Angiogenic activity of swine granulosa cells: effects of hypoxia and vascular endothelial growth factor Trap R1R2, a VEGF blocker

Domestic Animal Endocrinology 28 (2005) 308–319

Angiogenic activity of swine granulosa cells: effects of hypoxia and vascular endothelial growth factor Trap R1R2, a VEGF blocker Federico Bianco, Giuseppina Basini∗ , Francesca Grasselli Dipartimento di Produzioni Animali, Biotecnologie Veterinarie, Qualit`a e Sicurezza degli Alimenti – Sezione di Fisiologia Veterinaria, Universit`a di Parma, Via del Taglio 8, 43100 Parma, Italy Received 30 September 2004; received in revised form 17 December 2004; accepted 20 December 2004

Abstract The possible role played by hypoxia and vascular endothelial growth factor (VEGF) in the regulation of follicular angiogenesis was studied in a three-dimensional fibrin gel model. Granulosa cells from follicles > 5 mm were subjected to normoxia (19% O2 ), partial (5% O2 ) or total (1% O2 ) hypoxia and their culture media were collected and used to stimulate porcine Aortic Endothelial Cells (AOC) included in the fibrin matrix. A suspension of AOC on microcarrier beads was pipetted in a fibrinogen solution (1 mg/ml PBS) before the addition of 1250 IU thrombine (250 ␮l) to catalize the gel formation. Granulosa cell conditioned media were tested in the presence or absence of VEGF Trap R1R2 (150 ng/ml), a potent VEGF inhibitor, that had its efficacy tested by adding VEGF (100 ng/ml) to AOC culture. Endothelial cell proliferation was measured at 48, 96, 144, 192 h by means of Scion Image Beta. A significant (p < 0.01) increase of AOC proliferation at each time of measurement was induced by culture media from granulosa cells subjected to partial (except at the end of the first 48 h) and total hypoxia compared to control and normoxia conditions, and by VEGF. VEGF Trap significantly (p < 0.01) inhibited the stimulatory effect of media conditioned by granulosa cells cultured in hypoxic conditions. These data suggest that hypoxia stimulates angiogenic activity of granulosa cells possibly by means of VEGF which could represent the main effector in promoting endothelial cell proliferation. © 2004 Elsevier Inc. All rights reserved. Keywords: Granulosa cells; Angiogenesis; Hypoxia; VEGF; VEGF Trap R1R2

∗

Corresponding author. Tel.: +39 0521 902775; fax: +39 0521 902770. E-mail address: [email protected] (G. Basini).

0739-7240/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.domaniend.2004.12.004

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1. Introduction The formation of new vessels from pre-existing ones, namely angiogenesis, is a process of critical importance in both normal and pathological conditions. While most efforts have been addressed to the study of angiogenesis in tumors, the physiological pattern of this process is still to be fully investigated. With regard to the molecular mechanisms that control this event, vascular endothelial growth factor (VEGF) has been described as one of the most potent proangiogenic factor [1–3] which specifically promotes endothelial cell mitosis, permeability and survival [4]. VEGF biological activity results from the binding to its receptors on endothelial cells: the two best characterized are VEGF receptor 1 (VEGFR1 Flt, fms-like tyrosine kinase) and VEGF receptor 2 (VEGFR2 KDR, kinase insert domaincontaining receptor). Both receptors are highly related transmembrane tyrosine kinases that use their ectodomains to bind to VEGF, thus activating the intrinsic tyrosine kinase activity initiating the intracellular signalling [5]. Blockade of VEGF pathway has been achieved in different ways including antibodies against VEGF [6] or its receptors [7]. VEGF activity can also be efficiently inhibited by soluble VEGF decoy receptors, such as the VEGF Trap which comprises portions of the ligand binding Ig domains of VEGFR1 and/or VEGFR2 fused to the constant region (Fc portion) of human IgG1 [5]. VEGF Traps have been shown to suppress pathological angiogenesis in rodent models of cancer and ocular vascular disease [5,8] as well as luteal angiogenesis in the primates [9]. The ovary represents a valuable model for studying angiogenesis, since it is a site of active cyclical vessel growth and regression [10]. While follicles in the preantral stage have no vascular supply of their own, they later acquire a vascular sheath in the theca that develops during follicular maturation. Thecal vessels do not penetrate the basement membrane that lays between the theca and granulosa compartment, so the granulosa layer remains avascular until basement membrane breakdown at ovulation [11]. The follicular vascular sheath is reckoned to be necessary for the delivery of hormones, oxygen and nutrients to the follicle itself, and increased vascularization seems to play an instrumental role in follicle selection and maturation [12]. The role played by the avascular granulosa layer in the control of follicular angiogenesis is controversial. As a matter of fact, granulosa cells have been pointed out as the main component involved in VEGF production, at least in swine ovaries [13], but at the same time they produce factors inhibiting endothelial cells mitosis such as a high molecular-weight hyaluronic acid [14] and 2-methoxyestradiol, an estradiol metabolite [15]. It has been shown that hypoxia represents a proangiogenic stimulus and its relationship with VEGF production has been described [16]. In fact, the hypoxia inducible factor 1␣ (HIF-1␣), rapidly degraded by cellular peroxisomes under normoxic conditions, is stabilized under hypoxic conditions [17,18], thus promoting VEGF production [19]. In previous studies we have demonstrated that a progressive hypoxic environment is established during follicular growth; at the same time, we have observed an increase of VEGF production by granulosa cells during follicular maturation, pointing out a possible relationship between hypoxia and VEGF production [19]. The aim of the current study was to investigate the role of granulosa cells in follicular angiogenesis; in particular, the modulatory effects of hypoxia on their angiogenic activity

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have been studied. To this purpose, after incubation of granulosa cells in hypoxic conditions, we studied the effects of their conditioned media on porcine aortic endothelial cells (AOC) [20] growth in a three-dimensional fibrin gel matrix [21]. In order to verify the possible role played by VEGF produced by granulosa cells in these conditions, we evaluated AOC growth in the presence or absence of a VEGF inhibitor, the VEGF Trap R1R2. The effectiveness of VEGF Trap R1R2 in inhibiting VEGF action on AOC growth was verified using the same angiogenesis bioassay as employed for testing the effect of the conditioned media by adding exogenous VEGF to AOC cultures.

2. Materials and methods All reagents were obtained from Sigma (St. Louis, MO, USA) unless otherwise specified. 2.1. Granulosa cell culture Ovaries from about 20 sows were obtained from a local slaughterhouse, placed into cold PBS (4 ◦ C) supplemented with penicillin (500 IU/ml) and streptomycin (500 ␮g/ml), maintained in a freezer bag and transported to the laboratory within 1 h. Ovaries were washed twice with PBS, then with ethanol (70%), and finally with PBS at room temperature. Granulosa cells from follicles > 5 mm were aseptically harvested by aspiration with a 26-gauge needle and released in medium containing heparin (50 IU/ml), centrifuged for pelleting and then treated with 0.9% prewarmed ammonium chloride at 37 ◦ C for 1 min to remove red blood cells. Cell number and viability were estimated using a haemocytometer under a phase contrast microscope after vital staining with trypan blue (0.4%) of an aliquote of cell suspension. 106 cells/well were seeded in 24-well plates in 1 ml M199 supplemented with sodium bicarbonate (2.2 mg/ml), bovine serum albumin (0.1%), penicillin (100 UI/ml), streptomycin (100 ␮g/ml), amphotericin B (2.5 ␮g/ml), selenium (5 ng/ml) and transferrin (5 ␮g/ml). Cells were incubated at 37 ◦ C under humidified atmosphere (5% CO2 , 19% O2 ) for 24 h and then subjected for 18 h to normoxic (19% O2 ), partial (5% O2 ) or total hypoxic (1% O2 ) conditions. Total and partial hypoxia were achieved employing Anaerocult® A mini and Anaerocult® C mini (Merck KgaA, Darmstadt, Germany), respectively. In both cases, the system consisted of plastic pouches and a paper gas generating sachet. 2.2. Endothelial cell culture An immortalized porcine aortic endothelial cell line (AOC) [20] was generously provided by Jos´e Yelamos (Hospital Universitario Virgen de la Arrixaca, El Palmar, 30120 Murcia, Spain). In all experiments, AOC at 19th passage were used and seeded in culture medium (CM) composed of M199 supplemented with sodium bicarbonate (2.2 mg/ml), penicillin (100 UI/ml), streptomycin (100 ␮g/ml), amphotericin B (2.5 ␮g/ml), Fetal Calf Serum 20% (GIBCOTM , Invitrogen Corporation, UK) and incubated at 37 ◦ C under humidified atmosphere (5% CO2 ).

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2.3. Three-dimensional endothelial cell culture on a fibrin gel support The microcarrier-based fibrin gel angiogenesis assay was performed as described by Grasselli et al. [21] with some modifications. Briefly, 12.5 mg gelatin-coated cytodex-3 microcarriers in 1 ml PBS were incubated for 3 h to hydrate. After two washings in PBS and one in CM, the microcarriers were put in flasks containing 5 ml CM; AOC (5 × 105 ) were added and cultured for 24 h in order to let the endothelial cells coat the microcarriers. For the fibrin gel preparation, 40 ␮l microcarriers covered by AOC were pipetted into 6 well plates containing a solution of fibrinogen (1 mg/ml PBS, pH 7.6), to which 1250 IU thrombine (250 ␮l) were added. Fibrin gels were allowed to polymerize for 30 min at 37 ◦ C, then they were equilibrated for 60 min with 2 ml M199. Thereafter, the medium was totally changed by aspiration with a 26-gauge needle and AOC were subjected to different treatment (2 ml) depending on the experiments below described. A control fibrin gel was performed containing AOC incubated with non-conditioned medium. 2.3.1. Effect of granulosa cell conditioned media on AOC growth Culture media from granulosa cells incubated in normoxic, partial and total hypoxic conditions were added to fibrin gels in the presence or absence of VEGF Trap R1R2 (150 ng/ml). The VEGF Trap R1R2 (Regeneron Pharmaceuticals Inc., NY, USA) is a recombinant chimeric protein comprising portions of the extracellular, ligand binding domains of the human VEGF receptors Flt-1 (VEGFR1, Ig domain 2) and KDR (VEGFR2, Ig domain 3) expressed in the sequence with the Fc portion of human IgG1. AOC were cultured for 192 h, renewing treatment totally every 48 h as described above. 2.3.2. Effects of VEGF and VEGF Trap R1R2 on AOC growth Fibrin gels were treated with VEGF (100 ng/ml) (PeproTech EC Ltd., London, UK) in the presence or absence of VEGF Trap R1R2 (150 ng/ml). AOC culture were termined after 192 h, renewing treatment every 48 h as described above. 2.4. Quantification of AOC growth on fibrin gel matrix Endothelial cell proliferation in the fibrin gel matrix was evaluated by means of the public domain NIH Program Scion Image Beta 4.02 (Scion Corporation, MA, USA, http://rsb.info.nih.gov/nih-image/). Ten pictures were taken for each gel at 48, 96, 144 and 192 h; images were converted into gray scale, resized to 50% (Paintbrush Software, MS Office) and saved as Bitmap 24bit format compatible with Scion. The modified images were then imported into the program and measurements were made drawing the perimeter of the area occupied by AOC expressed as number of pixel. The area of the microcarrier (diameter 150 ␮m) was chosen as an internal calibration to transform the number of pixel in mm2 . 2.5. Statistical analysis Data are expressed as means ± S.E.M. of five independent experiments, and statistical analysis was performed by means of multifactorial ANOVA using Statgraphics package (STSC Inc., Rockville, MD, USA).

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When significant differences were found, means were compared by Scheffe’ F test; p values <0.05 were considered to be statistically significant.

3. Results 3.1. Effect of granulosa cell conditioned media on AOC growth A significant (p < 0.01) and constant increase of AOC proliferation with time was observed in all groups (Table 1); in fact, during the 192 h of culture the area covered by endothelial cells enhanced by 25% at each time of evaluation. However, when the AOC proliferation was compared at each time-point, there were some differences among groups. AOC incubated with media from granulosa cells subjected to normoxic conditions showed a growth rate similar to controls (AOC incubated with non-conditioned medium) at each time of evaluation (Table 1, Fig. 1). Media obtained in partial hypoxic conditions did not modify AOC growth during the first 48 h (Fig. 1(A)), while they significantly (p < 0.01) stimulated AOC proliferation during the subsequent incubations (Fig. 1(B)–(D)). Media from granulosa cells in total hypoxia significantly (p < 0.01) enhanced AOC growth as compared to controls at every time of evaluation (Fig. 1). The addition of VEGF Trap R1R2 significantly (p < 0.01) reduced AOC growth induced by partial and total hypoxia conditioned media compared to respective time-point controls, while it did not modify AOC growth induced by normoxic conditioned medium (Table 1, Fig. 2). 3.2. Effect of VEGF and VEGF Trap R1R2 on AOC growth A significant (p < 0.01) increase of AOC growth at each time was seen in the presence of VEGF (Table 2). This effect was significantly (p < 0.01) inhibited by the addition of VEGF Trap R1R2. On the contrary, VEGF Trap R1R2 alone did not modify AOC growth compared to control (Table 2, Figs. 3 and 4).

4. Discussion In the past years the attention toward angiogenesis has been mainly focused on finding effective means to inhibit vascular growth which likely plays a crucial role in the development of neoplasia [22]. Though many progresses have been made in the characterization of potential anti-angiogenic agents, there is still much to investigate on the mechanisms controlling the physiological process. The female reproductive system represents a milieu of cyclical angiogenesis occurring both within the ovary and the endometrium [10]. The preovulatory follicle provides a unique physiological example of rapid growth and neovascularization, processes that are generally characteristics of pathologies such as malignancy. On this basis, the ovarian follicle has been compared to a solid tumour for its anatomical and physiological features [23]. The follicle has a complex structure mainly consisting of two compartments, separated by the basal membrane, the theca and the granulosa layer, displaying different functions.

Culture medium 48 h 96 h 144 h 192 h

0.87 1.39 2.10 2.74

± ± ± ±

0.01 a 0.01 b 0.02 c 0.03 d

Normoxia 0.89 1.29 2.09 2.65

± ± ± ±

0.01 a 0.01 b 0.03 c 0.04 d

Normoxia + Trap 0.87 1.33 2.13 2.69

± ± ± ±

0.02 a 0.02 b 0.03 c 0.04 d

Partial hypoxia 1.06 1.76 2.65 3.48

± ± ± ±

0.02 a 0.03 b 0.03 c 0.04 d

Partial hypoxia + Trap 0.90 1.42 2.19 2.69

± ± ± ±

0.01 a 0.01 b 0.02 c 0.03 d

Total hypoxia 1.24 1.89 2.96 3.75

± ± ± ±

0.01 a 0.02 b 0.03 c 0.03 d

Total hypoxia + Trap 0.90 1.39 2.15 2.79

Values in the same column with different letters are significantly (p < 0.01) different. Data represent the area covered by AOC in the fibrin gel (mm2 ).

± ± ± ±

0.01 a 0.02 b 0.02 c 0.03 d

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Table 1 Effect of granulosa cell conditioned media on AOC growth

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Fig. 1. Area covered by AOC (mm2 ) in fibrin gel evaluated after 48 (A), 96 (B), 144 (C), and 192 (D) hours of incubation with culture medium (CM) or with media conditioned by granulosa cells grown in normoxia, partial (5% O2 ) or total (1% O2 ) hypoxia. Different letters indicate significant differences (p < 0.01) among the different treatments at each time of evaluation.

VEGF-mediated angiogenic activity occurs in the thecal peripheral blood vessels surrounding the follicle, with capillary sprouting and increased vascular permeability. Though avascular, the granulosa has been shown to play a major role in the production of angiogenic factors being thus actively involved in the neovascularization that takes place during follicular development. Since oxygen supply to the granulosa cell layer depends on diffusion through the basement membrane, increasing size follicles are assumed to be in an hypoxic milieu [24]. In fact, oxygen content in the follicular fluid of mature follicles has been documented to range from less than 1% to ∼ 5.5% [25]. Remarkably, the hypoxic conditions Table 2 Effect of VEGF and VEGF Trap-R1R2 on AOC growth Culture media 48 h 96 h 144 h 192 h

0.85 1.41 1.98 2.64

± ± ± ±

0.01 a 0.02 b 0.02 c 0.02 d

VEGF 1.06 1.86 2.47 3.23

± ± ± ±

0.01 a 0.02 b 0.02 c 0.04 d

VEGF + Trap

Trap

± ± ± ±

0.82 1.57 1.90 2.74

0.80 1.50 1.87 2.71

0.01 a 0.02 b 0.02 c 0.03 d

± ± ± ±

0.01 a 0.02 b 0.02 c 0.03 d

Values in the same column with different letters are significantly (p < 0.01) different. Data represent the area covered by AOC in the fibrin gel (mm2 ).

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Fig. 2. Phase contrast micrographs showing AOC growth at 192 h in fibrin gel matrix in the presence of media conditioned by granulosa cells cultured in (A) normoxia, (B) normoxia plus VEGF Trap R1R2, (C) partial hypoxia, (D) partial hypoxia plus VEGF Trap R1R2, (E) total hypoxia, (F) total hypoxia plus VEGF Trap R1R2.

associated with follicular growth appear to represent a powerful stimulus for the production of the main pro-angiogenetic factors such as VEGF [23], angiogenin [26] and IL8 [27] by granulosa cells. Therefore, it may be argued that media conditioned by granulosa cells cultured in normoxia, shown to be unable to modify AOC growth when compared to partial and total hypoxia, do not contain enough angiogenic factors to promote endothelial cell proliferation; on the contrary, granulosa cells cultured in partial and total hypoxic conditions display angiogenic activity resulting in a marked enhancement of AOC growth in a three-dimensional fibrin gel matrix. As for the normoxic conditioned medium AOC proliferation rate increased during time, but this rise at time-point was not significantly different from the respective control group treated with non-conditioned medium. Our rationale is that during follicular growth an increasing hypoxic condition establishes within the granulosa layer [19] modulating granulosa cell features. In fact we observed that different experimental pO2 levels differently affect steroid hormones production, on the contrary we documented that both

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Fig. 3. Area covered by AOC (mm2 ) in fibrin gel evaluated after 48 (A) 96 (B) 144 (C) and 192 (D) hours of incubation with culture medium (CM) or medium supplemented with VEGF (100 ng/ml) in the presence or absence of VEGF Trap R1R2 (150 ng/ml). Different letters indicate significant differences (p < 0.01) among the different treatments at each time of evaluation.

Fig. 4. Phase contrast micrographs showing AOC growth at 192 h in fibrin gel matrix in the presence of (A) Culture Medium, (B) VEGF, (C) VEGF plus VEGF Trap R1R2, (D) VEGF Trap R1R2.

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partial and total hypoxia stimulate VEGF production by swine granulosa cells at the same extent [19]; this would confirm the results obtained in the three-dimensional angiogenesis bioassay, where partial and total hypoxia conditioned media similarly influenced AOC growth in fibrin gel. As above mentioned, several molecules produced by granulosa cells are potentially involved in follicular angiogenesis; among these, the endocrine gland vascular endothelial growth factor (EG–VEGF), which has been described in the ovary and in other steroidogenic tissues, possesses a biological activity very similar to that of VEGF but with a completely different structure [28]. Although experimental evidence support the hypothesis of an important role of EG–VEGF in the control of ovarian angiogenesis, recent data demonstrate a suppressive effect of hypoxia on the EG–VEGF mRNA levels [29]: VEGF, IL-8 and angiogenin production, on the contrary, are greatly enhanced in hypoxic conditions via the Hypoxia Inducible Factor (HIF), whose ␣ subunit, degraded under normoxic condition, is stabilized in absence of oxygen [26,27,30]. On the basis of the data obtained by the use of a potent VEGF inhibitor, we can hypothesize that the enhancement of granulosa cell angiogenic activity in hypoxic conditions is mainly due to VEGF production: VEGF Trap R1R2, in fact, resulted effective in blocking AOC growth driven by media from granulosa cells incubated in hypoxia. VEGF Trap R1R2 also suppressed the proliferation of AOC treated with exogenous VEGF, further suggesting the major role played by VEGF in promoting new vessel growth within the follicle. Moreover, in vivo incubation with VEGF Trap has been shown to severely reduce thecal angiogenesis in the growing follicle with a resulting impairment of antral follicle development [31]. In conclusion, this in vitro study suggests that swine granulosa cells, though avascular, play a crucial role in the regulation of the angiogenic process which is necessary for the developing follicle. The onset of an hypoxic environment which gradually establishes within the follicle during its growth enables the granulosa layer to produce more VEGF, supporting the formation of a normal thecal vasculature and the survival of the follicle itself.

Acknowledgements We would like to thank Professor Yelamos (Department of Biochemistry, Molecular Biology and Immunology, Facultad de Medicina, Universidad de Murcia, Spain) for supplying AOC and Dr. Stanley Wiegand (Regeneron Pharmaceuticals, Inc., Tarrytown, USA) for providing the VEGF Trap. This research was supported by a MIUR COFIN grant.

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