Download VEGF and endochondral bone formation

59
Journal of Cell Science 113, 59-69 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
JCS0598
Vascular endothelial growth factor (VEGF) in cartilage neovascularization and
chondrocyte differentiation: auto-paracrine role during endochondral bone
formation
Mariella F. Carlevaro1, Silvia Cermelli1, Ranieri Cancedda1,2 and Fiorella Descalzi Cancedda1,3,*
1Istituto Nazionale per la Ricerca sul Cancro, Centro di Biotecnologie Avanzate, Genova, Italy
2Dipartimento di Oncologia, Biologia e Genetica, Universita’ di Genova, Italy
3Centro di Studio per la Neurofisiologia Cerebrale, Consiglio Nazionale delle Ricerche, Genova,
Italy
*Author for correspondence (e-mail: [email protected])
Accepted 25 October; published on WWW 9 December 1999
SUMMARY
Vascular endothelial growth factor/vascular permeability
factor (VEGF/VPF) induces endothelial cell migration and
proliferation in culture and is strongly angiogenic in vivo.
VEGF synthesis has been shown to occur in both normal
and transformed cells. The receptors for the factor have
been shown to be localized mainly in endothelial cells,
however, the presence of VEGF synthesis and the VEGF
receptor in cells other than endothelial cells has been
demonstrated. Neoangiogenesis in cartilage growth plate
plays a fundamental role in endochondral ossification.
We have shown that, in an avian in vitro system for
chondrocyte differentiation, VEGF was produced and
localized in cell clusters totally resembling in vivo cartilage.
The factor was synthesized by hypertrophic chondrocytes
and was released into their conditioned medium, which is
highly chemotactic for endothelial cells. Antibodies against
VEGF inhibited endothelial cell migration induced by
chondrocyte conditioned media. Similarly, endothelial cell
migration was inhibited also by antibodies directed against
the VEGF receptor 2/Flk1 (VEGFR2). In avian and
mammalian embryo long bones, immediately before
vascular invasion, VEGF was distinctly localized in growth
plate hypertrophic chondrocytes. In contrast, VEGF was
not observed in quiescent and proliferating chondrocytes
earlier in development.
VEGF receptor 2 colocalized with the factor both in
hypertrophic cartilage in vivo and hypertrophic cartilage
engineered in vitro, suggesting an autocrine loop in
chondrocytes at the time of their maturation to hypertrophic
cells and of cartilage erosion. Regardless of cell exposure
to exogenous VEGF, VEGFR-2 phosphorylation was
recognized in cultured hypertrophic chondrocytes,
supporting the idea of an autocrine functional activation of
signal transduction in this non-endothelial cell type as a
consequence of the endogenous VEGF production.
In summary we propose that VEGF is actively
responsible for hypertrophic cartilage neovascularization
through a paracrine release by chondrocytes, with invading
endothelial cells as a target. Furthermore, VEGF receptor
localization and signal transduction in chondrocytes
strongly support the hypothesis of a VEGF autocrine
activity also in morphogenesis and differentiation of a
mesoderm derived cell.
INTRODUCTION
(Folkman, 1997), neovascularization in cartilage is finely
modulated and is controlled by the balance of molecules with
opposite potentials. Various findings indicate that chondrocytes
are able to synthesize both angiogenesis inhibitors and
stimulators, depending on their culture condition and state of
differentiation: resting and proliferative cartilage has strong
anti-angiogenic effects (Moses et al., 1990, 1992; Pepper et al.,
1991); troponin I present in cartilage was recently
demonstrated to be a potent inhibitor of angiogenesis and
tumor metastasis (Moses et al., 1999); mineralized
hypertrophic chondrocytes in vitro elicit neovascularization
(Brown and McFarland, 1992). In order to produce angiogenic
activity, hypertrophic chondrocytes need to be in a correct
extracellular matrix microenvironment. An in vitro system for
During embryo limb bud development and endochondral
ossification, a totally cartilaginous bone rudiment is gradually
vascularized, beginning in the diaphysis and continuing in the
growth plate.
Towards the diaphysis, in the growth plate the different
stages of chondrocyte differentiation are detectable: resting,
proliferating, and hypertrophic chondrocytes, a zone of
mineralization and calcium deposition, and finally a zone of
blood vessel invasion and cartilage erosion where ossification
occurs. Angiogenesis appears to be a key step toward
ossification.
As in other physiological and pathological processes
Key words: VEGF, Angiogenesis, Vasculogenesis, Development,
Endochondral ossification
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M. F. Carlevaro and others
chondrocyte differentation gave evidence supporting this
hypothesis (Descalzi et al., 1995): single hypertrophic
chondrocytes in suspension have inhibitory effects on
endothelial cell migration and invasion; when supplemented
with ascorbic acid, a condition that allows organization of an
extracellular matrix totally resembling in vivo cartilage,
hypertrophic chondrocytes switch to the release of angiogenic
activities in the conditioned media and start synthesizing
transferrin, which was identified as one of the major angiogenic
factors in this tissue (Carlevaro et al., 1997).
Recently other proteins produced by chondrocytes were
recognized to have an angiogenic role. In particular, a molecule
of approximately 120 kDa, isolated from bovine chondrocytes,
was shown to be both a non-mitogenic chemoattractant for
endothelial cells in vitro and angiogenic in vivo (Alini et al.,
1996). Production of this molecule was regulated by vitamin
D3 metabolites. In MMP-9/gelatinase B knock out mice, delay
in vascularization, apoptosis and ossification in skeletal growth
plate have been reported, even if normal development of
hypertrophic chondrocyte was not impaired (Vu et al., 1998).
Vascular endothelial growth factor/vascular permeability
factor (VEGF/VPF) is known to be an angiogenic factor in vivo
and in vitro, mitogen for endothelial cells, with effects on
vascular permeability (Dvorak et al, 1995). VEGF plays a role
in tumor growth and in pathological angiogenesis, but also in
the control of blood vessel development and in vasculogenesis
in embryos (Risau, 1997).
VEGF is a dimeric glycoprotein, with a molecular mass
ranging from 17 to 22 kDa in reducing conditions. It has a
region of homology to platelet-derived growth factor (PDGF)
and it is closely related to placenta-derived growth factor (PlGF)
(Dvorak, 1995). Several isoforms, resulting from alternative
splicing, are expressed both in mammal (Ferrara et al., 1992)
and avian embryos (Flamme et al., 1995); soluble and cellmatrix associated forms have very similar biological activities.
For a long time, VEGF has been considered to be an
endothelial-specific factor, mostly based on the almost
exclusive distribution of its tyrosine kinase receptors, VEGFR1 (Flt-1) and VEGFR-2 (Flk-1) in endothelial cells (Jakeman et
al., 1992, 1993; Millauer et al., 1993; de Vries et al., 1992;
Terman et al., 1992).
Only in the last few years has attention been focused on
VEGF synthesis and VEGF receptor presence in nonendothelial tissues. As far as chondro-osteogenic lineage is
concerned, VEGF was described as a binding but not
proliferative agent for cultured osteoblasts, inducing migration,
alkaline phosphatase increase and PTH-dependent cAMP
accumulation, thus suggesting a possible role in osteoblast
differentiation (Midy and Plouet, 1992). Nevertheless, it should
be noted that the above experiments were performed in
conditions that did not exclude the contemporary presence of
endothelial cells in the cultures. VEGF production was induced
by prostaglandins E1 and E2 in osteosarcoma osteoblast-like
cells (Harada et al., 1994). Vitamin D3 was responsible for the
increase of VEGF messenger RNA in human osteoblasts (Wang
et al., 1996) and paracrine interactions through VEGF, vitamin
D3, endothelin-1 and IGF I were also suggested between
endothelial cells and osteoblasts (Wang et al., 1997). So far,
VEGF has only been incidentally associated to the chondrocyte
cell type. VEGF expression was observed in the cartilaginous
vertebral discs of 14-day-old mouse embryos, and binding sites
for the factor were evidenced in inter-vertebral vessels
(Jakeman et al., 1993).
The targeted inactivation of the VEGF gene induced
heterozygous embryonic lethality between days 11 and 12 of
mouse development. Together with other anomalies, forelimb
buds were positioned in the caudal region as in earlier stages
and lost their segmentation, and in the cranial region also the
branchial arches were not segmented and poorly developed
(Ferrara et al., 1996).
There is consistent evidence for VEGF being involved in
cartilage pathological neovascularization, with factor increase
in synovial fluids deriving from rheumatoid arthritis after
secretion by lining cells and macrophages (Koch et al., 1994).
Microvascular endothelial cells close to VEGF accumulation
sites in inflamed joints express mRNA for both flt-1 and flk-1,
indicating a role for VEGF in rheumatoid arthritis pathogenesis
(Fava et al., 1994).
To assess the possible involvement of VEGF in cartilage
neovascularization, we performed studies with the in vitro
model of avian chondrocyte differentiation we have established.
In parallel we have also investigated the factor and receptor
expression in developing bones of avian and mammalian
embryos. Hypertrophic chondrocytes cultured in the presence
of an organized extracellular matrix secreted VEGF. The factor
was partially responsible for the angiogenic, chemotactic and
chemoinvasive activity observed in the conditioned culture
media. The presence of VEGF receptor and functional signal
transduction in hypertrophic chondrocytes was considered in
the light of a possible additional differentiating or morphogen
effect of VEGF in endochondral bone formation.
MATERIALS AND METHODS
Cell culture
Details on cell culture conditions for chondrocytes have been
published previously (Castagnola et al., 1986; Descalzi Cancedda et
al., 1992). Briefly, chondrocytes derived from digestion of 6-day-old
chick embryo tibiae were plated on culture dishes in Coon’s modified
F12 medium supplemented with 10% FCS. In adhesion,
dedifferentiated cells were obtained and maintained as such for the
following 3 weeks. The chondrocytic phenotype was resumed
and chondrocyte differentiation to hypertrophy continued upon
transferring cells into suspension culture in agarose-coated dishes. In
3-4 weeks, a nearly homogeneous population of single isolated
hypertrophic chondrocytes was obtained; filtration through a nylon
filter eliminated residual cell clusters. Suspension culture of these
cells, in the presence of 100 µg/ml ascorbic acid and 10 mM βglycerophosphate, led to the formation of in vitro engineered
cartilage.
HMEC-1 immortalized endothelial cells, kindly supplied by Dr F.
J. Candal (National Center for Infectious Diseases, Center for Disease
Control, Atlanta, Georgia, USA), were maintained in MCDB 131
medium (Life Technologies, Grand Island, NY, USA), containing
10% FCS, dexamethasone 1 µg/ml (Sigma Chemical Co., St Louis,
MO, USA), and human recombinant EGF 10 ng/ml (Peprotech Inc.,
Rocky Hill, NJ, USA), and cultured according to published
procedures (Ades et al., 1992).
Preparation of conditioned medium from cultured
chondrocytes
After rinsing twice with phosphate buffered saline (PBS), cultures of
hypertrophic chondrocytes were incubated for 24 hours in F12
medium (5 ml for 100 mm dish) containing ascorbic acid 100 µg/ml.
VEGF and endochondral bone formation
Unmodified protein release by the cells upon 24 hour serum
deprivation was checked by metabolic labeling. Collected conditioned
media were centrifuged for 10 minutes at 3000 rpm to eliminate cell
debris and stored at −20°C, for future use in a Boyden chamber assay.
DNA determination in cell layers from which conditioned media
were obtained was performed, in order to measure activities released
by equivalent numbers of cells.
DNA measurement
Cell lysates, in 0.01% SDS in PBS, were digested overnight at 50°C
with proteinase K (Sigma Chemical Co., St Louis, MO, USA), 100
µg/ml, in 10 mM Tris-HCl, pH 7.8, 5 mM EDTA.
Each sample was diluted 1:20 in dye solution (10 mM Tris-HCl,
pH 7.6, 1 mM EDTA, 0.1 M NaCl, 0.1 mg/ml Hoechst 33258 from
Sigma Chemical Co.) and DNA content was determined with a DNA
fluorimeter (Hoefer, San Francisco, CA, USA).
Antibodies
For VEGF and VEGFR2 immunolocalization, rabbit anti-human and
anti-mouse polyclonal antibodies (Santa Cruz Biotechnology Inc.,
Santa Cruz, CA, USA) were used (Ahmed et al., 1997). Specificity of
antibodies was preliminarly checked.
AntiVEGF were rabbit anti-human polyclonal antibodies against
N-terminal peptide 1-140 (Santa Cruz catalog SC-507) and
antiVEGFR2 (Flk1) were rabbit anti-mouse polyclonal antibodies
against C-terminal peptide 1158-1345 (Santa Cruz catalog SC-504).
Cross reactivity with mouse is from the data sheets for SC-507,
while SC-504 is specifically elicited against the mouse protein Flk 1.
A recombinant Flk-1 protein fragment, supplied by Santa Cruz, was
used as positive control and tested in parallel to our samples whenever
SC-504 was used in western blots.
From a search by Blast 2.0 (available from the NCBI, National
Center for Biotechnology Information), the homology of the peptide
recognized by the antibodies and the avian corresponding sequences
were sufficient for us to hypothesize a cross reaction. Identities of
human VEGF with chick VEGF (retrieved as sp/p52582) were 69%
(94/135), while positivities were 82% (112/135), with a score of 218
bits and an E value of 2e-56. Identities between mouse Flk 1 and quail
Quek 1 precursor (retrieved as sp/p52583), the closest to chick,
amounted to 70% (123/175), while positivities were 141/175 (80%),
with 1% gaps (2/175). The score was 236, with an E value of 6e-62.
Cross reactivity with chicken VEGF and VEGFR2 was confirmed
by the fact that immunolocalization on mouse and chicken embryo
tibiae showed an identical pattern of distribution (Figs 1 and 6).
AntiExFABP were rabbit polyclonal antibodies against
recombinant chicken ExFABP (Gentili et al., 1998).
Anti-phosphotyrosine antibody was a mouse monoclonal IgG
(Santa Cruz catalog SC-508).
Immunohistochemistry
Sections of chick and mouse embryo limbs of different ages were
deparaffinized and treated with methanol:H2O2 (49:1) for 20 minutes,
to inhibit endogenous peroxidase. They were than digested with
hyaluronidase (1 mg/ml, in PBS) for 15 minutes, at 37°C, and washed
in PBS. Aspecific sites were saturated with normal goat serum, for 20
minutes at room temperature. Sections were incubated with the
specific antisera (all diluted 1:100, for 1 hour at room temperature),
washed several times in PBS, exposed to a biotinylated goat antirabbit IgG (1:200, 30 minutes, room temperature) (Jackson
immunoresearch Laboratories Inc., West Grove, PA) and, after
additional washing, exposed to peroxidase-conjugated egg white
avidin (Jackson Laboratories, Inc.) (1:500, 30 minutes, at room
temperature). After one wash in PBS and one in Na acetate buffer 50
mM, pH 5, antibody binding sites were detected through the enzyme
activity on 3-amino-9-ethyl-carbazole (AEC) substratum (0.4% in
dymethylformamide, 1 ml; Na acetate 50 mM, pH 5, 9 ml; H2O2 30%,
0.01 ml). After 5-10 minutes, at room temperature and in the dark,
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sections were counterstained with Harris’ hematoxylin, mounted
(Glycergel, Dako, Gløstrup, DK) and photographed in a Zeiss
Axiophot (Oberkochen, Germany).
Western blot analysis
Cell layers were lysed with 0.1% SDS in PBS. Aliquots of samples
corresponding to 1-2 mg of total proteins were loaded on a 15% SDSpolyacrylamide gel and electrophoresis was performed in reducing
conditions. After electrophoresis, the gel was blotted to a PVDF
membrane (Millipore Corporation, Bedford, MA, USA) following the
procedure described by Towbin et al. (1979).
The blot was saturated overnight with 3% skim milk powder
(Merck Biochemica, Darmstadt, Germany) in TTBS buffer (20 mM
Tris-HCl, pH 7.5, 500 mM NaCl, 0.05% Tween-20) and washed
several times with TTBS. It was then incubated (for 3 hours at room
temperature) with the rabbit polyclonal antibody directed against the
human VEGF (Santa Cruz).
After further washes, detection was performed by a biotinconjugated anti-rabbit IgG (Jackson Immunoresearch Laboratories
Inc., West Grove, PA) and avidin-HRP (Jackson Immunoresearch
Laboratories Inc.), followed by the non radioactive ECL detection
system (Amersham International plc, Little Chalfont, UK).
Immunoprecipitation
Hypertrophic chondrocytes were incubated for 2 hours in serum free
F12 medium. Stimulation was performed with human recombinant
VEGF (Peprotech Inc., Rocky Hill, NJ, USA), 10 ng/ml, 5 minutes at
37°C. Unstimulated cells in serum free medium were used as control.
After incubation, cells were washed twice with PBS containing 1 mM
Na orthovanadate. Lysates were obtained resuspending cells in 0.5-1
ml immunoprecipitation buffer with protease inhibitors (IP buffer: 50
mM Tris-HCl, 150 mM NaCl, 1 mM Na orthovanadate, 0.1 mM
ZnCl2, 1 mM PMSF, leupeptin 50 µg/ml, aprotinin 10 µg/ml,
pepstatin 10 µg/ml, Triton X-100 1%), incubated at 4°C, 10 minutes,
and centrifuged to eliminate cell debris. For preclearing, 50 µl of
Protein A-Sepharose beads (Protein A-Sepharose CL-4B, Pharmacia
Biotech AB, Uppsala, Sweden) were added for 30 minutes at 4°C.
Total protein content was assessed by the BCA method (Pierce,
Rockford, IL, USA). Samples were equalized in volume and
protein concentration, supplemented with specific antibodies
(immunopurified polyclonal antibodies against VEGFR2, Santa Cruz,
or anti-ExFABP as control), in the proportion of 1 µg immunoglobulin
for 500 µg total protein, and incubated for 1 hour on an orbital shaker,
at 4°C. After addition of 100 µl Sepharose beads (1 additional hour,
4°C, orbital shaker), samples were centrifuged (13000 rpm, 2 minutes)
for precipitation of immunocomplexes.
Pelleted beads were washed twice, first with IP buffer and then with
washing buffer (IP buffer without Triton X-100), resuspended in
sample buffer, and boiled to release immunocomplexes from the
beads. After an additional centrifugation, proteins in the supernatant
were resolved on a 10% SDS-PAGE, in reducing conditions. After
transferring to a PVDF membrane (Millipore Corporation, Bedford,
MA, USA), a western blot was performed as previously described,
using rabbit anti-human VEGFR2 or anti-phosphotyrosine mAb
(1:100) as primary antibody. Immunoreactive bands were detected
with the non radioactive ECL system (Amersham International plc,
Little Chalfont, UK).
Chemoinvasion and chemotaxis assays
Following described methods (Albini et al., 1987), Boyden chamber
chemoinvasion assays were performed with some modifications.
Polycarbonate filters (12 µm pore size, PVP-free, Nucleopore,
Concorezzo, Italy) were covered with Matrigel (an EHS murine
sarcoma extract containing basement membrane components;
Kleinman et al., 1986), at the concentration of 25 µg/filter. After air
drying the mixture was reconstituted into a solid gel.
As chemoattractants in the lower compartment of a Boyden
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M. F. Carlevaro and others
chamber, conditioned media from chondrocytes were used. Media
conditioned by distinct primary cultures of hypertrophic chondrocytes
were used in different experiments.
HMEC-1 cells are an immortalized human microvascular
endothelial cell line retaining morphologic, phenotypic and functional
characteristics of the corresponding normal human type (Ades et al.,
1992). Based on this, HMEC-1 cells were used for the experiments
reported in the present work. Endothelial cells (1.3×105 cells per
chamber) were placed in the Boyden chamber upper compartment
after harvesting with trypsin and washing with serum-free medium.
Migration assays were prolonged for 6 hours, at 37°C in 5% CO2.
Cells adhering to the upper surface of the filter were mechanically
removed, whereas those that had migrated beyond the filter to the
under-surface were stained (Toluidine Blue, Sigma Chemical Co.) to
allow microscopic quantitation. Triplicates of each point and at least
one repetition of each experiment were performed. Standard
significance was based on counting five to ten random fields on
each filter. Incubation of chemoattractants with antibodies and
corresponding control samples took place overnight at 4°C.
Chemotaxis assays were performed similarly to chemoinvasion, but
filters were coated with gelatin (5 µg/ml, type A, Sigma Chemical
Co.) instead of Matrigel.
Fig. 1. Immunohistochemical analysis of avian and mammal embryo
tibiae for the presence of vascular endothelial growth factor. Chick
(10 d., A-D) and mouse (16 d., E-G) embryos were stained with antiVEGF polyclonal antibodies (A-C and E-F), with preimmune rabbit
serum used as negative control (D and G). Hypertrophic chondrocytes
show clear positivity for VEGF (A-B-E-F), that is also localized in
‘borderline’ chondrocytes (C). In B and F enlargements of the areas
of hypertrophy in A and E are shown, while C shows a detail of the
borderline zone. Bars: 50 µm (A-D-E); 25 µm (B-C-F-G).
VEGF and endochondral bone formation
63
RESULTS
VEGF is detectable in embryonic hypertrophic
cartilage
To asses the role of VEGF in cartilage physiological
neovascularization during development, we investigated by
immunohistochemistry for the presence of the factor in
mammalian and avian embryo long bone growth plate at stages
prior and contemporary to cartilage hypertrophy. Cartilage
hypertrophy precedes vascularization of the bone rudiment.
The factor was differentially expressed during chondrocyte
differentiation. VEGF was present in fully mature hypertrophic
chondrocytes, sporadically present in prehypertrophic
chondrocytes, but totally absent in proliferating and quiescent
cells, both in chicken (Fig. 1A-D) and in mice (Fig. 1E-G).
Antibodies against VEGF also stained the ‘borderline’
chondrocytes (1C). ‘Borderline’ chondrocytes have been
described as chondrocytes that specifically retain a bone
forming potential and express osteoblast traits, as a result of
their interposed localization between bone and cartilage
(Bianco et al., 1998).
Choroid plexus and kidney glomeruli in mouse embryos of
15 days were used as positive controls to test specificity of the
reaction of the antibodies against VEGF (data not shown).
VEGF synthesis appeared to be precisely timed when
chondrocytes become hypertrophic, before the onset of
cartilage neovascularization, i.e. at day 10-11 in chicken and
at day 14-15 in mice.
Similarly to cartilage, in skeletal muscle, the VEGF was
expressed just before the onset of tissue vascularization (data
not shown). This finding is in agreement with the observations
made by Aitkenhead et al. (1998).
Hypertrophic chondrocytes produce VEGF in vitro
The observed VEGF synthesis by hypertrophic chondrocytes
in vivo was confirmed by in vitro studies performed taking
advantage of a culture system for avian chondrocyte
differentiation. In this culture VEGF was released by
chondrocytes maintained in conditions leading to the formation
of engineered hypertrophic cartilage. Hypertrophic
chondrocytes grown in suspension in the presence of ascorbic
acid for 14 days expressed all the markers of hypertrophy, and
were enclosed in a correctly folded extracellular matrix
containing cartilage specific proteoglycans and collagen fibrils.
We have previously shown that these cells synthesize a high
amount of ovotransferrin, which was demonstrated to hold
angiogenic effects both in vivo and in vitro (Carlevaro et al.,
1997). Here we report that immunohistochemical staining of
this in vitro engineered hypertrophic cartilage with specific
antibodies shows positivity for VEGF in the cells, around their
lacunae and in the interposed extracellular matrix (Fig. 2).
The VEGF synthesized in vitro by hypertrophic
chondrocytes has a molecular mass of 24 kDa. A western blot
analysis of the conditioned culture medium, resolved for its
protein content by electrophoresis in reducing conditions,
indicated that the immunoreactive species has a molecular
mass of approximately 24 kDa (Fig. 3, lane 2). An additional
doublet band of approximately 28 and 29 kDa was also evident.
This result is in agreement with the reported existence of
several VEGF isoforms in different species and in chick in
particular (Flamme et al., 1995). According to cDNA cloning
Fig. 2. Immunolocalization of VEGF in hypertrophic chondrocytes
cultured in suspension in the presence of ascorbic acid. (A) Avian
chondrocytes in culture for 14 days in the presence of ascorbic acid
produce VEGF, with specific staining for the factor in the cells and in
the surrounding matrix. (B) Negative control with preimmune rabbit
serum used as primary antibody. Bar, 20 µm.
results, possible avian VEGF isoforms are 122, 146, 166 and
190 amino acids long. Mice isoforms could be 120, 164 and
188 amino acids long. Five products of 121, 145, 165, 189 and
206 amino acids, respectively, are recognized in humans.
VEGF isoforms of quail have a predicted size of 14, 17, 19.4
and 22 kDa; a 3-5 kDa observed size increase estimated by
western blot with respect to the expected size can be easily
explained by peptide glycosylation (Flamme et al., 1998).
Paracrine response of endothelial cells to the
chondrocyte-derived VEGF
Serum free supernatants from hypertrophic chondrocytes act
powerfully in the activation of endothelial cell migration and
invasion. The addition to the chemoattractant of polyclonal
antibodies blocking the biological function of VEGF, at a
concentration of 200 ng/ml, reduced the chemotactic and
chemoinvasive effects of the media up to 50% (Fig. 4).
Immunopurified
polyclonal
antibodies
against
ovotransferrin (50 µg/ml) were used as a positive internal
control and inhibited cell migration in a way comparable to
anti-VEGF antibodies. Equal, ten- and one-hundred-fold
amounts of immunopurified antibodies against Ex-FABP, a
protein synthesized by hypertrophic chondrocytes but lacking
angiogenic activity, did not change the level of migration and
were used as an internal negative control (Fig. 4).
Immunopurified antibodies against an unrelated protein (antiTrkE, a kind gift from Dr E. Di Marco) also failed to give any
effect (data not shown).
64
M. F. Carlevaro and others
Fig. 3. Western blot analysis of VEGF in hypertrophic chondrocytes.
Proteins in conditioned media of cells in suspension with ascorbic
acid for 14 days were separated in reducing conditions on 15% SDSpolyacrylamide gel. About 2 mg of total proteins of each sample
were loaded. Immunodetection was performed with anti-VEGF
purified antiserum (lanes 1-2); preimmune rabbit serum was used as
negative control (lanes 3). Arrow refers to the recognized avian
VEGF form, of approximately 24 kDa. Human recombinant VEGF
(approximately 19 kDa) was used as positive control. Lane 1: human
recombinant VEGF (100 ng). Lanes 2-3: conditioned medium.
Since antibodies directed against both VEGF and
ovotransferrin failed to completely abolish chemotactic and
chemoinvasive activity in the conditioned medium, we checked
for a possible synergic effect of VEGF and ovotransferrin.
Contemporary treatment of media with both antibodies did not
show either additive or synergic properties (data not shown).
The addition in the Boyden chamber of anti-VEGFR2
neutralizing antibodies (200 ng/ml; Ahmed et al., 1997)
together with the conditioned medium halved endothelial cell
migration and invasion (Fig. 5). The inhibition of VEGF action
observed blocking its receptor VEGFR2, considered to be
endothelial specific, supports the concept that migration and
invasion of endothelial cells are regulated by VEGF produced
by chondrocytes via a paracrine angiogenic loop.
VEGF receptor 2 is colocalized with VEGF in in vivo
cartilage and in in vitro engineered cartilage
VEGF expression by hypertrophic chondrocytes has to be
viewed in the perspective of a role, in an avascular tissue such
as cartilage, other than that of an inducer of angiogenesis. In
the hypothesis of an additional VEGF autocrine activity,
possibly mediated by a receptor, immunohistochemical
analyses for the presence of VEGFR2 were performed on
sections of chicken and mice embryo tibiae (respectively at 10
and 16 days of development). A clear positive signal for the
receptor was detected in hypertrophic chondrocytes of both
species (Fig. 6A-D), whereas in the same growth plates
quiescent and proliferating chondrocytes were negative. The
receptor was distinctly associated with cell bodies and
membranes. A strong positivity was also detectable in articular
chondrocytes (data not shown). As in the case of VEGF
distribution, borderline chondrocytes showed some VEGFR2
expression (Fig. 6B1). It should be noted that limb skeletal
muscles were also stained by the antibodies directed against
the receptor (data not shown).
Antibodies against VEGFR2 specifically stained endothelial
Fig. 4. Antibody-mediated inhibition of the chemotactic migration
and invasion induced by chondrocyte-released VEGF. Chemotactic
(A) and chemoinvasive (B) responses of microvascular endothelial
cells (HMEC-1) to conditioned medium from hypertrophic
chondrocytes in suspension with ascorbic acid for 14 days. Medium
was preincubated without (first column) and with polyclonal
antibodies against VEGF, at the concentration of 200 ng/ml
(antiVEGF). Anti-ovotransferrin immunopurified antibodies (50
µg/ml) were used as internal positive control for inhibition
(antiOTF). Antibodies against ExFABP, a protein secreted by
chondrocyte and present in the medium, were used as an internal
negative control (antiExFABP, 200 ng/ml). Antibodies alone elicited
cell migration and invasion equivalent to background levels (data not
shown). F12 medium alone was used as control for background
random migration that has been subtracted. Assays were performed
in triplicate and repeated at least twice. Five fields were counted on
each triplicate filter. Bar indicates standard deviation. A typical
experiment is shown.
cells at the same stage (Fig. 6E-F). Therefore, both in
embryonic cartilage and in developing muscle, VEGF receptor
is expressed at the same time as VEGF, i.e. at the same time
when physiological neovascularization of tissues occurs during
development. This finding is in agreement with previous
findings by Aitkenhead et al. (1998). At earlier embryonic
VEGF and endochondral bone formation
65
chondrocytes in suspension culture expressed VEGFR2 on
their cell surface (Fig. 7).
Hypertrophic cartilage is a possible target for a
VEGF/VEGF receptor autocrine loop
The concerted expression of VEGF and VEGF receptors in a
non-endothelial and avascular tissue, such as hypertrophic
cartilage, suggests that VEGF does not only promote vascular
invasion of growth plate, but also acts as an autocrine signal
for the cell of the chondrogenic lineage.
We investigated functional signal transduction in
hypertrophic chondrocytes, grown in suspension in the
presence of ascorbic acid, both stimulated and not stimulated
with VEGF for 5 minutes. Proteins were first
immunoprecipitated from cell lysates with antibodies against
VEGFR2/Flk-1 and then analysed by western blot with
a monoclonal antibody against phosphotyrosine. An
immunoreactive band with an approximate molecular mass of
180 kDa, as reported for VEGFR2, was evident both in
stimulated and unstimulated cells (Fig. 8, lane 1-2). The
identification of this band was confirmed by western blot with
antiVEGFR2 antibodies (lane 3-4).
The phosphorylation of the tyrosine kinase receptor in
chondrocytes was independent of VEGF treatment. This
finding supports the concept of an active autocrine
VEGF/VEGFR2 loop in hypertrophic chondrocytes.
As expected when we substituted specific antibodies with
preimmune rabbit serum in the western blot analysis or with
antibodies against an unrelated protein in the initial
immunoprecipitation, we failed to detect any positive signal
(lanes 5-8).
DISCUSSION
Fig. 5. Inhibition by anti-VEGF receptor 2 blocking antibodies of
chemotactic migration and invasion induced by hypertrophic
chondrocyte conditioned media. Chemotactic (A) and chemoinvasive
(B) responses of microvascular endothelial cells (HMEC-1) to
conditioned medium from hypertrophic chondrocytes in suspension
with ascorbic acid for 14 days. Medium was preincubated without
(first column) and with polyclonal antibodies against VEGF receptor
2/Flk-1 (antiVEGFR2), at the concentration of 200 ng/ml. Antiovotransferrin immunopurified antibodies (50 µg/ml) were used as
internal positive control for inhibition (antiOTF). Antibodies alone
elicited cell migration and invasion equivalent to background levels
(data not shown). F12 medium alone was used as control for
background random migration that has been subtracted. Assays were
performed as in Fig. 4. Bars indicate standard deviation. A typical
experiment is shown.
stages (chick 7 day (d.), mouse 13 d.) the factor and its receptor
were not detectable in the mesenchymal condensation and
in the cartilage bone rudiment prior to hypertrophy; at later
stages (chick 12-14-16 d., mouse 18 d. and newborn) VEGF
production by chondrocytes diminished and a limited number
of receptors was detectable on the chondrocyte cell membrane
(not shown).
The VEGF receptor is also expressed during in vitro
differentiation of chondrocytes. In addition to secreting VEGF,
in the presence of ascorbic acid, avian hypertrophic
Vascular endothelial growth factor/vascular permeability
factor is a well characterized secreted angiogenic factor
acting on endothelial cells. It is produced by human and
animal tumor cells of different embryonal origin (ectodermal,
mesodermal and endodermal) and in non-neoplastic
pathologies characterized by angiogenesis, such as cutaneous
wound healing, ischemic myocardium and rheumatoid
arthritis (Dvorak et al., 1995). In early embryos and, at later
developmental stages, in fetal organs, VEGF is involved in
establishing the novel embryonic circulatory system by the
process of vasculogenesis (Risau, 1997), whereas VEGF
production in normal adult tissues, such as kidney, brain
and pituitary gland, has to be seen in the perspective of
the maintenance of steady state vasculature (Ferrara et al.,
1992).
An increasing number of observations suggests that VEGF,
for a long time considered to be endothelium specific on the
basis of its receptor localization, might instead have effects also
on non endothelial cell types, holding active signal transduction.
Active signal transduction through VEGF receptors plays a
fundamental role in normal trophoblast cells (Ahmed et al.,
1997), and in uterine smooth muscle (Brown et al., 1997), and
in rodent models Claffey et al. (1992) reported on the role of
VEGF during early adipogenesis and myogenesis and neuronal
transformation. Similarly VEGF/VEGFR are involved in
pathological processes such as germ cell tumors (Viglietto et al.,
66
M. F. Carlevaro and others
Fig. 6. Immunohistochemical
analysis of avian and
mammal embryo tibiae for
the presence of vascular
endothelial growth factor
receptor 2. Chick (10 d., AB1-B2) and mouse (16 d., CF) embryos were stained
with anti-VEGF receptor
2/Flk-1 polyclonal
antibodies. Hypertrophic
chondrocytes (hc,
arrowheads) and also
borderline cells (bc, empty
arrows) show clear positivity.
Proliferating chondrocytes
are negative (arrows, pc).
Endothelial cells (asterisks,
ec) are specifically stained by
the antibodies against
VEGFR2 (6E). Negative
control with preimmune
rabbit serum (6F). B1-B2 and
D are enlargements of the
areas highlighted,
respectively, in A and C.
Bars: 60 µm (A); 35 µm (B1B2); 50 µm (C); 25 µm (DE-F).
1996),
and
thyroid
hypervascularization
(Viglietto et al., 1997).
VEGF has also been
localized
in
normal
human skin explants. In
keratinocytes the distinct
isoforms of the factor are
differentially upregulated
by hypoxia; at the
same time, in dermal
microvessels, flt-1 receptor
is induced dramatically,
whereas
flk-1
is
downregulated (Detmar et
al., 1997).
The avian homologue
of VEGFR-2 is Quek-1,
whereas Quek-2 is similar to VEGFR-3/flt-4 (Eichman et al.,
1996). In 9 day avian embryos, the receptor Quek-1 was
expressed during tissue differentiation in several organs
(kidney, central nervous system, muscle), and VEGF
expression was detected in neural tube, thyroid gland and
cartilaginous skeleton before the onset of angiogenesis, and in
endothelial cells in the developing bone (Aitkenhead et al.,
1998).
So far, cartilage development toward endochondral bone has
been only marginally considered in the perspective of a
possible VEGF expression and activity. Very preliminary
observations indicated VEGF production in vertebrae
primordia (Jakeman et al., 1993).
VEGF was overexpressed in avian embryos and specifically
in cartilage (Flamme et al., 1995). Neither premature cartilage
angiogenesis nor embryonal limb pattern alterations were
observed; an increased vascular density with increased
permeability and oedema were observed instead. When
different avian VEGF isoforms were overexpressed in the avian
eye, neither the avascular cornea, nor the retina were
ectopically invaded by capillaries (Schmidt and Flamme,
1998). These observations could be explained by the presence
of angiogenesis inhibitors associated with the extracellular
matrix of the tissues which, in the case of cartilage, are
somehow overwhelmed during development by angiogenic
substances promoting the ingrowth of new vessels and
subsequent bone deposition.
The aim of our study was to assess a possible role played by
VEGF in cartilage maturation and endochondral ossification,
combining information derived from studies on the in vivo
VEGF and endochondral bone formation
Fig. 7. Immunolocalization of VEGF receptor 2 in cultured
hypertrophic chondrocytes. (A) Avian chondrocytes in suspension
for 14 days in the presence of ascorbic acid synthesize VEGF
receptor 2/Flk 1, with specific staining in the cells. (B) Negative
control with preimmune rabbit serum used as primary antibody. Bar,
20 µm.
distribution of the factor and its receptor in developing
embryos with studies performed taking advantage of an in vitro
chondrocyte differentiation system.
VEGF was localized in developing long bones of avian and
mammalian embryos, specifically in the hypertrophic cartilage,
temporally and spatially soon before vascular invasion of the
growth plate. Quiescent and proliferating chondrocytes failed
to release VEGF, whereas borderline chondrocytes expressed
the factor.
In vitro, hypertrophic chondrocytes embedded in a correctly
folded cartilaginous extracellular matrix, corresponding to
their in vivo counterpart, synthesize VEGF in a molecular
monomeric form of approximately 24 kDa. This avian 190
amino acid long isoform contains both a long (44 aa) and a
short (24 aa) basic domain, and confers on the factor an affinity
for heparin, binding to extracellular matrix and association
with the cell surface (Flamme et al., 1995; Park et al., 1993).
In principle such molecular features might interfere with
VEGF free diffusion and mitogenic action on endothelium;
nevertheless we have shown that a significant part of the
angiogenic activity released into the medium conditioned by
the cultured cells is abolished by specific blocking antibodies.
Conditioned culture media from hypertrophic chondrocytes
cultured in suspension in the presence of ascorbic acid were
earlier discovered to have chemotactic and chemoinvasive
potential towards human endothelial cells (Descalzi et al.,
1995). In this paper we have reported that VEGF released into
the media contributes to the angiogenic activity to an extent of
up to 50%. Ovotransferrin is another protein secreted by
chondrocytes whose angiogenic and chemoattractive potentials
67
Fig. 8. VEGF receptor 2 is in a phosphorylated state in hypertrophic
chondrocytes in suspension with ascorbic acid, independently from
stimulation with VEGF. Before immunoprecipitation, cells were
incubated in serum free medium either with (lane 2-4-6-8) or without
(lane 1-3-5-7) VEGF, for 5 minutes at 37°C. Cell extracts were
obtained with 1 ml of immunoprecipitation buffer and incubated with
anti-VEGF receptor 2 or antiExFABP; samples (1 mg of total
proteins) were then resolved by SDS-PAGE 10%, blotted and
followed by immunodetection. Lanes 1-2: immunoprecipitation with
antiVEGFR2, western blot with anti-phosphotyrosine. Lanes 3-4:
immunoprecipitation and western blot with antiVEGFR2. Lanes 5-6:
immunoprecipitation with antiExFABP, western blot with antiVEGFR2. Lanes 7-8: immunoprecipitation with antiVEGFR2,
western blot with preimmune rabbit serum. VEGF receptor 2/Flk 1
phosphorylation is evident from the band of the approximate
molecular mass of 180 kDa (lane1-2, arrow).
were recently discovered (Carlevaro et al., 1997). By the
contemporary addition of blocking antibodies against VEGF
and against ovotransferrin to the conditioned culture medium
prior to the Boyden chamber assay, we failed to show a
synergistic effect between the two molecules. Our data point
out the complex composition of our conditioned media,
probably containing additional active molecules still to be
isolated and interacting with mechanisms still to be elucidated.
In the present paper we have also hypothesized VEGF
autocrine action on growth plate chondrocytes regardless of the
angiogenic activity of the factor. We showed the receptor
tyrosine phosphorylation, i.e. its functional signal
transmission. At the moment we can only suggest possible
roles for this autocrine loop for maintaining chondrocyte
survival, and controlling cell differentiation and proliferation.
It should be noted that an in vivo administration of a
recombinant human antiVEGF monoclonal antibody for up to
13 weeks in adult monkeys resulted in a dose related increase
of hypertrophied chondrocytes, subchondral bony plate
formation and inhibition of vascular invasion of the growth
plate, together with effects on the female reproductive system.
The changes were reversible with interruption of the treatment
and indicated that VEGF is required for the physiological
neovascularization occurring in longitudinal bone growth
(Ryan et al., 1999).
Basic fibroblast growth factor, another angiogenic factor,
does also have the dual effect of regulating in vitro terminal
chondrocyte differentiation (Kato and Iwamoto, 1990) and of
accelerating vascular and bone cell invasion upon in vivo
infusion in epiphyseal growth plate (Baron et al., 1994); it has
been localized in avian growth plate (Twal et al., 1994) and
consistently demonstrated as an autocrine growth factor for
chondrocytes (Luan et al., 1996).
68
M. F. Carlevaro and others
A clear positive staining for the VEGF receptor was also
observed in articular chondrocytes. These cells will never come
in contact with endothelial cells during development, but they
are subjected to pathological neovascularization upon
inflammation in rheumatoid arthritis. Macrophages and lining
cells were proposed as a source of synovial fluid-derived
VEGF (Koch et al., 1994; Fava et al., 1994).
Our observation about parallel distributions of VEGF and
VEGF receptors in cartilage indicates that the mechanisms
hypothesized in chondrocytes might be active in different
organs. Observations in kidney, central nervous system, heart
and skeletal muscle of avian embryos (Aitkenhead et al., 1998)
coherently support the hypothesis of VEGF action as a
paracrine and autocrine factor in tissues other than endothelia
and of an involvement of this factor in embryonic
developmental processes closely linked to, but yet distinct from
angiogenesis and vasculogenesis.
During revision of this paper, Gerber et al. (1999)
demonstrated, in an animal model, the importance of VEGF in
growth plate morphogenesis and remodeling, accomplished by
a functional inactivation approach, through administration of a
soluble receptor chimeric protein sequestering VEGF. In
agreement with our findings about the protein, VEGF expression
by hypertrophic chondrocytes is indicated to be responsible for
directional growth of metaphyseal blood vessels.
By the same authors, VEGF receptor 2/Flk 1 messenger was
found physiologically in the growth plate of juvenile mice: in
endothelial cells, as expected, and at the cartilage-bone
junction. Our observations on embryonic cartilage confirm the
localization at the chondro-osseous junction of the protein;
however, it was possible to assess a clear signal for VEGFR2
in hypertrophic chondrocytes at times earlier to vascular
invasion and bone formation in vivo and in cultured
hypertrohic chondrocytes in vitro.
Partially supported by funds from Associazione Italiana per la
Ricerca sul Cancro and MURST. We thank Dr A. Albini for helpful
discussion. We also thank ms. Barbara Minuto for editing the
manuscript, ms. Paola Gregori for secretarial help, and ms. Raffaela
Arbicò for histological assistance.
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