Expression of the Hypoxically Regulated Angiogenic Factor Adrenomedullin Correlates with Uterine Leiomyoma Vascular Density1

2808 Vol. 6, 2808–2814, July 2000 Clinical Cancer Research

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 endothelial and smooth muscle proliferation indices were also examined. The overall aim was to clarify the role of angiogenesis 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.

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 incubated 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 the slides were incubated with streptavidin formed using a New Fuchsin Substrate System (Dako) accord- 1:200 (Dako) for 30 min. The final color was developed with the ing to the manufacturer’s instructions by incubation for 5–10 New Fuschin substrate system (Dako). min. Slides were counterstained with hematoxylin and mounted VEGF. Immunohistochemical staining for VEGF was with Apathy’s mounting medium. carried out using the streptavidin-biotin-alkaline phosphatase Ki67/CD34 Double Staining. Immunohistochemical (ABC) method, as described previously (22). After dewax- staining for CD34 was performed using the streptoavidin-biotin- ing and rehydrating the sections, the slides were incubated in alkaline phosphatase (ABC) method. Throughout the protocol, double-distilled water at 37°C for 10 min and in 200 ml of all antibody dilutions and washes were performed in TBS, with

PBSA (0.13 M NaCl, 0.002 M KCl, 0.01 M Na2HPO4, 0.002 M incubations being performed at room temperature. Antigen re- KH2PO4) containing 25 mg of protease type 24 (Sigma, Poole, trieval was performed by means of pressure cooking in 1.6 L United Kingdom) at 37°C for another 10 min. The slides were 0.01 M sodium citrate buffer (pH 6.0) for 90 s, followed by a then left in double-distilled water at room temperature for 30 30-min rinse in distilled water and 5 min in TBS. Prior to the min and finally washed in TBS. Prior to application of primary application of the primary antibody, the sections were blocked antibody, the sections were incubated with 5% NHS/TBS for 20 in 10% NHS. The sections were incubated at room temperature min to block nonspecific binding sites. The sections were then for 30 min with the primary CD34 antibody (Qbend 10, Nova- incubated with anti-VEGF antibody M293 (R & D Systems, castra, United Kingdom), diluted 1:25. Again, the sections were Abingdon, United Kingdom) diluted to 12.5 mg/ml in TBS/5% rinsed for 2 ϫ 5 min, followed by a 30-min incubation with the NHS at 4°C. After incubation with the primary antibody, the secondary biotinylated rabbit antimouse IgG (Sigma), diluted sections were washed in TBS and incubated with biotinylated 1:400. The sections were incubated with phosphate-conjugated rabbit antimouse IgG diluted to l:400 in TBS/5% NHS, washed streptavidin at a dilution of 1:200 for 30 min; the final color was again, and incubated with alkaline phosphatase-conjugated to developed with the New Fuschin substrate (Dako). streptavidin at dilution of 1:200 in TBS/5% NHS. The New The Vectastain ABC kit was used for the staining of Ki67. Fuchsin Substrate System was used to visualize sections. In the Prior to staining, double stain enhancer (Zymed) was added to negative controls, the primary antibodies were replaced with the the sections; endogenous peroxidase activity was quenched by

the application of 0.3% hydrogen peroxide, diluted in distilled phages exhibited high levels of ADM, as shown previously (18). water. The sections were washed and incubated in horse serum, The presence of a leiomyoma appeared to up-regulate the ex- after which the primary Ki-67 antibody (BioGenex, San Ramon, pression of ADM and VEGF in the endometrium as compared CA) at a dilution of 1:10 was added for 30 min. The sections with controls. The presence of aFGF and bFGF was noted in the were washed and incubated with the secondary biotinylated epithelium but not the stroma or endothelium of the endome- ABC antibody for 30 min, with the final color being developed trium. The expression of aFGF and bFGF in the endometrium with diaminobenzidine tetrahydrochloride substrate (Sigma). was unaffected by the presence of a leiomyoma. PDECGF/TP Determination of Vascular Density and Endothelial was detected in the endometrium throughout the menstrual Cell Proliferative Indices. The vascular density of myome- cycle, localizing to epithelial, stromal, and endothelial cells. trium and leiomyoma was determined by Chalkley counting Vascular Density and Endothelial Proliferation Index. (23). The three most vascular areas where the highest number of The vascular density of the various tissues was analyzed at the discreet microvessels stained were chosen. Vascular density was various stages of the menstrual cycle, menstrual, proliferative, then determined using a 25-point Chalkley eyepiece graticule at and secretory, by CD34 immunostaining, followed by Chalkley ϫ250. The graticule was rotated in the eyepiece to where the counting. The vascular density of the endometrium, myome- maximal number of vessels were overlaid by graticule dots. trium, and leiomyomata did not change significantly during the Individual density was then obtained by taking the mean of three menstrual cycle. The vascular density of the myometrium was graticule counts. significantly higher (P Ͻ 0.05) in leiomyoma-bearing uteri The endothelial cell proliferative index was determined at compared with controls. The vascular density of the endome- ϫ400. The endothelial cell proliferative index was calculated as trium was unchanged by the presence of a leiomyoma. The the percentage of all Ki-67 positively stained endothelial nuclei endothelial proliferation index in leiomyoma, myometrium, and that also had concomitant positive cytoplasmic staining in endometrium did not vary with the various stages of the men- CD34-positive cells. strual cycle. A significant correlation between vascular density Statistical Analysis. Analysis of microvascular density, and endothelial cell proliferation index was observed (P Ͻ determined by Chalkley counts, and the endothelial cell prolif- 0.05). Table 1 shows that endothelial cell proliferation is higher eration index used the nonparametric Mann-Whitney U test and in the leiomyoma and the endometrium compared with the Spearman Rank correlation coefficient. The intensity of immu- myometrium. This was expected because the leiomyoma and nostaining for angiogenic factors was scored on a scale of 1–3ϩ, endometrium are areas of active angiogenesis, whereas the with 3ϩ indicating a strong positive result, by two independent myometrium is not. observers. Kendall Tau Analysis was used for correlations be- Analysis by Kendall Tau correlations of the degree of tween microvascular density and angiogenic factor expression. staining for angiogenic factors ADM or VEGF with microvascular density and endothelial proliferation index showed a number of significant correlations (Fig. 2). The level of ADM RESULTS expression directly correlated with both the vascular density and The expression of five angiogenic polypeptides, ADM, proliferation index in leiomyoma (P Ͻ 0.05). No such correla- VEGF, aFGF, bFGF, and PDECGF/TP, were examined in uteri tion was seen with VEGF, aFGF, or bFGF expression (P Ͼ containing leiomyomas and in a control group bearing none 0.08). In myometrium, ADM expression correlated with the (Fig. 1). The vascular density and endothelial proliferative index proliferative index. ADM expression in leiomyomas and myo- of all tissue sections were determined in parallel. metrial vascular density were also significantly correlated (P Ͻ Leiomyoma. The most strongly expressed angiogenic fac- 0.01). tors were ADM and VEGF, both localizing principally to the endothelium and vascular smooth muscle. No change in expression was seen throughout the menstrual cycle. aFGF and bFGF were DISCUSSION present in leiomyomas with expression localized primarily to the This study has shown that ADM and VEGF are the most smooth muscle cells and not changing with the menstrual cycle. widely expressed angiogenic factors in uterine leiomyomas and Expression of PDECGF/TP was not detected in leiomyomas. that these tumors have a higher vascular density and endothelial Myometrium. Expression of ADM was found in the proliferative index than both the normal myometrium and en- myometrium, with staining localizing to the smooth muscle of dometrium. ADM and VEGF both share similarities in that they arteries and venules and endothelium with approximately equal are induced by hypoxia (24, 25) and increase vascular perme- intensity. The intensity of ADM expression in the myometrium ability, in addition to being angiogenic. It is well documented was generally weaker than in leiomyomas. ADM expression that they can be hypoxically regulated via a hypoxic response was up-regulated in the myometrium of leiomyoma-bearing motif present within their promoter sequences (26, 27). Similar uteri. Macrophages, arterial walls, venules, and smooth muscle to most tumors, uterine leiomyomata are generally considered to cells stained positive for VEGF, with increased expression in the be hypoxic (21). The two factors differ in that VEGF exhibits presence of a leiomyoma. aFGF and bFGF were only detected in estrogen-dependent induction (7, 25), but ADM does not (18). the myometrium of leiomyoma-bearing uteri. PDECGF/TP ex- Also, although the mitogenic activity of VEGF is highly re- pression was of weak intensity, localizing to the smooth muscle stricted (mainly to endothelial cells), ADM exhibits widespread cells. effects in both benign and malignant tissues (28). Thus, ADM Endometrium. ADM and VEGF localized to the epithe- could be a key player in the growth of leiomyomas. lium and endothelium of the endometrium; stromal macro- ADM expression is known to be up-regulated by the in-

Fig. 1 Immunohistochemical localization of adrenomedullin (A–C) using the alkaline phosphatase method, aFGF (D–F) using the alkaline phosphatase method, and bFGF (G–I) using the peroxidase anti-peroxidase method, in human leiomyomata and myometrium. A, ADM localizes to the smooth muscle fibers of the normal myometrium. B, ADM is present in the smooth muscle cells and vessels of the myometrium derived from a leiomyoma bearing uterus. C, ADM is strongly positive in the smooth muscle and vessels of the leiomyomas. D, normal myometrium is negative for the expression of aFGF. E, myometrium derived from a leiomyoma-bearing uterus is positive for the expression of aFGF, with the highest intensity in the blood vessels. F, leiomyomas are positive for the expression of aFGF. G, bFGF is present in the smooth muscle and vessels of the normal myometrium at a low intensity. H, myometrium derived from a leiomyoma-bearing uterus expresses bFGF specifically in the venules. I, bFGF is strongly expressed in the leiomyoma.

Table 1 Comparison of the endothelial proliferative index of the cytokines by estradiol and progesterone has been demonstrated endometrium, myometrium, and leiomyomata in mouse and human uterine tissues (32, 33). However, little Mean endothelial information is available concerning the expression of the inter- Tissue proliferation rate SE leukins and tumor necrosis factors in leiomyoma. Endometrium 10.729 1.16 Leiomyomata have classically been thought to show poor a Myometrium 3.97 0.36 vascularity (21). For the first time, we show this not to be the Leiomyoma 11.21 1.175a case and that the vascular density of leiomyomas is comparable a Leiomyomas examined had a significantly higher endothelial cell with that of normal myometrium and endometrium. We have proliferative index than the myometrium (P Ͻ 0.005). More than 100 fields were examined for each tissue type. also shown that the vascular density of myometrium (but not that of endometrium) was higher in leiomyomata-bearing uteri compared with controls. This may be attributable to excess release of angiogenic stimuli by the leiomyoma stimulating flammatory cytokines interleukin-1, tumor necrosis factor-␣, angiogenesis in the surrounding myometrium. Alternatively, it and tumor necrosis factor-␤ in cardiac tissue (myocytes and may be that women with an unusually high myometrial vascular nonmyocytes), rat endothelium, and rat and bovine vascular density or increased expression of ADM, VEGF, and aFGF and smooth muscle (29–31). Modulation of expression of these bFGF may have a predisposition for the development of uterine

Fig. 2 Expression of ADM correlates with vascular density in the myometrium (a) and leiomyoma (b). Vascular density was quantitated by Chalkley counting, and significance was determined by Kendall Tau analysis. Bars, SE.

leiomyoma. This is of interest in the light of the known familial egies for the treatment of non-life-threatening pathologies are predisposition to leiomyomas (34). receiving considerable attention. Vascular density and endothelial proliferative index were The high level of VEGF and ADM expression observed unaltered throughout the menstrual cycle in endometrium, myo- within leiomyomas presents a possible target for therapeutic metrium, and leiomyoma. This concurs with previous studies of intervention, using antibodies specific to VEGF and ADM to the endometrium and myometrium by others (35, 36). Vascular treat leiomyomas. Specific monoclonal antibodies capable of density correlated with the proliferative index in the endome- inhibiting VEGF are now available. Such antibodies have been trium, myometrium, and leiomyoma. shown to exert a potent inhibitory effect on both the growth of Fig. 2 shows the intensity of myometrial ADM immu- xenografted human tumors in mice (40) and in development of nostaining against median leiomyoma vascular density and the vasculature of the primate corpus luteum (41). Indeed, median myometrial vascular density. A striking finding of abrogation of VEGF signaling has been shown to lead to vas- this study was that expression of ADM but not that of VEGF cular apoptosis and tumor regression (42). Use of blocking or aFGF or bFGF correlated with the vascular density of the anti-VEGF antibodies has revealed remarkably few side effects leiomyomas. This points to an important role for ADM in of their use. The effect of blocking ADM action has yet to be uterine leiomyoma angiogenesis. We have shown previously evaluated. that ADM plays a key role in endometrial angiogenesis and It has been reported recently that the calcitonin receptor- hyperplasia seen in women receiving tamoxifen therapy for like receptor can function either as a calcitonin gene-related breast carcinoma (18). peptide or ADM receptor, depending on which RAMPs are This is the first report of expression of aFGF and bFGF in expressed. RAMPs are required to transport calcitonin receptor- leiomyoma. In this context, this finding is of interest in view of like receptor to the plasma membrane. RAMP2-transported re- the previous report of FGF receptor 1 in leiomyomas (37). The ceptors are ADM receptors, whereas RAMP1-transported recep- lack of correlation of aFGF and bFGF with vascular density and tors are calcitonin gene-related peptide receptors. This would proliferation in leiomyomas means that their roles in angiogen- suggest that antibodies to RAMP2 may also have potential as a esis in this tissue is unclear. therapeutic tool. Thus, as for other solid tumors, antiangiogen- No expression of PDECGF/TP was detected in leiomyo- esis may have potential in the future therapy of leiomyomas. mas, but it was present in the endometrium and myometrium. Patterns of expression in the endometrium were in agreement ACKNOWLEDGMENTS with previous reports (17). Other groups have also failed to We thank Dr. Andrew Graham, Nuffield Department of Pathology, detect PDECGF/TP expression in leiomyoma (16). It is unusual for assistance with image analysis. for PDECGF/TP not to be up-regulated in neoplastic tissue (38). It is of interest that PDECGF/TP is also not expressed in endometrial malignancies (17). REFERENCES Increased angiogenesis in leiomyomas raises the possibility 1. Andersen, J. Growth factors and cytokines in uterine leiomyomas. Semin. Reprod. Endocrinol., 14: 269–282, 1996. of therapeutic intervention with antiangiogenic agents. Al- 2. Vollenhoven, B. J., and Healy, D. L. Pathophysiology and medical though at present many promising drugs are in development treatment of uterine fibroids. In: I. T. Cameron, I. S. Fraser, and S. K. (39), they will need to be well tolerated before their use can be Smith (eds.), Clinical Disorders of the Endometrium and Menstrual envisioned in benign tumors. Nevertheless, antiangiogenic strat- Cycle, pp. 207–218. Oxford: Oxford University Press, 1998.

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