impaired angiogenesis and endochondral bone formation in mice

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

www.elsevier.com/locate/modo

* 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: [email protected] (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.


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


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-


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-


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-


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


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-


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-


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.

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impaired angiogenesis and endochondral bone formation in mice

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