61
Expression of vascular endothelial growth factor in the growth plate
is stimulated by estradiol and increases during pubertal development
Joyce Emons, Andrei S Chagin1, Torun Malmlöf1, Magnus Lekman1, Åsa Tivesten2,
Claes Ohlsson2, Jan M Wit, Marcel Karperien3,4 and Lars Sävendahl1
Department of Paediatrics, Leiden University Medical Center, 2300 ZA Leiden, The Netherlands
1
Department of Women’s and Children’s Health, Karolinska Institutet, SE-171 76 Stockholm, Sweden
2
Division of Endocrinology, Department of Internal Medicine, Sahlgrenska University Hospital, SE-41345 Gothenburg, Sweden
3
Department of Tissue Regeneration, University of Twente, 7522 NB Enschede, The Netherlands
4
Department of Endocrinology and Metabolism, Leiden University Medical Center, 2300 ZA Leiden, The Netherlands
(Correspondence should be addressed to J Emons who is now at Department of Paediatrics, LUMC, PO Box 9600, 2300 RC Leiden,
The Netherlands; Email:
[email protected])
Abstract
Longitudinal bone growth is regulated in the growth plate.
At the end of puberty, growth velocity diminishes and
eventually ceases with the fusion of the growth plate
through mechanisms that are not yet completely understood. Vascular endothelial growth factor (VEGF) has an
important role in angiogenesis, but also in chondrocyte
differentiation, chondrocyte survival, and the final stages of
endochondral ossification. Estrogens have been shown to
up-regulate VEGF expression in the uterus and bone of rats.
In this study, we investigated the relation between estrogens
and VEGF production in growth plate chondrocytes both
in vivo and in vitro. The expression of VEGF protein was
down-regulated upon ovariectomy and was restored upon
estradiol (E2) supplementation in rat growth plates.
In cultured rat chondrocyte cell line RCJ3.1C5.18, E2
dose dependently stimulated 121 and 189 kDa isoforms of
VEGF, but not the 164 kDa isoform. Finally, VEGF
expression was observed at both protein and mRNA levels
in human growth plate specimens. The protein level
increased during pubertal development, supporting a link
between estrogens and local VEGF production in the
growth plate. We conclude that estrogens regulate VEGF
expression in the epiphyseal growth plate, although the
precise role of VEGF in estrogen-mediated growth plate
fusion remains to be clarified.
Introduction
high concentration, estrogens inhibit growth and promote
epiphyseal fusion in the long bones (Ross et al. 1986, Metzger
& Kerrigan 1994, Klein et al. 1996).
A critical step in endochondral ossification is when blood
vessels enter from the primary spongiosum and osteoblasts
invade from the bone marrow to lay down trabecular bone
(Kember 1993, Hunziker 1994). Vascular endothelial growth
factor (VEGF) is a potent mediator of angiogenesis, but it has
also been shown to modulate chondrocyte differentiation and
survival, osteoblast differentiation, and osteoclast recruitment
(Zelzer et al. 2004, Zelzer & Olsen 2005, Dai & Rabie 2007).
VEGF is expressed by growth plate chondrocytes and
osteoblasts in different species including humans (Gerber
et al. 1999, Carlevaro et al. 2000, Garcia-Ramirez et al. 2000,
Haeusler et al. 2005). Human VEGF is present in six different
proteins, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E,
and VEGF-F, where VEGF-A has been shown to be
expressed in the growth plate and also believed to be most
important in the regulation of longitudinal bone growth.
VEGF-A has six alternatively spliced isoforms: VEGF-121,
Longitudinal growth occurs at the epiphyseal plate, a thin
layer of cartilage entrapped between epiphyseal and metaphyseal bones, at the distal ends of the long bones
(Kronenberg 2003). In the growth plate, immature cells lie
toward the epiphysis, called the resting zone, with flat more
mature chondrocytes in the proliferating zone and large
chondrocytes in the hypertrophic zone adjacent to this. At the
end of puberty, longitudinal growth ceases with total
replacement of avascular cartilage by highly vascularized
bone, eventually resulting in epiphyseal fusion. Estrogens are
known to be important hormones in the regulation of
growth plate maturation and epiphyseal fusion; they regulate
and can accelerate the programmed senescence of the growth
plate, leading to proliferative exhaustion of chondrocytes and
epiphyseal fusion (Weise et al. 2001, Chagin & Savendahl
2007). At a low concentration, estrogens are known to
increase growth velocity, an effect possibly mediated through
the GH–insulin-like growth factor 1 (IGF1) axis, while at a
Journal of Endocrinology (2010) 205, 61–68
0022–0795/10/0205–061 q 2010 Society for Endocrinology
Journal of Endocrinology (2010) 205, 61–68
Printed in Great Britain
DOI: 10.1677/JOE-09-0337
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and others . Estradiol stimulates VEGF expression
VEGF-145, VEGF-165, VEGF-183, VEGF-189, and
VEGF-206 (Robinson & Stringer 2001). The receptors
involved in VEGF-A signaling are VEGFR-1 (also known as
fms-like tyrosine kinase receptor 1, FLT1) and VEGFR-2
(also known as kinase insert domain-containing receptor,
KDR), with almost all responses being mediated through the
second receptor, which has also been detected at the
chondro-osseous junction in the mouse growth plate (Gerber
et al. 1999, Ferrara et al. 2003, Dai & Rabie 2007).
Inactivating VEGF in mice and monkeys resulted in
impaired trabecular bone formation and expansion of the
hypertrophic zone, indicating inhibition of cartilage resorption (Gerber et al. 1999, Ryan et al. 1999). In addition, Vegfa
conditional knockout mice driven by a Col2a1 promoter
showed delayed invasion of blood vessels into the primary
ossification center and delayed removal of terminal hypertrophic chondrocytes together with massive cell death in
chondrocytes throughout the growth plate, demonstrating
the importance of VEGFA in chondrocyte survival (Zelzer
et al. 2004).
In vitro VEGF expression can be up-regulated by factors
known to be important in the regulation of longitudinal bone
growth such as fibroblast growth factor, transforming growth
factor b, and IGF1 (Garcia-Ramirez et al. 2000). Studies on
rats showed that in uterus and bone tissue, VEGF expression is
up-regulated by estrogens (Hyder et al. 1996, Mekraldi et al.
2003). In humans, bone growth and estrogen levels increase in
parallel during earlier phases of puberty, while at the end of
puberty, growth ceases with a total replacement of cartilage by
bone resulting in the fusion of the growth plate.
We hypothesized that estrogens have the capacity to
stimulate local VEGF production in growth plate chondrocytes, and that this could be a possible mechanism involved
in the process of growth plate maturation and fusion in
humans. To address this, we performed in vivo studies
in ovariectomized rats supplemented with estradiol (E2) and
also in vitro studies in cultured rat chondrocytes exposed to E2,
and assessed chondrocyte-specific expression of VEGF.
In addition, we measured VEGF expression in growth plate
specimens obtained from humans in different pubertal stages.
Materials and Methods
Animals and study protocol
Female Sprague–Dawley rats were purchased from Scanbur
BK AB (Sollentuna, Sweden). The animals were housed in a
temperature- and humidity-controlled room under a 0600 h
light:0600 h darkness cycle, and allowed a soy-free diet
containing 0.7% of calcium and 0.5% of phosphorus (R70;
Lactamin AB, Kimstad, Sweden) and tap water ad libitum.
All procedures were approved by the Ethics Committee at
Göteborg University, and conformed to the National
Institutes of Health Guide for the Care and Use of Laboratory
Animals. The animals were randomly divided into three
Journal of Endocrinology (2010) 205, 61–68
groups: sham operationCvehicle treatment (sham, nZ12),
OVXCvehicle treatment (OVX, nZ10), and OVXCE2
treatment (E2, nZ11). At 12 weeks of age (body weight,
251G2 g), the rats were either sham-operated or OVX under
isoflurane anesthesia (Baxter Medical AB, Kista, Sweden),
and small silastic implants were placed subcutaneously in the
cervical region. The silastic implants were prepared as
described previously, releasing 2.5 mg/day of E2 (Vandenput
et al. 2002). Vehicle-treated animals received an empty
implant. E2 was obtained from Sigma Chemical. After 6
weeks of treatment, the animals were killed by excision of the
heart under isoflurane anesthesia, and the right proximal tibia
was fixed in 4% paraformaldehyde, decalcified, and
embedded in paraffin. Uterus size, correlating with estrogen
levels, was smaller in the ovariectomized rats and slightly
larger in the estrogen-supplemented animals compared with
the sham-operated animals, indicating supra-physiological
levels of estrogens in the estrogen-treated group. Study details
were described previously (Tivesten et al. 2004, 2006).
Patients and tissue preparation
Human proximal and distal femur growth plate tissues were
collected from 12 girls at different pubertal stages who were
undergoing surgery for different medical indications
(Table 1). One fetal sample was collected from a female
donor of 23 weeks of gestational age. The study protocol
was approved by the local medical ethics committees of the
Leiden University Center, Leiden, The Netherlands, and by
the Karolinska University Hospital, Stockholm, Sweden.
Informed consent was obtained from all patients and their
parents. Epiphyseal samples were either directly frozen in
liquid isopentane and stored at K80 8C or fixed in 10%
formalin, decalcified, and embedded in paraffin. All tissue
samples were processed in the same way.
Immunohistochemistry
All tissues were cut into 5-mm sections and mounted
on histological glass slides (Starfrost, Knittel Glaser,
Braunschweig, Germany), dried at 37 8C overnight, and
heated at 60 8C for 1 h before immunohistochemical
treatment. Immunohistochemistry was performed as
described previously (Nilsson et al. 2003), with the modification that antigen retrieval was achieved by incubating with
0.1% trypsin (Invitrogen) for 10 min at 37 8C. Anti-VEGF
antibody was obtained from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA, USA), and was used in a 1:200 dilution for
rat tissues and 1:50 dilution for human tissues. Secondary
anti-rabbit biotinylated antibody (Jackson ImmunoResearch
Lab, West Grove, PA, USA) was used in a 1:1000 dilution,
followed by incubation with avidin–biotin Vectastain ABC
reagent according to the manufacturer’s instructions (Vector
Laboratories, Burlingame, CA, USA). Digital images were
collected employing a Nikon Eclipse E800 microscope
equipped with an Olympus DP70 digital camera.
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and others 63
Table 1 Patient information and human tissues. The diagnosis of each patient, location of growth plate, age, in years, Tanner pubertal breast
stage (B1–B5), and experiment in which growth plate sample was used (IHC and/or qPCR) are given in the table
Diagnosis
Bone
Age (years)
Puberty
Experiment
Leg length difference
Hip luxations, femur head resection
Leg length difference
Leg length difference
Hip luxations, femur head resection
Cerebral palsy, femur head resection
Osteosarcoma in tibia
Upper limb amputation of the leg
Tall stature
Tall stature
Tall stature
Hip luxations, femur head resection
Fetal, selective abortion
Distal femur
Proximal femur
Distal femur
Distal femur
Proximal femur
Proximal femur
Distal femur
Distal femur
Distal femur
Distal femur
Distal femur
Proximal femur
Distal femur
9
12
12
14
13
15
8
9
10
12
15
14
23 weeks
B1–B2
B2
B2
B2–B3
B3
B4
B1
B1
B2
B3
B4
B4
Fetal
IHC
qPCR, IHC
IHC
IHC
qPCR, IHC
IHC
qPCR
qPCR
qPCR
qPCR
qPCR
qPCR
qPCR
Patient
1
2
3
4
5
6
7
8
9
10
11
12
13
IHC, immunohistochemistry.
Image analysis
Images of the central two-thirds of the rat growth plates were
captured in three visual fields. All pictures were taken at
200! magnification with a 2040!1536 resolution, and
were further analyzed in Image Pro Plus 5.0 software (Silver
Spring, MD, USA). Pictures were converted into grayscale-8
mode, and were inverted in order to obtain correct optical
density (OD) values in immunopositive areas. An automatic
bright object counting was performed to identify the
number of immunopositive objects above the defined
thresholds. Threshold level for cell size was defined as
objects with an area over 12 and 20 mm2 in the proliferative
and hypertrophic zones respectively. The total OD of the
immunopositive objects was calculated automatically, a
function referred as density sum in the Image Pro Plus
software. The analyzed areas were measured in mm2, and
results are expressed as the number of positive cells/mm2,
protein expression (OD arbitrary unit)/mm2, and protein
expression (OD arbitrary unit)/per cell. Data are presented
as meanGS.E.M.
by purification using an RNeasy kit according to the
manufacturer’s protocol (Qiagen), and the quality and
integrity of each sample were checked with the Agilent
2100 Bioanalyzer.
Real-time reverse transcription-PCR
RNA was reverse transcribed into cDNA using a First Strand
cDNA Synthesis kit for quantitative PCR (qPCR; Roche
Diagnostics Gmbh) according to the manufacturer’s instructions. Expression of VEGF-A and VEGFR-2 (KDR) mRNA
was quantified by real-time PCR using the Bio-Rad iCycler
with SYBR Green. QuantiTect Primer Assays were
purchased from Qiagen Benelux BV, and were used according
to the manufacturer’s protocol. Threshold cycles (Ct) were
estimated and averaged for the triplicates. Relative amounts of
mRNA were normalized to b2-microglobulin expression in
the same sample to account for variability in the initial
concentration and quality of total RNA and in the efficiency
of the reverse transcription reaction.
Western blotting
RNA isolation
Bone was removed from all epiphyseal samples, and 40-mm
thick sections were cut with a cryostat. Every fifth section
was followed by a 5-mm thick section, which was studied
with hematoxylin staining to ensure lack of bone
contamination. Total RNA isolation was performed with
an optimized method for RNA extraction from cartilage
as described by Heinrichs et al. (1994), except that the
protocol was started by homogenizing the sections in 1 ml
guanidine thiocyanate solution. RNA samples from bladder
and prostate tissue were obtained from Gentaur molecular
products (Brussel, Belgium). RNA extraction was followed
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Nontransformed clonal rat chondrogenic cells RCJ3.1C5.18
(C5.18 cells) were differentiated for 10 days (Lunstrum et al.
1999), and were subsequently treated for 24 h with a dose
range of E2. Cells were lysated, and the protein concentration
was measured by the Bradford protein assay (Bio-Rad
Laboratories AB). Proteins were separated on acrylamide
gels (Bio-Rad Laboratories), and transferred to polyvinylidene
fluoride membrane. Three different isoforms of VEGFA
were detected employing anti-VEGF rabbit Ab (1:1500;
sc-152, Santa Cruz Biotechnology Inc). Secondary goat antirabbit antibody was peroxidase labeled and used in a 1:10 000
dilution (Santa Cruz Biotechnology Inc). The resulting bands
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were confirmed by comparing the size of the protein in the
cell extract with that of the known molecular weight markers.
The antigen–antibody complexes were then detected by
chemiluminescence. After the films had been developed, blots
were stained with Coomassie blue to ensure equal loading
of total protein. Density measurements were normalized
per density of Coomassie blue staining (Chrysis et al. 2005).
Each experiment was repeated at least three times.
Statistical analysis
The human sections were blinded, and a relative staining
intensity was scored (score 0–3) for the proliferative and
hypertrophic zones of each growth plate. Scores were
displayed in a scatter plot, and a linear regression analysis
was performed to calculate significance. The same was done
for the qPCR results.
For the rat data, six growth plate sections for every animal
were analyzed by the image analysis protocol described above,
and means were calculated in terms of VEGF-positive cells/mm2
Figure 2 Quantification of VEGF staining in the proliferative and
hypertropic zones. VEGF staining was quantified with a computerized method for the proliferative zone. Panel A shows a
significant decrease in the amount of positive cells/mm2 growth
plate after ovariectomy, which was completely restored by 17bestradiol. Staining intensity per cell (OD arbitrary unit/cell) showed
no difference between all groups (panel B). The hypertrophic zone
showed the same trend as the proliferative zone; however, there
were no statistically significant differences (panel C). Staining
intensity per cell (OD arbitrary unit/cell) showed a significant
decrease after ovariectomy in the hypertropic zone, but there was
no restoration with estrogen supplementation (panel D). Data are
presented as meanGS.E.M. *P!0.05, ** P!0.01, and *** P!0.001.
and VEGF expression (OD arbitrary unit)/per cell for each
animal. Significance was calculated by one-way ANOVA
followed by Fisher’s protected least significant difference test.
Results
VEGF protein expression in the rat growth plate
Figure 1 VEGF immunohistochemistry staining. VEGF protein
expression was detected in the rat growth plate (panel A,
100! magnification) and the human pubertal growth plate (panel
B, 100! magnification). Panels Ai and Bi show 5! enlargements of
the proliferative zone in panels A and B. Panels Aii and Bii show
5! enlargements of the hypertrophic zone in panels A and B.
Pre-incubation of the primary antibody with recombinant VEGF
abolished the staining in both proliferative and hypertrophic
chondrocytes (panel C, 400!). Panel D shows a negative control
for the human growth plates. Bars indicate 100 mm.
Journal of Endocrinology (2010) 205, 61–68
Sham-operated rats were used as an internal control which
confirmed abundant VEGF expression in the growth plates,
in both the proliferative and hypertrophic zones (Fig. 1A).
Pre-incubation of the primary antibody with recombinant
VEGF abolished the staining in both proliferative and
hypertrophic chondrocytes (Fig. 1C). Staining was analyzed
by a computerized method for the proliferative and
hypertrophic zones (see Materials and Methods section).
To reveal any possible regulation of VEGF expression by
estrogens, we analyzed the number of VEGF-expressing
chondrocytes in rats upon ovariectomy and E2 supplementation. Ovariectomy resulted in a significant decrease in
the number of VEGF-positive cells in the proliferative zone
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Figure 3 VEGFA isoform western blot analysis in chondrocyte culture. Panel A illustrates that 17b-estradiol directly stimulated the expression
of isoforms VEGF-189, VEGF-164, and VEGF-121. The isoforms VEGF-121 and VEGF-189 showed a very strong response, and in contrast,
VEGF-164 showed just a marginal response. In panel B, the expression levels are calculated as a percentage of the VEGF expression in the
nontreated control.
(1173G93 vs 1556G100 cells/mm2 in sham-operated
animals; P!0.01), an effect that was completely restored
by E2 replacement (1713G81 vs 1173G93 cells/mm2 in
vehicle alone; P!0.001; Fig. 2A). A similar trend was
observed in hypertropic chondrocytes, albeit not statistically significant (Fig. 2C). The level of VEGF per cell
(expressed as OD arbitrary units/cell) did not differ
significantly between the groups in the proliferative zone
(Fig. 2B). However, in the hypertropic zone, ovariectomy
resulted in a significant decrease in the level of VEGF per
cell (35 747G1989 vs 43 240G1900 in sham-operated
animals; P!0.01), an effect which was not restored by E2
supplementation (36 601G1615 vs 35 747G1989 in vehicle
alone; PZ0.74; Fig. 2D).
VEGF-A isoform expression in cultured chondrocytes
In order to distinguish between direct and systemic effects of
estrogens on VEGF expression, we performed experiments in
the rat chondrogenic cell line RCJ3.1C5.18 (C5.18 cells),
which can be differentiated into hypertrophic chondrocytes
(Lunstrum et al. 1999). The cells were differentiated for
10 days and were then treated with E2 for 24 h. E2 dose
dependently stimulated the expression of VEGF-121 and
VEGF-189, while VEGF-164 expression was not affected
(Fig. 3). VEGF-189 was suppressed by low E2 concentrations,
and it was increased by high concentrations.
zone chondrocytes (Fig. 1B). When the relative staining
intensity was scored (score 0–3), a significant increase in
VEGF expression with progression of puberty was found
in both the proliferative (PZ0.022) and hypertrophic zones
(PZ0.017; Fig. 4 panels A and B). Negative controls showed
no staining (data not shown).
Studies of mRNA levels with qPCR confirmed VEGF
expression in pubertal as well as fetal human growth plates,
albeit expression of VEGF mRNA in the prepubertal growth
plate was w200-fold lower compared with the expression in
prostate and bladder tissues (positive controls). The Ct for
VEGF expression were subtracted from the b2-microglobulin
Ct in order to calculate the DCt. Average values for the
different groups were calculated and compared with the
prepubertal growth plate. The fetal growth plate showed a
4.2-fold higher expression of VEGF compared with the
prepubertal growth plate. The pubertal growth plate samples
(nZ6) showed on average a 1.6-fold higher expression of
VEGF mRNA compared with the prepubertal growth plate
(nZ2), but in contrast to VEGF protein expression, we did
not find a significant correlation between VEGF mRNA
levels assessed by qPCR and the stage of pubertal
development (PZ0.183, RZ0.238). The VEGF receptor,
VEGF protein and mRNA expressions in human growth plates
Growth plate biopsies were obtained from girls at different
stages of pubertal development. To verify how VEGF is
distributed in the human pubertal growth plate, we analyzed
VEGF expression levels in these rare tissue samples. Of the 13
collected growth plates, we analyzed six human growth plates
for VEGF protein expression and nine for VEGF mRNA
expression (Table 1). In all these human growth plates, VEGF
protein was detected in both proliferative and hypertrophic
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Figure 4 Scatter plot of VEGF expression in the human growth
plate. Relative intensity of VEGF protein expression was scored for
each growth plate and plotted in relation to the pubertal stage for
the proliferative zone (panel A) and the hypertrophic zone (panel B).
R2 values were respectively 0.766 (PZ0.022) for the proliferative
zone and 0.794 (PZ0.017) for the hypertrophic zone.
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VEGFR2, was also expressed at mRNA level in all our
growth plate samples that were analyzed. Similar to VEGF,
the average VEGFR2 mRNA level was 1.6-fold higher
in pubertal girls (nZ6) compared with the prepubertal
girls (nZ2), but there was no statistically significant
correlation with pubertal progression (PZ0.585, RZ0.045)
likely due to a high variation between samples and low
number of patients.
Discussion
Our in vivo and in vitro data demonstrate that E2 directly
stimulates the expression of VEGF in rat growth plate
chondrocytes. Furthermore, we confirmed that VEGF is
expressed in the human pubertal growth plate and that the
VEGF protein level increases with pubertal progression,
supporting a link between estrogens and local VEGF
production in the growth plate.
VEGF was previously detected mostly in hypertrophic
chondrocytes of human growth plate samples by immunohistochemistry (Haeusler et al. 2005). We observed VEGF
expression not only in the hypertropic zone, but also in the
proliferative zone, which is in line with the observations of
Horner et al. who studied VEGF protein expression in
neonatal human growth plate cartilage (Horner et al. 1999).
VEGF mRNA was also detected in the proliferative zone
of murine growth plate cartilage (Cramer et al. 2004). VEGF
expression in proliferating chondrocytes and the significant
change in expression with alternating estrogen levels were
observed in both human and rat growth plates in our study.
This slight divergence in results could be due to technical
issues attributed to immunohistochemistry such as antigen
retrieval or type of antibody.
We confirmed our immunohistochemistry data by qPCR
analysis. mRNA expression of VEGF and the VEGFR-2
was, to our knowledge for the first time, detected in
adolescent and pubertal human growth plates. VEGF
mRNA was previously detected in the fetal growth plate by
others (Garcia-Ramirez et al. 2000, Petersen et al. 2002).
Expression of VEGFR-2 was detected earlier at the chondroosseous junction in mice (Gerber et al. 1999), in epiphyseal
cartilage of pigs (Kim et al. 2009), in the avian growth plate
(Rath et al. 2007), and in hypertrophic chondrocytes of the
fetal growth plate (Petersen et al. 2002). In contrast to protein
levels, mRNA did not reveal a significant increase in VEGF
expression with progression of puberty. This discrepancy in
results could be due to a difference between mRNA and
protein expression, a difference in tissue preparation for
RNA extraction when the surrounding bone was removed or
alternatively due to a change in morphological organization
of the growth plate during progression of puberty (e.g. a
decreased hypertrophic layer).
This is the first report to demonstrate a link between
estrogens and VEGF expression in the epiphyseal growth
plate. In vivo treatment of rats with E2 increased the number of
Journal of Endocrinology (2010) 205, 61–68
growth plate chondrocytes expressing VEGF. In line with this,
in the hypertrophic zone of ovariectomized animals, the
number of VEGF-positive cells/mm2 growth plate was
decreased. In addition, in vitro data in a rat chondrocytic cell
line showed a dose-dependent stimulatory effect of E2 on the
expression of VEGF-121 isoform. VEGF-189 was suppressed
by low E2 concentrations and was stimulated by higher
concentrations. This might counterbalance the slight concomitant increase of VEGF-121, thereby protecting the
growth plate when exposed to low concentrations of E2.
Our growth plate findings are in line with previous reported
effects of estrogens on VEGF expression in bone, uterus, and
breast cancer tissues (Mekraldi et al. 2003, Kazi et al. 2005,
Garvin et al. 2006).
Systemic estrogen levels increase with puberty eventually
resulting in epiphyseal fusion by the end of puberty (Juul
2001), presumably due to acceleration of growth plate
senescence through proliferative exhaustion of chondrocytes
(Weise et al. 2001). From our results obtained in rats, we
hypothesized that estrogens not only accelerate senescence of
the growth plate, but also stimulate chondrocytes to secrete
VEGF, which might contribute to the process of epiphyseal
fusion. VEGF protein was detected in human pubertal growth
plates, and indeed, the expression level significantly increased
during pubertal progression. This observation in humans
supports our findings in rats, and strengthens our hypothesis
that estrogens stimulate VEGF expression in the growth plate.
A 200-fold lower VEGF mRNA level in the growth plate
was observed compared with the prostate or bladder tissues.
We believe that the observed stimulation of VEGF expression
by estrogens in avascular growth plate chondrocytes can
substantially affect the growth plate. Indeed, VEGF has an
important role in chondrocyte differentiation, chondrocyte
survival, and endochondral ossification (Zelzer et al. 2004,
Zelzer & Olsen 2005, Dai & Rabie 2007). In several studies,
inhibition of VEGF showed dramatic effects on the growth
plate, such as expansion of the hypertrophic zone and delayed
removal of terminal hypertrophic chondrocytes (Gerber et al.
1999, Zelzer et al. 2004). Conversely, one might speculate
that an increase in expression leads to a smaller hypertrophic
zone, a more rapid removal of terminal hypertrophic
chondrocytes, and eventually epiphyseal fusion. To our
knowledge, reports on an increase in VEGF expression have
not been published before.
Estrogen levels were higher in the estrogen-supplemented
rats compared with the sham-operated rats. Uterus size,
correlating with estrogen levels, was small in ovariectomized
rats compared with the sham-operated animals and was
slightly larger in the estrogen-supplemented animals,
indicating supra-physiological levels of estrogens in the
estrogen-treated group (Tivesten et al. 2004, 2006). Serum
levels of E2 were not measured in patients from whom
growth plate tissue samples were collected. However, serum
levels of E2 are well known to positively correlate with the
stage of pubertal development (Norjavaara et al. 1996).
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Estradiol stimulates VEGF expression .
The collection of human samples is small and originates
from patients having a variability of disorders. However,
human growth plate samples are extremely difficult to obtain.
We believe that even though patients suffered from diverse
disorders, the underlying mechanism of epiphyseal maturation and fusion will be the same for all growth plates.
Eventually, longitudinal growth stops in all patients, with only
few exceptions, at the end of puberty. The human data are in
line with both in vivo and in vitro rat data, thereby
strengthening our conclusion that estrogens stimulate VEGF
expression in the growth plate. Estrogen levels were not
analyzed in these patients, and our assumption of different
levels of estrogen exposure is based on the fact that tissue
samples were obtained from girls in different pubertal stages.
In summary, we demonstrated that VEGF protein
expression in the growth plate is elevated by estrogens
in vivo in ovariectomized rats and in vitro in a rat chondrocytic
cell lines. Our findings are supported by human expression
studies in girls in different pubertal stages. From this, we
conclude that estrogens stimulate VEGF expression in the
growth plate, although the exact role of VEGF in estrogenmediated growth plate fusion remains to be clarified.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived
as prejudicing the impartiality of the research reported.
Funding
This study was supported by ZonMw (project # 920-03-358),
The Netherlands; the Swedish Research Council (project 2007-54X15073-04-3); and a visiting scholarship award from the European Society
for Paediatric Endocrinology.
Acknowledgements
The authors thank the orthopedic surgeons in the Leiden University Medical
Center and at the Karolinska University Hospital in Stockholm for providing
the growth plate samples.
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Received in final form 28 December 2009
Accepted 19 January 2010
Made available online as an Accepted Preprint
19 January 2010
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