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Vascular Endothelial Growth Factor and Its Soluble Receptor in Ovarian Pathology

Posted on: Thursday, 18 August 2005, 03:01 CDT

Abstract

Objective. The soluble form of the vascular endothelial growth factor (VEGF) receptor, s-VEGFR-1, may negatively regulate the action of VEGF. Our purpose was to better understand the regulation of angiogenetic processes in ovarian cysts.

Methods. Seventy-three women, 36 with serous cystoadenoma, 30 with ovarian endometriosis and seven with cystoadenocarcinoma, were enrolled. We calculated both VEGF and s-VEGFR-1 levels in cystic fluid and a VEGF activity index by means of the ratio VEGF/s-VEGFR- 1. Student's t test was used for the statistical analysis.

Results. We found higher VEGF concentration in both endometriotic and malignant lesions than in serous cystoadenoma (p = 0.03 and 0.001, respectively). Also s-VEGFR-1 concentration was higher in endometrioma than in serous cysts (p = 0.005); however, there was no statistically significant difference between cystoadenoma and the malignant lesions (p = 0.15). VEGF activity index in cystoadenoma, endometriotic and malignant lesions was 0.61, 0.27 and 0.50, respectively.

Conclusions. VEGF certainly has an important role in both ovarian endometriosis and for cancer progression; however, the activity index may be better to investigate the real role of VEGF in the pathology we have considered.

Keywords: Vascular endothelial growth factor, soluble VEGF receptor-1, ovarian cyst, endometriosis, cystoadenocarcinoma

Introduction

The ovary causes pathology in every age of life; each kind of ovarian cell may develop dysfunctional pathology and benign or malignant neoplasm. Follicular and luteal cysts are dysfunctional neoformations, and they are so frequent that they can be considered paraphysiological. Serous cysts represent the most frequent benign ovarian neoformation (about 25%). Cystoadenocarcinoma is the malignant variant of the serous cyst, representing 90% of ovarian carcinomas. The mean age at diagnosis is about 63 years and unfortunately an early diagnosis is very rare, so we frequently find advanced stage pathology.

Another very important pathology because of its high incidence in young women is endometriosis, the ectopic localization of endometrial glandular and stromal cells [1]. Although endometriosis is thought to be a benign gynecological disorder, it is associated with significant pain and morbidity. It occurs in about 10% of women of reproductive age and in up to 50% of women with infertility [2]. The ovary is the most common location of endometrial implants, being involved in 40-60% of cases [3]. The pathogenesis of this pathology is probably related to retrograde menstruation.

Many studies [4-6] have drawn attention to the activation of peritoneal macrophages, local cytokines and growth factors, and to the peritoneal neovascularization required to make the cells able to implant. Furthermore, neoangiogenetic processes are considered very important in the progression of the disease [7]. Angiogenesis is a physiological process present in adults only in the reproductive tract.

Kamat and colleagues hypothesized that vascular endothelial growth factor (VEGF) is a candidate to provoke the increased permeability that occurs shortly before ovulation and furthermore that VEGF contributes to the angiogenesis and connective tissue stroma generation that accompany corpus luteum/ corpus albicans formation [8]. Gordon and associates found that VEGF in the epithelial lining of benign ovarian neoplasms may contribute to fluid formation in ovarian cysts, increasing vascular permeability [9]. Also, neoplastic and metastatic tissues are angiogenesis- dependent; in fact, neoplastic cells need the presence of new blood vessels not only to acquire metabolites but also to reach the general circulation [10]. The hypothesis that tumor growth is angiogenesis-dependent is particularly relevant for ovarian cancer. Histological studies have confirmed that ovarian cancer is richly vascularized and have shown a correlation between microvascular density and tumor aggressiveness [11].

VEGF is one of the most important and most investigated of the factors involved in angiogenesis. It is a 34-46 kDa homodimeric glycoprotein which is a highly specific mitogen for vascular endothelial cells [12], is capable of inducing angiogenesis [13], is a potent inducer of vascular permeability [14] and is a survival factor for newly formed blood vessels [15]. The VEGF family at present comprises four different isoforms, each one with a specific affinity for the VEGF receptors and with a particular action.

VEGF-A^sub 165^ is the predominant variant, produced by a variety of normal and transformed cells; it is induced by hypoxia [16]. VEGF- B is strongly expressed in the heart of the developing embryo and in adult cardiac and skeletal muscle [17,18]; its production is not induced by hypoxia [19]. VEGF-C is induced by pro-inflammatory cytokines but not by hypoxia [19] and it generally induces a lymphangiogenic response. VEGF-D shares similar receptor-binding specificity to VEGF-C.

The action of VEGF is mediated by two receptors, both of which bind the VEGF family with high affinity. These receptors are expressed predominantly on endothelial cells [20], but additionally are found on non-endothelial cells (i.e., peripheral blood monocytes, malignant melanoma cells lines, ovarian carcinoma tumor cells, trophoblasts, peritoneal fluid macrophages) [1]. VEGFR-1 is an fms-like tyrosine kinase receptor [21] that is indispensable during embryonic development [22], but its significance is uncertain in adults. VEGFR-2, if blocked, inhibits tumor angiogenesis and growth, so it is possible to think of a role in mediating the effect of VEGF in the tumor angiogenic process [23].

In the last few years, a soluble form of VEGF receptor, s-VEGFR- 1, has been discovered. This receptor binds especially VEGF-A with high affinity and has been located in a number of tissues, including human endometrium [24] and placenta [25]. This soluble receptor may negatively regulate the action of VEGF on two levels: first, it may bind and sequester VEGF, thereby reducing its bioavailability; and second, by occupying the VEGF receptors it will prevent VEGF occupancy and the resulting signal transduction [26].

A study by McLaren and coworkers [27] suggested that the high VEGF levels found in the peritoneal fluid of women with endometriosis could have a crucial role in the pathogenesis of the disease. Our recent study demonstrated a correlation between the VEGF levels in cystic fluid and ovarian endometriosis, showing that VEGF-mediated angiogenesis could be a necessary event for the formation and progression of this kind of cyst [28].

VEGF is an angiogenetic mediator also in the neoplastic ovarian pathology. Olson and collaborators identified the malignant epithelium of the ovarian neoplasm as a possible producer of VEGF [29]. Because VEGF has been implicated as a regulator of angiogenesis in ovarian cancer [30], the production of VEGF may reflect the angiogenetic activity of this neoplasm [31]. High VEGF levels have been found in ascitic fluid of women with ovarian cancer, suggesting that VEGF has a causal role in the formation of ascitic fluid and in tumor progression [32].

In a recent study, Neulen and associates [33] reconsidered the role of VEGF and its regulation in the angiogenetic processes, evaluating the importance of s-VEGFR-1 with respect to ovarian response to gonadotropin stimulation. They calculated an activity index by taking a ratio between the amount of VEGF and s-VEGFR-1. The data indicated that a delicate balance between VEGF and its naturally occurring antagonist s-VEGFR-1 is necessary to allow an adequate ovarian reaction to gonadotropin therapy. Excess of bioactive VEGF increases the risk for ovarian hyperstimulation syndrome. An excess of s-VEGFR-1 results in poor response and reduced chances of conception.

Based on this observation we have speculated about the role of the VEGF soluble receptor in the modulation of angiogenetic VEGF- mediated process in ovarian pathology such as endometriosis and epithelial cancer.

Materials and methods

Patients

From September 2000 to January 2002 we retrospectively enrolled 73 patients hospitalized in the Obstetric and Gynecological University Hospital, Pisa, Italy for ovarian pathology. The Committee on Human Research at the University of Pisa approved the study and informed consent was obtained from each patient.

We considered 36 premenopausal, non-pregnant women with ultrasound diagnosis of serous cystoadenoma who underwent resolutive laparoscopic surgery, with histological agreement to the benignity.

Another 30 patients referred to our clinic for infertility problems associated with pelvic pain had ultrasound imaging results suggestive of endometriosis. All patients were free from hormonal therapy for almost 3 months (no estrogen/progestin or gonadotropin- releasing hormone agonist). The diagnosis of endometriosis was confirmed by laparoscopic vision, during excision of the endometriomata, and by histological examination. All of these patients were evaluated for endometriotic invasion and revealed Stage IU-IV3 based on the revised classification of the American Fertility Society (AFS) [34].

Seven patients with malignant lesions, confirmed on the basis of the histological response of surgical specimens, were also enrolled. All were in Stage III-IV of the disease according to the \classification of the International Federation of Obstetrics and Gynecology (FIGO).

Interventions

For patients with benign and endometriotic lesions laparoscopic surgery was performed around the 10th day of the cycle, having previously obtained an accurate ultrasonographic evaluation of the lesion [35].

For those patients with a lesion suggestive to be malignant, the aggressive surgery recommended for ovarian cancer was performed.

All specimens were sent to an independent pathologist for histological examination. Cystic fluid was collected via a Verres needle during the surgical session, immediately after the enucleation.

Samples

The cystic fluid was immediately centrifuged at 2000g for 10 min at room temperature; the supernatant was separated and stored at - 20C until the assay.

Determination of VEGF

VEGF levels were detected by an enzyme-linked immunosorbent assay (ELISA) that measures the 'free' forms of the growth factor (CYTElisa(TM) VEGF; Peninsula Laboratories Inc., Belmont, CA, USA); the kit has a sensitivity of 12.5 pg/ml, an intraassay variability of 7.7% and an inter-assay variability of 10.7%.

Determination of s-VEGFR-1

The soluble form of the VEGF receptor was detected in our samples by an ELISA which measures the total amount of such receptor (Human sVEGFR-1 (total) ; RELIATech, Braunschweig, Germany). The kit has a sensitivity of < 0.16 ng/ml, a range of detection from 0.3 to 10 ng/ ml, an intra-assay variation of 8% and an inter-assay variation of 7- 8%.

Table I. Clinical characteristic and laboratory data of the patients.

Activity index

To better evaluate the biological significance of the proteins we have tested, the ratio between the amount of VEGF and the amount of its soluble receptor (VEGF/s-VEGFR-1) was calculated for each of the pathologies we considered.

Statistical analysis

Differences between the groups were evaluated with the Student t test. Results are expressed as mean - standard deviation. We considered a probability of p < 0.05 to be statistically significant.

Results

The clinical characteristics and laboratory data of the patients are shown in Table I; the laboratory data are summarized in Figures 1, 2 and 3.

The VEGF concentration in the fluid of serous cystoadenoma was 478.3 95.0 pg/ml, and the s-VEGFR-1 level in the same patients was 781.4 342.1 pg/ml. In endometriomata, the levels of VEGF and s- VEGFR-1 were 1365 184 pg/ml and 5020 1799 pg/ml respectively, whereas concentrations found in the cystic fluid from cystoadenocarcinoma were 1721 188 pg/ml for VEGF and 3452 3393 pg/ ml for sVEGFR-1 (Figure 1) .

Statistical analysis revealed a significant difference in VEGF concentration between serous cystoadenoma and endometrioma (p = 0.03) and also between serous cystoadenoma and cystoadenocarcinoma (p = 0.001). There was no statistically significant difference in cystic fluid VEGF concentration between endometriotic and cystoadenocarcinoma lesions (p = 0.24).

For s-VEGFR-1, there was a statistically significant difference between its levels in serous cystoadenoma and in endometriomata (p = 0.005); however, we did not find a statistically significant difference between s-VEGFR-1 levels in serous cystoadenoma and cystoadenocarcinoma (p = 0.15) (Figure 2).

Figure 1. Levels of vascular endothelial growth factor (VEGF) in cystic fluid, as determined by enzyme-linked immunosorbent assay, of patients divided for pathology. Serous cysts contained markedly lower VEGF levels than endometriomata (p = 0.03) and malignant cysts (p = 0.001). The difference between endometriosis and cancer was not statistically significant (p = 0.24).

Figure 2. Levels of soluble receptor for vascular endothelial growth factor (s-VEGFR-1) in cystic fluid, as determined by enzyme- linked immunosorbent assay, of patients divided for pathology. s- VEGFR-1 levels were higher in endometriomata than in serous cysts (p = 0.005); the levels in cystoadenocarcinoma were not statistically different compared with those in serous cysts (p = 0.15) and endometriosis (p = 0.24).

Considering the high levels found in the pathologies with respect to the control patients (serous cysts), we also calculated an index to estimate the biological activity of VEGF. In serous cysts we found an index of 0.61, an intermediate index of 0.50 was found in cystoadenocarcinoma and, finally, in endometriotic lesions we found a lower index of 0.27 (Figure 3).

Discussion

In ovarian physiology, recent studies [8,9] have declared the important action of VEGF in follicular growth and in the formation of corpus luteum. In fact, both VEGF and its mRNA are expressed in granulosa and theca cells in the late phase of follicular growth and in the postovulatory age [8]. The literature of the past 10 years contains many studies that hypothesize a connection between angiogenesis and ovarian cysts. Indeed, intratumoral arterial blood flow has been found in 94% of benign ovarian tumors by use of color Doppler [35], and Gordon and colleagues have demonstrated VEGF activity in the epithelial tissue of benign ovarian neoplasm [9]. Furthermore, immunohistochemistry and molecular biology have revealed the presence of VEGF and its receptors in benign, borderline and malignant neoplasms [36].

Figure 3. VEGF activity index, derived as the ratio of the amount of vascular endothelial growth factor (VEGF) to that of its soluble receptor (s-VEGFR-1). High activity was found in serous and in malignant cysts; lower activity was found in endometriomata.

In agreement with literature results concerning the angiogenetic switch of malignant cells [10], in the present study we found higher VEGF levels in malignant lesions that represented a statistically significant increase compared with the levels in serous cysts (p = 0.001). This result seems to confirm the important role of angiogenetic processes for neoplastic tissue; furthermore we may suggest the involvement of VEGF in this angiogenesis. Regarding the VEGF receptor, we found high levels of s-VEGFR-1 in the cystic fluid of adenocarcinoma but unfortunately with a very high range of variability, probably related to the low number of patients, which makes the value of uncertain significance.

We also evaluated endometriotic lesions and found high VEGF levels with respect to control patients (serous cysts) (p = 0.03), confirming our previous study regarding the role of VEGF in the formation, growth and maintenance of ovarian endometriomata [28]. However, contrary to expectations, we also found high levels of s- VEGFR-1 in the cystic fluid of endometriomata that seems to suggest a downregulation of VEGF-mediated angiogenesis in these processes.

Starting from this observation and considering literature data [33], we calculated an activity index to better investigate the biological significance of the high VEGF levels found. The index derives from the ratio between the amount of VEGF and that of its soluble receptor found in the cystic fluid.

We found a higher activity index in the benign pathology (0.61); based on the level of VEGF and on this index we hypothesized that in the benign pathology the low concentration found does not represent the real activity of the growth factor. Probably in this condition the high activity is related to the intense neovascularization, in most part caused by inflammation and only for the residual amount related to hypoxia.

The intermediate index (0.50) found in the neoplastic lesions derives from the very high concentration of the growth factor found in the cystic fluid, but the high standard deviation of the s-VEGFR- 1 determinations make this result very difficult to understand. In trying to interpret the results, it is important to stress that we have evaluated cystoadenoma and advanced epithelial ovarian cancer (Stage III-IV) and this probably constitutes a large study bias; unfortunately this bias is related to the great difficulty in making an early diagnosis of epithelial ovarian cancer. Probably we need a greater number of patients and more time to evaluate them; furthermore, it would be interesting to investigate patients in different stages of pathology, but it is very rare to have an early diagnosis of ovarian cancer. At present, from our results, it seems that s-VEGFR-I may negatively regulate VEGF action, but the block is not complete. If confirmed by further studies, this result could reinforce the important role that VEGF plays also in the advanced phases of ovarian cancer (FIGO Stage III-IV).

In the endometriotic lesions we found an index of 0.27 that derives from high levels of both VEGF and sVEGFR-I. Based on this result, we suppose that in the endometriotic cysts we evaluated (advanced stage, IIIIV, of AFS) the angiogenetic processes are still present but probably not completely mediated by VEGF.

Finally we propose to reconsider the real involvement of VEGF in these stages of pathology, especially for endometriosis. Further investigations should be done to evaluate the cystic fluid levels of VEGF (including different isoforms, if possible) and s-VEGFR-I in different stages of endometriosis, to gain a better understanding of the role and regulation of this cytokine in both the early phases of the pathology and the formation and growth of ovarian endometriotic cysts.

Acknowledgement

We gratefully acknowledge Professor Angiolo Gadducci for his assistance and kind collaboration.

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P. G. ARTINI1, F. CRISTELLO1, M. MONTI1, V. CELA1, C. BATTAGLIA2, G. D'AMBROGIO1, & A. R. GENAZZANI1

1 Department of Reproductive Medicine and Child Development, Division of Obstetrics and Gynecology, 'S. Chiara' Hospital, University of Pisa, Pisa, Italy, and 2 Third Department of Obstetrics and Gynecology, University of Bologna, Bologna, Italy

Correspondence: P. G. Artini, Department of Reproductive Medicine and Child Development, Division of Obstetrics and Oynecology, University of Pisa, Via Roma 56, 56126 Pisa, Italy. Tel: 39 50554104. Fax: 39 50551293. E-mail: g.artini@obgyn.med.unipi.it

Copyright CRC Press Jul 2005

Source: Gynecological Endocrinology

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