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Available online 3 November 2006
overned in part by angiogenesis. Although it has been demonstrated that
stage, cartilage extracellular matrix (ECM) is mineralized by
deposition of apatitic crystals. Next, blood vessels emerging
ossification and expands the hypertrophic zone of cartilage
making longer bones. Additionally, VEGF stimulates prolifera-
tion and differentiation of osteoblasts via the production of
osteogenic factors by endothelial cells [52]. Recently, a VEGF
Bone 40 (2007) 568from perichondrial tissues invade the growth plate. Finally,Introduction
Cartilage formation is a multi-step process initiated with the
condensation of mesenchymal cells. These cells then differ-
entiate into chondrocytes with the appearance of characteristic
markers such as Sox-9, type II, IX and XI collagens and
aggrecan [23]. Chondrocytes undergo a final step of differentia-
tion in which they become post-mitotic and hypertrophic and
express new extracellular macromolecules including type X
collagen, osteopontin and alkaline phosphatase [23]. At this
precursor cells of bone marrow origin ensure the replacement of
mineralized cartilage by newly formed bone.
It is well known that vascular invasion is a critical step for
endochondral and membranous ossification. In this context,
vascular endothelial growth factor (VEGF), one of the most
potent mediators of angiogenesis, is expressed by hypertrophic
chondrocytes of the growth plate [17]. Its inhibition by soluble
VEGF receptor treatment decreases angiogenesis as well as
trabecular bone formation and repair [17,48]. The absence of
vascular invasion in the growth plate delays endochondralvascular endothelial growth factor (VEGF-A) is crucial during endochondral ossification, little is known about the involvement of the other VEGF
family members. Thus, we examined the expression and production of these members on primary chondrocytes and ATDC5 chondrogenic cells.
VEGF-A, VEGF-B, VEGF-C and VEGF-D were shown to be expressed and synthesized demonstrating that numerous angiogenic factors can be
produced by chondrocytes. In ATDC5 VEGF-A, VEGF-B and VEGF-C were over-expressed in the presence of chondrogenic and bone
morphogenetic protein (BMP)-2 treatment suggesting that these factors play an important role during chondrogenesis. In addition, neuropilin-1,
VEGF receptor-2 and VEGF receptor-3 gene expression were observed with an increase in VEGF-R2 expression under chondrogenic and BMP-2
treatment, suggesting that VEGF proteins could act in an autocrine/paracrine manner in addition to their angiogenic function. In conclusion, we
demonstrated for the first time that chondrocytes secreted the four members of the VEGF family. We also showed that VEGF-B, VEGF-C and
VEGF-D were secreted as processed proteins. The up-regulation of VEGF-B and VEGF-C at the mRNA and protein levels under chondrogenic
stimulation strongly suggests a major role for these proteins in growth plate physiology.
' 2006 Elsevier Inc. All rights reserved.
Keywords: VEGF; Growth plate chondrocyte; ATDC5; RT-PCR; Western-blotting; ChondrogenesisDuring long bone development, cartilage replacement by bone is gAbstractVEGF and VEGF receptors are diff
Gilles Bluteau
a
, Marion Julien
a
, David Mag
Guy Daculsi
a
, Jé
a
INSERM UMRS 791, University of Nantes, Laboratoire d’Ingén
1 Place Alexis Ricordeau, 4
b
CNRS UMR 5086, University Claude Bernard Lyon I, Institut de Biologie
Received 7 June 2006; revised 30 Au

Corresponding author. Fax: +33 2 40 08 37 12.
E-mail address: Jerome.guicheux@nantes.inserm.fr (J. Guicheux).
8756-3282/$ - see front matter ' 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.bone.2006.09.024entially expressed in chondrocytes
a
, Frédéric Mallein-Gerin
b
, Pierre Weiss
a
,
me Guicheux
a,


Ostéoarticulaire et Dentaire, LIOAD, School of Dental Surgery,
42 Nantes cedex 1, France
himie des Protéines, 7 Passage du Vercors, 69367 Lyon cedex 07, France
t 2006; accepted 26 September 2006
–576
www.elsevier.com/locate/bonechemoattractive activity was also observed for primary
osteoblasts as well as for bone marrow-derived mesenchymal
progenitor cells [15,31]. Finally, VEGF is involved in

onemigration, survival and stimulation of cells of the monocyte/
macrophage lineage to differentiate into functional osteoclasts
[20,32]. All these data highlight the relationship between
angiogenesis and bone formation.
The VEGF family is comprised of several proteins encoded
by five different genes: Vegf/Vegf-A, Vegf-B, Vegf-C, Vegf-D
and Vegf-E. Each of these genes generates one or several VEGF
isoforms by alternative splicing. Vegf/Vegf-A generates at least
five isoforms, e.g. VEGF-
121
,-
145
,-
165
,-
189
,-
206
[39]. Vegf-B/
VRF (VEGF-related factor) leads to the VEGF-B
167
and VEGF-
B
186
isoforms [35,39], Vegf-C/VRP (VEGF-related protein)
leads to one isoform [35,39] and Vegf-D/FIGF (c-fos-induced
growth factor) encodes two isoforms, e.g. VEGF-D
326
and
VEGF-D
358
[6]. Finally, VEGF-E generates one isoform and
has been identified in the genome of the Orf virus (NZ-7 strain)
[34]. Each of these proteins is capable of stimulating
angiogenesis in vivo [30,38,45,53]. Three different type III
tyrosine kinase receptors mediate the biological effects of
VEGF. VEGF-A and -B activate VEGF-R1/Flt-1. VEGF-A, -C
and -D activate VEGF-R2/KDR/Flk-1 in humans and VEGF-C
and -D bind and activate VEGF-R3/Flt-4 [35,39]. Additionally,
neuropilin-1 is a co-receptor for VEGF-R2 and enhances the
binding of VEGF-A
165
[46].
Despite a large body of evidence demonstrating the role of
VEGF-A in endochondral ossification, the other VEGF family
members have been poorly studied. In this context, the aim of
the present work was to assess the expression of all four VEGF
members and their receptors during the differentiation process
of chondrocytes. For this purpose, we used primary chondro-
cytes from rib growth plates as well as a well-documented in
vitro cellular model (namely ATDC5 cells). ATDC5 cells are
derived from a mouse teratocarcinoma and mimic the multistep
process of chondrocyte differentiation in culture under appro-
priate conditions, from type II to type X collagen synthesis and
until the mineralization of the ECM [2,28]. ATDC5 cells have
been extensively used to decipher the involvement of various
growth factors in endochondral ossification [28,42,44].
In this work we demonstrate the expression of the four VEGF
genes in mouse primary chondrocytes as well as in the ATDC5
cell line. We also show the synthesis/secretion of two VEGF-A
isoforms, the two VEGF-B and VEGF-D proteins as well as the
synthesis and cleavage of VEGF-C and VEGF-D under
chondrogenic stimulation. ATDC5 cells also expressed three
receptors for VEGF: neuropilin-1, VEGF-R2 and VEGF-R3.
Finally we demonstrate the regulation of VEGF-A, -B and -C by
BMP-2, a growth factor known to stimulate the differentiation of
chondrocytes, at both the mRNA and protein levels.
Materials and methods
Reagents
Recombinant human BMP-2 was a gift from Dr. J.M. Wozney (Genetic
Insitute, Cambridge, MA). Cell culture plastic ware was purchased from
Corning-Costar (Corning BV Life Sciences, Schiphol-Rijk, The Netherlands).
G. Bluteau et al. / BFetal calf serum (FCS) was obtained from D. Dutscher (Brumath, France).
DMEM/F12 was obtained from ICN Biomedicals (Orsay, France). α-MEM,
L-glutamine, antibiotics (P/S), trypsin/ethylenediamine tetraacetic acid (T/E),Trizol® Reagent, Taq polymerase, dNTPs, NuPAGE™ 12% Bis–Tris gel and
PVDF membranes were obtained from Life Technologies Ltd. (Paisley, UK).
ITS (10 g/ml insulin, 10 g/ml transferrin and 10ng/ml sodium selenite) and
ethylene glycol-bis (β-aminoethyl ether)-N, N, N′, N′-tetraacetic acid
(EGTA) were from Sigma-Aldrich Co (St Quentin Fallavier, France). RQ1
RNase-free DNase, AMV-RT, random hexaprimers and RNAsin were
obtained from Promega (Charbonnières, France). Oligo-dT
14
VN (V=A, G,
C; N=A, T, G, C) were synthesized by MWG Biotech (Ebersberg, Germany).
The anti-actin antibody (MAB1501R) was from Chemicon International
(Euromedex, Mundolsheim, France). Antibodies directed against VEGF-A
(sc-507); VEGF-B (sc-13083) and VEGF-C (sc-9047) were from Santa Cruz
Biotechnology Inc (Santa Cruz, California, USA). The anti-VEGF-D antibody
(3565-100) was from Biovision (Clinisciences, Montrouge, France). The
chicken anti-rabbit secondary antibody (sc-2963, HRP-conjugated) was from
Santa Cruz Biotechenology Inc (Santa Cruz, California, USA). All other
chemicals were from standard laboratory suppliers and of the highest purity
available.
Cell culture
Primary chondrocytes were isolated from the ventral part of rib cages of
new born mice as described [25]. Briefly, ribs were digested first with a
pronase solution (2 mg/ml) and then with a collagenase solution (2 mg/ml)
until complete dissolution. Cells were then seeded in 25 cm
2
flasks at 2×10
6
cells/flask and cultured for 48 h in DMEM/F12 containing 10% FCS and
supplemented with 1% glutamine and 1% P/S. ATDC5 cells were cultured in
DMEM/F12 supplemented with 5% FCS, 1% P/S and 1% L-glutamine for
proliferation. Cells were then recovered at 70–80% confluency with T/E and
seeded at 15,000 cells/cm
2
in 25 cm
2
flasks. For differentiation, chondrocytes
were cultured in the presence of ITS for the indicated times. RAW264.7
mouse macrophages (a kind gift from Dr. Pierre Jurdic, Lyon, France)
were cultured in α-MEM supplemented with 10% FCS, 1% P/S and 1%
L-glutamine. Cells were seeded at a final density of 40,000 cells/cm
2
and recovered when 70–80% confluent. All cells were maintained at
37°C in a humidified atmosphere containing 5% CO
2
and the culture
medium was renewed every 2 days.
RNA isolation and RT-PCR
After the indicated times, total RNA was extracted with Trizol® Reagent
according to the manufacturer's instructions. Briefly, lysis of the cells in Trizol®
was followed by centrifugation at 10,000×g for 15 min at 4°C in the presence of
chloroform. The upper aqueous phase was collected and the RNA was
precipitated by addition of 100% ethanol and a high salt precipitation solution
(0.8 M sodium citrate and 1.2 M NaCl), and centrifugation at 7500×g for 5 min
at 4°C. RNA pellets were washed with ice-cold 75% ethanol, dried and
reconstituted in sterile water. RQ1 RNase-free DNase treatment (1–2 h at 37°C)
was followed by phenol–chloroform extraction and precipitation with 2 volumes
of 100% ethanol and 1/10th volume of 3 M sodium acetate (pH 5.2). RNA
pellets were rinsed twice with 75% ethanol, dried, resuspended in sterile water
and quantified by spectrometry.
Samples (1–2 g) were reverse-transcribed using AMV-RT and random
hexaprimers or oligo-dT
14
VN (V=A, G, C; N=A, T, G, C). Template cDNA
was then amplified in a typical 50 l PCR reaction containing 1.5 mM MgCl
2
,
1 M of the respective primers and 2.5units of Taq polymerase. Primer
sequences and annealing temperatures are detailed in Table 1. Amplifications
were carried out in an Eppendorf master cycler (VWR, Brumath, France) under
the following conditions: denaturation for 3 min at 95°C, followed by cycles of
20 s denaturation, 20 s annealing at the primer-specific temperature and 10–20 s
elongation at 72°C. Primers were designed using Primer 3 (http://frodo.wi.mit.
edu/cgi-bin/primer3/primer3_www.cgi) except those for VEGF-A and VEGF-
R3 [10]; ALP [37]; type II and type X collagen [51] and aggrecan [49]. All
results show PCR products obtained in the linear range of amplification. For this
purpose, each cycle number was determined by serial PCR reactions with
decreasing cycle numbers so as to obtain a faint reproducible signal. The ratio of
56940 (2007) 568–576collagen IIB/collagen IIAwas determined by densitometry after normalization to
β-actin levels as an internal control (Leica Q500, Cambridge, UK). All PCR
products were cloned (pGEM®-Teasy vector systems, Promega, Charbonnières,