1 Department of Cell Biology, Harvard Medical School, Boston, MA 02115,
USA
2 Department of Surgical Research, Children's Hospital and Harvard Medical
School, Boston, MA 02115, USA
3 Department of Molecular Oncology, Genentech, South San Francisco, CA 94080,
USA
4 Molecular Biology Section, Division of Biology, University of California, San
Diego, CA 92093, USA
5 Endocrine Unit, Massachusetts General Hospital and Harvard Medical School,
Boston, MA 02114, USA
* Author for correspondence (e-mail: bjorn_olsen{at}hms.harvard.edu)
Accepted 30 December 2003
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SUMMARY |
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Key words: Conditional knockout mice, VEGFA, Chondrocyte survival, Bone development, Angiogenesis, HIF1, HIF1, VEGF
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Introduction |
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Vascular endothelial growth factor A (VEGFA) is an important regulator of
angiogenesis during endochondral ossification. Inhibition of VEGFA by
administration of soluble chimeric VEGFA receptor protein to 24-day-old mice
inhibited blood vessel invasion into the hypertrophic zone of long bone growth
plates and resulted in impaired trabecular bone formation and the expansion of
the hypertrophic zone (Gerber et al.,
1999). Further support for the role of VEGFA in angiogenesis of
hypertrophic cartilage came from studies of mice expressing only the VEGFA120
isoform of VEGFA (Maes et al.,
2002
; Zelzer et al.,
2002
).
VEGFA120 is one of three isoforms of VEGFA in the mouse
(Ferrara et al., 1992;
Shima et al., 1996
). VEGFA120
does not bind heparan sulfate, while the other two isoforms, VEGFA164 and
VEGFA188, possess one or two heparin-binding domains, respectively, allowing
interactions with heparan sulfate (Ferrara
and Davis-Smyth, 1997
; Park et
al., 1993
). Unlike heterozygous Vegfa null mice, mice
that express only the 120 isoform survive through embryonic development
(Carmeliet et al., 1999
).
Studies of blood vessel invasion into the primary ossification centers in
VEGFA120 mice demonstrated a delay in vessel invasion and alterations in the
extent of expression of cartilage differentiation markers at E14.5, indicating
a role for VEGFA in cartilage angiogenesis and maturation
(Maes et al., 2002
;
Zelzer et al., 2002
). The
skeletons of VEGFA120 mice showed decreased mineralization and a reduction in
the expression of osteoblastic markers in membranous and endochondral bone.
This suggests that VEGFA has a direct effect on the activity of osteoblasts
(Zelzer et al., 2002
). There
is also evidence from in-vitro experiments that VEGFA may regulate bone
formation through a direct effect on osteoblasts
(Deckers et al., 2000
;
Midy and Plouet, 1994
).
Thus, VEGFA appears to have several functions during bone formation. In
this study, we report for the first time that VEGFA is critical for
chondrocyte survival. We demonstrate that VEGFA, in addition to its
upregulated expression in hypertrophic chondrocytes of long bone growth
plates, is expressed at moderate levels in epiphyseal chondrocytes, and that
these chondrocytes undergo massive cell death in the absence of VEGFA. Based
on the striking similarity in the cartilage phenotypes resulting from
inactivation of either Vegfa or hypoxia-inducible factor 1
(Hif1a) in chondrocytes, we conclude that the chondrocyte survival
function of VEGFA is controlled by HIF1
.
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Materials and methods |
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Skeletal preparations
Cartilage and bones in whole mouse embryos (E18.5) were visualized after
staining with Alcian Blue and Alizarin Red S (Sigma) and clarification of soft
tissue with potassium hydroxide (McLeod,
1980).
Histology and immunohistochemistry
For histological analysis, embryonic limbs and heads were fixed in 4%
paraformaldehyde and embedded in paraffin for sectioning using standard
procedures. Sections of 7 µm thickness were stained with hematoxylin and
eosin (H & E). For CD31 immunohistochemistry, embryos were fixed in 4%
paraformaldehyde, followed by 20% sucrose infiltration. Tissues were embedded
in OCT (Tissue-Tek®) and 7 µm cryostat sections were made. Sections
were incubated with monoclonal rat anti-mouse CD31 (BD PharMingen). Sections
were incubated with biotinylated anti-rat IgG (Vector Laboratories), and an
ABC kit (Vector Laboratories) was used for detection.
In situ hybridization and TUNEL assay
In situ hybridization was carried out on paraffin sections with
33P-labeled anti-sense RNA essentially as described
(Hartmann and Tabin, 2000;
Zelzer et al., 2002
). For
TUNEL assay, paraffin sections were permeabilized with 0.1% Triton X-100 in
0.1% sodium citrate. TUNEL assay was performed using a Roche In situ Cell
Death Detection kit (Roche) according to the manufacturer's
recommendations.
Culture of primary epiphyseal chondrocytes
Epiphyseal chondrocytes were isolated from the knee joint region of newborn
mice. Skin, muscles and soft tissue were removed. DMEM containing 0.5 mg/ml
hyaluronidase (Sigma H-3506) was added and the tissue was digested for 30
minutes at 37°C on a rotating shaker. The medium was then replaced by
medium containing 1 mg/ml trypsin (Sigma T-1426) and incubation continued for
30 minutes at 37°C on a rotating shaker. The trypsin containing medium was
removed and cartilage was incubated twice with medium containing 1 mg/ml
bacterial collagenase (Sigma C-9263). Medium from the first incubation was
discarded after 20 minutes and then chondrocytes were isolated by collagenase
digestion for 2 hours at 37°C on a rotating shaker. The cells were passed
through a cell strainer (Falcon 352340) and were centrifuged at 400
g for 5 minutes. The cells were suspended in DMEM containing
10% FBS and 2% Penicillin/Streptomycin/Glutamine, plated at a density of 5
x 105 cells/well in a 24-well plate and incubated at 37°C
overnight.
Gene-specific RT-PCR analysis
Total RNA was extracted from cultured primary mouse epiphyseal chondrocytes
using Rneasy kit (Qiagen 74104). Five micrograms of total RNA were treated
with DNA-Free RNA Kit (Zymo Research) and reverse transcribed with Superscript
II First Strand Synthesis System for RT-PCR (Invitrogen). All genes were
amplified using Taq DNA polymerase (Roche) for 35 cycles and PCR products were
fractionated by gel electrophoresis. The primers used for the PCR
amplification were 5'CACAGATAAGCCCACCAGAG3' and
5'GACAACCACCGCAATGA3' for Cd31;
5'GGCTCAGGGTCGAAGTTAAAAGTGCCT3' and
5'TAGGATTGTATTGGTCTGCCGATGGGT3' for VEGFAr1;
5'CTCTGTGGGTTTGCCTGGCGATTTTCT3' and
5'GCGGATCACCACAGTTTTGTTCTTGTT3' for Vegfr2;
5'GCTGGCAGGAGGAGGAA3' and 5'TCCCGCTGTCTGTCTGGTTA3' for
Vegfr3; 5'TCCCGCCTGAACTACCCTGAA3' and
5'GCCTTGCGCTTGCTGTCATC3' for neuropilin 1;
5'CCCCGAACCCAACCAGAAGA3' and 5'GAATGCCATCCCAGATGTCCA3'
for neuropilin 2.
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Results |
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Lack of VEGFA in chondrocytes leads to impaired embryonic bone development
Staining of skeletons of mice that were heterozygous for both
floxed-Vegfa and Col2a1-Cre alleles (unaffected) and mice
that were heterozygous for Col2a1-Cre and homozygous for the
floxed-Vegfa alleles (conditional knockout, CKO) with Alizarin Red
and Alcian Blue showed that the Alizarin Red-stained zones were reduced in the
Vegfa CKO mice at E18.5, suggesting a reduction in mineralization
(Fig. 1). This reduction was
observed in long bones in the limbs (Fig.
1B), in ribs (Fig.
1D), in sternum (Fig.
1F), and in the bones of the vertebral column
(Fig. 1H). Furthermore, the
cartilaginous parts were smaller and stained less intensely for Alcian Blue in
the CKO tissues, suggesting a cartilage abnormality.
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Loss of VEGFA in chondrocytes results in massive cell death
Histological studies of Vegfa CKO femurs at E16.5 revealed a zone
of large, balloon-like cells, poorly stained and with a `ghost'-like
appearance, at the center of the epiphysis
(Fig. 4A,B). There was some
variation in the severity of the abnormalities, with the distal limb bones
showing the highest degree of abnormality. In the distal bones at E18.5,
extensive regions of dead cells were observed
(Fig. 4C,D). These regions of
cell death were located in the central regions of the skeletal elements,
starting at an articular surface and continuing through the resting to the
proliferating zones of chondrocytes and ending in a misshapen growth plate.
Depending on the severity of the phenotype, the growth plate was dramatically
affected and hypertrophic chondrocytes of normal shape were almost completely
missing. Outside the center of the growth plates, proliferating cells appeared
to accumulate (Fig. 4D). These
zones of proliferating cells were much more extended in Vegfa CKO
than in unaffected growth plates.
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Discussion |
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Hif1 and Vegfa are part of a chondrocyte survival pathway
HIF1 regulates the transcription of a broad range of genes that are
involved in a variety of processes such as glucose metabolism, angiogenesis
and cell survival (Pugh and Ratcliffe,
2003
; Semenza,
2003
). Several stimuli, such as hypoxia, hormones and growth
factors, induce stabilization of the HIF1 heterodimeric transcription complex.
This complex is composed of HIF1
and HIF1ß. HIF1ß is
constitutively expressed, whereas HIF1
is tightly regulated
(Maxwell et al., 1993
;
Semenza and Wang, 1992
;
Wang and Semenza, 1993
;
Wang and Semenza, 1995
;
Zelzer et al., 1998
); the
tumor-suppressor protein von Hippel-Lindau (VHL) protein is a key element in
this regulation (Bruick and McKnight,
2001
; Ivan et al.,
2001
; Jaakkola et al.,
2001
; Masson et al.,
2001
; Yu et al.,
2001
).
HIF1 is known to be a regulator of VEGFA in many systems
(Shima et al., 1995
). However,
in hypertrophic chondrocytes of Hif1a CKO growth plates,
Vegfa expression was comparable to the expression in unaffected,
control growth plates at E15.5 (Fig.
6). This suggests that Vegfa expression is not dependent
on Hif1a in hypertrophic chondrocytes, at least at that stage of
development. Runx2 and Ctgf are two genes that may be
involved in the regulation of VEGFA at that stage or later, since
Vegfa expression in the hypertrophic zones of Runx2 and
Ctgf null embryos is decreased
(Ivkovic et al., 2003
;
Zelzer et al., 2001
)
(Fig. 11). In epiphyseal
chondrocytes Hif1a may well be a major regulator of Vegfa
expression, since the expression of Vegfa was dramatically reduced at
E18.5 in Hif1a CKO bones (Fig.
6F). The important role of Hif1a in regulating expression
of Vegfa in epiphyseal chondrocytes is further demonstrated by the
recent analysis of the phenotype of mice that are homozygous for VHL
null alleles in chondrocytes (Pfander et
al., 2004
). In these VHL conditional knockout mice, levels of
hydroxylated HIF1
protein are elevated and expression of Vegfa
is enhanced in epiphyseal cartilage.
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Although VEGFA is best known for its activity as an angiogenic factor
(Ferrara et al., 2003), it has
been shown to be a survival factor for endothelial and hematopoietic stem
cells (Benjamin et al., 1999
;
Gerber et al., 1998a
;
Gerber et al., 2002
;
Gerber et al., 1998b
), and
here we describe a role for VEGFA in supporting chondrocyte survival. We
cannot at present rule out the possibility that Vegfa expression in
the epiphyseal chondrocytes regulates some aspect of vascularization in the
vicinity of the epiphysis (with secondary consequences for the survival of
epiphyseal chondrocytes), but we favor the view that VEGFA has a direct role
in chondrocyte survival. First, epiphyseal chondrocytes express three
receptors for VEGFA, namely Vegfr3, Nrp1 and Nrp2,
suggesting that VEGFA can initiate signaling in these cells. Interestingly, it
was recently demonstrated that Nrp1 can, independently of other receptors,
initiate cell signaling (Bachelder et al.,
2001
; Foster et al.,
2003
; Wang et al.,
2003
). Secondly, cell death in the Prx1/Vegfa
CKO epiphyseal cartilage was observed at E16.5
(Fig. 5H), the stage at which
we identified cell death in the Col2a1/Vegfa CKO mice,
suggesting that VEGFA is needed for chondrocyte survival at a specific
developmental time. This finding is particularly important, since the
cartilaginous elements in the Prx1/Vegfa CKO are
significantly smaller than in the Col2a1/Vegfa CKO mice,
suggesting that the cell death cannot be a simple consequence of a deficient
diffusion of nutrients and oxygen into the epiphysis. Consistent with this
interpretation is the recent report that incubation of chondrocytes in hypoxic
conditions did not cause cell death
(Pfander et al., 2003
).
Finally, when we analyzed vascularization in the vicinity of the cartilaginous
elements to examine a possible reduction in vessel numbers before cell death
occurs in the Vegfa CKO mice, we failed to observe any obvious
difference between unaffected and Vegfa CKO limbs.
Several studies have recently suggested a role for VEGFA during organ
development as a mediator of cell-cell interactions between endothelial and
parenchymal cells (Cleaver and Melton,
2003). We have therefore considered the possibility that the role
of VEGFA in supporting survival of epiphyseal chondrocytes may somehow involve
an interaction with endothelial cells in the vicinity of the cartilage. During
development of pancreas and liver there is a close physical interaction
between endothelial and endodermal cells and VEGFA was demonstrated to have an
important role in interactions that led to development of these organs
(Lammert et al., 2001
;
Matsumoto et al., 2001
). Since
chondrocyte differentiation and cartilage formation take place in an
endothelium-free environment, any involvement of endothelial cells in the
HIF1
/VEGFA chondrocyte survival pathway would have to include
long-range interactions.
VEGFA regulation of bone vascularity
Blood vessel invasion into the primary ossification center is a key step in
bone development. In this study we have provided further in-vivo evidence that
VEGFA is required for angiogenesis into the primary ossification center and
the maintenance of blood vessel growth in developing bones. At E15, vessels
are invading hypertrophic cartilage, and as development proceeds, the vessels
continue to grow under the growth plates as hypertrophic chondrocytes are
being removed. In the Vegfa CKO bones, there is a delay in blood
vessel invasion into the primary ossification center
(Fig. 2), and accumulation of
terminally differentiated chondrocytes in growth plates suggests a reduction
of vessel sprouting and cartilage removal
(Fig. 3). Since the angiogenic
process in the Vegfa CKO bones was not completely abolished, we
consider the possibility that VEGFA is not the only factor regulating the
process of endochondral angiogenesis, although it is also possible that the
Cre was less than fully efficient in generating a chondrocyte-specific
Vegfa null phenotype. However, comparing the phenotypes of the
Vegfa CKO and the Vegfa120 mice clearly demonstrates that
the 120 isoform cannot compensate for the loss of the other VEGFA isoforms as
an angiogenic regulator in endochondral bones, since the conditional loss of
Vegfa in chondrocytes results in an angiogenic phenotype similar to
the phenotype observed in mice that express only the 120 isoform
(Fig. 2) (Maes et al., 2002;
Zelzer et al., 2002
). In
contrast, the 120 isoform is clearly sufficient to compensate for the loss of
other isoforms as a survival factor for chondrocytes, since the massive cell
death observed in Vegfa CKO bones was not observed in
Vegfa120 mice (Maes et al.,
2002
; Zelzer et al.,
2002
). Runx2 null bones represent the most severe example
of a deficient angiogenic process in developing bones, since the hypertrophic
zone is not invaded by blood vessels
(Zelzer et al., 2001
). This
lack of endochondral angiogenesis in Runx2 null mice makes the mice
useful for further studies aimed at identifying other components of the
mechanism that regulates skeletal vascularization.
In conclusion, in this study we provide further in-vivo evidence for the
important role of VEGFA in blood vessel invasion into hypertrophic cartilage
during bone development. More importantly, we describe for the first time a
connection between VEGFA and chondrocyte survival during skeletal development.
The similarities in the phenotypes of Hif1a and Vegfa null
bones, together with the in-vivo finding that HIF1 is a regulator of
Vegfa expression, establishes that these two genes are part of a
pathway that regulates chondrocyte survival.
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ACKNOWLEDGMENTS |
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