The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 1 359-362
Copyright © 1999 by The Endocrine Society
Expression of Platelet-Derived Endothelial Cell Growth Factor (PD-ECGF) Related to Angiogenesis in Ovarian Endometriosis
Jiro Fujimoto,
Hideki Sakaguchi,
Reiko Hirose and
Teruhiko Tamaya
Department of Obstetrics and Gynecology, Gifu University
School of Medicine, Gifu City 500-8705, Japan
Address all correspondence and requests for reprints to: Jiro Fujimoto, M.D., Ph.D., Department of Obstetrics and Gynecology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu City 500-8705, Japan.
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Abstract
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Platelet-derived endothelial cell growth factor (PD-ECGF) is expressed
in the lining epithelial cells of ovarian endometriomas, and in
interstitial cells of the subepithelial area with angiogenesis. The
expression of PD-ECGF persists in endometriotic endometrium during the
menstrual cycle. This might suggest that PD-ECGF contributes to the
growth of ovarian endometriomas via subepithelial angiogenesis
independently of the sex steroidal milieu.
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Introduction
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ANGIOGENESIS is essential to the
advance of pelvic endometriosis (1, 2, 3, 4, 5). The following angiogenic
factors have been recognized as positive factors in the advance of
endometriosis: basic fibroblast growth factor (FGF) (6, 7, 8), vascular
endothelial growth factor (VEGF) (3, 9, 10), and interleukin (IL)-8 (2, 11, 12, 13). Platelet-derived endothelial cell growth factor (PD-ECGF) was
cloned as a novel angiogenic factor (45 KDa polypeptide) from human
platelet (14). Thereafter, PD-ECGF was completely identified with
thymidine phosphorylase (TP) (15, 16). PD-ECGF/TP does not stimulate
the growth of endothelial cells, but rather promotes chemotaxis of
them, and induces angiogenesis in vivo with the activation
of TP as an enzyme (17, 18). Among normal tissues, PD-ECGF is expressed
in lymph nodes, peripheral lymphocytes, spleen, lung, liver, placenta
(19), and uterine endometrium (20, 21). Among solid tumors, PD-ECGF is
expressed in malignant gliomas, thyroid tumors, cancers of the breast,
esophagus, stomach, colon, pancreas, gall bladder, kidney, bladder,
lung (19), uterine cervix (22) and endometrium (23), and ovary
(24).
Immunohistochemical staining for factor VIII-related antigen, which is
synthesized by vascular endothelial cells, is specific for the
endothelial cells of blood vessels (25) and is useful for detecting
tumor angiogenesis (26). Therefore, to know the contribution of PD-ECGF
to angiogenesis in the growth of ovarian endometrioma, we studied the
expression between PD-ECGF with its messenger RNA (mRNA) and factor
VIII-related antigen expressions, as well as the level of PD-ECGF
during the menstrual cycle.
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Subjects and Methods
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Subjects
Informed consent for the following studies was obtained from all
patients and from the Research Committee for Human Subjects, Gifu
University School of Medicine. Twenty patients, ranging in age from 29
to 41 yr, underwent surgery for ovarian endometriosis at the Department
of Obstetrics and Gynecology, Gifu University School of Medicine,
between August 1995 and May 1998. None of the patients had received any
preoperative therapy. The eutopic uterine endometria were obtained from
the endometrial cavity and submitted for histopathological examination
(27). Basal body temperature and estradiol and progesterone in
peripheral blood were measured to confirm the histological endometrial
dating. The inner wall of the ovarian endometrioma was peeled and
snap-frozen in liquid nitrogen for reverse transcription-polymerase
chain reaction (RT-PCR)-Southern blotting for PD-ECGF mRNA expression
and enzyme immunoassay for determination of human PD-ECGF antigen.
Three parts of each wall were collected, and each part was studied in
triplicate.
Immunohistochemistry
For formalin-fixed paraffin-embedded tissues, 4-µm sections
were cut with a microtome and dried overnight at 37C on a
silanized-slide (Dako, Carpinteria, CA). Samples were
deparaffinized in xylene at room temperature for 80 min and washed with
a graded ethanol/water mixture, then with distilled water. The samples
for PD-ECGF antigen were soaked in a phosphate buffer (PBS), and those
for factor VIII-related antigen were treated with 0.3 µg/mL trypsin
in PBS at room temperature for 20 min. The protocol for a DAKO LSAB2
Kit, Peroxidase (Dako) was followed for each sample. In the described
procedures, mouse anti-human PD-ECGF antigen 654-1 [10 µg/mL, Nippon
Roche, Kamakura, Japan (28)] and rabbit anti-factor VIII-related
antigen (Zymed, San Francisco, CA) were used at dilutions
of 1:100, and 1:2, respectively, as the first antibodies. The addition
of the first antibody, mouse anti-human PD-ECGF antigen 654-1 or rabbit
anti-factor VIII-related antigen, was omitted in the protocols for
negative controls of PD-ECGF or factor VIII-related antigens,
respectively.
RT-PCR-Southern blotting for PD-ECGF mRNA
Total RNA was isolated from the tissue by the acid guanidium
thiocyanate-phenol-chloroform extraction method (29). Total RNA (3
µg) was reverse transcribed with Moloney murine leukemia virus
reverse transcriptase (MMLV-RTase, 200 units, Gibco BRL,
Gaithersburg, MD) in a buffer of 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 2.5
mM MgCl2, 0.1 mg/mL bovine serum albumin (BSA), 10 mM DTT
(dithiothreitol), and 0.5 mM deoxynucleotides to generate complementary
DNAs (cDNAs) using random hexamer (50 ng, Gibco BRL) at
37C for 60 min (RT total RNA). The RT reaction mixture was heated at
94C for 5 min to inactivate MMLV-RTase. The template for negative
controls of RT-PCR was not reverse transcribed (RT negative total RNA).
Twenty-eight cycles of PCR for PD-ECGF mRNA, consisting of denaturation
for 1 min at 94C, annealing for 1 min at 55C, and extension for 1
min at 72C, were carried out with RT or RT negative total RNA, 0.1 µM
specific primers and Vent DNA polymerase (New England Biolabs, Beverly, MA) in a buffer of 10 mM KCl, 20 mM Tris-HCl,
pH 8.8, 10 mM (NH4)2SO4, 2 mM
MgSO4, 0.1% Triton X-100, and 0.15 mM deoxynucleotide
phosphates using the IWAKI thermal sequencer TSR-300 (Iwaki Glass, Tokyo, Japan). The oligodeoxynucleotides of specific
primers in PCR were synthesized according to the published information
on cDNA for PD-ECGF (30) as follows: sense primer for PD-ECGF mRNA:
5'-AGTCGGATGGCCATCAGCAT-3' (in exon 2); antisense primer for PD-ECGF
mRNA: 5'-TGGAATGCTTGTCCACAAGC-3' (in exon 3).
PCR products were applied to 1.2% agarose gel, and
electrophoresis was performed at 50–100 V. PCR products were
capillary-transferred to an Immobilon transfer membrane
(Millipore Corp., Bedford, MA) for 16 h. The membrane
was dried at 80C for 30 min and was irradiated with ultraviolet light
to tightly fix the PCR products. PCR products on the membrane were
prehybridized in 1 M NaCl, 50 mM Tris-HCl, pH 7.6, and 1% sodium
dodecyl sulfate at 42C for 1 h, and hybridized in the same
solution with the biotinylated oligodeoxynucleotide probe
(5'-AAGCGGACATCAGGGGCTTC-3') synthesized from the sequences of PD-ECGF
cDNAs between the specific primers at 65C overnight. Specific
bands hybridized with the biotinylated probe were detected with Plex
Luminescent Kits (Millipore Corp.), and X-ray film was
exposed on the membrane at room temperature for 10 min.
Enzyme immunoassay for determination of human PD-ECGF antigen
All steps were carried out at 4C. Tissues (wet weight: 10–20
mg) were homogenized in HG buffer (5 mM Tris-HCl, pH 7.4, 5 mM NaCl, 1
mM CaCl2, 2 mM ethylene glycol-bis-(ß-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 1 mM MgCl2, 2 mM DTT, 25
µg/mL aprotinin, and 25 µg/mL leupeptin) with a Polytron
homogenizer (Kinematics, Luzern, Switzerland). This
suspension was centrifuged in a microfuge at 12,000 rpm for 3 min to
obtain the supernatant. The protein concentration of samples was
measured by the method of Bradford (31) to standardize PD-ECGF antigen
levels.
PD-ECGF antigen levels in the sample were determined by the sandwich
enzyme immunoassay described by Nishida, et al. (28). The levels of
PD-ECGF were standardized with corresponding cellular protein
concentrations.
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Results
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Immunohistochemical staining for PD-ECGF antigen revealed that
PD-ECGF was expressed in lining epithelial cells in ovarian
endometriomas, and in interstitial cells of the subepithelial area in
all 20 endometriomas. PD-ECGF was expressed in the glandular cells, and
diffusely in the stroma, in all 20 corresponding eutopic uterine
endometria, as shown in Fig. 1A
. There
was no significant difference, in the strength of immunohistochemical
staining for PD-ECGF antigen, between the epithelial and interstitial
cells during the menstrual cycle. Furthermore, immunohistochemical
staining for factor VIII-related antigen revealed vascular endothelial
cells in the subepithelial area in all 20 endometriomas, as shown Fig. 1B, indicating subepithelial angiogenesis was activated.
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Figure 1. Correlation between PD-ECGF expression and
angiogenesis. A, Immunohistochemical staining for PD-ECGF in an ovarian
endometrioma and the corresponding eutopic uterine endometrium (x200).
The protocol for a DAKO LSAB2 Kit, Peroxidase was followed. In the
procedures, mouse anti-human PD-ECGF antigen 654-1 as the first
antibody was used at a dilution of 1:100. The addition of the first
antibody was omitted in the protocol for negative controls (data not
shown). PD-ECGF was expressed in the glandular cells, and diffusely in
the stroma in the corresponding eutopic uterine endometrium of the
ovarian endometriomas. In this endometrioma, PD-ECGF was expressed in
lining epithelial cells, and in interstitial cells of the subepithelial
area. B, Immunohistochemical staining for factor VIII-related antigens
in an ovarian endometrioma (x200). The protocol for a DAKO LSAB2 Kit,
Peroxidase was followed. In the procedures, rabbit anti-factor
VIII-related antigen as the first antibody was used at a dilution of
1:2. The addition of the first antibody was omitted in the protocols
for negative controls (data not shown). Factor VIII-related antigen was
expressed in vascular endothelial cells in the subepithelial area of
the ovarian endometriomas. C, RT-PCR-Southern blot analysis for PD-ECGF
mRNA in the inner wall of the ovarian endometrioma (n = 20). Total
RNA (3 µg) isolated from the tissue was reverse transcribed (RT total
RNA) except for negative controls (RT negative total RNA) as described
in Subjects and Methods. PCRs as the templates of RT and RT
negative total RNA were carried out. After electrophoresis, the PCR
products were analyzed using Southern blotting as described in
Subjects and Methods. The Southern blot revealed a single
specific band (240 bp) in the lane for endometriotic endometria, but no
band in the lane for negative controls.
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Consistent with the immunohistochemical results, enzyme immunoassay
revealed that PD-ECGF protein expression (as shown in Fig. 2) and its mRNA expression (as shown in
Fig. 1C
) in ovarian endometriotic endometria (n = 20) were not
significantly altered during the menstrual cycle.
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Figure 2. Levels of PD-ECGF in ovarian endometriosis during
the menstrual cycle. Three parts of the inner wall of each ovarian
endometrioma were collected, and each part was studied in triplicate.
The levels of PD-ECGF were determined by a sandwich enzyme immunoassay
as described in Subjects and Methods. Each level (mean
± SD) was determined from three parts of each wall in
triplicate (n = 20).
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Discussion
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Newly developed capillary network formation from the original
vessel is designated as neovascularization. Generally, turnover of
capillary endothelial cells is extremely slow, to the order of months
or years, in physiological neovascularization, while the turnover in
ovary and uterine endometrium is rapidly altered along with the ovarian
cycle. Sex steroidal regulation of PD-ECGF on endometrial angiogenesis
has been demonstrated (21). In normal uterine endometrium, PD-ECGF
expression is slightly decreased a few days before ovulation, and is
once again induced several days after ovulation following a rise in
progesterone level in peripheral blood. Therefore, PD-ECGF expression
might be up-regulated by progesterone under estrogen-primed conditions
in the secretory phase (21). There is no appreciable change in the
intensity of immunoreactivity for PD-ECGF in the epithelial cells,
while the intensity is relatively sparse and weak in the stromal cells
at the proliferative and early secretory phases (20). Furthermore, a
marked increase is found in decidualized endometrial stromal cells
(20).
In the present study, PD-ECGF was expressed in lining epithelial cells
of ovarian endometriomas and in interstitial cells of the subepithelial
area. Consistent with subepithelial PD-ECGF expression, factor
VIII-related antigen was expressed in vessel cells of the subepithelial
area, which might be recognized as endometriomal angiogenesis. In
general, the response to estrogen and progesterone is partially damaged
in endometriosis (32, 33). There is no specific alteration in PD-ECGF
expression in ovarian endometriotic endometrium during the menstrual
cycle. Therefore, the intact response to progesterone of PD-ECGF
expression might be damaged in ovarian endometriotic endometrium, while
normal uterine endometrium conserves the response (21). In normal
uterine endometrium, transient progestational angiogenesis in the
subepithelial stromal cells is abolished with menstruation. On the
other hand, PD-ECGF persistently expresses in the subepithelial area of
endometriotic endometrium. This might contribute to the growth of
ovarian endometrioma via subepithelial angiogenesis independent of the
sex steroidal milieu.
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Footnotes
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Received for publication July 21, 1998. Revision received September 23, 1998. Accepted for publication October 1, 1998.
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Abstract
Introduction
