2808 Vol. 6, 2808–2814, July 2000 Clinical Cancer Research Expression of the Hypoxically Regulated Angiogenic Factor Adrenomedullin Correlates with Uterine Leiomyoma Vascular Density1 Stephen Hague, Lyna Zhang, Martin K. Oehler, found in leiomyomas and the myometrium of leiomyoma- Sanjiv Manek, Ian Z. MacKenzie, Roy Bicknell, bearing uteri. As such, ADM is identified as a novel target and Margaret C. P. Rees2 for antiangiogenic therapy of these benign, clinically prob- lematic uterine tumors. Nuffield Department of Obstetrics and Gynecology [S. H., L. Z., M. K. O., I. Z. M., M. C. P. R.], Molecular Angiogenesis Laboratory, Imperial Cancer Research Fund, Institute of Molecular Medicine[S. H., L. Z., M. K. O., R. B.], and Department of Cellular INTRODUCTION Pathology [S. M.], University of Oxford, John Radcliffe Hospital, Uterine leiomyomas are the most common benign tumors Oxford OX3 9DU, United Kingdom affecting adult women. They are present in one of three women over the age of 30 (1, 2). Leiomyomas are a significant cause of ABSTRACT menorrhagia, pelvic pain, infertility, and pregnancy loss and are one of the commonest indications for hysterectomy. Their Uterine leiomyomas are the most prevalent benign tu- growth is controlled by estrogen, because they only occur after mor type in women of reproductive age and are one of the puberty and regress after the menopause. The increased mitotic most common indications for hysterectomy. The expression of five angiogenic factors, adrenomedullin (ADM), vascular activity in leiomyomas during the luteal phase of the menstrual endothelial growth factor (VEGF), acidic fibroblast growth cycle suggests that their growth is stimulated by progesterone as factor, basic fibroblast growth factor, and platelet-derived well as estrogen (3, 4). Estradiol and progesterone receptors are endothelial cell growth factor/thymidine phosphorylase, present in leiomyomas and are often overexpressed when com- were examined in 91 uteri collected throughout the men- pared with that in adjacent myometrium (4–6) . strual cycle; 52 of which contained leiomyomata, and the Specific biological effects of ovarian steroids are known to remainder were normal controls. The microvascular density be mediated through the actions of polypeptide growth factors and endothelial proliferative indices were then determined (7), some of which are angiogenic (7). Growth of leiomyomas is for each of the uterine sections. ADM and VEGF were the known to be controlled by a series of hormone-regulated most widely expressed angiogenic factors in the leiomyomas. polypeptides. Thus, various growth factors have been identified 3 Furthermore, the expression of ADM and VEGF in the in leiomyomas that include epidermal growth factor, VEGF, endometrium and myometrium was up-regulated in leiomy- insulin-like growth factor, platelet-derived growth factor, bFGF, ␤ oma-bearing uteri compared with controls. Although acidic transforming growth factor- , and an Mr 18,000 leiomyoma- fibroblast growth factor and basic fibroblast growth factor derived mitogenic protein (8–14). bFGF shows a mitogenic were expressed in leiomyomas and endometrium in all of the effect on cultured leiomyoma cells (15). Other growth factors uterine samples examined, they were only expressed in the such as the peptide ADM and the angiogenic enzyme TP have myometrium of leiomyomata-bearing uteri. Endothelial been identified in uterine tissues but not systematically studied proliferation in leiomyomas was statistically greater than in leiomyomas (16–19). ADM was first identified as a hypo- that of the myometrium and endometrium, both within and tensive peptide isolated from a human pheochromocytoma cell between uteri (P < 0.05). The vascular density in the myo- line (20) and recently shown to be an endothelial cell growth metrium but not the endometrium was significantly in- and angiogenic factor (18). Expression of ADM in the endome- creased in leiomyoma-containing uteri (P < 0.05). Expres- trium has been shown to be induced by tamoxifen but not sion of ADM alone correlated directly with vascular density estrogen (18). and endothelial cell proliferation index in leiomyomas and This study was undertaken to examine determine expres- myometrium and may account for the high vascularity sion of the angiogenic factors ADM, VEGF, aFGF, bFGF, and PDECGF/TP in uterine leiomyomata. Vascular density and en- dothelial and smooth muscle proliferation indices were also examined. The overall aim was to clarify the role of angiogen- esis in the etiology of uterine leiomyomata. Received 11/2/99; revised 3/27/00; accepted 3/30/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by The Sir Jules Thorn Charitable Trust, The 3 The abbreviations used are: VEGF, vascular endothelial growth factor; Imperial Cancer Research Fund, and Astra-Zeneca. ADM, adrenomedullin, aFGF, acidic fibroblast growth factor; bFGF, 2 To whom requests for reprints should be addressed, at Nuffield De- basic fibroblast growth factor; PDECGF/TP, platelet-derived endothe- partment of Obstetrics and Gynaecology, University of Oxford, John lial growth factor/thymidine phosphorylase; NHS, normal human se- Radcliffe Hospital, Oxford OX3 9DU, United Kingdom. rum; SS, swine serum; RAMP, receptor activity-modifying protein. Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2000 American Association for Cancer Research. Clinical Cancer Research 2809 MATERIALS AND METHODS same subtype of mouse immunoglobulin (Sigma) at the same Tissue Preparation. Formalin-fixed, wax-embedded concentration. specimens of endometrium, myometrium, and leiomyoma from aFGF and bFGF. Immunohistochemical staining for hysterectomy samples were selected from the archival files of aFGF was performed using the streptavidin-biotin-alkaline the Histopathology Department of the John Radcliffe Hospital. phosphatase (ABC) method using the Vectastain ABC kit for The 91 hysterectomy samples examined were obtained from alkaline phosphatase (rabbit IgG) according to the manufactur- women of ages 30–49 years. All women had a history of regular er’s protocol using rabbit anti-bovine aFGF polyclonal antibody menstrual cycles (26–30 days) and had used neither oral nor (R & D Systems) at a dilution of 1:200 in TBS/20% SS. The intrauterine contraception or had not received any hormonal peroxidase anti-peroxidase method was used for bFGF. Prior to treatment for at least 6 months prior to surgery. The stage of the the application of the primary antibody, the sections were incu- bated with 20% SS in TBS for 20 min. Rabbit anti-bovine bFGF menstrual cycle at which the tissue was obtained was deter- polyclonal antibody (Sigma) diluted to 84 ng/ml in TBS/20% SS mined from the patient’s menstrual history and endometrial was added for 60 min. After washing in TBS, the sections were histology (21). The presence or absence of leiomyomas was incubated with peroxidase-conjugated swine antirabbit immu- confirmed by histopathological examination (S. M.). noglobulin (Dako, Cambridge, United Kingdom) at a dilution of Specimens were obtained from 39 women without 1:50 in TBS/20% SS for 30 min, washed again in TBS, and leiomyomas undergoing hysterectomy for a subjective com- incubated with rabbit peroxidase anti-peroxidase at a dilution of plaint of menorrhagia. Eleven patients were menstruating (days 1:50 in TBS/20% SS for 30 min. Sections were washed in TBS 1–7), 14 patients were from the follicular phase, and 14 were in twice, developed with diaminobenzidine tetrahydrochloride the luteal phase. Fifty-two uterine specimens containing (Dako) at 0.6 mg/ml in TBS containing 3 ␮l/ml of hydrogen leiomyomas were collected, of which 22 were menstrual, 16 peroxide for 10 min. All slides were counterstained with hema- were follicular phase, and 14 luteal phase. Sections of endome- toxylin (Sigma) and mounted in Apathy’s mounting medium trium and myometrium were obtained from all specimens and (BDM Merck, Poole, United Kingdom). Negative controls had were present from leiomyomas. the same concentration of rabbit immunoglobulin in place of the Immunohistochemistry. All sections were dewaxed us- primary antibody. ing Citroclear (HS Supplies, Aylesbury, United Kingdom); re- PD-ECGF/TP. Immunohistochemical staining for PD- hydrated sequentially in absolute, 95, 70, and 20% ethanol and ECGF/TP was performed using the alkaline phosphatase-anti distilled water; and rinsed in TBS (0.05 M Tris base, 0.15 M alkaline phosphatase method, as described previously (17). Prior NaCl, 10ϫ; pH 7.6) prior to staining. to application of the primary antibody, the sections were incu- ADM. Immunohistochemistry was carried out as de- bated with 20% normal rabbit serum to block nonspecific pro- scribed previously (18). Briefly, slides were incubated with 5% tein binding sites. Primary antibody (PGF44Cl) was added to the goat serum (Dako) to reduce nonspecific background staining, slides for 30 min. The slides were then washed twice in TBS and followed by 1:800 anti-ADM (Peninsula Laboratories, Liver- incubated for an additional 30 min with rabbit antimouse im- pool, United Kingdom). A second biotinylated swine antirabbit munoglobulin at a 1:50 dilution. The sections were washed antibody (Dako) at a dilution of 1:400 was then applied for 30 again in TBS/10% NHS. Chromogen development was per- min, after which