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Relationship Between Platelet-Derived
Growth Factor Expression in Leiomyomas
and Uterine Volume Changes After
Gonadotropin-ReleasingHormone
Agonist Treatment
ANDREA DI LIETO, MD, GAETANO DE ROSA, MD,MARIANNA DE FALCO, MD, FRANCESCA IANNOTTI, MD,STEFANIA STAIBANO, MD, FABRIZIO POLLIO, MD,MARIANGELA SCARAMELLINO, MD,AND GAETANO SALVATORE, MD The unopposed estrogen effect is the main cause of leiomyoma
obtained fibroid samples. Uterine volume decreased significantly
growth and is at the basis of the clinical use of gonadotropin-releasing
after treatment, whereas just a poor modification was found in the
hormone (GnRH) agonists. Platelet-derived growth factor (PDGF)
controls. The decrease in the uterine volume was found to be statis-
has been indicated as the main growth factor involved, in vitro in the
tically related to PDGFexpression. Thus PDGFlevels decreased in
proliferation response of leiomyoma smooth muscle cells to estrogen
treated patients as compared with controls. The decreased PDGF
stimulation. The aim of this article is to evaluate the mitogenic action
production in leiomyomas after GnRH analogue treatment and the
of PDGFin vivo by studying the relationship between PDGFexpres-
relationship between decreased PDGFexpression and greater shrink-
sion in leiomyomas and post-GnRH analogue treatment changes in
age in uterine volume suggest that PDGFmight have a mitogenic action
uterine volume. Thirty-nine patients suffering from uterine leiomyo-
on leiomyomas in vivo. HUM PATHOL 33:220-224. Copyright 2002,
mas were treated with leuprorelin acetate depot 3.75 mg for three
Elsevier Science (USA). All rights reserved.
cycles; 31 untreated patients were enrolled as control group. Uterine
Key words: platelet-derived growth factor, leiomyoma, gonado-
volume was determined twice by ultrasonography in each patient, the
tropin-releasing hormone analogue.
first time at admission and the second time after treatment in the
Abbreviations: GnRH, gonadotropin-releasing hormone; PDGF,
study group and after 3 months in the control group. The change in
platelet-derived growth factor; EGF, epidermal growth factor; IGF,
the uterine volume was then evaluated. Patients underwent surgery,
insulin-like growth factor; E2, 17-␤-estradiol; MAP, mitogen-activated
and PDGFimmunohistochemical detection was performed on the
protein.
Uterine leiomyomas are benign smooth muscle of sex hormones at their target organs; therefore, they cell tumors of the myometrium arising during the fe- decrease sex hormone synthesis and secretion in long- male reproductive period.1 The growth of these neo- term treatment,9 with a subsequent pharmacologic hy- plasms was shown to be dependent on the action of poestrogenic state. A significant reduction in cellular estrogen2 and progesterone3,4 and in some cases proliferation was found in leiomyomas after GnRH ag- associated with recently investigated chromosomal changes.5,6 The unopposed estrogen effects on the Sex steroid hormones seem to mediate their ac- growth of leiomyomas are at the basis of the clinical use tions on leiomyomas also through the autocrine and of gonadotropin-releasing hormone (GnRH) agonists paracrine effects of growth factors produced locally by to reduce fibroid volume and to control symptoms such smooth muscle cells and fibroblasts, such as epidermal as menorrhagia, anemia, dysmenorrhea, and pelvic growth factors (EGF and EGF-R),11-14 insulin-like pressure,1,7,8 especially before a surgical procedure for growth factors I and II (IGF-I and IGF-II),14-17 and uterine leiomyomas. GnRH agonists induce pituitary desensitization, through the downregulation of GnRH Literature data on the effects of PDGF on uterine receptors and the reduction of luteinizing and follicu- fibroid progression are rather scarce. PDGF is synthe- lar-stimulating hormones,7 and also inhibit the actions sized and secreted by many cell types, including acti-vated vascular endothelial cells and macrophages, fi-broblasts, and smooth muscle cells under appropriate From the Department of Obstetrical-Gynaecological and Urolog- conditions.19 The expression of the PDGF-␤ receptor in ical Science and Reproductive Medicine and the Department of numerous benign and malignant mesenchymal lesions Biomorphological and Functional Science, Pathology Section, Uni- suggests that PDGF might be involved in promoting versity Federico II, Naples, Italy. Accepted for publication November19, 2001.
Address correspondence and reprint requests to Andrea Di Li- Human myometrial tissue and myometrial smooth eto, MD, Via L. Giordano, 120, 80127 Napoli, Italy.
muscle cells in primary culture locally produce EGF Copyright 2002, Elsevier Science (USA). All rights reserved.
Some authors have demonstrated that both leiomyo- PDGF EXPRESSION IN GnRH-a TREATED LEIOMYOMAS (Di Lieto et al) mas and normal myometrium synthesize PDGF and times in 10 mM of citrate buffer). After endogenous peroxi- have assumed that this growth factor can act in an dase activity was blocked, sections were preincubated with autocrine or paracrine fashion.21 The PDGF-␤ receptor nonimmune mouse serum (1:20; Dakopatts, Glostrup, Den- was found to coexist with PDGF in human leiomyo- mark) diluted in PBS/BSA (1%) for 25 min at room temper-ature to prevent nonspecific bindings of antibodies. After mas,19 and so changes in the synthesis or in the turn- three washings with Tris buffer saline, the anti-PDGF (clone over of this growth factor apparently play a role in P-GF.44C; Novocastra, New Castle Upon Tyne, England) pri- mary antibody was added, at 1:100 dilution, for an overnight In a recent work,22 we demonstrated for the first incubation at 4°C. As a positive control, the immunoreactivity time that estrogen in vitro plays a key role in mediating in a sample of human palatine tonsil was evaluated. Negative the mitogenic effects on leiomyoma cells, by triggering controls were performed in each run by substituting the rapid and transient activation of the mitogen-activated primary antibody with nonimmune serum (Dako Antibody protein (MAP)– kinase pathway. Interestingly, the early Diluent; Dakopatts). All of the slides were washed twice in downstream signal transduction events determined Tris-HCl buffer after each step. Sequential 20-min incuba- tions with biotinylated link antibody and peroxidase-labelled streptavidin (L.U. Dako LSAB kit, HRP) were then per- induced PDGF secretion. PDGF was identified as the formed. The chromgen 3,3Ј-diaminobenzidine (DAB; Vector main growth factor involved in the proliferation re- Laboratories, Burlingame, CA) was developed by the addition sponse of leiomyoma muscle cells to estrogen stimula- of 0.3% hydrogen peroxide. Nuclear counterstaining by he- tion, upregulated by E2 and downregulated by anties- matoxylin was performed; then sections were mounted and cover slipped with a xylene-based mounting media (Entellan; Basing on these findings about PDGF’s in vitro Merck, Darmstadt, Germany). Immunostained sections were action on human leiomyoma cells, in the present study analyzed using a double-headed Leitz light microscope (ϫ40) we evaluated the relationship between immunohisto- in the most evenly and heavily labeled areas of the lesions, chemical detection of PDGF in leiomyomas and post- selected by inspection at low-power magnification with the aid GnRH analogue treatment changes in uterine volume.
of a random table. A final 1000 cells were evaluated for eachcase and for each antibody. The average fraction of positivecells was determined and ranked on a semiquantitative scalebased on the percentage of cells with a definite nuclear MATERIALS AND METHODS
staining, as follows: grade 0 (Ͻ 5%), grade 1 (5% to 25%),grade 2 (26% to 50%), and grade 3 (51% to 75%). All of the Thirty-nine premenopausal women (mean age, 36.1 Ϯ sections were examined without knowledge of the clinical and 3.2 [SD]; parity, 2.2 Ϯ 1.8) suffering from uterine leiomyomas and referring to our Department of Obstetrical-Gynaecologi- Patients were classified on the basis of the immunohisto- cal and Urological Science and Reproductive Medicine gave chemical grade for PDGF expression. The change in uterine their informed consent to take part in the study. Patients were volume (increase or shrinkage), defined as the ratio of the treated with leuprorelin acetate depot 3.75 mg by monthly second scanned volume minus the first scanned volume to subcutaneous injection for three cycles, starting within the the first scanned volume, was calculated for each immunohis- first 7 days of the menstrual cycle, and then underwent uter- tochemical grade. To assess the significance of the relation- ine surgery. Thirty-one premenopausal women (mean age, ship between the posttreatment modifications in the uterine 37.3 Ϯ 3.7; parity, 1.9 Ϯ 1.3) were enrolled as control group; volume and the immunohistochemical grade for PDGF ex- they received no GnRH agonist therapy and underwent sur- pression, the change in uterine volume for each grade was gery directly during the follicular phase of the menstrual compared with that of the other grades.
cycle. No patient in the study or control group took hormonal Data were analyzed by Fisher’s exact test (␣ ϭ 0.05) and therapy, delivered, or underwent uterine surgery within 12 the Student t test (monodirectional hypothesis, ␣ ϭ 0.05).
months of the study or had malignant neoplasms.
Patients were submitted to bimanual examination, rou- tine hematologic examinations, and ultrasonography. All of the patients were scanned twice by transabdominal ultra-sonography, the first time at admission in both groups, and All of the patients completed the therapy, thus the second time after treatment in the study group and after meeting the criteria to participate in the study. There about 3 months in the control group. The uterine volume wascalculated using the formula of an ellipsoid.
All of the patients underwent surgery. In the study group, 21 patients underwent laparomiomectomy, 10 underwent laparohisterectomy, and 8 underwent histeroscopic miomec-tomy. In the control group, 22 patients underwent laparo- histerectomy and 9 underwent laparomiomectomy.
The surgical specimens were formalin fixed and paraffin embedded, and then 4-␮m-thick serial sections were cut from each block. One section was stained by hematoxylin and eosin to confirm the histopathologic diagnosis. Immunocytochem- istry was then performed on the remaining sections mounted on poly-L-lysine– coated glass slides. To improve the stainingpattern, these sections were treated with the high-tempera- NOTE. Data presented as mean (ϮSD).
ture antigen unmasking technique for paraffin sections (pre- *Ratio of the second scanned volume minus the first scanned incubation in a microwave oven at 650 watts for 5 min, three volume to the first scanned volume.
FIGURE 1.
Immunohistochemical grades for PDGF expression in myomas of treated (o) and untreated (■) patients.
FIGURE 3.
Immunohistochemical staining for PDGF in a GnRH agonist–treated leiomyoma (grade 0). (Original magnification was no significant difference between the groups withregard to age, parity, and uterine size. The uterinevolume decreased after Gn-RH analogue treatment. Inthe study group it was 571.3 Ϯ 266.7 cm3 (mean Ϯ SD) with a mere appearance of increased cellular density, at the first ultrasonographic scanning and 413.4 Ϯ 217 but in fact without nuclear atypia or mitotic activity.
cm3 after Gn-RH analogue administration, with a In terms of the immunohistochemical detection of shrinkage of 0.27 Ϯ 0.17. The difference between the PDGF within the cytoplasm of smooth muscle cells and first and second evaluations was found to be statistically fibroblasts, in the study group 11 patients (28.2%) were (Student t test, ␣ ϭ 0.05) significant. In the control classified as grade 0, 16(41.1%) as grade 1, 8 (20.5%) group, uterine volume was 540.4 Ϯ 250.8 cm3 at the as grade 2, and 4 (10.2%) as grade 3. In the control first evaluation and 601.1 Ϯ 241.3 cm3 at the second group, 2 patients (6.5%) were classified as grade 0, 3 evaluation, with a statistically (Student t test) nonsignif- icant increase of 0.1 Ϯ 0.2 (Table 1). In every patient, (32.2%) as grade 3 (Figs 1, 2, and 3). The difference in the uterine volume was different in the first and the mean immunohistochemical grade between the study group and the control group was found to be statisti- The histologic features of the leiomyomas exam- cally (Student t test) significant.
ined were those of typical leiomyomas, including inter- The mean changes in uterine volume after GnRH lacing bundles of smooth muscle cells and a variablecontent of fibroblasts and blood vessels. The histologic analogue treatment were 0.36 Ϯ 0.19 (mean Ϯ SD) in appearance of treated leiomyomas were similar in the grade 0, 0.32 Ϯ 0.18 in grade 1, 0.14 Ϯ 0.03 in grade 2, treated cases and the untreated cases. Only in nine and 0.09 Ϯ 0.006in grade 3 (Table 2).
cases (23%) was considerable nuclear crowding found, Patients with a lower PDGF expression (i.e., a low immunohistochemical grade) showed a higher volumedecrease after GnRH analogue administration. The re-lationship between the grades with regard to the per-centage of shrinkage in uterine volume after treatmentwas found to be statistically (Student t test) significant,except for the comparison between grade 0 and grade1 (Table 3).
change*(mean Ϯ SD) 0.36 Ϯ 0.19 0.32 Ϯ 0.18 0.14 Ϯ 0.03 0.09 Ϯ 0.006 FIGURE 2.
Immunohistochemical staining for PDGF in an un- *Ratio of the second scanned volume minus the first scanned treated leiomyoma. (Original magnification ϫ250.) volume to the first scanned volume.
PDGF EXPRESSION IN GnRH-a TREATED LEIOMYOMAS (Di Lieto et al) GnRH analogue–treated uterine leiomyomas27 re- ported cellular atrophy due to a decrease in myofila- ments, mithocondrial swelling, and emergence of thelysosomal body. Finally, the decrease in cell prolifera- tion, indicated by reduced expression of proliferating cell nuclear antigen and of nuclear antigen Ki67, was demonstrated in uterine leiomyomas after GnRH ana- The decrease in uterine volume observed in our study seems to be significantly related to the PDGF Abbreviations: S, significant; NS, nonsignificant.
NOTE. Student t test, monodirectional hypothesis, ␣ ϭ 0.05.
expression in leiomyoma cells. The comparison be-tween the immunohistochemical grades with regard tothe uterine volume changes was found to be statistically DISCUSSION
significant, except for the relationship between grade 0and grade 1.
The presence of RNA transcripts for PDGF21,23 and At the beginning of the study, the two groups for PDGF receptors20 has been demonstrated in normal statistically belonged to the same population. The myometrium. In vitro studies20 have shown that myo- mean immunohistochemical grade was significantly metrial smooth muscle cells in culture are responsive to higher in controls than in treated patients. In the con- the mitogenic action of PDGF, independent of the trol group, a high PDGF expression prevailed (with phases of the menstrual cycle. The detection of PDGF most patients classified as grade 2 or 3), whereas the in human leiomyomas19,21 and the coexpression of treated patients were mostly classified as grade 0 or 1, PDGF and PDGF-␤ receptor19 suggested the involve- thus showing low PDGF expression. This brings us to ment of this growth factor with mitogenic effects in the hypothesize that GnRH analogue treatment actually caused reduced PDGF synthesis in leiomyoma cells.
In a recent study22 we reported the results of in PDGF expression was significantly reduced in those vitro experiments demonstrating that E2 acts by induc- treated patients who exhibited a greater uterine vol- ing the production of growth factors and determines ume decrease. Then, the “nonresponders” mechanism transduction events related to the estrogen-induced might be related to a low tissue and receptor response PDGF secretion. Moreover, we demonstrated that to GnRH analogue; this is made more evident by the PDGF had a main role in the proliferative activity of absence of reduced PDGF levels after treatment.
leiomyoma smooth muscle cells. Antiestrogen cell treat- The assumption of GnRH analogue–induced de- ment is correlated with the disappearance of the mito- crease in PDGF synthesis by leiomyoma cells and the genic factor, PDGF, in leiomyoma smooth muscle cell– demonstration that this lower expression is significantly conditioned media, whereas the E2 mitogenic action is related to the post-GnRH analogue treatment shrink- almost completely neutralized by anti-human PDGF age in the uterine volume suggest that PDGF might antibodies, suggesting the direct involvement of this have a mitogenic in vivo action on leiomyomas. In growth factor in leiomyoma proliferation.
addition, we confirm the presence of PDGF within the Because the interaction between estrogen and cytoplasm of leiomyomatous smooth muscle cells and PDGF in leiomyoma progression has been demon- of fibroblasts, but not in the extracellular matrix.21 The strated in vitro, and considering the pharmacologic absence of deposition in the extracellular matrix might effects of GnRH analogues, an in vivo mitogenic action indicate that PDGF has a local autocrine or paracrine of PDGF on leiomyomas is suggested by a significant action. Even if further studies on a greater number of relationship between the shrinkage in uterine volume patients are necessary, our results can integrate the and the decrease in PDGF synthesis, as a result of the findings about the PDGF role on leiomyoma progres- GnRH analogue–induced hypoestrogenic state.
sion that have been well demonstrated in vitro.
All of the data collected in the present study con- firm that GnRH analogue treatment causes a significantshrinkage in uterine volume.24,25 The histologic fea- REFERENCES
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