Original Article

F. Grasselli1, G. Basini1, M. Tirelli1, V. Cavalli1, S. Bussolati1, C. Tamanini2


ANGIOGENIC ACTIVITY OF PORCINE GRANULOSA CELLS CO-CULTURED WITH ENDOTHELIAL CELLS IN A MICROCARRIER-BASED THREE-DIMENSIONAL FIBRIN GEL


1 Dipartimento di Produzioni Animali, Biotecnologie Veterinarie, Qualita' e Sicurezza degli Alimenti-Sezione di Fisiologia-Universita' degli Studi di Parma-Via del Taglio 8, 43100 PARMA - ITALY
2 DIMORFIPA- Sezione di Fisiologia- Universita' degli Studi di Bologna- Via Tolara di Sopra 50, Ozzano Emilia, BOLOGNA- ITALY


  To verify the possible role played by pig granulosa cells in the ovarian angiogenic process, we have developed a reliable in vitro system which allows the evaluation of endothelial sprouting and capillary growth in three-dimensional matrices. Granulosa cells collected from porcine follicles of different size were co-cultured with porcine aortic endothelial cells (PAEC) in a microcarrier-based fibrin gel system; after 2 and 5 days of co-culture, we determined the number and length of all endothelial sprouts; moreover, these parameters were quantified only in capillary-like structures, which were defined as continuous multicellular sprouts at least 200 µm long. In granulosa cells- PAEC co-cultures we observed an increase of angiogenic activity as compared to controls (PAEC alone). Granulosa cells from follicles of different size regulate angiogenesis differently: cells from the small follicle group significantly enhanced endothelial sprouting, while those from the large follicle group favoured mainly capillary elongation. Our observations seem therefore to suggest that the development and growth of thecal vascular bed is controlled by paracrine factors of granulosa cell origin that may induce the formation of a primitive capillary plexus during the early phases of antral follicle growth, which will be remodelled in more advanced phases of follicular development.

Key words:    angiogenesis, porcine, granulosa cells, endothelial cells, capillary growth



INTRODUCTION

The angiogenic process has been extensively investigated during the last 10-15 years, mainly because of its close association with numerous pathological conditions, including tumour growth and progression. However, a better understanding of the mechanisms regulating new vessel growth can be accomplished by studying angiogenesis in physiological conditions, a rare event in most normal adult tissues (1), which are characterized by a relative stability and a very low mitotic activity of the endothelial population (2). One of the few exceptions is represented in adult females by ovarian and other reproductive tissues, whose cyclic growth is a self-limiting and tightly regulated process, supported by a rapid microvascular growth and development. It is now well established that angiogenesis is a critical component of follicular and luteal function and some experimental evidences support the concept of a strict relationship between the maintenance of the theca vascular bed and follicular health (3-5) even if this concept seems to be still controversial. An active role of the angiogenic process in the control of follicular growth and development has also been hypothesized (6; 7). The formation of capillaries from a pre-existing vascular network is favoured by many angiogenic promoters, among which VEGF/VPF and bFGF play a crucial role. Also in the ovary, capillary remodelling is likely to be mainly regulated by these factors (8-10), which would act in a co-ordinated and complementary manner with other pro-angiogenic factors, such as angiogenin (11), EG-VEGF (12) and a variety of cytokines. At this regard, the role of the avascular granulosa layer in controlling angiogenesis in the developing follicles appears still controversial. Mattioli et al. (6) suggested that, at least in pig, granulosa cells represent the main component involved in the production of VEGF, which appears to be positively related with follicular development (13; 14); on the other hand, other experimental evidences suggest that granulosa cells are also the source of angiogenesis inhibitors, such as high-molecular-weight hyaluronic acid (15) and 2-methoxyestradiol (16). The difficulty in evaluating the overall contribution of granulosa cells to follicular angiogenesis can be partly due to the paucity of appropriate experimental systems which might allow a reliable quantification of angiogenic activity, taking into account the multiple steps involved in the 'angiogenesis cascade': to our knowledge, potential angiogenic activity of cultured granulosa cells has been screened by studying endothelial cell proliferation or chemotaxis (17; 18), while only scarce data are available about bioassays which could appropriately quantify capillary growth in vitro, following both 'sprouting' mechanisms, as well as vessel elongation (19).

In order to investigate the possible involvement of granulosa cells in the regulation of angiogenesis, we set up a reliable bioassay which allows to study the growth of capillary-like structures in a three-dimensional matrix: in this model, porcine granulosa cells from different size follicles were co-cultured with porcine aortic endothelial cells (PAEC) in a microcarrier-based system, and capillary growth was evaluated and quantified at different times.


MATERIALS AND METHODS

Materials
Cell culture plastic were obtained from COSTAR (Broadway, Cambridge, MA, USA), Medium 199 and Foetal calf Serum (FCS) were purchased from Gibco (Milan, Italy), collagen S type 1 from Roche Diagnostics (Mannheim, Germany). All other reagents were obtained from Sigma Chemical (St Louis, MO, USA).

Granulosa cell culture
Ovaries from adult sows were collected in iced PBS at a local abattoir and immediately transported to the laboratory. Follicles were classified as healthy or atretic on the basis of morphological criteria and those with haemorragic, opaque or "milky" follicular fluid were excluded (20; 21). Granulosa cells were aseptically collected by aspiration of the follicular fluid from healthy follicles grouped on the basis of the external diameter as small (<3 mm), medium (3-5 mm) or large (>5 mm). After two washings with culture medium, cell pellet was treated with 0.9% prewarmed ammonium chloride at 37°C for 1 min to remove red blood cells. Cell number and viability (85-90%) were estimated using a haemocytometer under a phase-contrast microscope after vital staining with trypan blue dye of an aliquot of cell suspension. Cells were then seeded in collagen (5µg/cm2) pre-treated 6-well plates at the density of 106 cells/ml and cultured for 48 h in M199 supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml), BSA (0.1%), FCS (1%), transferrin (5µg/ml) and sodium selenite (5 ng/ml) at 37°C under humidified conditions (5%CO2).

Endothelial cell culture
Endothelial cells were harvested from pig aortas and cultured using standard techniques; after trypsinization, cells of passages 7-10 were allowed to attach onto cytodex-3 microcarrier beads for 4 h and subsequently were grown for 24 h at 37°C in M199 supplemented with FCS (2%).

Co-culture of granulosa cells and porcine aortic endothelial cells (PAEC) in a serum free fibrin gel
The microcarrier-based fibrin gel angiogenesis assay was performed as described by Nehls et al. (22), with some modifications. After the preparation of the gelatin-coated cytodex-3 microcarriers according to the recommendations of the supplier, 40 µl of the microcarriers coated with PAEC were pipetted into a 1.710 ml fibrinogen solution (1 mg/ml fibrinogen in PBS, pH 7.6) and added to each group of granulosa cells (small follicles = SF, medium follicles = MF, large follicles = LF) after culture media were discarded; clotting was then induced by addition of 1.250 U thrombin in 250 µl PBS . After fibrin gels were allowed to polymerize for 30 min at 37 °C, they were equilibrated for 60 min with M199 and thereafter fresh medium was added to the fibrin matrices. Co-cultures of granulosa cells and PAEC were incubated at 37°C in 5% CO2 for 5 days, and the culture media were renewed every other day.

Using the same three-dimensional fibrin gels, PAEC were also incubated with culture medium alone and used as control.

Quantification of Cell Migration and Capillary Formation
Two and five days after polymerization of the gels, angiogenesis was evaluated by using an inverted microscope equipped with fluorescence.

Since Nehls and Drackenhan (19) have observed that some endothelial sprouts appear only transiently and do not always represent a result of a co-ordinated invasion of the surrounding fibrin matrix by endothelial cells, we chose to distinguish "endothelial sprouts" (ES) from "capillary-like structures" (CLS). All endothelial cell extensions invading the fibrin matrix from the microcarriers were recorded as ES; among these, the continuous sprouts longer than 200 µm and composed of at least three tightly connected endothelial cells were defined as CLS. In order to confirm the multicellularity of these structures , the fibrin gels were stained at day 5 by the nuclear dye bis-benzimide (Hoechst 33258, 20 µg/ml in PBS for 60 min) and examined by the fluorescence microscope.

In our model, angiogenesis was quantified on the basis of different parameters, taking into account that new vessel growth in adult tissues can be accomplished by sprouting or non-sprouting mechanisms (23). Sprouting was evaluated by counting the number of ES and CLS originating from 4 randomly chosen microcarriers; the process of capillary remodelling or pruning was instead quantified by measuring the length of ES and CLS.

Statistical analysis
Data are expressed as means ± SEM of five independent experiments, and statistical analysis was performed by means of multifactorial ANOVA using Statgraphics package (STSC Inc., Rockville, MD, USA). When significant differences were found, means were compared by Scheffe F test. P values <0.05 were considered to be statistically significant.


RESULTS

After the first 48 h of culture, the formation of ES was observed in all co-cultures containing PAEC and granulosa cells; no difference in either the number or the length of the sprouts was observed in the SF, MF and LF as compared to the PAEC control group (Fig. 1); as regards to CLS formation, we recorded a significant reduction in the number but an increase in the length in the PAEC/LF granulosa cells as compared to the other groups (Fig. 2 and Fig. 3).

Fig. 1. Quantification at day 2 (grey bars) and at day 5 (black bars) of the number (A) and length (B) of endothelial sprouts observed in the PAEC control group and in PAEC co-cultured with granulosa cells from small, medium and large follicles. Values are the mean ± SEM of 5 different experiments. Asterisks indi-cate significantly (at least p<0.05) different values compared to controls.

From day 2 to 5, capillary growth progressed in all groups, as shown by the increase in number (p<0.001) and length (p<0.001) of CLS.

Fig. 2. Phase contrast micro-graphs showing the formation of capillary-like structures from PAEC-coated microcarriers at day 2 after polymerization of the fibrin gels in controls (A) and in co-cultures with granulosa cells from small (B), medium (C) and large (D) follicles. Bar = 50 µm.

At day 5 PAEC co-cultured with SF and MF granulosa cells showed a higher number of endothelial sprouts than controls (Fig. 1, a); nevertheless, ES resulted of comparable length to those in the control group, while they appeared longer in the LF group (Fig. 1, b). The quantification of the CLS gave similar results, in that the presence of SF granulosa cells increased their number, without affecting their length (Fig. 3, a). Conversely, LF granulosa cells significantly stimulated elongation as compared to the other experimental groups, even if they did not modify either the number of sprouts or the number of CLS (Fig. 3, b; Fig. 4). Neither the number nor the length of CLS resulted affected by the presence of MF cells.

Fig. 3. Quantification at day 2 (grey bars) and at day 5 (black bars) of the number (A) and length (B) of capillary-like structures observed in the PAEC control group and in PAEC co-cultured with granulosa cells from small, medium and large follicles. Values are the mean ± SEM of 5 different experiments. Asterisks indi-cate significantly (at least p<0.05) different values compared to controls.

The examination by fluoroscence microscopy, performed at day 5 after staining with bis-benzimide of all fibrin gels, confirmed the multicellularity of the capillary sprouts (Fig. 5).

Fig. 4. Phase contrast micro-graphs showing the formation of capillary-like structures from PAEC-coated microcarriers at day 5 after polymerization of the fibrin gels in controls (A) and in co-cultures with granulosa cells from small (B), medium (C) and large (D) follicles. Bar = 50 µm.

Fig. 5. Representative phase contrast (A) and corresponding bis-benzimide fluorescence (B) micrograph of capillary-like formation at day 5 in a co-culture of PAEC and granulosa cells. Note that capillary-like structures formed by migrating endothelial cells are multicellular (arrows).


DISCUSSION

Our observations document that the co-culture of endothelial and granulosa cells in a three-dimensional fibrin gel represents a reliable and simple method to investigate the involvement of the granulosa layer in angiogenesis. Different bioassays have been developed in order to study the possible angiogenic activity of various intra-ovarian factors, but the results have proven to be contradictory, probably owing to the fact that the traditional two-dimensional systems employed do not adequately mimic the complexity of the angiogenic process in situ.

The development and the re-organization of the vascular network in the growing follicle is likely to be driven by specific angiogenic factors whose production within the ovary has been experimentally demonstrated. VEGF expression in granulosa cells (10; 13; 24) would support the concept of a key role played by the granulosa layer in the regulation of thecal vasculature growth, but the existence of substantial species differences in the mechanisms of ovarian new vessel growth has been recently hypothesized (25). Our results confirm that pig granulosa cells are involved in the regulation of angiogenesis possibly by means of paracrine factors (VEGF and/or other substances) whose production appears related to follicular development: in fact, by quantifying microvessel formation we have shown that cells from different size follicles seem to be predominantly involved in the control of different events of the angiogenic process (endothelial cell proliferation, migration and guided migration). A positive relationship between VEGF production and follicle size has been previously documented (6) and should account for the marked increase of VEGF levels in culture media of cells from follicles >5 mm (14). The data reported by Mattioli et al. (6) about the 'switching on' of VEGF production by granulosa cells at a threshold follicle size of 4 -5 mm can probably explain the lack of specific angiogenic effects exerted by the medium follicle group [3-5 mm]: in fact, while cells from small follicles appeared to stimulate endothelial sprouting and those from large follicles capillary elongation, the angiogenic response elicited in our model by cells from medium follicles is more difficult to interpretate, probably as a consequence of a poor homogeneity of this group in terms of VEGF production.

Although the angiogenic signal provided by VEGF undoubtedly plays a crucial role also in the ovary, only the existence of additional VEGF-independent pathways could explain the different contribution to angiogenesis by the three types of granulosa cells. In fact, VEGF is known to represent a multipotent angiogenic stimulator, being effective in inducing endothelial cell proliferation, migratory and sprouting activity (26-28); therefore, it is tempting to speculate that the development of the theca vascular network would be the result of an integrated network of regulatory substances, whose relative balance may change according to the stage of follicular development. We may hypothesize that, during the early phases, granulosa layer would produce angiogenic factors essential for the initiation of capillary tube formation, thus favouring mainly sprouting; as the follicle grows, other regulators should be mainly involved and might mediate the re-organization of endothelial cells into more complex vascular structures. Future studies addressing the identification of possible additional factors contributing to ovarian angiogenesis, as well as the clarification of their interrelationships in relation to follicular development will be probably helpful in elucidating the complex signalling mechanisms regulating this process.

Acknowledgments: PAEC were prepared by Dr. Chiara Bernardini and kindly supplied by Prof. Maria Laura Bacci from the Section of Fisiologia Veterinaria- Dipartimento di Morfofisiologia Veterinaria e Produzioni Animali, University of Bologna, Italy.

Contract grant sponsor: MURST COFIN



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R e c e i v e d:  December 19, 2002
A c c e p t e d: July 23, 2003

Author’s address: Prof. Francesca Grasselli, Dipartimento di Produzioni Animali, Biotecnologie Veterinarie, Qualita' e Sicurezza degli Alimenti- Via del Taglio 8- 43100 PARMA - ITALY, Tel:+39-0521/032775, Fax:+39-0521/032770
E-mail: francesca.grasselli@unipr.it