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Impaired angiogenesis and endochondral bone formation in mice lackingthe vascular endothelial growth factor isoforms VEGF164 and VEGF188
Christa Maesa, Peter Carmelietb, Karen Moermansa, Ingrid Stockmansa, Nico Smetsa,Desire Collenb, Roger Bouillona, Geert Carmelieta,*
aLaboratory of Experimental Medicine and Endocrinology, KU Leuven, Leuven, B-3000, BelgiumbThe Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, KU Leuven, Leuven, B-3000, Belgium
Received 13 September 2001; received in revised form 19 October 2001; accepted 19 October 2001
Abstract
Vascular endothelial growth factor (VEGF)-mediated angiogenesis is an important part of bone formation. To clarify the role of VEGF
isoforms in endochondral bone formation, we examined long bone development in mice expressing exclusively the VEGF120 isoform
(VEGF120/120 mice). Neonatal VEGF120/120 long bones showed a completely disturbed vascular pattern, concomitant with a 35% decrease
in trabecular bone volume, reduced bone growth and a 34% enlargement of the hypertrophic chondrocyte zone of the growth plate.
Surprisingly, embryonic hindlimbs at a stage preceding capillary invasion exhibited a delay in bone collar formation and hypertrophic
cartilage calcification. Expression levels of marker genes of osteoblast and hypertrophic chondrocyte differentiation were significantly
decreased in VEGF120/120 bones. Furthermore, inhibition of all VEGF isoforms in cultures of embryonic cartilaginous metatarsals, through
the administration of a soluble receptor chimeric protein (mFlt-1/Fc), retarded the onset and progression of ossification, suggesting that
osteoblast and/or hypertrophic chondrocyte development were impaired. The initial invasion by osteoclasts and endothelial cells into
VEGF120/120 bones was retarded, associated with decreased expression of matrix metalloproteinase-9. Our findings indicate that expression
of VEGF164 and/or VEGF188 is important for normal endochondral bone development, not only to mediate bone vascularization but also to
allow normal differentiation of hypertrophic chondrocytes, osteoblasts, endothelial cells and osteoclasts. q 2002 Elsevier Science Ireland
Ltd. All rights reserved.
Keywords: Vascular endothelial growth factor; Vascular endothelial growth factor isoforms; Endochondral ossification; Bone development; Angiogenesis
1. Introduction
Angiogenesis is a crucial part of bone formation. During
endochondral ossification, an avascular cartilage template is
replaced by highly vascularized bone tissue. Chondrocytes in
the bone model core first become hypertrophic and produce a
calcified cartilaginous matrix, while perichondrial cells
differentiate into osteoblasts forming a mineralized bone
collar (Caplan, 1988). In contrast to immature chondrocytes,
which secrete angiogenic inhibitors (Moses et al., 1999;
Shukunami et al., 1999), hypertrophic cartilage switches to
production of angiogenic stimulators, thereby becoming a
target for capillary invasion and angiogenesis (Alini et al.,
1996; Carlevaro et al., 1997; Engsig et al., 2000; Vu et al.,
1998). This process is accompanied by apoptosis of termin-
ally differentiated chondrocytes, resorption of the cartilage
matrix by invading osteoclasts/chondroclasts and deposition
of mineralized matrix by osteoblasts (Erlebacher et al.,
1995). Genetic models with defects in normal functioning
of bone cells have been associated with impaired angiogen-
esis: absence of osteoblast differentiation and impaired chon-
drocyte hypertrophy (Komori et al., 1997; Otto et al., 1997),
disturbed chondrocyte differentiation (Colnot et al., 2001;
Lanske et al., 1999; Schipani et al., 1997), and defects in
matrix resorption (Holmbeck et al., 1999; Vu et al., 1998;
Zhou et al., 2000) were reported to affect bone vasculariza-
tion. The importance of angiogenesis in bone is also reflected
in processes of repair and pathology. Bone fracture healing
requires the restoration of blood supply (Ferguson et al.,
1999; Glowacki, 1998) and defects in bone vasculature
have been reported in osteoporosis and rickets (Green et
al., 1987; Hunter et al., 1991; Reeve et al., 1988).
Recent studies have suggested that vascular endothelial
growth factor (VEGF), a potent angiogenic stimulator, may
play an important role during endochondral bone formation.
Mechanisms of Development 111 (2002) 61–73
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0925-4773(01)00601-3
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* Corresponding author. Legendo, Onderwijs en Navorsing, Campus
Gasthuisberg, Herestraat 49, B-3000, Leuven, Belgium. Tel.: 132-16-
346-023; fax: 132-16-345-934.
E-mail address: geert.carmeliet@med.kuleuven.ac.be (G. Carmeliet).
Hypertrophic chondrocytes, but not resting or proliferating
chondrocytes, express VEGF in vivo (Carlevaro et al. 2000;
Gerber et al., 1999; Horner et al., 1999) and osteoblastic cells
in vitro express VEGF and its receptors (Deckers et al., 2000;
Harper et al., 2001). In addition, VEGF can induce migration
and differentiation of osteoblastic cells (Deckers et al., 2000;
Midy and Plouet, 1994). Furthermore, VEGF was shown to
stimulate the formation, survival and resorption activity of
osteoclasts in vitro (Nakagawa et al., 2000; Niida et al., 1999)
and to be a chemoattractant for osteoclasts invading into
developing long bones (Engsig et al., 2000). Evidence for
an important physiological role for VEGF in bone was
found recently, as administration of a soluble VEGF receptor
chimeric protein in juvenile mice suppressed blood vessel
invasion at the growth plate and concomitantly inhibited
endochondral bone formation (Gerber et al., 1999). Remark-
ably, inactivation of matrix metalloproteinase-9 (MMP-9)
resulted in a comparable bone phenotype (Vu et al., 1998),
suggesting the involvement of proteinases as well as VEGF
in the processes of angiogenesis, growth plate morphogen-
esis and endochondral ossification.
However, the gene for VEGF encodes at least three
spliced isoforms in mice: VEGF120, VEGF164 and VEGF188
(Breier et al., 1992). VEGF120 is a freely soluble protein that
fails to bind heparin. The longer isoforms show increasing
binding to heparan sulfate-containing proteoglycans on the
cell surface and the extracellular matrix, from where they
can be released by the action of proteolytic enzymes
(Ferrara and Davis-Smyth, 1997). All VEGF isoforms are
capable of binding the tyrosine kinase receptors Flt-1
(VEGFR1) and Flk-1/KDR (VEGFR2), which are expressed
on endothelial cells (Neufeld et al., 1999). Recently neuro-
pilin 1 (NRP1) has been identified as a new isoform-specific
VEGF receptor, binding VEGF164 but not VEGF120 (Soker et
al., 1998). Although the heparin binding capacities and
receptor binding characteristics of the VEGF isoforms
differ, little is known about their differential functions in
vivo. Therefore, mice were generated that express exclu-
sively the VEGF120 isoform (VEGF120/120 mice) by Cre/
loxP-mediated removal of exons 6 and 7 encoding the
isoforms of 164 and 188 amino acids. VEGF120/120 mice
had impaired postnatal myocardial angiogenesis, resulting
in ischemic cardiomyopathy and death by cardiac failure
before postnatal day 14 (Carmeliet et al., 1999), which
precluded investigation of adult mice. In the present study
we investigated bone development in VEGF120/120 perinatal
mice and show that combined inactivation of VEGF164 and
VEGF188 resulted in impaired bone vascularization, growth
plate morphogenesis and endochondral ossification. In addi-
tion, analysis of early embryonic VEGF120/120 mice revealed
a delay in the initial steps of long bone development. Also,
inhibition of VEGF in cultures of embryonic cartilaginous
metatarsals retarded the ossification process. Thus, we
present evidence that deletion of the VEGF164 and
VEGF188 isoforms disturbs the normal differentiation and
regulated activity of hypertrophic chondrocytes, osteo-
blasts, endothelial cells and osteoclasts, which is essential
for normal bone development.
2. Results
2.1. Impaired angiogenesis and endochondral ossification
in VEGF120/120 mice
A survey of the skeletal structure of VEGF120/120 mice
revealed that the long bones were shorter and thinner as
compared to wild-type (WT) littermates (Fig. 1). The differ-
ence in tibia size between VEGF120/120 and WT mice was
C. Maes et al. / Mechanisms of Development 111 (2002) 61–7362
Fig. 1. Impaired bone growth in VEGF120/120 mice. (A) Staining with Alcian
Blue and Alizarin Red S of WT and VEGF120/120 tibia at E18.5, showing
size difference. (B,C) Quantification of tibia length (B) and width (C) of
WT and VEGF120/120 mice, as a function of age. Values are means ^ SEM.
*P , 0:05; **P , 0:01; ***P , 0:001 (t-test, versus WT; n ¼ 3–11).
significant from embryonic day (E) 16.5 on. By postnatal
day (P) 0.5, the reduction of tibia length and width was 10%
(P , 0:01) and 15% (P , 0:001), respectively, suggesting a
defect in the process of endochondral ossification.
Immunohistochemical staining for CD34, performed on
E18.5 and P0.5 tibia, showed that bone vascularization was
completely disturbed in VEGF120/120 mice (Fig. 2A,B).
Metaphyseal blood vessels were oriented rather randomly
in VEGF120/120 mice, in contrast to the orderly directional
growth of blood vessels towards the longitudinal septae of
the terminal row of hypertrophic chondrocytes in WT
bones. Blood vessels showed considerable dilatation and
blood vessel density was reduced by 28% in VEGF120/120
mice as compared to WT littermates at P0.5 (P , 0:01)
(Fig. 2C). Concomitantly, intercapillary distance was signif-
icantly greater in VEGF120/120 than in WT bones (119 mm vs.
86 ^ 5 mm (n ¼ 4) at P0.5, P , 0:01). Comparable results
were obtained in E18.5 bones, the decrease in blood vessel
density at this stage being 36% and the intercapillary
distance enlarged to 122% of WT.
The disturbed vascularization was associated with altera-
tions in bone mineralization, as assessed by Von Kossa
C. Maes et al. / Mechanisms of Development 111 (2002) 61–73 63
Fig. 2. Histological and histomorphometrical analysis of bone vascularization (A–C), mineralization (D–F) and growth plate morphology (G-I). (A,B)
Immunostaining for CD34 of neonatal (P0.5) WT (A) and VEGF120/120 (B) proximal tibia, showing severe dilatation and irregular pattern of blood vessels
in VEGF120/120 mice. (C) Quantification of capillary density in WT and VEGF120/120 proximal tibia at E18.5 and P0.5, demonstrating a significant reduction in
the mutant mice. (D,E). Von Kossa staining of WT (D) and VEGF120/120 (E) tibia sections at P0.5. (F) Quantification of TBV at E18.5 and P0.5 was performed
in a defined area of the proximal tibia metaphysis. VEGF120/120 mice exhibit a significant decrease in TBV compared to WT. (G,H) Toluidine staining of P0.5
WT (G) and VEGF120/120 (H) tibia, showing the enlargement of the hypertrophic chondrocyte zone of the growth plate. (I) Quantification of the length of the
hypertrophic cartilage zone in the proximal tibia of WT and VEGF120/120 mice. Values are expressed as means ^ SEM. *P , 0:05; **P , 0:01; ***P , 0:001
(t-test, versus WT; n ¼ 4–8). Scale bar: 160 mm.
staining (Fig. 2D,E). Trabecular bone volume (TBV) was
significantly reduced in the proximal tibia of E18.5 and P0.5
VEGF120/120 mice, being approximately 65% of WT TBV
(Fig. 2F). Bones from VEGF120/120 mice showed consider-
ably decreased trabeculae size (Fig. 2D,E). In addition, at
P5, total calcium content in femur was significantly reduced
in VEGF120/120 versus WT mice (10.1 ^ 0.5 mg (n ¼ 6) vs.
14.6 ^ 0.6 mg (n ¼ 3), P , 0:01).
Since bone length is determined in part by the activity of
the growth plate during endochondral bone formation, we
examined growth plate morphology in VEGF120/120 animals.
The zone of hypertrophic cartilage of E18.5 and P0.5
VEGF120/120 mice showed a significant expansion of 23
and 34%, respectively (P , 0:05) (Fig. 2G–I). Cartilage
calcification seemed normal, as the proportion of hyper-
trophic chondrocytes calcifying their extracellular matrix
was identical in VEGF120/120 and WT (data not shown). No
manifest alterations were detected in the proliferating,
maturing and prehypertrophic zones of the growth plate in
VEGF120/120 mice, suggesting that early stages of chondro-
cyte development were normal.
Taken together, inactivation of VEGF164 and VEGF188
impaired angiogenesis in long bones, which was associated
with reduced bone lengthening and widening, decreased
trabecular bone volume and an expansion of the hypertrophic
chondrocyte zone of the growth plate. These data indicate
that the VEGF isoforms play a pivotal role in the coordina-
tion of the key events of endochondral bone formation.
2.2. Isoform-specific role of VEGF in bone
Possibly, the skeletal abnormalities in VEGF120/120 mice
could be due to a VEGF dosage effect, as the angiogenic
activity of VEGF is highly dose-dependent (Carmeliet et
al., 1996; Ferrara et al., 1996). Interestingly however, total
VEGF mRNA level was not different between the two geno-
types, due to a more than 2-fold increase of the VEGF120
mRNA level in VEGF120/120 bones, as revealed by real-time
quantitative RT-PCR analysis (Fig. 3C,D). In WT bones,
VEGF164 constituted about 50–70% of total VEGF levels,
VEGF120 constituting about 30–50% and VEGF188 less than
1% (data not shown), as approximated by real-time RT-PCR.
In addition, immunostaining for total VEGF showed no
differences in VEGF expression pattern between VEGF120/
120 and WT bones at P0.5 (not shown) and E14.5 (Fig. 3A,B).
In both genotypes, VEGF was expressed abundantly in
hypertrophic chondrocytes, as well as in a few prehyper-
trophic chondrocytes and in some cells localized in the peri-
chondrium. Furthermore, quantitative RT-PCR showed
similar mRNA levels of the VEGF receptors Flt-1 and
NRP1 and of the VEGF family members PlGF, VEGF-B
and VEGF-C in VEGF120/120 and WT E16.5 femurs (data
not shown).
2.3. Delayed cartilage calcification and bone collar
formation in VEGF120/120 embryos
To investigate whether the VEGF120/120 bone phenotype
was exclusively related to a defect in vascularization, we
examined early stages of endochondral ossification. At
E14.5, no sign of blood vessel invasion into the cartilage
mold was observed in any of the genotypes, as evidenced by
the lack of staining with antibodies against the endothelial
cell marker CD31 (PECAM-1) (data not shown). At this
stage, the diaphysis of WT long bones consisted primarily
of hypertrophic chondrocytes whose extracellular matrix
had started to be calcified, as visualized on Von Kossa
stained sections (Fig. 4A). A perichondrial mineralized
bone collar surrounded the midshaft cartilage. Strikingly,
the initial calcification process was considerably reduced
in VEGF120/120 embryos, with only scarcely detectable calci-
fication in the matrix surrounding the hypertrophic chondro-
cytes (Fig. 4B). In addition, the bone collar was
underdeveloped in VEGF120/120 mice, as reflected by its
decreased length and thickness. Thus, inactivation of
VEGF164 and VEGF188 resulted in a delay of the calcification
process at the start of endochondral ossification, preceding
the initial capillary invasion of the bone.
C. Maes et al. / Mechanisms of Development 111 (2002) 61–7364
Fig. 3. VEGF expression. (A,B) Immunostaining for VEGF on sections through the diaphysis of E14.5 WT (A) and VEGF120/120 (B) hindlimbs, showing
abundant VEGF expression in hypertrophic chondrocytes (hc). Staining is also seen in some prehypertrophic chondrocytes (phc) and in cells localized in the
perichondrium (pe). Scale bar: 100 mm. (C,D) Quantitative RT-PCR analysis of total VEGF (C) and VEGF120 (D) mRNA on E16.5 femurs from WT and
VEGF120/120 mice. WT values were set at 100%. Values are shown as means ^ SEM. *P , 0:05.
2.4. Molecular analysis of chondrocyte and osteoblast
differentiation in VEGF120/120 mice
The decreased cartilage calcification and bone collar
formation in VEGF120/120 embryos led us to assess whether
the regulation of chondrocyte and osteoblast development
was disturbed, by investigating expression of stage-specific
differentiation markers of these cell types in embryonic
hindlimbs. Chondrocyte differentiation was investigated
via in situ hybridization for collagens type II and type X.
Collagen II expression in E14.5 long bones was found in
resting, proliferating, mature and early hypertrophic chon-
drocytes, with no significant differences between VEGF120/
120 and WT animals (Fig. 5A,B). The expression signal of
collagen X, a specific marker of hypertrophic chondrocytes,
covered the whole diaphysis in VEGF120/120 femurs, while in
WT mice an area was noticed in the midshaft center where
cells had ceased expressing collagen X (Fig. 5C,D). This
observation indicated that the middiaphyseal cells in the
WT bones had already attained a more progressed develop-
mental stage, in contrast to the hypertrophic chondrocytes in
the VEGF120/120 mice, which were still actively producing
collagen X. These data demonstrate that at this stage the
terminal differentiation of hypertrophic chondrocytes was
delayed in VEGF120/120 mice, explaining the retarded carti-
lage calcification.
In situ hybridization for core binding factor a1 (Cbfa1/
Osf2), a transcriptional activator of osteoblast differentia-
tion (Ducy et al., 1997) and a regulator of chondrocyte
C. Maes et al. / Mechanisms of Development 111 (2002) 61–73 65
Fig. 5. Expression patterns of markers of chondrocyte and osteoblast development. (A–D) In situ hybridisation for collagen type II (A,B) and type X (C,D) on
adjacent sections through femurs from WT (A,C) and VEGF120/120 (B,D) littermates at E14.5. Scale bar: 100 mm. The pattern of collagen II transcripts, marking
proliferating and prehypertrophic chondrocytes, is similar in both genotypes (A,B). In WT, expression of collagen X, a marker of hypertrophic chondrocytes, is
observed in two areas adjacent to the diaphysis center that is devoid of collagen X expression (C). In contrast, in VEGF120/120 bones collagen X expression is
detected in the center (D), indicating a delay in terminal chondrocyte differentiation. (E–H) Radioactive in situ hybridisation for Cbfa1 on E15.5 WT (E,G) and
VEGF120/120 (F,H) tibia, showing darkfield (E,F) and brightfield (G,H) images. Scale bar: 150 mm. Photographs were made using identical settings for both
genotypes. Pattern of expression is similar in both genotypes, yet staining intensity is strongly reduced in VEGF120/120 bones. (I,J) Immunostaining for
osteocalcin on E15.5 tibia of WT (I) and VEGF120/120 (J) littermates. Scale bar: 50 mm. In WT, abundant osteocalcin expression is found in the perichondrial
bone collar region, while VEGF120/120 bones show weak expression in the perichondrial area.
Fig. 4. Von Kossa staining of sections through the diaphysis of WT (A) and
VEGF120/120 (B) tibia at E14.5, illustrating impaired hypertrophic cartilage
calcification and bone collar formation in the VEGF120/120 avascular carti-
laginous bone model. Scale bar: 50 mm.
hypertrophy (Takeda et al., 2001), was performed on E15.5
VEGF120/120 and WT hindlimbs. Cbfa1 expression was
found in both genotypes in the perichondrial region where
the bone collar was being formed and in the cartilaginous
center of the bone (Fig. 5E–H). Although the pattern of
expression was identical, staining intensity was strongest
in WT bones, suggesting a reduced level of expression in
VEGF120/120 mice. Similar results were obtained for osteo-
calcin, which is considered as a specific marker of mature
osteoblasts (Ducy et al., 2000). Immunostaining displayed a
normal expression pattern, the osteocalcin protein being
most prominently localised in the bone collar in both geno-
types at E15.5, yet staining was less abundant in VEGF120/120
bones (Fig. 5I,J). These histological findings were
confirmed by quantitative real-time RT-PCR analysis of
various genes associated with osteoblast development.
Cbfa1 as well as collagen I mRNA expression levels were
reduced in VEGF120/120 femurs as compared to WT at E16.5,
but not at E18.5 (Fig. 6A,B). Furthermore, mRNA levels of
osteopontin and osteocalcin were significantly reduced in
VEGF120/120 bones, both at E16.5 and E18.5 (Fig. 6C,D).
Taken together, our data indicate that deletion of
VEGF164 and VEGF188 resulted in alterations in hyper-
trophic chondrocyte and osteoblast development.
2.5. Blocking VEGF impairs the ossification process in
embryonic metatarsal cultures
As mentioned above, VEGF120/120 embryos exhibited a
retarded calcification of the diaphyseal hypertrophic carti-
lage and formation of the bone collar, and a reduced expres-
sion of genes associated with hypertrophic chondrocyte and
osteoblast differentiation. These findings suggested that
VEGF might exert direct actions on chondrocytes and/or
osteoblasts. To establish further this possible role we
analyzed the effect of blocking VEGF activity on ossifica-
tion in E16.5 metatarsals grown in organ culture. Treatment
of metatarsals with soluble VEGF receptor, mFlt-1/Fc
chimera, retarded the onset of the ossification center
which was seen in control metatarsals after 1–2 days of
culture (Fig. 7), and reduced the length of the ossification
center after 3 days with 20% (P ¼ 0:003; n ¼ 10). This
inhibition was in agreement with the VEGF120/120 in vivo
results, suggesting that absence of VEGF164 and/or
VEGF188 signaling results in an impaired progression of
the osteogenic and/or chondrogenic program that leads to
the formation of the primitive bone collar and, later, of
trabecular bone.
2.6. Cartilage resorption and invasion by osteoclasts and
endothelial cells are retarded in VEGF120/120 mice
The terminal stage of hypertrophic chondrocyte develop-
ment is associated with invasion and resorption of the calci-
C. Maes et al. / Mechanisms of Development 111 (2002) 61–7366
Fig. 6. Quantitative RT-PCR analysis of genes related to osteoblast
(collagen I, osteocalcin) or osteoblast and chondrocyte (Cbfa1, osteopontin)
differentiation in E16.5 and E18.5 femurs (n ¼ 9–13). Expression of Cbfa1
(A) and collagen I (B) mRNA is decreased in VEGF120/120 bones at E16.5,
but not at E18.5, while levels of osteopontin (C) and osteocalcin (D) mRNA
expression are significantly reduced at both ages. Values are shown as
means ^ SEM and represent the relative expression level, determined as
the ratio of the respective signal to the signal obtained for the HPRT gene.
*P , 0:05; **P , 0:01; versus WT.
Fig. 7. Effect of inhibition of VEGF on ossification in embryonic metatar-
sals grown in organ culture. Alcian Blue and Alizarin Red S staining of
E16.5 metatarsals cultured for 1.5 days without (A) or with soluble VEGF
receptor mFlt-1/Fc chimera (B). Formation of an ossification center was
initiated in control metatarsals, while treatment with mFlt-1/Fc delayed this
process. Scale bar: 250 mm.
fied cartilage core. Given the delay in hypertrophic cartilage
differentiation and calcification in VEGF120/120 mice, we
investigated whether VEGF164 and VEGF188 deficiency
disturbed the cartilage resorption process. At E16.5, it was
apparent that resorption of hypertrophic cartilage and
formation of the bone marrow cavity were strongly
decreased in VEGF120/120 mice, as the length of the resorbed
diaphyseal area was 40% reduced in mutant versus WT tibia
(Fig. 8). To further explore the molecular basis of the
impaired cartilage resorption, two fundamental aspects of
this process were analyzed, namely expression of MMP-9
(immunohistochemistry) and recruitment of and invasion by
osteoclasts (TRAP staining) and endothelial cells (CD31
immunostaining) in the developing long bones, on sets of
subsequent sections. Before the formation of the marrow
cavity MMP-9 expression was found in the mesenchyme
surrounding the bone rudiment, where at this stage only a
few TRAP-positive cells appeared (not shown), as has also
been reported by others (Blavier and Delaisse, 1995).
Subsequently, when resorption of the cartilage mold started,
MMP-9 protein was not only detected in the bone collar
area, but also along the resorption front adjacent to the
hypertrophic chondrocytes in the core of the diaphysis, as
can be seen in E15.5 WT tibia (Fig. 9A). At the stage of
bone marrow cavity formation, as in E15.5 WT femur,
MMP-9 was expressed at the cartilage resorption front and
to a lesser extent in the primitive marrow cavity (Fig. 9B),
where large TRAP-positive multinucleate osteoclasts and
endothelial cells were abundant (Fig. 9C–E). Analysis of
VEGF120/120 littermates revealed that resorption of calcified
hypertrophic cartilage and invasion by osteoclasts and
endothelial cells were strongly retarded. MMP-9 expression
in E15.5 mutant tibia was confined to cells in the perichon-
C. Maes et al. / Mechanisms of Development 111 (2002) 61–73 67
Fig. 8. Bone marrow cavity formation is delayed in VEGF120/120 long bones.
(A,B) Hematoxylin and eosin staining of WT (A) and VEGF120/120 (B) tibia
at E16.5. Scale bar: 100 mm. (C) Analysis of bone marrow cavity length in
WT and VEGF120/120 tibia at E16.5 shows a significant reduction in
VEGF120/120 mice. Values are means ^ SEM. **P , 0:01 (n ¼ 4).
Fig. 9. Histological sections through the diaphysis of WT (A–E) and VEGF120/120 (F–J) tibias (A,F) and femurs (B–E,G–J) at E15.5, showing the retardation in
MMP-9 expression, cartilage resorption and invasion by osteoclasts and endothelial cells in VEGF120/120 bones. Sets of adjacent sections were immunostained
for MMP-9 (A,B,F,G), or stained for TRAP activity (C,E,H,J) or CD31 immunoreactivity (D,I) to visualize (pre)osteoclasts and endothelial cells, respectively.
MMP-9 immunoreactivity in WT tibia is found along the initial cartilage resorption front and in the area of cellular invasion (A), whereas staining in VEGF120/
120 tibia is only observed in the perichondrial cell layer (F). In WT femur, the resorption process has progressed to the formation of the primitive bone marrow
cavity showing abundant MMP-9 expression (B), TRAP-positive osteoclasts (C) and endothelial cells (D). In VEGF120/120 femur the resorption process is
retarded, as shown by restricted MMP-9 immunoreactivity (G) and localization of osteoclasts (H) and endothelial cells (I), although the patterning seems
normal. (E,J) Magnified views of the area indicated by a rectangle in TRAP-stained sections, showing large multinucleate osteoclasts. Scale bars: (A–D,F–I)
100 mm; (E,J) 25 mm.
drial cell layer and no invasion was observed (Fig. 9F). In
the femur, a small resorption area was apparent with expres-
sion of MMP-9 at the resorption front (Fig. 9G). At that
time, large mature TRAP-positive osteoclasts had just
started to invade the calcified cartilage core, together with
endothelial cells (Fig. 9H–J). The distribution of TRAP-
positive cells and endothelial cells correlated closely during
initial invasion of the calcified cartilage and marrow cavity
development. These histological findings were confirmed by
quantitative RT-PCR analysis. Corresponding to the delay
in the resorption process, MMP-9 mRNA levels were 41%
reduced in VEGF120/120 versus WT bones at E16.5
(P ¼ 0:012; n ¼ 10). By E18.5 however, MMP-9 mRNA
reached normal levels in VEGF120/120 mice. Taken together,
these findings indicate that VEGF120/120 mice exhibited a
delay in the key steps in cartilage resorption of developing
long bones, namely expression of MMP-9 and invasion of
TRAP-positive cells and endothelial cells into the calcified
cartilage. Correspondingly, the degradation of the cartilage
model and the development of the marrow cavity were
retarded, although the normal patterning of these events
was not impaired.
3. Discussion
In this study we demonstrate that loss of the VEGF164 and
VEGF188 isoforms completely disturbed bone vasculariza-
tion, and concomitantly resulted in a decreased trabecular
bone volume and an abnormal growth plate morphology in
perinatal mice. In addition, examination of VEGF120/120
embryos showed that bone development was retarded
even at a stage prior to the initial capillary invasion of the
cartilaginous bone model. Furthermore, inhibition of VEGF
in cultures of normal embryonic cartilaginous metatarsals,
through the administration of a soluble receptor chimeric
protein (mFlt-1/Fc), delayed ossification. These data indi-
cate that expression of VEGF164 and/or VEGF188 is impor-
tant for normal angiogenesis, endochondral ossification and
growth plate development, and suggest the involvement of
direct VEGF effects on bone cells during the initial stages of
endochondral bone formation.
3.1. VEGF164 and VEGF188 are essential for the coupling of
bone angiogenesis and endochondral ossification
The combined lack of VEGF164 and VEGF188 caused an
abnormal development of the bone vascularity, resulting in
an irregular blood vessel pattern with a reduced number of
vessels that were highly dilated. The reduced trabecular
bone volume was likely a consequence of the impaired
angiogenesis resulting in an insufficient supply of oxygen,
nutrients and growth factors. Osteoblasts respond to
hypoxia by producing VEGF (Akeno et al., 2001; Stein-
brech et al., 1999), which on its turn is a participant of the
finely regulated cross-talk between bone endothelial cells
and osteoblasts that is only beginning to be characterized
(Collin-Osdoby, 1994; Streeten and Brandi, 1990; Villars et
al., 2000; Wang et al., 1997). Angiogenesis is well known to
be a prerequisite for osteogenesis, and the vasculature is
considered as a source of bone marrow stromal cells,
which are progenitors of skeletal tissue components (Bianco
and Robey, 2000). Another striking feature in VEGF120/120
mice is the enlargement of the hypertrophic chondrocyte
zone in the growth plate. A comparable phenotype, that
was attributed to delayed chondrocyte apoptosis, was
observed in mice with a targeted mutation in the gene for
the proteolytic enzyme MMP-9 (Vu et al., 1998), in mice
with conditional deletion of a single VEGF allele in cells
expressing collagen type II (Haigh et al., 2000) and in juve-
nile mice with inhibition of VEGF by injection of soluble
VEGF receptor chimeric protein (mFlt-(1–3)-IgG) (Gerber
et al., 1999). These studies suggested that MMP-9 and
VEGF provide a molecular link between matrix solubiliza-
tion, capillary invasion of the growth plate and hypertrophic
chondrocyte apoptosis. The growth plate was however more
severely enlarged in these models as compared to the
VEGF120/120 phenotype, indicating that the VEGF120 isoform
is able to partially induce cartilage resorption, possibly by
affecting MMP-9 expression at the growth plate. Yet, in the
study of Gerber et al. (1999), inhibition of other VEGF-
related molecules binding Flt-1, such as PlGF and VEGF-
B, may have contributed to the phenotype as well.
The data presented here provide direct evidence that
angiogenesis mediated by VEGF isoforms is an essential
signal in the regulation of trabecular bone formation and
growth plate morphogenesis during the process of endo-
chondral ossification.
3.2. Inactivation of VEGF164 and VEGF188 inhibits the initial
embryonic stages of endochondral ossification
During early embryonic bone development, chondrocyte
hypertrophy coincides with differentiation of mesenchymal
cells in the diaphyseal perichondrium into osteoblasts to
form a mineralized ‘bone collar’ around the cartilage core
(Caplan, 1988). The molecular players in this coordination
are becoming characterized. Cbfa1 and Indian hedgehog
have been implicated in the common regulation of osteo-
blast and chondrocyte differentiation and bone collar forma-
tion (St-Jacques et al., 1999; Takeda et al., 2001). In
addition, Cbfa1 has recently been implicated in the regula-
tion of VEGF gene expression during endochondral bone
formation (Zelzer et al., 2001). In embryonic VEGF120/120
bones both bone collar formation and cartilage matrix calci-
fication were decreased at a stage preceding vascularization.
The proliferation and maturation of chondrocytes seemed
normal, while terminal differentiation of hypertrophic chon-
drocytes was retarded, as evidenced by collagen type II and
type X expression. The levels of expression of several genes
associated with osteoblast and hypertrophic chondrocyte
differentiation were reduced. Thus, our data indicate that
in addition to the strongly impaired endochondral ossifica-
C. Maes et al. / Mechanisms of Development 111 (2002) 61–7368
tion in vascularized bones of perinatal mice, absence of
VEGF164 and VEGF188 also resulted in retardation in bone
development during the initial steps of endochondral ossifi-
cation, preceding capillary invasion. At this developmental
stage, VEGF is being produced by hypertrophic chondro-
cytes and perichondrial cells, possibly osteoblasts. These
cells express also receptors for VEGF, more precisely,
hypertrophic chondrocytes express NRP1, whereas peri-
chondrial cells produce Flt-1 and Flk-1 (Colnot and
Helms, 2001; Zelzer et al., 2001). Our findings suggest
direct effects of VEGF164- and/or VEGF188-signaling on
hypertrophic chondrocytes and/or osteoblasts, via an auto-
crine or paracrine loop, contributing to the process of timely
coordinated osteoblast and hypertrophic chondrocyte differ-
entiation. This hypothesis is supported by the in vitro
experiments showing reduced ossification in WT metatar-
sals when cultured with a soluble VEGF receptor 1 chimeric
protein (mFlt-1/Fc). The effect of inactivation of PlGF and
VEGF-B can however not be excluded in these experiments.
Recent observations in vitro also support a role for VEGF
signaling in osteoblasts, particularly during the late stages of
their differentiation when highest expression of VEGF and
its receptors is found (our unpublished results; Deckers et
al., 2000).
Following the initial bone collar formation, the calcified
extracellular matrix surrounding hypertrophic chondrocytes
becomes degraded by invading chondroclasts and/or osteo-
clasts. Vascular invasion occurs and the cartilaginous matrix
is then replaced with a bone matrix secreted by invading
osteoblasts. As shown, the cartilage resorption front is asso-
ciated with MMP-9 expression, whereas TRAP-positive
cells and endothelial cells fill the marrow cavity. The coor-
dinate invasion of resorptive and endothelial cells was
delayed in VEGF120/120 embryonic bones, resulting in a
retardation in the initial cartilage resorption, neovasculari-
zation and marrow cavity formation. The delayed recruit-
ment of resorptive cells in VEGF120/120 mice may be coupled
to the slower differentiation of hypertrophic chondrocytes
and osteoblasts. Alternatively, the lack of VEGF165 and/of
VEGF188 may alter the formation, migration, resorptive
activity and/or survival of osteoclasts, as VEGF has been
shown to exert these effects (Engsig et al., 2000; Nakagawa
et al., 2000; Niida et al., 1999) and osteoclasts express the
VEGF receptors Flt-1, Flk-1 and NRP1 (Harper et al., 2001;
Nakagawa et al., 2000).
Taken together, our findings strongly suggest that VEGF
isoforms exert direct actions on bone cells during endochon-
dral bone formation and show that the timely invasion of
resorptive and endothelial cells during early bone develop-
ment is dependent on the expression of VEGF164 and/or
VEGF188.
3.3. Specific differential functions of VEGF isoforms during
endochondral ossification?
In view of the strong dose-dependency of VEGF angio-
genic activity (Carmeliet et al., 1996; Ferrara et al., 1996), it
would be reasonable to assign the defective bone develop-
ment in the VEGF120/120 mice to a reduction in total VEGF
levels. However, total VEGF mRNA amounts were equiva-
lent in VEGF120/120 and WT bones, as shown by quantitative
RT-PCR. In addition, immunohistochemical analysis did
not indicate any difference in total VEGF expression pattern
between VEGF120/120 and WT mice. The abnormal pheno-
type might also be explained by the 2-fold increase in
VEGF120 since overexpression studies have shown that
elevated levels of VEGF120 induces irregular, dilated vessels
and hypervascularization (Cheng et al., 1997; Flamme et al.,
1995; Larcher et al., 1998). On the other hand, it has
recently been shown in 24-day-old mice that administration
of soluble VEGF receptor, inhibiting all VEGF isoforms,
results in a comparable bone phenotype to that described
here (Gerber et al., 1999). Therefore, the most likely
hypothesis is that the VEGF164 and/or VEGF188 isoforms
are essential for normal bone angiogenesis and the coupling
to endochondral bone formation.
The various VEGF isoforms might exert specific func-
tions during endochondral bone development due to their
differential localization, as the longer isoforms bind more
avidly to heparan sulfate-rich extracellular matrix, or to
their different receptor binding characteristics (Ferrara,
2001). NRP1, a VEGF164-specific receptor (Soker et al.,
1998), is expressed in vivo by osteoblasts (Harper et al.,
2001) and by hypertrophic chondrocytes (Colnot and
Helms, 2001). In addition, overexpression of NRP1 resulted
in hindlimb anomalies in mouse embryos (Kitsukawa et al.,
1995). Lack of activation of NRP1 due to the absence of
VEGF164 and/or impaired signaling through the receptors
Flt-1 and Flk-1 by the absence of heterodimerization
between the various VEGF isoforms may have caused or
contributed to the abnormal VEGF120/120 phenotype. The
relative importance of specific VEGF isoforms in different
biological functions may also be reflected by their tissue-
specific expression patterns. This feature may explain the
severe dysfunction of the heart in neonatal VEGF120/120
mice, since VEGF120 constitutes only about 5% of the
total VEGF levels in WT hearts (Carmeliet et al., 1999).
In contrast, in bone tissue VEGF120 makes up about 30–
50% of the total VEGF amount in WT mice. Ongoing
studies characterizing the bone phenotypes in mice expres-
sing exclusively the VEGF164 or VEGF188 isoforms will
further determine their common and distinct roles in bone
angiogenesis, bone formation and remodeling. In view of
the recent advances in VEGF gene therapy protocols for
therapeutical angiogenesis, where VEGF120 and VEGF164
now seem to be used indiscriminately, it will be important
to detect potential side-effects of specific VEGF isoforms on
other organs, including bone.
In conclusion, we have shown that expression of VEGF164
and/or VEGF188 is essential for normal endochondral bone
development, not only to mediate the establishment of the
bone vascularization but also to allow normal differentiation
C. Maes et al. / Mechanisms of Development 111 (2002) 61–73 69
and function of hypertrophic chondrocytes, osteoblasts,
endothelial cells and osteoclasts.
4. Experimental procedures
4.1. Animals
Mice expressing exclusively the VEGF120 isoform
(VEGF120/120 mice) (Carmeliet et al., 1999) were bred in
our animal housing facilities (Proefdierencentrum Leuven,
Belgium) under conventional conditions. Intercrossing of
heterozygous animals gave rise to homozygous VEGF120/
120 and WT mice. The day pups were born was stated as
postnatal day 0 (P0). The age of embryos was stated as
embryonic day (E), where E0.5 is the morning of the day
a vaginal plug was observed following overnight mating.
Deletion of the genomic sequences encoding VEGF164 and
VEGF188 was tested by Southern blot analysis of genomic
DNA from tail or liver biopsies. The experiment was
conducted after obtaining formal approval by the ethical
committee of the Katholieke Universiteit Leuven.
4.2. Histology, skeletal preparation and bone calcium
measurement
Neonatal long bones (P0.5 or P5) were fixed in 1% paraf-
ormaldehyde overnight and decalcified in 0.5 M EDTA (pH
7.4)/PBS for 7 days at 48C prior to dehydration, embedding
in paraffin and sectioning (5 mm). Additional bones were
fixed in Burckhardt’s solution, embedded undecalcified in
methyl-methacrylate and sectioned at 4 mm using a tungsten
carbide 508 knife. Embryonic bones were paraffin-
embedded without decalcification. Staining with hematox-
ylin and eosin, toluidine blue or Von Kossa’s stain for
mineralization was performed using standard protocols.
Osteoclasts were visualized on paraffin sections reacted
for tartrate-resistant acid phosphatase (TRAP) activity
essentially as described by Rice et al. (1997) and counter-
stained with Light Green SF Yellowish. For skeletal
preparations, embryos were skinned, eviscerated and the
skeletons stained with Alcian Blue (cartilage) and Alizarin
Red S (bone) using standard procedures (McLeod, 1980).
Total calcium content was determined by microcolorimetry
(Sigma) in HCl-dissolved ash dilutions of P5 femurs,
obtained by burning the bones in a muffle furnace for 24 h
at 1008C, followed by 24 h at 6008C.
4.3. Immunohistochemistry
Sections were deparaffinized, rehydrated, incubated when
necessary in Antigen Retrieval solution (DAKO) for 20 min
at 958C, and washed in 0.01 M Tris–HCl, 0.15 M NaCl, pH
7.6 (TBS). Sections were immersed in 0.3% H2O2 in metha-
nol for 20 min, followed by three washes in TBS. Unspecific
binding was blocked by incubating the sections for 30 min
in either TBS with 2% BSA or 0.1 M Tris–HCl, 0.15 M
NaCl, pH 7.6 (TNT) with 0.5% Blocking Reagent (NEN),
depending on the primary antibody used. Subsequently,
sections were incubated overnight with primary antibody
diluted in the corresponding blocking solution, followed
by three washes with TNT containing 0.05% Tween-20.
The following antibodies were used: rat anti-mouse CD31
and CD34 biotinylated antibodies (Pharmingen), and rabbit
polyclonal antibodies against human VEGF (VEGF(A-20),
Santa Cruz Biotechnology), mouse MMP-9 (Lijnen et al.,
1998) and osteocalcin (Verhaeghe et al., 1989). When non-
biotinylated primary antibodies were used, incubation with
a biotinylated swine anti-rabbit secondary antibody
(DAKO) was performed for 1 h. After three additional
washes, slides were exposed to horseradish peroxidase-
conjugated streptavidin (NEN) for 30 min. When necessary,
signal detection was indirectly using Tyramide Signal
Amplification-Indirect Kit (NEN). Antibody binding was
visualized with diaminobenzidine (DAB) and sections
were lightly counterstained with hematoxylin.
4.4. Histomorphometric analysis
Histomorphometric analysis of mineralization and vascu-
larization was conducted on tibia of E18.5 and P0.5 animals
using a Kontron Image Analyzing system (Kontron Electro-
nik, KS400 V 3.00, Germany). Measurements were done on
three to six sections (each at least 15 mm apart) from four to
eight mice per group, stained for Von Kossa or CD34,
respectively. Trabecular bone volume (TBV, as a percen-
tage of tissue volume) and capillary density were deter-
mined in a defined area of the proximal tibia,
encompassing most of the metaphysis. Intercapillary
distance was measured at three distances distal to the growth
plate, and expressed as the width of the bone divided by the
number of blood vessels at the respective depth. Measure-
ments of tibia length and width, bone marrow cavity length
and growth plate characteristics were done on at least four
sections that were more that 15 mm apart. The length of the
zones of hypertrophic chondrocytes and cartilage calcifica-
tion was measured at three sites, equally distributed along
the width of the proximal growth plate.
4.5. In situ hybridization
Digoxigenin (DIG)-11-UTP-labeled sense and antisense
riboprobes were prepared with DIG RNA Labeling Kit and
the corresponding SP6/T7 or T3 RNA polymerase (Roche)
according to the manufacturer’s instructions. In situ hybri-
dizations using the collagen II plasmid (a kind gift from
Frank Luyten, Division of Rheumatology, Leuven,
Belgium) and the collagen X plasmid (a generous gift
from Henry Kronenberg, Harvard Medical School, Boston,
MA) were performed on paraffine sections by standard tech-
niques. Hybridisation was carried out at 558C overnight with
approximately 1 mg/ml probe. Washes were at 608C. Immu-
nological detection was done with DIG Nucleic Acid Detec-
C. Maes et al. / Mechanisms of Development 111 (2002) 61–7370
tion Kit (Roche) according to the manufacturer’s instruc-
tions.
A plasmid containing Cbfa1/Osf2 cDNA (kindly donated
by Patricia Ducy, University of Texas, Texas) was used to
generate 35S-labeled Cbfa1 sense and antisense riboprobes.
In situ hybridization was carried out on paraformaldehyde-
fixed paraffin sections using a modified protocol of Wilk-
inson (1992). Hybridisation was overnight at 558C and
washes were at 628C.
4.6. Isolation of RNA and real-time quantitative RT-PCR
analysis
Total RNA from E16.5 and E18.5 femurs (n ¼ 9–13) was
prepared by snap freezing freshly dissected bones in liquid
nitrogen followed by extraction in TRIZOL (Gibco-BRL).
Subsequently, RNA samples were treated with RNase-free
DNase (Roche) for 15 min at 378C and cDNA was synthe-
sized by using reverse transcriptase Superscript II RT
(Gibco-BRL). Real-time quantitative PCR was performed
according to the manufacturer’s protocol (Perkin-Elmer).
Specific forward (F) and reverse (R) oligonucleotide
primers, and probes (P) with fluorescent dye (FAM) and
quencher (TAMRA) were used for total VEGF, VEGF120,
placenta growth factor (PlGF), Cbfa1, collagen Ia1, osteo-
calcin, osteopontin, MMP-9 (Table 1), Flt-1, NRP1, VEGF-
B, VEGF-C (Carmeliet et al., 1999). Expression levels of
these genes were normalized for the hypoxanthine transfer-
ase (HPRT) gene (Carmeliet et al., 1999).
To determine the absolute copy number of the target
transcripts, a calibration curve was generated, using plasmid
cDNA templates prepared by RT-PCR with gene-specific
primers. The PCR products were cloned using the pGEM-
T Easy Vector Systems (Promega) and the sequence of the
amplicons was verified. Purified plasmid cDNA templates
were measured at 260 nm and copy numbers were calcu-
lated using the following equation: 1 mg of 1000 bp DNA
represents 9:1 £ 1011 molecules. Serial dilutions of the plas-
mid cDNA, in log steps from 108 copies down to 10 copies,
were used to create a calibration curve by plotting the
threshold cycle (Ct) versus the known copy number. The
copy numbers for the unknown samples were determined by
the ABI Prism 7700 Sequence Detection System (software
version 1.7), according to the calibration curve. Unknown
samples and calibrator dilutions were quantified simulta-
neously in the same run in triplicate, together with the
appropriate non-template controls. Quantitative results are
presented as copies of target gene per copy of HPRT.
4.7. Metatarsal cultures with mFlt-1/Fc chimera
Metatarsal rudiments were dissected from E16.5 WT
embryos and stripped of skin. The middle three metatarsals
were kept together as triads and cultured for 1–3 days on a
Falcon insert membrane (pore size 0.4 mm) in 12-well plates
(Becton Dickinson) in 1 ml of BGJb culture medium
(Gibco-BRL) supplemented with 0.1% bovine serum albu-
min (BSA), 25 mg/ml ascorbic acid and 10 mM b-glycer-
C. Maes et al. / Mechanisms of Development 111 (2002) 61–73 71
Table 1
Oligonucleotide sequences used in real-time quantitative RT-PCRa
Gene Sequence
Total VEGF F 5 0-AGTCCCATGAAGTGATCAAGTTCA-3 0
R 5 0-ATCCGCATGATCTGCATGG-3 0
P 5 0-FAM-TGCCCACGTCAGAGAGCAACATCAC-TAMRA-3 0
VEGF120 F 5 0-TGCAGGCTGCTGTAACGATG-3 0
R 5 0-CCTCGGCTTGTCACATTTTTCT-3 0
P 5 0-FAM-TGTCTTTCTTTGGTCTGCATTCACATCGG-TAMRA-3 0
PlGF F 5’-TTCAGTCCGTCCTGTGTCCTT-3 0
R 5’-GCACACAGTGCAGACCTTCA-3 0
P 5 0-FAM-ACCACAGCAGCCACTACAGCGACTCA-TAMRA-3 0
Cbfa1 F 5 0-TACCAGCCACCGAGACCAA-3 0
R 5 0-AGAGGCTGTTTGACGCCATAG-3 0
P 5 0-FAM-CTTGTGCCCTCTGTTGTAAATACTGCTTGCA-TAMRA-3 0
Collagen Ia1 F 5 0-TGTCCCAACCCCCAAAGAC-3’
R 5 0-CCCTCGACTCCTACATCTTCTGA-3’
P 5 0-FAM-ACGTATTCTTCCGGGCAGAAAGCACA-TAMRA-3’
Osteocalcin F 5 0-GGCCCTGAGTCTGACAAAGC-3 0
R 5 0-GCTCGTCACAAGCAGGGTTAA-3 0
P 5 0-FAM-ACAGACTCCGGCGCTACCTTGGAGC-TAMRA-3 0
Osteopontin F 5 0-CCCATCTCAGAAGCAGAATCTCC-3’
R 5 0-TTCATCCGAGTCCACAGAATCC-3’
P 5 0-FAM-AAGCAATTCCAATGAAAGCCATGACCACAT-TAMRA-3’
MMP-9 F 5 0-CCAAAGACCTGAAAACCTCCAA-3 0
R 5 0-GTAGAGACTGCTTCTCTCCCATCAT-3 0
P 5 0-FAM-CAGCTGGCAGAGGCATACTTGTACCGCTA-TAMRA-3 0
a F, forward primer; R, reverse primer; P, probe.
ophosphate (all from Sigma). In addition, mouse VEGFR1
(Flt-1)/Fc chimera (R&D Systems) was added to a final
concentration of 4 mg/ml. In each experiment, the counter
metatarsal triad from the same embryo was used as control,
with addition of vehicle (PBS containing 0.1% BSA). After
the culture, metatarsals were stained with Alcian Blue and
Alizarin Red S. The length of the ossification center of the
metatarsals was measured using a Kontron Image Analyzing
Computer and the values for mFlt-1/Fc treated bones were
expressed as percentage of the corresponding control meta-
tarsal triads.
4.8. Statistical analysis
Results are expressed as mean ^ SEM. Data were
analyzed by two-sample Student t-test, using a statistical
software program (NCSS). Differences were considered
significant at P , 0:05.
Acknowledgements
The authors thank R. Van Looveren, P. Windmolders, S.
Torrekens, A. Van den Hoeck and G. Luyckx for technical
assistance. We are very grateful to A. Zwijsen and T. Van de
Putte (Laboratory for Molecular Biology, Belgium) for help
and advice with radioactive in situ hybridisation. Przemys-
law Tylzanowski and the Laboratory of Skeletal Develop-
ment and Joint Disorders (Belgium) are greatly
acknowledged for help on artwork. This work was
supported by FWO (G.0225.00 and G.0125.00), GOA/
2001/09, and an uncommitted grant from Chugai. C.M. is
a fellow of the IWT.
References
Akeno, N., Czyzyk-Krzeska, M.F., Gross, T.S., Clemens, T.L., 2001.
Hypoxia induces vascular endothelial growth factor gene transcription
in human osteoblast-like cells through the hypoxia inducible factor-
2alpha. Endocrinology 142, 959–962.
Alini, M., Marriott, A., Chen, T., Abe, S., Poole, A.R., 1996. A novel
angiogenic molecule produced at the time of chondrocyte hypertrophy
during endochondral bone formation. Dev. Biol. 176, 124–132.
Bianco, P., Robey, P.G., 2000. Marrow stromal stem cells. J. Clin. Invest.
105, 1663–1668.
Blavier, L., Delaisse, J.M., 1995. Matrix metalloproteinases are obligatory
for the migration of preosteoclasts to the developing marrow cavity of
primitive long bones. J. Cell Sci. 108, 3649–3659.
Breier, G., Albrecht, U., Sterrer, S., Risau, W., 1992. Expression of vascular
endothelial growth factor during embryonic angiogenesis and endothe-
lial cell differentiation. Development 114, 521–532.
Caplan, A.I., 1988. Bone development. In: Evered, D., Harnett, S. (Eds.).
Cell and molecular biology of vertebrate hard tissues. Wiley, Chiche-
ster, Cibia Foundation Symposium, Vol. 136, pp. 3–21.
Carlevaro, M.F., Albini, A., Ribatti, D., Gentili, C., Benelli, R., Cermelli,
S., Cancedda, R., Descalzi Cancedda, F., 1997. Transferrin promotes
endothelial cell migration and invasion: implication in cartilage neovas-
cularization. J. Cell Biol. 136, 1375–1384.
Carlevaro, M.F., Cermelli, S., Cancedda, R., Descalzi Cancedda, F., 2000.
Vascular endothelial growth factor (VEGF) in cartilage neovasculariza-
tion and chondrocyte differentiation: auto-paracrine role during endo-
chondral bone formation. J. Cell Sci. 113, 59–69.
Carmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S., Kieckens, L., Gertsen-
stein, M., Fahrig, M., Vandenhoeck, A., Harpal, K., Eberhardt, C.,
Declercq, C., Pawling, J., Moons, L., Collen, D., Risau, W., Nagy,
A., 1996. Abnormal blood vessel development and lethality in embryos
lacking a single VEGF allele. Nature 380, 435–439.
Carmeliet, P., Ng, Y.-S., Nuyens, D., Theilmeier, G., Brusselmans, K.,
Cornelissen, I., Ehler, E., Kakkar, V.V., Stalmans, I., Mattot, V.,
Perriard, J.-C., Dewerchin, M., Flameng, W., Nagy, A., Lupu, F.,
Moons, L., Collen, D., D’Amore, P.A., Shima, D.T., 1999. Impaired
myocardial angiogenesis and ischemic cardiomyopathy in mice lacking
the vascular endothelial growth factor isoforms VEGF164 and VEGF188.
Nat. Med. 5, 495–502.
Cheng, S.-Y., Nagane, M., Huang, H.-J.S., Cavenee, W.K., 1997. Intracer-
ebral tumor-associated hemorrhage caused by overexpression of the
vascular endothelial growth factor isoforms VEGF121 and VEGF165
but not VEGF189. Proc. Natl. Acad. Sci. USA 94, 12081–12087.
Collin-Osdoby, P., 1994. Role of vascular endothelial cells in bone biology.
J. Cell. Biochem. 55, 304–309.
Colnot, C.I., Helms, J.A., 2001. A molecular analysis of matrix remodeling
and angiogenesis during long bone development. Mech. Dev. 100, 245–
250.
Colnot, C., Sidhu, S.S., Balmain, N., Poirier, F., 2001. Uncoupling of
chondrocyte death and vascular invasion in mouse galectin 3 null
mutant bones. Dev. Biol. 229, 203–214.
Deckers, M.M.L., Karperien, M., van der Bent, C., Yamashita, T., Papa-
poulos, S.E., Lowik, C.W.G.M., 2000. Expression of vascular endothe-
lial growth factors and their receptors during osteoblast differentiation.
Endocrinology 141, 1667–1674.
Ducy, P., Zhang, R., Geoffroy, V., Ridall, A.L., Karsenty, G., 1997. Osf2/
Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89,
747–754.
Ducy, P., Schinke, T., Karsenty, G., 2000. The osteoblast: a sophisticated
fibroblast under central surveillance. Science 289, 1501–1504.
Engsig, M.T., Chen, Q.-J., Vu, T.H., Pedersen, A.-C., Therkidsen, B., Lund,
L.R., Henriksen, K., Lenhard, T., Foged, N.T., Werb, Z., Delaisse, J.-
M., 2000. Matrix metalloproteinase 9 and vascular endothelial growth
factor are essential for osteoclast recruitment into developing long
bones. J. Cell Biol. 151, 879–889.
Erlebacher, A., Filvaroff, E.H., Gitelman, S.E., Derynck, R., 1995. Toward
a molecular understanding of skeletal development. Cell 80, 371–378.
Ferguson, C., Alpern, E., Miclau, T., Helms, J.A., 1999. Does adult fracture
repair recapitulate embryonic skeletal formation? Mech. Dev. 87, 57–
66.
Ferrara, N., 2001. Role of vascular endothelial growth factor in regulation
of physiological angiogenesis. Am. J. Physiol. Cell. Physiol. 280,
C1358–C1366.
Ferrara, N., Davis-Smyth, T., 1997. The biology of vascular endothelial
growth factor. Endocr. Rev. 18, 4–25.
Ferrara, N., Carver-Moore, K., Chen, H., Dowd, M., Lu, L., O’Shea, K.S.,
Powell-Braxton, L., Hillan, K.J., Moore, M.W., 1996. Heterozygous
embryonic lethality induced by targeted inactivation of the VEGF
gene. Nature 380, 439–442.
Flamme, I., von Reutern, M., Drexler, H.C.A., Syed-Ali, S., Risau, W.,
1995. Overexpression of vascular endothelial growth factor in the
avian embryo induces hypervascularization and increased vascular
permeability without alterations of embryonic pattern formation. Dev.
Biol. 171, 399–414.
Gerber, H.-P., Vu, T.H., Ryan, A.M., Kowalski, J., Werb, Z., Ferrara, N.,
1999. VEGF couples hypertrophic cartilage remodeling, ossification
and angiogenesis during endochondral bone formation. Nat. Med. 5,
623–628.
Glowacki, J., 1998. Angiogenesis in fracture repair. Clin. Orthop. 355,
S82–S89.
Green, J.R., Reeve, J., Tellez, M., Veall, N., Wootton, R., 1987. Skeletal
blood flow in metabolic disorders of the skeleton. Bone 8, 293–297.
C. Maes et al. / Mechanisms of Development 111 (2002) 61–7372
Haigh, J.J., Gerber, H.-P., Ferrara, N., Wagner, E.F., 2000. Conditional
inactivation of VEGF-A in areas of collagen2a1 expression results in
embryonic lethality in the heterozygous state. Development 127, 1445–
1453.
Harper, J., Gerstenfeld, L.C., Klagsbrun, M., 2001. Neuropilin-1 expression
in osteogenic cells: down-regulation during differentiation of osteo-
blasts into osteocytes. J. Cell. Biochem. 81, 82–92.
Holmbeck, K., Bianco, P., Caterina, J., Yamada, S., Kromer, M., Kuznet-
sov, S.A., Mankani, M., Robey, P.G., Poole, A.R., Pidoux, I., Ward,
J.M., Birkedal-Hansen, H., 1999. MT1-MMP-deficient mice develop
dwarfism, osteopenia, arthritis, and connective tissue disease due to
inadequate collagen turnover. Cell 99, 81–92.
Horner, A., Bishop, N.J., Bord, S., Beeton, C., Kelsall, A.W., Coleman, N.,
Compston, J.E., 1999. Immunolocalisation of vascular endothelial
growth factor (VEGF) in human neonatal growth plate cartilage. J.
Anat. 194, 519–524.
Hunter, W.L., Arsenault, A.L., Hodsman, A.B., 1991. Rearrangement of the
metaphyseal vasculature of the rat growth plate in rickets and rachitic
reversal: a model of vascular arrest and angiogenesis renewed. Anat.
Rec. 229, 453–461.
Kitsukawa, T., Shimono, A., Kawakami, A., Kondoh, H., Fujisawa, H.,
1995. Overexpression of a membrane protein, neuropilin, in chimeric
mice causes anomalies in the cardiovascular system, nervous system
and limbs. Development 121, 4309–4318.
Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K., Deguchi, K.,
Shimizu, Y., Bronson, R.T., Gao, Y.-H., Inada, M., Sato, M., Okamoto,
R., Kitamura, Y., Yoshiki, S., Kishimoto, T., 1997. Targeted disruption
of Cbfa1 results in a complete lack of bone formation owing to matura-
tional arrest of osteoblasts. Cell 89, 755–764.
Lanske, B., Amling, M., Neff, L., Guiducci, J., Baron, R., Kronenberg,
H.M., 1999. Ablation of the PTHrP gene or the PTH/PTHrP receptor
gene leads to distinct abnormalities in bone development. J. Clin.
Invest. 104, 399–407.
Larcher, F., Murillas, R., Bolontrade, M., Conti, C.J., Jorcano, J.L., 1998.
VEGF/VPF overexpression in skin of transgenic mice induces angio-
genesis, vascular hyperpermeability and accelerated tumor develop-
ment. Oncogene 17, 303–311.
Lijnen, H.R., Van Hoef, B., Lupu, F., Moons, L., Carmeliet, P., Collen, D.,
1998. Function of the plasminogen/plasmin and matrix metalloprotei-
nase systems after vascular injury in mice with targeted inactivation of
fibrinolytic system genes. Arterioscler. Thromb. Vasc. Biol. 18, 1035–
1045.
McLeod, M.J., 1980. Differential staining of cartilage and bone in whole
mouse fetuses by alcian blue and alizarin red S. Teratology 22, 299–
301.
Midy, V., Plouet, J., 1994. Vasculotropin/vascular endothelial growth
factor induces differentiation in cultured osteoblasts. Biochem.
Biophys. Res. Commun. 199, 380–386.
Moses, M.A., Wiederschain, D., Wu, I., Fernandez, C.A., Ghazizadeh, V.,
Lane, W.S., Flynn, E., Sytkowski, A., Tao, T., Langer, R., 1999. Tropo-
nin I is present in human cartilage and inhibits angiogenesis. Proc. Natl.
Acad. Sci. USA 96, 2645–2650.
Nakagawa, M., Kaneda, T., Arakawa, T., Morita, S., Sato, T., Yomada, T.,
Hanada, K., Kumegawa, M., Hakeda, Y., 2000. Vascular endothelial
growth factor (VEGF) directly enhances osteoclastic bone resorption
and survival of mature osteoclasts. FEBS Lett. 473, 161–164.
Neufeld, G., Cohen, T., Gengrinovitch, S., Poltorak, Z., 1999. Vascular
endothelial growth factor (VEGF) and its receptors. FASEB J. 13, 9–22.
Niida, S., Kaku, M., Amano, H., Yoshida, H., Kataoka, H., Nishikawa, S.,
Tanne, K., Maeda, N., Nishikawa, S.-I., Kodama, H., 1999. Vascular
endothelial growth factor can substitute for macrophage colony-stimu-
lating factor in the support of osteoclastic bone resorption. J. Exp. Med.
190, 293–298.
Otto, F., Thornell, A.P., Crompton, T., Denzel, A., Gilmour, K.C., Rose-
well, I.R., Stamp, G.W.H., Beddington, R.S.P., Mundlos, S., Olsen,
B.R., Selby, P.B., Owen, M.J., 1997. Cbfa1, a candidate gene for clei-
docranial dysplasia syndrome, is essential for osteoblast differentiation
and bone development. Cell 89, 765–771.
Reeve, J., Arlot, M., Wootton, R., Eduard, C., Tellez, M., Hesp, R., Green,
J.R., Meunier, P.J., 1988. Skeletal blood flow, iliac histomorphometry,
and strontium kinetics in osteoporosis; a relationship between blood
flow and corrected apposition rate. J. Clin. Endocrinol. Metab. 66,
1124–1131.
Rice, D.P.C., Kim, H.-J., Thesleff, I., 1997. Detection of gelatinase B
expression reveals osteoclastic bone resorption as a feature of early
calvarial bone development. Bone 21, 479–486.
Schipani, E., Lanske, B., Hunzelman, J., Luz, A., Kovacs, C.S., Lee, K.,
Pirro, A., Kronenberg, H.M., Juppner, H., 1997. Targeted expression of
constitutively active receptors for parathyroid hormone and parathyroid
hormone-related peptide delays endochondral bone formation and
rescues mice that lack parathyroid hormone-related peptide. Proc.
Natl. Acad. Sci. USA 94, 13689–13694.
Shukunami, C., Iyama, K., Inoue, H., Hiraki, Y., 1999. Spatiotemporal
pattern of the mouse chondromodulin-I gene expression and its regula-
tory role in vascular invasion into cartilage during endochondral bone
formation. Int. J. Dev. Biol. 43, 39–49.
Soker, S., Takashima, S., Miao, H.Q., Neufeld, G., Klagsbrun, M., 1998.
Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-
specific receptor for vascular endothelial growth factor. Cell 92, 735–
745.
St-Jacques, B., Hammerschmidt, M., McMahon, A.P., 1999. Indian hedge-
hog signaling regulates proliferation and differentiation of chondrocytes
and is essential for bone formation. Genes Dev. 13, 2072–2086.
Steinbrech, D.S., Mehrara, B.J., Saadeh, P.B., Chin, G., Dudziak, M.E.,
Gerrets, R.P., Gittes, G.K., Longaker, M.T., 1999. Hypoxia regulates
VEGF expression and cellular proliferation by osteoblasts in vitro.
Plast. Reconstr. Surg. 104, 738–747.
Streeten, E.A., Brandi, M.L., 1990. Biology of bone endothelial cells. Bone
Mineral. 10, 85–94.
Takeda, S., Bonnamy, J.-P., Owen, M.J., Ducy, P., Karsenty, G., 2001.
Continuous expression of Cbfa1 in nonhypertrophic chondrocytes
uncovers its ability to induce hypertrophic chondrocyte differentiation
and partially rescues Cbfa1-deficient mice. Genes Dev. 15, 467–481.
Verhaeghe, J., Van Herck, E., Van Bree, R., Van Assche, F.A., Bouillon,
R., 1989. Osteocalcin during the reproductive cycle in normal and
diabetic rats. J. Endocrinol. 120, 143–151.
Villars, F., Bordenave, L., Bareille, R., Amedee, J., 2000. Effect of human
endothelial cells on human bone marrow stromal cell phenotype: role of
VEGF? J. Cell. Biochem. 79, 672–685.
Vu, T.H., Shipley, J.M., Bergers, G., Berger, J.E., Helms, J.A., Hanahan,
D., Shapiro, S.D., Senior, R.M., Werb, Z., 1998. MMP-9/gelatinase B is
a key regulator of growth plate angiogenesis and apoptosis of hyper-
trophic chondrocytes. Cell 93, 411–422.
Wang, D.S., Miura, M., Demura, H., Sato, K., 1997. Anabolic effects of
1,25-dihydroxyvitamin D3 on osteoblasts are enhanced by vascular
endothelial growth factor produced by osteoblasts and by growth
factors produced by endothelial cells. Endocrinology 138, 2953–2962.
Wilkinson, D.G., 1992. Whole mount in situ hybridisation to vertebrate
embryos. In: Wilkinson, D.G. (Ed.). In Situ Hybridisation: A Practical
Approach, IRL Press, Oxford, UK, pp. 75–83.
Zelzer, E., Glotzer, D.J., Hartmann, C., Thomas, D., Fukai, N., Soker, S.,
Olsen, B.R., 2001. Tissue specific regulation of VEGF expression
during bone development requires Cbfa1/Runx2. Mech. Dev. 106,
97–106.
Zhou, Z., Apte, S.S., Soininen, R., Cao, R., Baaklini, G.Y., Rauser, R.W.,
Wang, J., Cao, Y., Tryggvason, K., 2000. Impaired endochondral ossi-
fication and angiogenesis in mice deficient in membrane-type matrix
metalloproteinase I. Proc. Natl. Acad. Sci. USA 97, 4052–4057.
C. Maes et al. / Mechanisms of Development 111 (2002) 61–73 73