1 Hubrecht Laboratory, Netherlands Institute for Developmental Biology,
Uppsalalaan 8, 3584CT Utrecht, The Netherlands
2 Institute of Human Genetics, International Centre for Life, University of
Newcastle, Newcastle upon Tyne, NE1 3BZ, UK
3 Division of Cellular Biochemistry, The Netherlands Cancer Institute,
Plesmanlaan 121, 1066CX Amsterdam, The Netherlands
4 Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine and
Department of Medicine, Lund University Hospital, 221 00 Lund, Sweden
Author for correspondence (e-mail:
christin{at}niob.knaw.nl)
Accepted 13 October 2004
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SUMMARY |
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Key words: HHT, TGFß, Endoglin, Yolk sac, ACVRL1
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Introduction |
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The TGFß superfamily controls cell proliferation, differentiation,
migration and adhesion. The prototype of the family, TGFß1, has multiple
roles in development and homeostasis of the vascular system. It regulates
endothelial cell proliferation, production of extracellular matrix (ECM),
vascular tone and interactions between endothelium and smooth muscle cell
layers in the vessel wall (Pepper,
1997). It is also implicated in vascular remodelling.
TGFß signalling involves heteromeric complex formation of type I and
type II receptors, initiated by ligand binding, and subsequent phosphorylation
of downstream target proteins known as Smads (R-Smads), which then complex
with the common mediator Smad (Smad4). This complex is then imported to the
nucleus and activates target genes. In most cells types TGFß signals via
the ALK5 receptor, phosphorylating Smad2 and Smad3. In contrast, TGFß
appears to signal through two pathways in endothelial cells: either via ALK1
through activation of Smad1 and Smad5, or via the conventional ALK5 mediated
activation of Smad2 and Smad3 (Goumans et
al., 2002). In addition to the TGFß serine/threonine kinase
receptors, TGFß also binds an accessory receptor, endoglin, in
endothelial cells. It is believed that the function of endoglin involves
alteration in TGFß signalling, as it has been show to modulate TGFß
responses in endothelial cells and rat myoblasts
(Barbara et al., 1999
;
Lebrin et al., 2004
;
Letamendia et al., 1998
;
McAllister et al., 1994
;
Pece et al., 1997
).
Multiple studies in mice have implicated TGFß as a potent mediator of
angiogenesis (Goumans et al.,
1999). Mice deficient in TGFß1, ALK5 or TßRII die at
E10.5 as a result of an inadequate yolk sac capillary network with poor
adhesiveness between endothelial and mesothelial cell layers
(Dickson et al., 1995
;
Larsson et al., 2001
;
Oshima et al., 1996
). Targeted
inactivation of ALK1 causes severe arteriovenous malformations resulting from
fusion of major arteries and veins and loss of arterial-specific hematopoiesis
(Urness et al., 2000
), while
in endoglin (Eng) mutant embryos the primary abnormality appears to
be defective remodelling of the primary vascular plexus that results in
abnormal yolk sac and embryonic blood vessel development
(Arthur et al., 2000
;
Bourdeau et al., 1999
;
Li et al., 1999
). It has been
postulated that Eng haploinsuficiency is the mechanism underlying HHT1
(Marchuk, 1998
;
Pece et al., 1997
;
Shovlin, 1997
). The
heterozygous mice show clinical signs of HHT, such as nose bleeds and
cutaneous telangiectases (Bourdeau et al.,
1999
; Torsney et al.,
2003
). However, this phenotype is highly dependent on the genetic
background of the inbred strain as additional modifier genes, contributed by
the 129/Ola strain are necessary to generate the vascular anomalies associated
with HHT (Bourdeau et al.,
2000
).
TGFß signalling is required for both the first stage of vascular
development, vasculogenesis, when the primary capillary network is formed
(Dickson et al., 1995) as well
as the second stage, angiogenesis, that involves remodelling the primary
endothelial network into a mature circulatory system
(Folkman and D'Amore, 1996
;
Pepper, 1997
). During blood
vessel assembly, endothelial cells recruit mesenchymal progenitors and induce
their differentiation into vascular smooth muscle cells (VSMC) or pericytes
via contact-dependent TGFß activation. However, the molecular basis of
interactions between endothelial and mesenchymal cells that lead to TGFß
activation and smooth muscle cell differentiation are not well understood.
Here, we have examined vasculogenesis in the yolk sacs of Eng mutant mice and in mice in which TGFß signalling, via TßRII or ALK5, has been disrupted specifically in endothelial cells. The yolk sac is a particularly useful model for studying blood vessel formation, as it is composed of only a limited number of cell types, is easily accessible and amenable to short-term culture. We show that the lack of an intact TGFß pathway in endothelial cells of the yolk sac results in reduced phosphorylation of Smad2 in the adjacent mesothelial layer, by affecting accessible TGFß protein levels. These cells then fail to differentiate to vascular smooth muscle cells, a process that can be rescued in part by short term culture of the yolk sac in the presence of TGFß1. Our data provide a molecular basis for understanding HHT and could explain the development of the weak-walled and fragile vessels that are characteristic of this disease.
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Materials and methods |
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lacZ staining and immunohistochemistry
After isolation, E9.5 yolk sacs were divided in half, cultured in
Dulbecco's minimum essential medium (DMEM) with or without TGFß1 (1 or 5
ng/ml) or BMP2 (10 ng/ml), for 1, 3 or 8 hours, as indicated, at 37°C and
then fixed for 30 minutes at room temperature in 2% paraformaldehyde (PFA).
Prior to immunostaining, lacZ reporter expression was visualised
using ß-gal staining, as described by Nagy et al.
(Nagy et al., 2003). For
whole-mount immunohistochemistry, yolk sacs from E9.5 embryos were dehydrated
serially to 100% methanol, treated for 5 hours with 5% hydrogen peroxide in
methanol, rehydrated to PBS at room temperature, permeabilised with 0.1%
Triton X-100 (Merck) in PBS for 8 minutes. After washing with PBS, the yolk
sacs were blocked for 1 hour, at room temperature in TNB blocking solution
from the Tyramide Signal Amplification (TSA) Biotin System (PerkinElmer, Life
Sciences). After a 1 hour incubation with anti-PECAM1 (1:100, BD Biosciences),
anti-endoglin (1:100, USBiological), Flk1 (1:100, SantaCruz), affinity
purified anti-Phosphorylated Smad2 (PSmad2)
(Persson et al., 1998
),
anti-TGFß1 (1:100, recognizing total TGFß1, SantaCruz), anti-smooth
muscle actin (1:400, Sigma), anti-caldesmon (1:500, Sigma), anti-fibronectin
(1:100, Sigma) or anti-collagen type I (1:100, Rockland) in TNB blocking
solution, yolk sacs were washed in 0.05% Tween/PBS and treated for 1 hour with
the secondary antibody (biotin-conjugated rabbit anti-rat IgG for PECAM1 and
endoglin, biotin-conjugated swine anti-rabbit for PSmad2, TGFß1,
fibronectin and collagen type I or biotin-conjugated goat anti-mouse IgG for
Flk-1 and
-sma, DAKO, 1:250) in blocking solution, at room temperature.
Embryos were then treated with ABComplex/HRP (DAKO), followed by 10 minutes
incubation in biotinil tyramide diluted 1:50 in tyramide diluent supplied in
TSA Biotin System. After washing in 0.05% Tween/PBS, yolk sacs were treated
for 1 hour, at room temperature, with strepavidin-HRP (1:250) in TNB.
Peroxidase activity was detected using 3,3'-diaminobenzidine tablet set
(Fast DAB, Sigma), according to manufacturer's instructions. Stained yolk sacs
were fixed overnight in 2% PFA/0.1% gluteraldehyde in PBS at 4°C, embedded
in plastic and sectioned.
Paraffin wax embedded sections of E9.5 embryos and yolk sacs were treated as above but using 1.2% hydrogen peroxide for 15 minutes. For antigen retrieval for PSmad1 paraffin sections were treated by boiling in Tris/EDTA pH 9.0 buffer while for PSmad2 paraffin section were treated by boiling in 10 mM citrate buffer (pH 6.0). Anti-PSmad1 and anti-PSmad2 were used as primary antibodies and biotin-conjugated goat anti-rabbit IgG (1:250, DAKO) as secondary antibody.
Score of positive endothelial cells in the yolk sac
The number of PSmad1/5/8 and PSmad2 positive endothelial cells was
determined as described by Lebrin et al.
(Lebrin et al., 2004).
Western blotting
Protein isolation for western blotting has been described previously
(Fauré et al., 2000).
Analysis was as described by Larsson et al.
(Larsson et al., 2001
), using
the total lysate of two yolk sacs. Anti-TGFß1 (1:200, SantaCruz) was used
as primary antibody and HRP-conjugated goat anti-rabbit IgG as secondary
antibody (1:10000, BD Biosciences).
In situ hybridization
Whole-mount in situ hybridisation was performed as described before
(Roelen et al., 2002). The
digoxigenin-labelled (Boehringer Mannheim) TGFß1 antisense probe was a T3
polymerase transcript from a 600 bp KpnI-ApaI fragment
(Millan et al., 1991
).
Separation of yolk sac mesoderm and endoderm
The yolk sac mesoderm and endoderm of E9.5 embryos were mechanically
separated after incubating in trypsin/pancreatin as described by Roelen et al.
(Roelen et al., 1994).
RNA extraction and real time PCR analysis
One half of each E9.5 yolk sac was cultured with TGFß1 (1 ng/ml) for 3
hours, the other without as before, and were collected in 100 µl Ultraspec
(Biotecx). RNA was extracted according to the manufacturer's protocol; 10
µg PolyI (Sigma) was added as a carrier. Samples were DNase I treated to
eliminate genomic DNA and 1 µg RNA was reversed transcribed as described
before (Roelen et al., 1994).
RNA samples which had not been reverse transcribed served as negative controls
for genomic DNA contamination and RNA isolated from the endoderm layer was
used as an additional negative control.
Real-time PCR was performed in a MyiQTM single-color real time detection system (BioRad). The PCR primers were tested for both primer dimer formation and efficiency. To normalise for the amount of mRNA used as starting material, cDNA of ß-actin was amplified. Routinely, a three-step program, followed by melt curve, was used. Data was collected and real-time analysis carried out during the extension period. Primers were as follows: ß-actin forward primer, 5'-CCTGAACCCTAAGGCCAACCG-3' and reverse 5'-GCTCATAGCTCTTCTCCAGGG-3', annealing temperature 60.2°C; smooth muscle actin forward primer, 5'-CAGAGCAAGAGAGGGATCCTGA-3' and reverse 5'-TAGATAGGCACGTTGTGAGTCACA-3', annealing temperature 60.2°C; TGFß forward primer, 5'-ATGGAGCTGGTGAAACGGAA-3' and reverse primer 5'-ACTGCTTCCCGAATGTCTGA-3', annealing temperature 61.2°C.
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Results |
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Crucial to the present study was the unambiguous identity of ECs in the yolk sac. We therefore first used ß-gal staining to identify Eng-positive cells and immunohistochemistry for PECAM1, Flk1 and Eng to show that ECs were present as an extremely thin layer surrounding the developing vessels. The EC-specific immunostaining was clearer in individual cells that had been sectioned through the nucleus, although they were in all sections easily distinguishable from the adjacent mesothelial cell layer, which completely lacked these markers (Fig. 1).
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To confirm the specificity of PSmad2 staining, we examined yolk sacs from
Tgfbr2 mutant embryos resulting from intercrosses of PGK-Cre
transgenics with floxed Tgfbr2 mice
(Lallemand et al., 1998;
Leveen et al., 2002
). As
expected, PSmad2 staining was absent in both endothelial and mesothelial layer
of the yolk sac (Fig. 2I) and
could not be restored by incubation with TGFß1
(Fig. 2J).
This demonstrated that TßRII/ALK5 receptor complexes on the surface of
both cell types in the Eng mutants can be activated by exogenous
TGFß1 in short-term culture. The mesothelial cell layer, which does not
express Eng (Fig.
1A,D), showed reduced PSmad2 levels when loss of endoglin
disrupted TGFß signalling in the endothelial layer. We also observed
downregulation of ALK5 expression in ECs from Eng+/
embryos (Lebrin et al., 2004).
This genetic adaptation might occur in order for ECs to survive the
potentiation of ALK5-induced growth arrest
(Goumans et al., 2003
).
Smad2 activation is absent in endothelial cells of Eng knockout embryos
To investigate whether loss of PSmad2 in Eng knockout mice was
restricted to the yolk sac vasculature or was a more general feature of the
Eng mutant mice, we also examined the embryo proper. Previously, we
have shown PSmad2 scattered throughout the embryonic mesenchyme, somites, body
wall, surface ectoderm, blood vessels, blood, fore- and hindgut, endo- and
myocardium of the heart at E8.5 but by E10.5 most endothelial cells of
arteries veins and capillaries had become PSmad2 negative
(Sousa Lopes et al.,
2003).
We again used PECAM1 staining in parallel with PSmad2 staining to distinguish embryonic ECs unequivocally from adjacent cells (Fig. 3A-D). As expected, at E9.5 PSmad2 was observed in endothelial cells of both arteries and veins of wild-type embryos at this stage. However, activated Smad2 was no longer detected in any Eng/ ECs throughout the embryonic blood vasculature (Fig. 3). These results are in agreement with the observation in the extra-embryonic yolk sac (Fig. 2D,F). Interestingly, Smad2 was still phosphorylated in the endocardium of the heart of endoglin mutant embryos (Fig. 3K,K'), probably via ALK4 or ALK7, although the expression of these two receptors has not been documented at these stages.
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TGFß expression is reduced in yolk sac of Eng knockout mice
One explanation for these findings might be that disruption of TGFß
signalling in endothelial cells by loss of Eng resulted in decreased levels of
TGFß1 in the yolk sac and, consequently, insufficient ligand to activate
paracrine TGFß signalling to neighbouring mesothelial cells. Whole-mount
in situ hybridization and immunohistochemistry for TGFß1 mRNA and protein
were thus carried out on yolk sacs from wild-type and Eng mutant
embryos. In situ hybridization showed that Tgfb1 mRNA is not detected
in the mesothelial layer but it is expressed strongly by endothelial cells in
the yolk sac. Its expression appeared unaltered in Eng knockout yolk
sacs (Fig. 6A,B); this was
confirmed by real time RT-PCR (Fig.
6G). However, using an antibody specific for TGFß1 we
observed that TGFß1 protein levels were significantly reduced in the
mesothelium of yolks sac lacking endoglin
(Fig. 6E,F) compared with
wild-type yolk sacs (Fig.
6C,D). This was confirmed by western blotting
(Fig. 6H). It is therefore most
likely that loss of Eng reduces availability of TGFß1 protein to the
mesothelial cells and as a consequence these cells are no longer able to
differentiate fully into smooth muscle cells.
|
Whole-mount immunohistochemistry for PSmad2 was carried out on half yolk sacs collected at E9.5 from these embryos treated or not with TGFß1 for 1 hour, as for the yolk sacs from the Eng mutants. Phosphorylated Smad2 was again absent in the endothelial and mesothelial cells of conditional knockouts lacking either TßRII or ALK5 in ECs of the yolk sacs (Fig. 7B,C), although in wild-type yolk sacs Smad2 is clearly phosporylated (Fig. 7A,D). However, after treatment with TGFß the nucleus of mesothelial cells of both mutants became positive for PSmad2 (Fig. 7E,F; Fig. 2L) again demonstrating the activity of intact TßRII/ALK5 complexes in the mesothelial cells even when either TßRII or ALK5 had been deleted in the adjacent ECs. These results demonstrated that the mesothelial layer indeed interacts closely with the endothelial layer and more importantly demonstrated that disabling any one of several receptors affecting TGFß signalling in ECs has a knock-on effect on TGFß responses in the adjacent mesothelium.
|
As mRNA levels of TGFß1 are not affected in the Eng knockout mice yet TGFß1 protein (probably taken up in an active form) is no longer detected in mesothelial cells, TGFß production is most likely to have been affected at the post-transcriptional level. Either the amounts of latent protein are reduced or activation of the latent TGFß is altered. We were, however, unable to determine whether this was the case directly as TGFß levels in protein extracts or short-term conditioned medium from yolk sacs were below the detection limit of available bioassay or ELISAs.
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Discussion |
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In the present study we analysed yolk sacs from endoglin deficient mice and from mice lacking TßRII or ALK5 specifically in endothelial cells. The data showed that loss of an intact TGFß signalling pathway in endothelial cells has a strong impact on Smad2 phosphorylation in the adjacent mesothelial layer. Rescue by TGFß1 suggested a TGFß-mediated interaction between the endothelial layer and the mesothelial layer of the yolk sac.
During blood vessel assembly, endothelial cells recruit mesenchymal
progenitors and induce their differentiation into smooth muscle cells or
pericytes. In the Eng mutant, yolk sac expression of -smooth
muscle actin and caldesmon are clearly affected, indicating that the
differentiation of mesothelial cells into smooth muscle cells is impaired, as
has also been also shown by Li et al. (Li
et al., 1999
). Importantly, however, we found that reduced
-sma expression in mesothelial cells could be rescued by treatment with
TGFß1. Microarray analysis in ECs, infected with constitutively active
(ca)ALK5 showed that caldesmon was one of the genes upregulated by the
TGFß signalling pathway (Ota et al.,
2002
). A number of other transcription factors are involved in
VSMC development. Mice lacking Fli1
(Hart et al., 2000
),
Mef2c (Lin et al.,
1998
), Smad5 (Yang et
al., 1999
), Hand2
(Yamagishi et al., 2000
),
Hand1 (Morikawa and Cserjesi,
2004
) and Cx43
(Hirschi et al., 2003
) also
show reduced smooth muscle development around the vessel. It is not known
whether TGFß processing or availability is affected in these mutants
although in Cx43 mutant mice, it was shown that the mechanism by
which gap junctions mediate endothelial-induced mural differentiation involves
the activation of TGFß (Hirschi et
al., 2003
). Nevertheless, disruption of TGFß signalling maybe
a common feature of some mice mutants with defective yolk sac
vasculogenesis.
The low levels of -sma in endoglin knockout,
tie-1-Cre-TßRII and
tie-1-Cre-ALK5 embryos suggests VSMC differentiation is
defective in mice with disrupted TGFß signalling in ECs. Several studies
have demonstrated that TGFß is activated upon endothelial-induced
mesenchymal cell differentiation and that it upregulates
-smooth muscle
actin expression (Assoian and Sporn,
1986
; Majack,
1987
; Merwin et al.,
1991
; Morisaki et al.,
1991
). Chen and Lechleider also showed that activation of Smad
pathway is necessary for smooth muscle cell differentiation from neural crest
cells (Chen and Lechleider,
2004
). We examined both TGFß1 mRNA and protein expression and
found the TGFß1 protein levels are indeed affected, demonstrating that
ECs are the crucial source of TGFß in VSMC recruitment to blood vessels.
Furthermore, the importance of endothelium-derived TGFß in VSMC
recruitment suggested that TGFß acts mainly through a paracrine route.
Bourdeau et al. (Bourdeau et al., 2001) have shown that the 129/Ola mouse
strain has lower levels of plasma TGFß1 compared with C57Bl/6 and is more
susceptible to HHT. Plasma TGFß was further reduced in Eng
heterozygous mice and as ECs are the major source of circulating TGFß,
this reduction would be compatible with our present findings. However, these
results have not been reproduced by others and determination of levels of
circulating TGFß has been notoriously difficult and subject to artefacts.
Whether TGFß receptor knockout mice exhibit lower levels of TGFß1
remains to be determined.
Autocrine regulation of TGFß1 expression has been described previously
in a number of different normal and transformed cells
(Van Obberghen-Schilling et al.,
1988). This autoregulation might explain why affecting TGFß
signalling pathway also affects TGFß1 protein. The lack of endoglin,
TßRII or ALK5 in ECs could thus lead to reduced activation of TGFß1
in all cases via an autocrine mechanism.
The composition and organization of vascular extracellular matrix is also
responsible for the mechanical properties of the vessel wall, forming complex
networks of structural proteins. Fibronectin and collagen are major components
of the ECM and promote cell adhesion and spreading, cell migration and
cytoskeletal organization (Hynes,
1991). It has been shown that TGFß upregulates expression of
ECM components, such as fibronectin and collagen, via both Smad-dependent
(Verrecchia et al., 2001
) and
Smad-independent pathways (Hocevar et al.,
1999
; Runyan et al.,
2004
). Reduced levels of TGFß protein may affect fibronectin
deposition between the endoderm and mesoderm layers of the yolk sac as has
already been described in chimaeric mice generated from ES cells expressing
dnTßRII (Goumans et al.,
1999
) and in more general terms affect ECM deposition. However, in
the yolk sac of Eng mutant mice, we were unable to detect any
differences in fibronectin or collagen expression, as has also been reported
for the embryo proper (Arthur et al.,
2000
).
Our studies indicate a general mechanism through which defects in
components of TGFß signalling pathway in ECs affect yolk sac
vasculogenesis and would explain why the phenotypes of many of these mutant
mice are so similar and also similar to the Tgfb1 mutant mice itself.
All may be mediated by altered TGFß protein levels. Deletion/mutation of
other genes that give rise to a vascular phenotype in the yolk sac
(Carvalho et al., 2002) may
act up or downstream of TGFß signalling. One example includes tissue
factor (Tf; F3 Mouse Genome Informatics) gene, a
procoagulant receptor, also involved in vascular integrity by affecting the
maturation of the muscular wall around endothelial cells
(Carmeliet and Collen, 1998
)
and stimulated by TGFß1 (Ranganathan
et al., 1991
). Another is latent TGFß-binding protein 1,
which seems to play an important role in the targeting and release of
TGFß1 in response to arterial injury
(Sinha et al., 2002
).
Compared with vascular endothelial cells in normal tissues, a stronger
staining for endoglin was detected in vascular endothelial cells in tissues
undergoing active angiogenesis, such as in regenerating and inflamed tissues
or tumours (Bodey et al.,
1998a; Bodey et al.,
1998b
; Miller et al.,
1999
; Seon et al.,
1997
; Wang et al.,
1994
). Increased endoglin expression was also observed in ECs of
microvessels from pathological skin lesions and in established atherosclerotic
lesions (Burrows et al.,
1995
).
In individuals with HHT, maximum achievable levels of endoglin expression would be significantly lower than in normal subjects. Should similar mechanisms operate as described here for the endoglin mutant mice, then the ECs in individuals with HHT may make less TGFß protein available than their normal counterparts. As a consequence, less TGFß1 may be present in the circulation as ECs are the major source of circulating TGFß1, but more importantly, less TGFß1 may be available locally to induce differentiation of pericytes or smooth muscle cells and recruit them to the neovasculature leaving the vessels weak and susceptible to further damage and haemorrhage, typical of individuals with HHT.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Center for Regenerative Medicine and Technology,
Massachusetts General Hospital, Boston, MA 02114, USA
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arthur, H. M., Ure, J., Smith, A. J., Renforth, G., Wilson, D. I., Torsney, E., Charlton, R., Parums, D. V., Jowett, T., Marchuk, D. A. et al. (2000). Endoglin, an ancillary TGFß receptor, is required for extraembryonic angiogenesis and plays a key role in heart development. Dev. Biol. 217, 42-53.[CrossRef][Medline]
Assoian, R. K. and Sporn, M. B. (1986). Type ß transforming growth factor in human platelets: release during platelet degranulation and action on vascular smooth muscle cells. J. Cell Biol. 102,1217 -1223.[Abstract]
Barbara, N. P., Wrana, J. L. and Letarte, M.
(1999). Endoglin is an accessory protein that interacts with the
signaling receptor complex of multiple members of the transforming growth
factor-ß superfamily. J. Biol. Chem.
274,584
-594.
Bodey, B., Bodey, B., Jr, Siegel, S. E. and Kaiser, H. E. (1998a). Immunocytochemical detection of endoglin is indicative of angiogenesis in malignant melanoma. Anticancer Res. 18,2701 -2710.[Medline]
Bodey, B., Bodey, B., Jr, Siegel, S. E. and Kaiser, H. E. (1998b). Overexpression of endoglin (CD105): a marker of breast carcinoma-induced neovascularization. Anticancer Res. 18,3621 -3628.[Medline]
Bourdeau, A., Dumont, D. J. and Letarte, M.
(1999). A murine model of hereditary hemorrhagic telangiectasia.
J. Clin. Invest. 104,1343
-1351.
Bourdeau, A., Faughnan, M. E. and Letarte, M. (2000). Endoglin-deficient mice, a unique model to study hereditary hemorrhagic telangiectasia. Trends. Cardiovasc. Med. 10,279 -285.[CrossRef][Medline]
Burrows, F. J., Derbyshire, E. J., Tazzari, P. L., Amlot, P., Gazdar, A. F., King, S. W., Letarte, M., Vitetta, E. S. and Thorpe, P. E. (1995). Upregulation of endoglin on vascular endothelial cells in human solid tumors: implications for diagnosis and therapy. Clin. Cancer Res. 1,1623 -1634.[Abstract]
Carmeliet, P. and Collen, D. (1998). Tissue factor. Int. J. Biochem. Cell Biol. 30,661 -667.[CrossRef][Medline]
Carvalho, R. L. S., Driessens, M. H. E. and Mummery, C. (2002). Vasculogenesis and angiogenesis: lessons from genetics in mice. Comm. Theoret. Biol. 7, 381-416.[CrossRef]
Chen, S. and Lechleider, R. J. (2004).
Transforming growth factor-ß-induced differentiation of smooth muscle
from a neural crest stem cell line. Circ. Res.
94,1195
-1202.
Dickson, M. C., Martin, J. S., Cousins, F. M., Kulkarni, A. B.,
Karlsson, S. and Akhurst, R. J. (1995). Defective
haematopoiesis and vasculogenesis in transforming growth factor-ß1 knock
out mice. Development
121,1845
-1854.
Faure, S., Lee, M. A., Keller, T., ten Dijke, P. and Whitman,
M. (2000). Endogenous patterns of TGFß superfamily
signaling during early Xenopus development.
Development 127,2917
-2931.
Folkman, J. and D'Amore, P. A. (1996). Blood vessel formation: what is its molecular basis? Cell 87,1153 -1155.[Medline]
Goumans, M. J., Zwijsen, A., van Rooijen, M. A., Huylebroeck,
D., Roelen, B. A. and Mummery, C. L. (1999).
Transforming growth factor-ß signalling in extraembryonic mesoderm is
required for yolk sac vasculogenesis in mice.
Development 126,3473
-3483.
Goumans, M. J., Valdimarsdottir, G., Itoh, S., Rosendahl, A.,
Sideras, P. and ten Dijke, P. (2002). Balancing the
activation state of the endothelium via two distinct TGF-ß type I
receptors. EMBO J. 21,1743
-1753.
Goumans, M. J., Valdimarsdottir, G., Itoh, S., Lebrin, F., Larsson, J., Mummery, C., Karlsson, S. and ten Dijke, P. (2003). Activin receptor-like kinase (ALK)1 is an antagonistic mediator of lateral TGFß/ALK5 signaling. Mol. Cell 12,817 -828.[Medline]
Gustafsson, E., Brakebusch, C., Hietanen, K. and Fassler, R.
(2001). Tie-1-directed expression of Cre recombinase in
endothelial cells of embryoid bodies and transgenic mice. J. Cell
Sci. 114,671
-676.
Guttmacher, A. E., Marchuk, D. A. and White, R. I., Jr
(1995). Hereditary hemorrhagic telangiectasia. N.
Engl. J. Med. 333,918
-924.
Hart, A., Melet, F., Grossfeld, P., Chien, K., Jones, C., Tunnacliffe, A., Favier, R. and Bernstein, A. (2000). Fli-1 is required for murine vascular and megakaryocytic development and is hemizygously deleted in patients with thrombocytopenia. Immunity 13,167 -177.[Medline]
Hirschi, K. K., Burt, J. M., Hirschi, K. D. and Dai, C.
(2003). Gap junction communication mediates transforming growth
factor-ß activation and endothelial-induced mural cell differentiation.
Circ. Res. 93,429
-437.
Hocevar, B. A., Brown, T. L. and Howe, P. H.
(1999). TGF-ß induces fibronectin synthesis through a c-Jun
N-terminal kinase-dependent, Smad4-independent pathway. EMBO
J. 18,1345
-1356.
Huber, P. A. (1997). Caldesmon. Int. J. Biochem. Cell Biol. 29,1047 -1051.[CrossRef][Medline]
Hynes, R. O. (1991). The complexity of platelet adhesion to extracellular matrices. Thromb. Haemost. 66, 40-43.[Medline]
Johnson, D. W., Berg, J. N., Baldwin, M. A., Gallione, C. J., Marondel, I., Yoon, S. J., Stenzel, T. T., Speer, M., Pericak-Vance, M. A., Diamond, A. et al. (1996). Mutations in the activin receptor-like kinase 1 gene in hereditary haemorrhagic telangiectasia type 2. Nat. Genet. 13,189 -195.[Medline]
Lallemand, Y., Luria, V., Haffner-Krausz, R. and Lonai, P. (1998). Maternally expressed PGK-Cre transgene as a tool for early and uniform activation of the Cre site-specific recombinase. Transgenic Res. 7,105 -112.[CrossRef][Medline]
Larsson, J., Goumans, M. J., Sjostrand, L. J., van Rooijen, M.
A., Ward, D., Leveen, P., Xu, X., ten Dijke, P., Mummery, C. L. and
Karlsson, S. (2001). Abnormal angiogenesis but intact
hematopoietic potential in TGF-ß type I receptor-deficient mice.
EMBO J. 20,1663
-1673.
Lebrin, F., Goumans, M.-J., Jonker, L., Carvalho, R. L. C.,
Valdimarsdottir, G., Thorikay, M., Mummery, C., Arthur, H. M. and ten
Dijke, P. (2004). Endoglin promotes endothelial cell
proliferation and TGF-ß/ALK1 signal transduction. EMBO
J. 23,4018
-4028.
Letamendia, A., Lastres, P., Botella, L. M., Raab, U., Langa,
C., Velasco, B., Attisano, L. and Bernabeu, C. (1998).
Role of endoglin in cellular responses to transforming growth factor-ß. A
comparative study with betaglycan. J. Biol. Chem.
273,33011
-33019.
Leveen, P., Larsson, J., Ehinger, M., Cilio, C. M., Sundler, M.,
Sjostrand, L. J., Holmdahl, R. and Karlsson, S.
(2002). Induced disruption of the transforming growth factor
ß type II receptor gene in mice causes a lethal inflammatory disorder
that is transplantable. Blood
100,560
-568.
Li, D. Y., Sorensen, L. K., Brooke, B. S., Urness, L. D., Davis,
E. C., Taylor, D. G., Boak, B. B. and Wendel, D. P.
(1999). Defective angiogenesis in mice lacking endoglin.
Science 284,1534
-1537.
Lin, Q., Lu, J., Yanagisawa, H., Webb, R., Lyons, G. E.,
Richardson, J. A. and Olson, E. N. (1998). Requirement of the
MADS-box transcription factor MEF2C for vascular development.
Development 125,4565
-4574.
Majack, R. A. (1987). ß-type transforming growth factor specifies organizational behavior in vascular smooth muscle cell cultures. J. Cell Biol. 105,465 -471.[Abstract]
Marchuk, D. A. (1998). Genetic abnormalities in hereditary hemorrhagic telangiectasia. Curr. Opin. Hematol. 5,332 -338.[Medline]
McAllister, K. A., Grogg, K. M., Johnson, D. W., Gallione, C. J., Baldwin, M. A., Jackson, C. E., Helmbold, E. A., Markel, D. S., McKinnon, W. C. and Murrell, J. (1994). Endoglin, a TGF-ß binding protein of endothelial cells, is the gene for hereditary haemorrhagic telangiectasia type 1. Nat. Genet. 8, 345-351.[Medline]
Merwin, J. R., Newman, W., Beall, L. D., Tucker, A. and Madri, J. (1991). Vascular cells respond differentially to transforming growth factors ß1 and ß2 in vitro. Am. J. Pathol. 138,37 -51.[Abstract]
Millan, F. A., Denhez, F., Kondaiah, P. and Akhurst, R. J. (1991). Embryonic gene expression patterns of TGF ß1, ß2 and ß3 suggest different developmental functions in vivo. Development 111,131 -143.[Abstract]
Miller, D. W., Graulich, W., Karges, B., Stahl, S., Ernst, M., Ramaswamy, A., Sedlacek, H. H., Muller, R. and Adamkiewicz, J. (1999). Elevated expression of endoglin, a component of the TGF-ß-receptor complex, correlates with proliferation of tumor endothelial cells. Int. J. Cancer 81,568 -572.[CrossRef][Medline]
Morikawa, Y. and Cserjesi, P. (2004).
Extra-embryonic vasculature development is regulated by the transcription
factor HAND1. Development
131,2195
-2204.
Morisaki, N., Kawano, M., Koyama, N., Koshikawa, T., Umemiya, K., Saito, Y. and Yoshida, S. (1991). Effects of transforming growth factor-ß1 on growth of aortic smooth muscle cells. Influences of interaction with growth factors, cell state, cell phenotype, and cell cycle. Atherosclerosis 88,227 -234.[Medline]
Nagy, A., Gertsenstein, M., Vintersten, M. and Behringer, R. R. (2003). Manipulating the Mouse Embryo A Laboratory Manual. New York, NY: Cold Spring Harbor Laboratory Press.
Oh, S. P., Seki, T., Goss, K. A., Imamura, T., Yi, Y., Donahoe,
P. K., Li, L., Miyazono, K., ten Dijke, P., Kim, S. and Li, E.
(2000). Activin receptor-like kinase 1 modulates transforming
growth factor-ß1 signaling in the regulation of angiogenesis.
Proc. Natl. Acad. Sci. USA
97,2626
-2631.
Oshima, M., Oshima, H. and Taketo, M. M. (1996). TGF-ß receptor type II deficiency results in defects of yolk sac hematopoiesis and vasculogenesis. Dev. Biol. 179,297 -302.[CrossRef][Medline]
Ota, T., Fujii, M., Sugizaki, T., Ishii, M., Miyazawa, K., Aburatani, H. and Miyazono, K. (2002). Targets of transcriptional regulation by two distinct type I receptors for transforming growth factor-ß in human umbilical vein endothelial cells. J. Cell Physiol. 193,299 -318.[CrossRef][Medline]
Pece, N., Vera, S., Cymerman, U., White, R. I., Jr, Wrana, J. L.
and Letarte, M. (1997). Mutant endoglin in hereditary
hemorrhagic telangiectasia type 1 is transiently expressed intracellularly and
is not a dominant negative. J. Clin. Invest.
100,2568
-2579.
Pepper, M. S. (1997). Transforming growth factor-ß: vasculogenesis, angiogenesis, and vessel wall integrity. Cytokine Growth Factor. Rev. 8, 21-43.[CrossRef][Medline]
Persson, U., Izumi, H., Souchelnytskyi, S., Itoh, S., Grimsby, S., Engstrom, U., Heldin, C. H., Funa, K. and ten Dijke, P. (1998). The L45 loop in type I receptors for TGF-ß family members is a critical determinant in specifying Smad isoform activation. FEBS Lett. 434,83 -87.[CrossRef][Medline]
Ranganathan, G., Blatti, S. P., Subramaniam, M., Fass, D. N.,
Maihle, N. J. and Getz, M. J. (1991). Cloning of
murine tissue factor and regulation of gene expression by transforming growth
factor type ß 1. J. Biol. Chem.
266,496
-501.
Roelen, B. A., Lin, H. Y., Knezevic, V., Freund, E. and Mummery, C. L. (1994). Expression of TGF-ß s and their receptors during implantation and organogenesis of the mouse embryo. Dev. Biol. 166,716 -728.[CrossRef][Medline]
Roelen, B. A., de Graaff, W., Forlani, S. and Deschamps, J. (2002). Hox cluster polarity in early transcriptional availability: a high order regulatory level of clustered Hox genes in the mouse. Mech. Dev. 119,81 -90.[CrossRef][Medline]
Runyan, C. E., Schnaper, H. W. and Poncelet, A. C.
(2004). The phosphatidylinositol 3-kinase/Akt pathway enhances
Smad3-stimulated mesangial cell collagen I expression in response to
transforming growth factor-ß1. J. Biol. Chem.
279,2632
-2639.
Seon, B. K., Matsuno, F., Haruta, Y., Kondo, M. and Barcos, M. (1997). Long-lasting complete inhibition of human solid tumors in SCID mice by targeting endothelial cells of tumor vasculature with antihuman endoglin immunotoxin. Clin. Cancer Res. 3,1031 -1044.[Abstract]
Shovlin, C. L. (1997). Molecular defects in rare bleeding disorders: hereditary haemorrhagic telangiectasia. Thromb. Haemost. 78,145 -150.[Medline]
Shovlin, C. L. and Letarte, M. (1999).
Hereditary haemorrhagic telangiectasia and pulmonary arteriovenous
malformations: issues in clinical management and review of pathogenic
mechanisms. Thorax 54,714
-729.
Sinha, S., Heagerty, A. M., Shuttleworth, C. A. and Kielty, C. M. (2002). Expression of latent TGF-ß binding proteins and association with TGF-ß 1 and fibrillin-1 following arterial injury. Cardiovasc. Res. 53,971 -983.[CrossRef][Medline]
Sousa Lopes, S. M., Carvalho, R. L., van den Driesche, S., Goumans, M. J., ten Dijke, P. and Mummery, C. L. (2003). Distribution of phosphorylated Smad2 identifies target tissues of TGFß ligands in mouse development. Gene Expr. Patterns. 3,355 -360.[CrossRef][Medline]
Torsney, E., Charlton, R., Diamond, A. G., Burn, J., Soames, J.
V. and Arthur, H. M. (2003). Mouse model for
hereditary hemorrhagic telangiectasia has a generalized vascular abnormality.
Circulation 107,1653
-1657.
Urness, L. D., Sorensen, L. K. and Li, D. Y. (2000). Arteriovenous malformations in mice lacking activin receptor-like kinase-1. Nat. Genet. 26,328 -331.[CrossRef][Medline]
Valdimarsdottir, G., Goumans, M. J., Rosendahl, A., Brugman, M.,
Itoh, S., Lebrin, F., Sideras, P. and ten Dijke, P.
(2002). Stimulation of Id1 expression by bone morphogenetic
protein is sufficient and necessary for bone morphogenetic protein-induced
activation of endothelial cells. Circulation
106,2263
-2270.
Van Obberghen-Schilling, E., Roche, N. S., Flanders, K. C.,
Sporn, M. B. and Roberts, A. B. (1988). Transforming growth
factor ß1 positively regulates its own expression in normal and
transformed cells. J. Biol. Chem.
263,7741
-7746.
Verrecchia, F. and Mauviel, A. (2002).
Transforming growth factor-ß signaling through the Smad pathway: role in
extracellular matrix gene expression and regulation. J. Invest.
Dermatol. 118,211
-215.
Verrecchia, F., Chu, M. L. and Mauviel, A.
(2001). Identification of novel TGF-ß/Smad gene targets in
dermal fibroblasts using a combined cDNA microarray/promoter transactivation
approach. J. Biol. Chem.
276,17058
-17062.
Wang, J. M., Kumar, S., Pye, D., Haboubi, N. and al-Nakib, L. (1994). Breast carcinoma: comparative study of tumor vasculature using two endothelial cell markers. J. Natl. Cancer Inst. 86,386 -388.[Medline]
Yamagishi, H., Olson, E. N. and Srivastava, D.
(2000). The basic helix-loop-helix transcription factor, dHAND,
is required for vascular development. J. Clin. Invest.
105,261
-270.
Yang, X., Castilla, L. H., Xu, X., Li, C., Gotay, J., Weinstein,
M., Liu, P. P. and Deng, C. X. (1999). Angiogenesis
defects and mesenchymal apoptosis in mice lacking SMAD5.
Development 126,1571
-1580.
Zambrowicz, B. P., Imamoto, A., Fiering, S., Herzenberg, L. A.,
Kerr, W. G. and Soriano, P. (1997). Disruption of
overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to
widespread expression of beta-galactosidase in mouse embryos and hematopoietic
cells. Proc. Natl. Acad. Sci. USA
94,3789
-3794.