
2005; Kim et al.,
2000a). In addition, β1 integrins, and 5β1 in
particular, are crucial for vascular network formation in embryoid bodies
(Bloch et al., 1997;
Francis et al., 2002).
Finally, Itga5-/- or fibronectin-/-
mice are embryonic lethal owing to cardiovascular and neuronal defects,
although the precise role of these genes in vascular development remains
unclear because of their widespread embryonic expression
(Francis et al., 2002;
George et al., 1997;
George et al., 1993;
Yang et al., 1999;
Yang et al., 1993).
Direct in vivo genetic evidence for an angiogenic role for endothelial
β1 integrins is precluded because Itgb1-/- mice die
at embryonic day (e) 5.5 owing to implantation defects, prior to vascular
development (Fassler and Meyer,
1995; Stephens et al.,
1995). In this study, we deleted Itgb1 in a cell
lineage-specific manner, using both Tie2-Cre (Tie2 is also
known as Tek-Mouse Genome Informatics) and Tie1-Cre, to
bypass the early implantation defects of Itgb1 complete null embryos,
and gained significant insights into its role in EC adhesion, migration,
proliferation and survival during vascular development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), Itgb1flox/flox
[β1flox/flox
(Graus-Porta et al., 2001)],
Tie1-Cre (Gustafsson et al.,
2001) and Tie1-GFP
(Iljin et al., 2002) have been
described. Genotyping was performed by PCR for 40 cycles: denaturation at
95°C, 1 minute; annealing at 56°C, 1 minute; extension at 72°C, 1
minute. The Cre primers were 5'-GCCTGCATTACCGGTCGATG-CAACGAGTG and
5'-CTGGCAATTTCGGCTATACGTAACAGGGTG. For routine genotyping, the
Itgb1flox/flox primers were 5'-GGAAAG-TAGGCTGGACATGG
and 5'-TGCCACTCCAAACATAGAGC. For detection of the wild-type, floxed and
recombined Itgb1 alleles in the same reaction,
5'-TTCTGCAAGTGTGGTGTGAAG and 5'-TGCCACTC-CAAACATAGAGC were used.
The GFP primers were 5'-GCCG-ACCACTACCAGCAGAACACCCCCATC and
5'-ATTTTATGTTTCAGGTTCAGGGGGAGGTGT. Animals were treated in accordance
with the guidelines of the University of California San Francisco,
Institutional Animal Care and Use Committee.
Immunological staining of embryo whole-mounts and sections
Embryo harvest, processing and immunostaining were performed as described
(Braren et al., 2006). The
following antibodies were used at 1:100 dilution: anti-CD31 (MEC13.3, BD
Biosciences, San Jose, CA), anti-VE-cadherin (11D4.1, BD Biosciences),
anti-β1 integrin (Ha2/5, biotinylated, BD Biosciences) and anti-BrdU
(Zymed/Invitrogen, Carlsbad, CA). Polyclonal anti-laminin (1:1000) and
anti-fibronectin (1:2500) antibodies were obtained from Dr Alex Morla
(University of Chicago, IL). Secondary reagents were obtained from Invitrogen
and 4',6-diamidino-2-phenylindole (DAPI)-containing Vectashield mounting
medium was from Vector Laboratories (Burlingame, CA).
Primary EC isolation and culture
Embryonic cells were prepared as described
(Braren et al., 2006) and
cultured in F12 containing 10% FBS, penicillin/streptomycin, 150 µg/ml
endothelial mitogen (Biomedical Technologies, Stoughton, MA), 25 mM HEPES pH
7.4, 0.1 mM 2-mercaptoethanol, non-essential amino acids, sodium pyruvate and
glutamine. For immortalization, cells were infected with a retrovirus
containing polyoma middle T antigen (pMIG-PyMT-IRES-GFP, obtained from Dr
Alana Welm at UCSF, CA), which selectively immortalizes embryonic ECs
(Balconi et al., 2000). Further
purification was performed with anti-CD31 antibodies and anti-rat IgG
conjugated magnetic Dynabeads (Invitrogen), and purity was confirmed by
anti-CD31 and anti-VE-cadherin immunofluorescence and DiI-Ac-LDL (Biomedical
Technologies) uptake (data not shown). Immortalized ECs were cultured on 0.1%
gelatin (Sigma, St Louis, MO) in DMEM:F12 containing 10% FBS,
penicillin/streptomycin, 50 µg/ml heparin (Sigma) and 25 µg/ml
endothelial mitogen. Adenoviral-mediated deletion of β1 integrins was
performed with Ad-GFP or Ad-Cre-GFP (obtained from Dr Hillary Beggs at UCSF)
at a multiplicity of infection of 100. Infections were performed in serum-free
medium for 2 hours. Up to four rounds of infection were performed to achieve
>95% β1 deletion by FACS (data not shown).
P-Sp explants
P-Sp explants obtained from e8.5 embryos were cultured and monitored as
described (Braren et al., 2006;
Takakura et al., 1998). EC
fluorescence was derived from a Tie1-GFP transgene bred into the
Tie2-Cre;Itgb1flox/+ line. Anti-CD31 immunostaining was
performed on cultures lacking GFP.
In vitro capillary morphogenesis
ECs were seeded onto Growth Factor Reduced Matrigel (BD Biosciences) in
24-well plates and cultured in a timelapse chamber at 37°C in normal
growth medium in a humidified mixture of 5% CO2/95% air.
Phase-contrast images were captured at 15-minute intervals, 1-24 hours after
plating. Function-blocking anti-β1 (Ha2/5), -β3 (2C9.G2) and
-v (H9.2B8) integrin antibodies, used at 10 µg/ml, were from BD
Biosciences.
Cell adhesion, spreading, migration and focal contact formation
Timelapse analyses of primary ECs were performed as described
(Braren et al., 2006). Imaging
and measurements were performed using Slidebook software (Intelligent Imaging
Innovations, Denver, CO). Cell adhesion and modified Boyden migration (ChemoTx
101-8, Neuro Probe, Gaithersburg, MD) studies were performed as described
(Carlson et al., 2001), except
that DMEM:F12 containing 0.5% BSA (Sigma) was the medium. Immunofluorescence
for focal contacts was performed as described
(Carlson et al., 2001) after
overnight culture of primary ECs on 10 µg/ml fibronectin-coated glass
coverslips. The following antibodies, diluted 1:100, were used: anti-β1
integrin (HMβ1-1-biotinylated, BioLegend, San Diego, CA), anti-β3
integrin (2C9.G2-Alexa Fluor 647, BioLegend), anti-FAKpY397 (BD
Biosciences) and anti-paxillin (Zymed/Invitrogen). ECs were identified with
anti-CD31 at 1:100. The sources of ECM were: human fibronectin (Roche,
Indianapolis, IN); natural mouse laminin (Invitrogen); rat collagen I (Upstate
Biotechnology/Millipore, Billerica, MA); mouse collagen IV (Sigma);
growth-factor-reduced Matrigel (BD Biosciences); and human vitronectin
(Chemicon/Millipore, Billerica, MA).
Cell proliferation and survival
In vivo analyses were performed as described
(Braren et al., 2006), except
that BrdU was injected at 100 µg/g body weight 2 hours prior to dissection
at e9.0. Apoptosis was detected with a Fluorescein In Situ Apoptosis Detection
Kit (Chemicon/Millipore) in combination with anti-CD31 immunofluorescence. In
vitro EC growth was measured by counting cells with a hemacytometer every day
for 1 week after seeding 30,000 cells/well in 0.1% gelatin- or 1 µg/ml
vitronectin-coated 12-well tissue culture plates. BSA (0.5% in PBS,
heat-inactivated for 10 minutes at 85°C) was added to vitronectin-coated
wells to block the additional adsorption of serum components prior to cell
seeding. In vitro proliferation was assessed by culturing ECs on ECM-coated
glass coverslips with 125 µM BrdU (Sigma) for 6 hours prior to staining
with anti-BrdU antibodies (Zymed/Invitrogen). In vitro survival was assessed
by culturing ECs on ECM-coated glass coverslips overnight prior to staining
with YO-PRO-1 (Vybrant Apoptosis Assay Kit #4, Invitrogen). In both assays,
the coverslips were mounted with Vectashield containing DAPI, and the
percentage of ECs positive for BrdU or YO-PRO-1 was calculated after manual
counting.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Cre was also active in a subset of hematopoietic cells in this line
(Braren et al., 2006). No live
Tie2-Cre;Itgb1flox/flox (from now on referred to as
`Tie2-Cre mutant') mice were born, indicating that the mutation was
embryonic lethal. Since Cre activity is observed as early as e7.5 in our
Tie2-Cre line (Braren et al.,
2006), we analyzed e8.5 embryos for β1 integrin deletion by
several methods. First, we performed genomic PCR analysis of whole embryos
using primers that flank the loxP sites and are capable of detecting wild-type
Itgb1, floxed Itgb1, and the recombined Itgb1
alleles. We found that only embryos carrying two floxed alleles and
Tie2-Cre demonstrated recombination
(Fig. 1A). Next, we performed
immunofluorescence with antibodies against CD31 (Pecam1 - Mouse Genome
Informatics), a pan-EC marker, and β1 integrins to determine the cell
type specificity of gene deletion. β1 integrins were expressed in ECs of
the primary head veins, dorsal aorta and yolk sac blood islands, as well as in
other tissues in littermate controls, which comprised embryos lacking
Tie2-Cre or having only one floxed Itgb1 allele
(Fig. 1B). Conversely, β1
integrins were absent from ECs of Tie2-Cre mutants despite their
persistent expression in non-EC tissues. Finally, we digested e8.5 embryos,
plated the cells onto fibronectin-coated dishes and stained the mixed
population with anti-CD31, anti-β1 integrin and anti-β3 integrin
antibodies. Whereas control ECs displayed prominent β1 integrin focal
contact staining, only about 10% of Tie2-Cre mutant ECs had
detectable β1 integrins in focal contacts
(Fig. 1C,D). We also observed
more-prominent focal contact staining of β3 integrins in
Tie2-Cre mutant ECs as compared with control ECs, suggesting
compensation for the absence of β1. These findings indicate that
Tie2-Cre mediates efficient deletion of β1 integrins from ECs in
embryos by e8.5.
Tie2-Cre mutant embryos were indistinguishable from controls upon
dissection at e8.5 (data not shown). At e9.0, Tie2-Cre mutants were
of normal size but appeared to have slightly enlarged pericardial sacs (see
Fig. S1 in the supplementary material). By e9.5, almost all Tie2-Cre
mutants displayed enlarged pericardial sacs indicative of edema, which is a
common phenotype of mutations that affect the vasculature at this stage
(Conway et al., 2003).
Approximately half of e9.5 Tie2-Cre mutants were also
growth-retarded, a phenotype that became more obvious as the embryos neared
death around e10.5. No live Tie2-Cre mutants were observed at e11.5
(see Table S1 in the supplementary material).
|
). We
bred Tie1-Cre;Itgb1flox/+ males with
Itgb1flox/flox females and found that among eight litters
of newborn pups, only one live Tie1-Cre;Itgb1flox/flox
(from now on referred to as `Tie1-Cre mutant') mouse was born,
indicating that this genotype was also embryonic lethal. Timed embryo
dissections yielded no Tie1-Cre mutants at e14.5 or e12.5. At e11.5,
four of four Tie1-Cre mutants were growth retarded and apparently
dead (lacked a heartbeat). e10.5 Tie1-Cre mutants were also slightly
growth retarded, and the majority of embryos displayed hemorrhaging in the
head veins (see Fig. S1 in the supplementary material). Genomic PCR analysis
of e10.5 embryos demonstrated recombination of Itgb1 in embryos
carrying two floxed Itgb1 alleles and Tie1-Cre
(Fig. 1E). Unlike
Tie2-Cre mutants, the size and appearance of e9.5-10
Tie1-Cre mutants were similar to those of controls, perhaps because
β1 deletion was incomplete in Tie1-Cre mutant ECs at e9.5
(Fig. 1F). Thus, most
Tie1-Cre mutants die around e11.5, approximately 1 day later than
Tie2-Cre mutants (see Table S1 in the supplementary material).
EC disorganization occurs throughout the entire cardiovascular system in the absence of EC β1 integrins
The embryonic yolk sac undergoes extensive angiogenic remodeling from
e8.5-10.5 and we therefore examined this tissue by anti-CD31
immunofluorescence. Although we observed rather homogenous capillary plexuses
at e8.5 in both control and Tie2-Cre mutant yolk sacs, mutant
capillaries were slightly thinner than controls in some regions (data not
shown). More strikingly, subsequent arteriovenous remodeling of yolk sac blood
vessels was severely affected in the Tie2-Cre mutants. Whereas large
tubular vessels were evident in control yolk sacs at e9.0, mutant blood
vessels failed to organize into tubular structures and instead assembled into
sac-like structures (Fig. 2).
At e9.5 and e10.5, a hierarchical network of arteries, veins and capillaries
had developed in control yolk sacs, whereas Tie2-Cre mutant yolk sacs
contained a disorganized network of thin capillaries and sac-like structures
containing CD31-positive ECs. Connections appeared to be missing among mutant
blood vessels, and we frequently observed individual or small clusters of ECs
between vessels at e9.5 and e10.5. We also examined the yolk sac vasculature
of Tie1-Cre mutants and found that blood vessels were disorganized,
irregularly shaped and frequently contained prominent EC protrusions in
between vessels (Fig. 2). Taken
together, these findings suggest that β1 integrins are required within
ECs for proper vascular morphogenesis and that in their absence, ECs are
disorganized and detached from one another.
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Next, we examined the patterning and structure of embryonic blood vessels. Tie2-Cre mutant dorsal aortae were formed and patent at e9.0 and the capillary beds appeared similar to controls (Fig. 3A,B). However, at e9.5 (Fig. 3D) and e10.5 (data not shown), Tie2-Cre mutant dorsal aortae had narrowed, capillaries were disorganized and intersomitic vessels failed to form (Fig. 3D).
Several prominent vascular defects were observed in Tie1-Cre mutants at e10.5. First, the endothelium of Tie1-Cre mutant blood vessels was frequently discontinuous (Fig. 4B,B'), unlike the continuous monolayer observed in controls (Fig. 4A,A'). This phenotype was most clearly observed when the basement membrane to which ECs adhere was labeled with anti-fibronectin antibodies (Fig. 4C,D), and was detected in approximately 75% of all dorsal aortae and cardinal vein sections analyzed (Fig. 4E). Second, the fibronectin staining was more diffuse and less prominent in mutant basement membranes than in controls, where it stained brightly beneath the continuous EC monolayer (Fig. 4C). A similar reduction in the intensity of fibronectin staining was observed in the vascular walls of Tie2-Cre mutants (data not shown). Third, cranial blood vessels in mutants were frequently dilated (Fig. 4B), unlike controls. Finally, we detected blood vessel patterning defects in mutant neural tubes. Whereas numerous capillaries that co-stained prominently with laminin were present within control neural tubes (Fig. 4A,F,H), mutant neural tubes were completely devoid of capillaries (Fig. 4B,G,H), even though the laminin boundary between the mesenchyme and neural tube was present (Fig. 4G). In summary, deletion of β1 integrins via Tie2- or Tie1-Cre leads to widespread EC disorganization resulting in embryonic lethality at mid-gestation.
Abnormal vascular morphogenesis is due to EC-intrinsic defects upon β1 integrin deletion
Fluid shear stress resulting from blood flow is known to contribute to
vascular remodeling in the chick and mouse yolk sacs
(le Noble et al., 2004). In
order to eliminate the influence of shear stress and examine the EC-intrinsic
effects of β1 integrin deletion on capillary morphogenesis, we performed
two in vitro angiogenesis assays. In the first, we monitored vascular
development in para-aortic splanchnopleural (P-Sp) explants
(Takakura et al., 1998). We
dissected the P-Sp region of e8.5 control and Tie2-Cre mutant embryos
and cultured it on top of a feeder layer of OP9 mouse stromal cells. We then
monitored vascular development in cultures expressing GFP in ECs via a
Tie1-GFP transgene (Iljin et al.,
2002) in real time with timelapse fluorescence videomicroscopy. In
cultures lacking Tie1-GFP, we used endpoint whole-mount anti-CD31
immunostaining. Vascular network formation began in control cultures when
spindle-shaped tip ECs sprouted outwards from the P-Sp. The tip ECs retained
contact with ECs at their rear, and the sprouts grew more or less as
single-file lines of ECs. The complexity of control networks was increased
when two or more sprouts came into contact with each other, and multiple ECs
within each sprout made stable connections with neighboring ECs
(Fig.5A,C and see Movies 1, 2
in the supplementary material). In contrast to the ordered development of EC
networks in control P-Sp cultures, Tie2-Cre mutant ECs, although
highly motile, were rounded and did not maintain their migration paths
(Fig. 5B,D and see Movies 1, 2
in the supplementary material). Mutant ECs migrated in clusters and either
failed to make EC-EC connections or made transient ones. We also observed that
mutant ECs died more frequently than control ECs (see Movies 1, 2 in the
supplementary material). The inability of mutant ECs to make and maintain
stable EC-EC connections led to the appearance of EC clusters at the endpoint
of the cultures (Fig. 5D),
which contrasted with the vascular networks that formed in control cultures
(Fig. 5C).
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v plus anti-β3 integrin antibodies,
phenocopied the response of mutant
ECsβ1flox/flox
(Fig. 5G). To verify that the
anti-v and anti-β3 integrin antibodies were function-blocking, we
included the combination in a cell adhesion assay and found that they
significantly inhibited the adhesion and spreading of control
ECsβ1flox/flox on vitronectin (data not
shown). The EC disorganization observed in the P-Sp and Matrigel assays
supports our in vivo findings and indicates that β1 integrins are
required in an EC-intrinsic manner for angiogenesis.
Itgb1-null ECs are defective on collagens and laminin, but not on fibronectin, in vitro
The embryonic vascular ECM consists of fibronectin, laminin and collagen,
and β1 integrins are thought to be the primary EC receptors for each of
these proteins (George et al.,
1997; Kalluri,
2003). We therefore examined the requirement for β1 integrins
for interactions with these ECM proteins in vitro. To determine whether
β1 integrins are required for EC adhesion, we plated control and mutant
ECsβ1flox/flox onto ECM-coated plates in
serum-free medium. We found that whereas control
ECsβ1flox/flox adhered well to all tested
ECM, mutant ECsβ1flox/flox failed to
adhere to laminin, collagen I, collagen IV or Matrigel, even though they
adhered to fibronectin or vitronectin as efficiently as the controls
(Fig. 6A). Next, we tested
whether β1 integrins are required for EC haptotaxis, or migration in
response to an immobilized substrate, in the absence of serum. Control and
mutant ECsβ1flox/flox were plated onto
ECM-coated filters and allowed to migrate for 4 hours. Fibronectin, laminin,
Matrigel or vitronectin each supported the migration of control
ECsβ1flox/flox
(Fig. 6B). Conversely, mutant
ECsβ1flox/flox were incapable of migrating
on laminin or Matrigel, even though they migrated as efficiently as controls
on fibronectin and significantly more so than controls on vitronectin.
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β1 integrins regulate EC growth by affecting cell survival but not proliferation
Integrins regulate cell proliferation and survival in adherent cell types
(Giancotti and Ruoslahti,
1999). To test whether decreased proliferation or survival
contribute to the vascular defects of mutants, we performed BrdU incorporation
and TUNEL assays on control and Tie2-Cre mutant embryos at e9.0.
Approximately 25.1±1.8% of control and 23.9±1.4% of
Tie2-Cre mutant ECs incorporated BrdU within 2 hours
(Fig. 7A). We observed a slight
increase in mutant EC apoptosis as 0.85±0.4% of control and
1.35±0.3% of Tie2-Cre mutant ECs were TUNEL positive
(Fig. 7B). Mutant ECs were
substantially more apoptotic than controls at e9.5, but mutant non-ECs were
highly apoptotic, which might indicate that systemic defects also contributed
to cell death at this stage (data not shown).
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) and an indirect
ligand for β1, β3 and β5 integrins owing to its high affinity
for fibronectin present in serum (Engvall
and Ruoslahti, 1977); or (2) on vitronectin, a ligand for β3
and β5, but not β1, integrins. We found that whereas mutant ECs grew
significantly slower than control ECs on gelatin, their growth was unaffected
on vitronectin (Fig. 7C).
Mutant ECs incorporated BrdU into DNA as efficiently as controls on both types
of ECM (Fig. 7D). Mutant ECs
were significantly more permeable than control ECs to the apoptotic cell dye
YO-PRO-1 on gelatin, but not on vitronectin
(Fig. 7E). These findings,
considered together with the P-Sp results, suggest that β1 integrin
deletion impairs EC growth primarily through effects on cell survival
pathways. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Integrins are multi-functional proteins, and our results suggest that
β1 integrins primarily regulate EC adhesion, migration and survival
during embryonic angiogenesis. For example, we demonstrate a discontinuous
endothelium in the mutant blood vessels, isolated ECs in between blood vessels
in mutant yolk sacs, and clusters of rounded endocardial cells in mutant
hearts. These phenotypes are consistent with the adhesion defects that we
observe in Itgb1-null ECs in vitro. Furthermore, ECs do not invade
the neuroepithelium in Tie1-Cre mutants. Since capillary growth into
the neural tube occurs exclusively via sprouting angiogenesis
(Kurz et al., 1996), it is
likely that Itgb1-null ECs are defective in cell migration. Cell
migration is a dynamic process not readily tractable in mouse embryos, and
therefore we examined this behavior in vitro. Itgb1-null ECs do not
migrate on laminin or Matrigel and fail to maintain their migration paths in
P-Sp explants. A rounded cellular phenotype in vivo, and similar cell adhesion
and migration defects in vitro, are observed when β1 integrins are
deleted in vascular smooth muscle cells, indicating that they also control
these processes in the other major cell type of the vascular wall in vivo
(Abraham et al., 2008). In
addition, we observe a slight increase in EC apoptosis in e9.0
Tie2-Cre mutant embryos and more substantial EC apoptosis in mutant
P-Sp explants and cultured ECs. Whereas others have reported no effect of
Itgb1 deletion on vascular smooth muscle cell survival
(Abraham et al., 2008), our
results in ECs are consistent with findings in mammary epithelial cells, which
are 1.5-fold more apoptotic than controls when Itgb1 is deleted in
vivo (Li et al., 2005), and
are substantially more apoptotic when β1 integrin is blocked with
antibodies in vitro (Boudreau et al.,
1995). Finally, we find that proliferation is unaffected by
β1 integrin deficiency, which is consistent with results from β1
integrin-deficient enteric neural crest cells
(Breau et al., 2006).
Conversely, keratinocyte (Raghavan et al.,
2000), mammary epithelial cell
(Li et al., 2005) and cerebral
granule cell (Blaess et al.,
2004) proliferation is reduced, and vascular smooth muscle cell
proliferation is increased (Abraham et al.,
2008) in the absence of β1 integrins. It is possible that
β3 integrins, which we find to be upregulated in the absence of β1,
can compensate for β1 integrin function in EC proliferation, but not
survival. Altogether, our results offer a mechanistic explanation for the
multitude of studies indicating that inhibition of β1 integrins with
antibody- or peptide-based approaches blocks angiogenesis in vivo
(Garmy-Susini et al., 2005;
Kim et al., 2000b;
Pozzi et al., 2000;
Senger et al., 1997).
|
). The main EC collagen receptors are 1β1 and
2β1, and therefore it is not surprising that Itgb1-null
ECs fail to adhere to collagens. Similarly, the main EC receptors for laminin,
3β1 and 6β1, are deleted in our system. However,
vβ3 and 6β4 are also expressed in ECs and have
demonstrated functions as laminin receptors. For example, the G domain of the
4 laminin chain, which is pro-angiogenic, binds vβ3 and
3β1, and EC adhesion to this domain is blocked by
anti-vβ3 or -β1 antibodies
(Gonzales et al., 2001;
Gonzalez et al., 2002). In
addition, β4 integrins colocalize with laminin in tumor blood vessels,
and blood vessels in mice expressing a truncated β4 integrin lacking the
intracellular domain do not develop in Matrigel implants to the extent that
they do in control mice (Nikolopoulos et
al., 2004). These results suggest that vβ3 and
6β4 interactions with laminin are pro-angiogenic. However, we find
that Itgb1-null ECs express β3 integrins and adhere and migrate
on vitronectin, the main ligand for vβ3 and vβ5, yet
fail to mount appropriate angiogenic responses. Therefore, vβ3 and
6β4 interactions with laminin, if they indeed exist in embryonic
ECs, are not sufficient to support embryonic angiogenesis.
Although Itga5-/- or fibronectin-/-
mice are embryonic lethal (Francis et al.,
2002; George et al.,
1997; George et al.,
1993; Yang et al.,
1999; Yang et al.,
1993), it was unclear from these studies whether the
cardiovascular phenotypes were due to gene deletion within ECs or supporting
tissues. Since the phenotypes of our mutants are similar to these mutants in
many regards, it is likely that EC-intrinsic defects play a major role in the
lethality of Itga5-/- or
fibronectin-/- embryos. For example, cranial blood vessels
in Itga5-/- embryos are dilated, branch less frequently
and contain less fibronectin than wild-type vessels
(Francis et al., 2002), which
is similar to what we observe in our mutants. In addition, ECs within
Itga5-/- or fibronectin-/- embryoid
bodies are disorganized and fail to form networks
(Francis et al., 2002), which
reflect our observations of Itgb1-null ECs in P-Sp cultures or grown
on Matrigel. Somewhat unexpectedly, we find that EC adhesion, migration and
focal contact formation on fibronectin appear to be independent of β1
integrin expression. These data are consistent with studies of
Itga5-/- embryonic cells
(Yang and Hynes, 1996), but
differ from data obtained with Itgb1-/- embryonic cells
(Fassler et al., 1995;
Stephens et al., 1993), which
do not adhere or migrate on fibronectin. Despite the apparent lack of a
requirement for β1 integrins in EC interactions with fibronectin in
vitro, fibronectin is a prominent component of embryonic vascular basement
membranes. Since we show EC adhesion and migration defects in our mutant
embryos, we hypothesize that compensatory pathways that support fibronectin
interactions with Itgb1-null ECs in vitro fail to compensate fully
for β1 interactions with fibronectin in vivo. This might be due to
additional in vivo factors, such as hemodynamics, a three dimensional
environment and the complex ECM composition of vascular basement membranes,
which are difficult to model in vitro but are essential to consider when
assessing the function of EC β1 integrin. Our results, considered
together with the phenotypes of Itga5-/- or
fibronectin-/- embryos and tumor models in which these
molecules are inhibited (Garmy-Susini et
al., 2005; Kim et al.,
2000b), strongly support the notion that EC β1 integrin
interactions with fibronectin are required for angiogenesis.
|
; Tanjore et al.,
2008). Lei et al., using different Tie2-Cre and
Itgb1flox/flox lines, report that deletion of β1
integrin is lethal around e9.5, with growth retardation and gross vascular
defects that include dilation and reduced branching in the head region as well
as abnormal patterning in the yolk sac
(Lei et al., 2008). Using the
same Tie2-Cre line as Lei et al. and a different
Itgb1flox/flox line than that used by both Lei et al. and
us, Tanjore et al. report that mutants die by e10.5 and are growth retarded.
Large vessels develop equally well in mutants and controls, but mutants
display less sprouting and branching of smaller vessels
(Tanjore et al., 2008).
Although the general conclusion regarding the requirement for β1
integrins within ECs is similar among the published reports and ours, our work
provides several unique morphological, cellular and molecular findings. First,
a major phenotype in both our Tie2- and Tie1-Cre mutants is
the histological evidence of EC adhesion defects, seen most prominently in the
hearts of Tie2-Cre mutants and in the dorsal aortae and cardinal
veins of Tie1-Cre mutants. By contrast, Tanjore et al. report a lack
of detectable histological abnormalities in mutant embryos at e9.5, and
instead conclude that general hypoxia is the major contributor to lethality
(Tanjore et al., 2008).
Second, we provide evidence from P-Sp explant and in vitro analyses that
β1 integrin-deficient ECs are defective in cell adhesion and migration.
Third, we identify that cell survival defects contribute to the slowed growth
of Itgb1-null ECs in vitro and are likely to contribute to the demise
of Tie2-Cre mutants in vivo. Finally, we demonstrate reduced
fibronectin in mutant blood vessels, and suggest a crucial role of EC β1
integrin for interactions with this molecule, despite the apparent
compensation by β3 integrins, which also bind to fibronectin. Thus, our
results not only demonstrate that β1 integrins within ECs are required
for vascular development, but also extend the published reports by providing
mechanistic insights into the functions of EC β1 integrins.
Our findings have implications that extend beyond embryonic vascular
development and might be of therapeutic importance. For example,
anti-angiogenic compounds targeting 5β1 are currently in
development for cancer therapy
(Ramakrishnan et al., 2006).
The data presented here provide genetic evidence of an EC-intrinsic
requirement for β1 integrins during angiogenesis, and contribute a
mechanistic explanation for the anti-angiogenic actions of β1
integrin-targeted therapies. Moreover, they support the further development of
targeted therapies to block EC β1 integrin function during pathological
angiogenesis or to promote its activity in ischemia or other diseases in which
the growth of new blood vessels is desirable.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/12/2193/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Current address: Department for Diagnostic Radiology, Technical University
Munich, Munich 80290, Germany
|
REFERENCES |
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