Decreased levels of insulin-like growth factor-1 and vascular endothelial growth factor relevant to the ossification disturbance in femoral heads spontaneous hypertensive rats.

Abstract

Ossification disturbance in femoral head reportedly is seen in the Spontaneously Hypertensive rats (SHR) between ages of 10 and 20 weeks. We investigated serum and tissue levels of insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) in SHR relevant to the ossification disturbance and osteonecrosis of the femoral head. Serum levels of IGF-1 and VEGF were significantly lower in SHR than in Wistar Kyoto rats (WKY) at weeks 5, 10, 15 and 20 (p<0.005). The incidence of histological ossification disturbance of the femoral head was higher in SHR (59%) than in WKY (40%) at week 20. Lower serum and local levels of VEGF in SHR appeared to be related to the incomplete ossification of the femoral heads. Immunohistochemical study showed significantly lower numbers of IGF-1 and VEGF positive chondrocytes in the femoral epiphyseal cartilage of SHR than in those of WKY at weeks 10, 15 and 20. Our results suggest that local and/or systemic levels of IGF-1 and VEGF between ages of 5 and 20 weeks might play roles in the pathogenesis of ossifi cation disturbance of the femoral head in SHR.

Full text

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egg-Calve-Perthes’disease (LCPD) is charac-
terized by ossifi cation disturbance and subse-
quent osteonecrosis of the femoral head. The
reported incidence of LCPD is approximately
1 : 9,000 children (1 : 8,000 boys and 1 : 30,000 girls)
and 80ｵ of the cases are found between the ages of
4 and 9 years [1ﾝ6]. If LCPD is untreated or not
treated appropriately, ossifi cation disturbance devel-
ops fl attening of the femoral head due to osteonecro-
sis, which can leave a deformity that result in osteo-
arthritis of the hip in adult life. Although the
prognosis is clear, the pathogenesis of LCPD is still
uncertain.
　　The spontaneously hypertensive rats (SHR) is
widely used as an animal model for hypertension.
SHR exhibit a variety of hormonal imbalances,
including prolactin, follicle-stimulating hormone, tes-
tosterone [7-9], and parathyroid hormone [10],
which are known to infl uence the process of chondro-
genesis and bone remodeling. It is also known that
the SHR exhibits osteonecrosis of the femoral head
between the ages of 10 and 20 weeks, the period
Decreased Levels of Insulin-like Growth Factor-1 and
Vascular Endothelial Growth Factor Relevant
to the Ossifi cation Disturbance in Femoral Heads
Spontaneous Hypertensive Rats
Takamitsu Komiyamaa, Keiichiro Nishidaa,b＊, Masanori Yorimitsua,
Hideyuki Doia, Shinichi Miyazawaa, Ai Kitamuraa, Aki Yoshidaa, Yoshihisa Nasua,
Nobuhiro Abea, and Toshifumi Ozakia
Departments of aOrthopaedic Surgery, and bHuman Morphology, Okayama University Graduate School of Medicine,
Dentistry and Pharmaceutical Sciences, Okayama 700 ﾝ8558, Japan
L
Ossifi cation disturbance in femoral head reportedly is seen in the Spontaneously Hypertensive rats
(SHR) between ages of 10 and 20 weeks. We investigated serum and tissue levels of insulin-like
growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) in SHR relevant to the ossifi -
cation disturbance and osteonecrosis of the femoral head. Serum levels of IGF-1 and VEGF were sig-
nifi cantly lower in SHR than in Wistar Kyoto rats (WKY) at weeks 5, 10, 15 and 20 (p ＜ 0.005). The
incidence of histological ossifi cation disturbance of the femoral head was higher in SHR (59ｵ) than in
WKY (40ｵ) at week 20. Lower serum and local levels of VEGF in SHR appeared to be related to the
incomplete ossifi cation of the femoral heads. Immunohistochemical study showed signifi cantly lower
numbers of IGF-1 and VEGF positive chondrocytes in the femoral epiphyseal cartilage of SHR than
in those of WKY at weeks 10, 15 and 20. Our results suggest that local and/or systemic levels of
IGF-1 and VEGF between ages of 5 and 20 weeks might play roles in the pathogenesis of ossifi cation
disturbance of the femoral head in SHR.
Key words : spontaneous hypertensive rats, insulin like growth factor-1, vascular endothelial growth factor,
ossifi cation disturbance, osteonecrosis
Acta Med. Okayama, 2006
Vol. 60, No. 3, pp. 141ﾝ148
CopyrightⒸ 2006 by Okayama University Medical School.
http :/ /www.lib.okayama-u.ac.jp/www/acta/
Original Article
Received August 9, 2005 ; accepted November 30, 2005.
＊Corresponding author. Phone : ＋81ﾝ86ﾝ235ﾝ7273 ; Fax : ＋81ﾝ86ﾝ223ﾝ9727
E-mail : knishida@md.okayama-u.ac.jp (K. Nishida)

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preceding skeletal maturity. Although the mechanism
of osteonecrosis seen in SHR remains unclear, SHR
is accepted for the animal model of human LCPD
because radiographic appearances of the femoral
heads of SHR resemble those of LCPD [11-13].
Recent radiographic and histological analyses demon-
strated that the osteonecrosis in SHR is preceded by
a disturbance of the cartilage mineralization and
ossifi cation, despite normal hypertrophy of the
epiphyseal cartilage, suggesting that abnormal devel-
opment of the femoral epiphysis occurs much earlier
than the osteonecrosis [14-16]. Recent report sug-
gested that the occlusion of the epiphyseal vessels
during the process of enchondral ossifi cation might
induce the ischemic necrosis of the femoral head,
which might lead to the breakdown of the vulnerable
immature epiphysis in SHR by the mechanical forces
[16].
　　Two of the known factors required for enchondral
ossifi cation are insulin-like growth factor (IGF-1) and
vascular endothelial growth factor (VEGF). IGF-1 is
an essential factor for cell function and diff erentia-
tion of chondrocytes during enchondral ossifi cation
[17]. Neidel et al. reported persistent, low serum
levels of IGF-1 in prepubertal children with LCPD
[18]. More recently, Kikkawa et al. reported that
peak expression of IGF-1 in epiphyseal chondrocytes
in SHR is delayed when compared with Wistar Kyoto
rats (WKY) during the ages of 1 to 6 weeks [14].
Although the precise relationship is unclear in chon-
drocytes, IGF-1 is known to regulate production of
VEGF in other cell types [8, 19-22]. VEGF is
responsible for hypertrophic cartilage neovascular-
ization through a paracrine release by chondrocyres,
and promotes the invasion of endothelial cells into
the cartilaginous tissue [23]. In the current study,
we used SHR, and the closely related WKY, which
do not develop spontaneous osteonecrosis of the fem-
oral head. We hypothesized that if the inhibition of
vascular invasion into the epiphyseal cartilage and
ossifi cation disturbance of the SHR femoral head are
related to the low serum or low tissue levels of
IGF-1 and VEGF, then the levels of both factors
should be diff erent in the 2 types of rats during the
developmental period preceding skeletal maturity.
Materials and Methods
　　Animals.　 SHR were originally raised by
selective inbreeding of WKY. We used SHR (n＝40)
(Charles River Inc., Yokohama, Japan) as the dis-
ease models of epiphyseal ossifi cation disturbance of
cartilage, and WKY (n＝20) (Charles River Inc.,
Yokohama, Japan) as normal control animals. All
animals were used in accordance with the Policies
and Guidelines for Care and Use of Laboratory
Animals of Okayama University, and all eff orts were
made to minimize animal suff ering. The rats were
euthanized by systemic perfusion of a mixture of 2ｵ
paraformaldehyde and 2ｵ glutaraldehyde in 0.1 M
phosphate-buff ered saline (PBS) under general anes-
thesia at weeks 5, 10, 15 and 20 (SHR : each 10,
WKY : each 5), and the serum samples were taken
and stored at －80 ℃ until analysis.
　　Radiographic study.　 The femoral head sam-
ples were perfusion-fi xed with a mixture of 2ｵ para-
formaldehyde and 2ｵ glutaraldehyde in 0.1 M PBS.
The radiographs of excised samples were taken by
Softex CNB-2 (MFG and EXP, Softex Co. Ltd.,
Tokyo, Japan ; 52 kV, 40 mA, exposure time 10 s).
All the radiographic analyses were carried out on
printed pictures magnifi ed at×100. In the current
study, the radiographic appearances of the femoral
heads were divided into 3 types ; Round (no sign of
deformation or fracture), Flat (deformation of femo-
ral head without bone fracture), and Crushed (defor-
mation and bone fracture of femoral head) (Fig. 1).
　　Measurement of IGF-1 and VEGF concen-
trations in the serum samples.　 Serum samples
were taken from both WKY and SHR at weeks 5,
10, 15 and 20. The concentration of VEGF was
measured by ELISA (Quantikine Mouse VEGF
ELISA Kit, R&D systems Inc., Minneapolis, MN,
USA), and IGF-1 by radioimmunoassay (Rat IGF-1
Radioimmunoassay Kit, Diagnostic Systems
Laboratories Inc., Webster, TX, USA). The serum
levels of IGF-1 and VEGF in SHR at each stage
were analyzed separately to further examine the dif-
ference between histological stages, and the correla-
tion with ossifi cation of the femoral heads.
　　Histological examination.　 Samples were
decalcifi ed in 4ｵ ethylene diamine tetra-acetic acid
(EDTA) and 10ｵ sucrose in cacodylate buff er (pH
142
Acta Med. Okayama　Vol. 60, No. 3
Komiyama et al.

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7.3) for 7ﾝ10 days. The coronal araffi n sections
(4.5 μm) were stained by hematoxylin and eosin.
Histological appearances of the femoral heads were
scored from 0 to 2, according to the area of ossifi ca-
tion ; Stage 0 : ossifi cation of less than 1/3 of the
femoral head, Stage 1 : ossifi cation from 1/3 to 2/3
of the femoral head, Stage 2 : ossifi cation of 2/3 or
more of the femoral head (Fig. 2). Blinded assess-
ments of histological and radiographic data were
made independently by 2 of the authors (TK and KN).
Diff erences in opinions were resolved by consensus.
　　Immunohistochemistry.　 The paraffi n sec-
tions were incubated with 0.3ｵ H₂O₂ that diluted
with PBS for 30 min at room temperature to inacti-
vate endogenous peroxidase. Each section was
treated with PBS containing 2ｵ BSA for 30 min at
room temperature to block non-specifi c binding to
sections and eliminate non-specifi c staining. The
slides were then incubated with rabbit polyclonal
anti-IGF-1 (5 μg/ml) or goat polyclonal anti-VEGF
(5 μg/ml) (Santa Cruz Biotechnology, Santa Cruz,
CA, USA) at 4 ℃ overnight, and incubated for
30 min at room temperature with the secondary anti-
body (DAKO LSAB Kit, DAKO, Kyoto, Japan).
After 3 washings by PBS, the sections were
immersed in 0.1ｵ 3,3’-diaminobenzidine tetrahydro-
chloride (DAB) and 1ｵ hydrogen peroxide. The sec-
tions were then counterstained with methylgreen,
and observed by light microscopy.
　　Quantitative analysis for IGF-1 and VEGF
positive cells.　 The population of IGF-1 and
VEGF positive chondrocytes within the femoral
heads was examined in SHR (n＝10) and WKY (n＝5)
at weeks 10, 15 and 20. The positive-cell numbers
were counted within the 3 defi ned square areas
(medial, central, lateral, 20 × 20 μm) of Stage 0
and 1 femoral epiphyseal cartilage of the rats at
weeks 10, 15 (SHR ; n＝10, WKY ; n＝10) and 20
143
VEGF in SHR Femoral Heads June 2006
RoundFlat Crushed
Fig. 1　　Radiographic appearances of the femoral heads of SHR (Softex). Femoral heads were divided into 3 types : Round, Flat,
Crushed.
Stage 0 Stage 1Stage 2
Fig. 2　　Histology of the epiphysis of femoral head of SHR (HE stain). Femoral heads were classifi ed into the following 3 stages
according to the pathological fi ndings. Stage 0, ossifi cation less than 1/3 of femoral head ; Stage 1, ossifi cation from 1/3 to 2/3 of the
femoral head ; Stage 2, ossifi cation more 2/3 of the femoral head (original magnifi cation×10).

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(SHR ; n＝10, WKY ; n＝4), and averaged. There
was no diff erence in ossifi cation pattern in the femo-
ral heads between SHR and WKY at week 5.
　　Statistical analysis.　 The serum levels of
IGF-1 and VEGF were expressed as mean＋/－SD.
Diff erences between WKY and SHR were statisti-
cally analyzed by Mann-Whiteney U test or by
Student’s t-test appropriately using Macintosh Stat-
View version 6 (SAS Institute Inc., Cary, NC,
USA).
Results
　　Radiological study.　 The radiographic
morphology of the femoral heads of WKY was Round
throughout the study, except for one femoral head
(10ｵ) at week 20, which showed a Flat appearance.
In contrast, Flat femoral heads were seen in 40ｵ
and in 60ｵ of SHR at weeks 10 and 15,
respectively. Femoral heads with a Crushed
appearance were seen in 5ｵ and in 15ｵ of SHR at
weeks 15 and 20, respectively (Table 1).
　　Serum levels of IGF-1 and VEGF.　 Serum
levels of IGF-1 were signifi cantly lower in SHR than
in WKY at weeks 5, 10 and 15. There was no
signifi cant diff erence in serum levels of IGF-1
between 2 lines of rats at week 20. The serum levels
of VEGF were signifi cantly lower in SHR than in
WKY at each measurement point (p＜0.005) (Table
2). Analysis by ELISA showed the serum levels of
IGF-1 were not signifi cantly diff erent among the
stages at weeks 10 and 20. Although the serum
levels of IGF-1 associated with Stage 1 and 2
cartilage were signifi cantly lower than those of Stage
0 in SHR at week 15 (p＜0.01, p＜0.05, respectively)
(Fig. 3), the levels were much lower than those with
WKY at the same age. The serum levels of VEGF
were not signifi cantly diff erent among the stages in
SHR at week 10. However, at week 15, the serum
levels of VEGF in SHR with Stage 0 and 1 femoral
heads were signifi cantly lower than those with Stage
2 (p＜0.05 and p＜0.005, respectively). At week 20,
the serum levels of VEGF of Stage 0 were
signifi cantly lower than those of Stages 1 and 2 in
SHR (p＜0.005) (Fig. 3).
　　Histology.　 The number of femoral heads in
each stage of ossifi cation was summarized in Table 3.
The femoral heads of SHR at week 5 and WKY at
weeks 5, 10 and 15 showed no ossifi cation (Stage 0).
At week 10, Stage 0 ossifi cation was seen in 8 of 20
femoral heads (40ｵ), and Stage 1 ossifi cation was
seen in 4 of 20 femoral heads (20ｵ) of SHR. Two of
the latter had osteonecrosis with empty lacunae.
Stage 2 ossifi cation was seen in 8 of 20 femoral
heads (40ｵ) of SHR, 2 of which had osteonecrosis.
At week 15, all 3 stages of ossifi cation of the
femoral head were seen : Stage 0 in 4 (21.1ｵ),
144
Acta Med. Okayama　Vol. 60, No. 3
Komiyama et al.
Table 1　　Number of femoral heads in each radiographic type
10 weeks
15 weeks
20 weeks
SHRWKYSHE WKYSHR WKY
Round
Flat
Crushed
1210 71079
1
0
8
0
0
0
120
0
10
13
Total
2010 2010 2010
Table 2　　Serum levels of IGF-1 and VEGF
　IGF-1 (ng/ml)
5 weeks
10 weeks
15 weeks
20 weeks
SHR (n=40)
WKY (n=20)
633.8± 86.2
1156.4±127.9*
1082.9±152.3
1586.0±231.5*
1115.0±154.0
1414.0±227.9*
988.9±106.9
1164.4±206.5
(*p<0.005)
　VEGF (pg/ml)
5 weeks
10 weeks
15 weeks
20 weeks
SHR (n=40)
WKY (n=20)
61.7±30.6
67.1± 6.1*
58.2±2.5
71.9±5.3*
58.9±4.5
72.7±4.4*
59.2±3.8
73.4±5.6*
(*p<0.005)

Page 5

Stage 1 in 6 (31.6ｵ), and Stage 2 in 9 (47.4ｵ) of
19 SHR femoral heads. Seven of 9 Stage 2 SHR
femoral heads (77.8ｵ) showed osteonecrosis. At
week 20, the incidence of Stage 1 and 2 ossifi cation
of the WKY femoraldeads (10 of 10, 100ｵ) was
higher than those in SHR (13 of 17 heads ; 76.4ｵ)
and 12 of 17 femoral heads (70.6ｵ) of SHR was
showed osteonecrosis. Extensive crushing precluded
further histological analysis of 4 of the SHR femoral
heads at weeks 15 (1 head) and (3 heads).
　　Immunohistochemistry.　 The local expression
of IGF-1 showed a signifi cant and transient increase
at week 15 followed by a decrease at week 20 in
SHR and WKY. The populations of IGF-1 positive
chondrocytes in the femoral heads of WKY were
50.8±20.5ｵ, 87.5±5.2ｵ, and 7.0±16.9ｵ at weeks
10, 15, and 20, respectively. The positive reactions
for IGF-1 were signifi cantly lower in hypertrophic
chondrocytes in the femoral heads of SHR at weeks
10, 15 and 20 (p＜0.001) (Fig. 4). The populations
of VEGF positive chondrocytes were signifi cantly
lower than those in WKY throughout the
experimental period (Fig. 5).
Discussion
　　The histological analysis of the current study
showed that osteonecrosis was found in 4 of 20 and 7
145
VEGF in SHR Femoral HeadsJune 2006
Stage 0Stage 1 Stage 2
1700
1500
1300
1100
900
700
500
70
65
60
55
50
10 w15 w20 w 10 w 15 w20 w
(ng/ml)
(pg/ml)
Serum levels of IGF-1 in SHR
Serum levels of VEGF in SHR
*p<0.05 **p<0.005
Fig. 3　　Serum levels of IGF-1 and VEGF in each ossifi cation stage at weeks 10, 15 and 20 in SHR. At week 15, serum levels of IGF-1
of Stage 0 were signifi cantly higher than those of Stages 1 and 2. Serum levels of IGF-1 were not signifi cantly diff erent among the stage
at weeks 10 and 20. Serum levels of VEGF were not signifi cantly diff erent among the stages at week 10. At week 15, serum levels of
VEGF of Stage 2 were signifi cantly higher than those of Stages 0 and 1. At week 20, serum levels of VEGF of Stage 0 were signifi cantly
lower than those of Stages 1 and 2.
Table 3　　Number of femoral heads in each stage of ossifi cation
5 weeks
10 weeks
15 weeks
20 weeks
SHRWKYSHRWKYSHRWKYSHRWKY
Stage 0
Stage 1
Stage 2
20
0
0
10
0
0
810
0
0
4
6
9(7)
10
0
0
4(2)
6(5)
7(5)
0
4
6
4(2)
8(2)
Total
2010 20101910 1710
( ) : Osteonecrosis