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Endocrinology, doi:10.1210/en.2003-1620
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Endocrinology Vol. 145, No. 6 2896-2905
Copyright © 2004 by The Endocrine Society

Vascular Endothelial Growth Factor and Its Receptor, Flk-1/KDR, Are Cytoprotective in the Extravascular Compartment of the Ovarian Follicle

James Greenaway, Kristin Connor, Hanne G. Pedersen, Brenda L. Coomber, Jonathan LaMarre and Jim Petrik

Department of Biomedical Sciences (J.G., K.C., B.L.C., J.L., J.P.), University of Guelph, Guelph, Ontario, Canada N1G 2W1; Department of Clinical Medicine-Reproduction (H.G.P.), Royal Agricultural and Veterinary University, 1870 Frederiksberg C, Denmark

Address all correspondence and requests for reprints to: Jim Petrik, Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1. E-mail: jpetrik{at}uoguelph.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Vascular endothelial growth factor (VEGF) is a potent mitogen and cytoprotective factor for vascular endothelial cells. Although VEGF is ubiquitously expressed, its role in nonvascular tissues is poorly understood. VEGF interacts with various cell surface receptors to mediate its cellular effects. It previously has been thought that the VEGF receptor Flk-1/KDR, its main signaling receptor, was expressed exclusively by endothelial cells. However, in the present study using bovine and rodent models, we demonstrate that VEGF and Flk-1/KDR are coexpressed in ovarian granulosa cells. VEGF and Flk-1/KDR mRNA and protein were both detectable in follicle tissue sections and in vitro cultured granulosa cells. Expression of both ligand and receptor increased in healthy follicles throughout follicular development. VEGF treatment of serum-starved and cytokine-exposed granulosa cells resulted in enhanced survival, and this cytoprotection was ameliorated when Flk-1/KDR signaling was inhibited. Reduced expression of Flk-1/KDR was also associated with the onset and progression of follicle atresia, suggesting involvement in follicular health in vivo. The results of this study demonstrate for the first time expression of Flk-1/KDR in ovarian granulosa cells and identify a novel extravascular role for VEGF and its receptor in ovarian function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VASCULAR ENDOTHELIAL GROWTH factor (VEGF) is a hypoxia-inducible proangiogenic growth factor that stimulates receptor tyrosine kinases and acts because a potent mitogen for vascular endothelial cells (1). Several models have demonstrated the angiogenic function of VEGF in vivo (2, 3). Along with the proliferative effects on endothelial cells, VEGF has recently been shown to act because a cytoprotective agent, protecting these cells from apoptosis (4, 5). VEGF exerts its cellular effects through interaction with its tyrosine kinase receptors Flt-1 (VEGF-R1) and Flk-1/KDR (VEGF-R2), which bind the ligand with high affinity (6). Flk-1/KDR is the principal mediator of the angiogenic effects of VEGF, and its importance is highlighted by the failure of flk-1 null mice to develop organized blood vessels, resulting in lethality between embryonic d 8.5 and 9.5 (7). The role of Flt-1 is less apparent. Reports have indicated that Flt-1 has limited signaling activity and may act because a decoy or scavenger receptor (8), whereas other studies have implicated Flt-1 in mediation of endothelial cell proliferation (9), chemotaxis (10), and cell survival (11). In early development, Flt-1 is important in vascular modeling and flt-1 null mutants die at midgestation with vascular overgrowth and disorganization (12).

During ovarian follicular development, vascularization occurs in a tightly regulated fashion. Primary primordial follicles do not have their own blood supply but rely on nutrient delivery from the stroma (13). After recruitment of a primary follicle and initial follicle development, a distinct theca cell (TC) layer is formed and endothelial cells are recruited from neighboring blood vessels (14). It is thought that the level of vascularization may be a decisive factor in selection of the dominant follicle by increasing the supply of nutrients, growth factors, and hormones and therefore supporting the growth of the dominant follicle (15). After ovulation occurs, there is rapid angiogenesis resulting in the mature corpus luteum having the highest blood flow of any tissue in the body (16).

VEGF is differentially expressed during follicular and luteal development. The growth factor is weakly expressed in early follicles and becomes more pronounced in granulosa and TCs as the follicle develops (14, 17). There is an increase in the number of small, preantral follicles formed and accelerated follicle growth in rodents stimulated with exogenous VEGF (18). In the early corpus luteum (CL), VEGF expression is elevated, and this expression decreases because the CL undergoes degradation and regression (19).

The role of VEGF in the ovary is multifactorial. It has been well established that VEGF interacts with receptors Flk-1 and Flt-1 to stimulate angiogenesis in the follicle and CL (20, 21). Neutralization of VEGF results in suppressed blood vessel formation and compromised function of the follicle and CL (22). VEGF was originally known as vascular permeability factor due to its ability to increase the permeability of blood vessels (23, 24). Dysregulated vascular permeability of blood vessels in the ovary contributes to reproductive disorders, such as the ovarian hyperstimulation syndrome (OHSS), and altered VEGF and VEGF receptor expression has been implicated in this pathology (25). Additionally, VEGF expression is thought to be important in recruitment of follicles to enter the ovarian cycle (26).

The role of VEGF in nonendothelial cells in extravascular tissues is poorly understood. A recent report has shown that VEGF is important in providing neuroprotection after induced brain injury and that its effects are independent of angiogenesis or other vascular events (27). Similarly, VEGF is cytoprotective for myocytes after ischemic injury (28). VEGF also stimulates chemotaxis in nonvascular cells such as microglial cells (29) and osteoblasts (30). The mechanisms by which VEGF elicits these effects in extravascular cells and tissues is not yet known. Although the VEGF ligand is expressed by a host of cell types, the VEGF receptors are thought to be primarily restricted to endothelial cells. Limited reports indicate that Flk-1/KDR may be expressed by other cell types such as hematopoietic stem cells (31) and neural progenitor cells (32) in which they stimulate neurogenesis (33). The ovary is a unique structure in that it undergoes cyclical angiogenesis in the adult. During follicle and luteal development, previously avascular areas within the follicle rapidly vascularize to support development and activity of the CL. We speculate that, similar to other tissues, VEGF may play a survival role in nonvascular tissue in the ovary. We therefore characterized the expression and localization of VEGF and its kinase receptor, Flk-1/KDR, within the follicle. The results demonstrate coexpression of VEGF and Flk-1/KDR in granulosa cells of follicles and in in vitro cultured granulosa cells. VEGF interacts with Flk-1/KDR and exerts cytoprotective effects on granulosa cells, indicating for the first time a functional VEGF system in extravascular areas of the follicle. These results shed new light on ovarian biology and the role of VEGF in follicle and luteal development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissues and cell lines
For protein and RNA experiments, granulosa cells were collected via ultrasound-guided transvaginal aspiration from normal, nonpregnant Holstein cattle and from ovaries of normal, nonpregnant Holstein cattle at slaughter. Synchronization of ovarian cycles was achieved by administering 5 ml (5 mg/ml) prostaglandin-F2{alpha} (Lutalyse; Pfizer, La Jolla, CA) to diestrus animals, followed by daily ultrasound evaluation to determine follicle size and morphology. Granulosa cells were aspirated from small (<0.5 cm diameter), medium (0.5–1.0 cm diameter), and large (>1.0 cm diameter) follicles. A minimum of 10 follicles from each size were aspirated, and the follicular fluid and granulosa cell lysates were pooled. Follicles from slaughterhouse ovaries were selected based on normal morphology and were separated based on the same size criteria. Bovine aortic endothelial cells (BAECs) were collected from the same slaughterhouse animals using standard techniques, and fetal bovine fibroblasts (bFs) were obtained through explant culture of skin sections from midgestation bovine fetuses. For each experiment, samples were collected from a minimum of six different animals. Slaughterhouse ovaries were used to supplement transvaginal aspirates and ensure sufficient sample volume for protein experiments, particularly from small and medium follicles. Initial experiments were performed to verify that no differences existed in proteins of interest between transvaginal aspirates and slaughterhouse samples from follicles of similar size. Established cell lines of spontaneously immortalized rat granulosa cells (SIRGCs) were kindly provided by Dr. R. Burghardt (Texas A&M University, College Station, TX). A minimum of three different SIRGC lines was used for each experiment. Cells were seeded at high density (2.5 x 105/ml), which resulted in all cultures reaching 80% confluence by 48 h. All procedures were approved by the Animal Care Committee of the University of Guelph, in accordance with the Canadian Council of Animal Care guidelines on the care and use of experimental animals.

Cell culture
To determine the effect of VEGF and Flk-1/KDR on apoptosis and active caspase-3 expression, freshly isolated bovine granulosa cells (GCs), BAECs, bFs, and SIRGCs were plated into culture flasks or onto glass coverslips in DMEM/12 supplemented with 10% fetal bovine serum, 2% penicillin/streptomcyin (Pen/Strep, Life Technologies, Inc., Grand Island, NY). At 80% confluence, cells were changed to serum-free DMEM/F12 with 2% Pen/Strep for 24 h, followed by serum-free culture in the presence or absence of the cytotoxic cytokine TNF{alpha} (100 ng/ml; Calbiochem-Novabiochem, San Diego, CA), growth factors recombinant human (rh)VEGF (50 ng/ml; R&D Systems, Minneapolis, MN), rhIGF-I (Gro-Pep; 30ng/ml), and/or the Flk-1/KDR inhibitor SU1498 (Calbiochem-Novabiochem; 30 ng/ml) for 24 h. After culture, cells were washed twice in PBS and fixed in 10% (vol/vol) neutral buffered formalin for 1 h at room temperature.

Immunofluorescence
Cultured cells and dissected follicles were processed for immunofluorescence and double-label immunofluorescence. Bovine granulosa cells were isolated from slaughterhouse ovaries and cultured in DMEM/F12 with 10% fetal calf serum and 2% Pen/Strep until 80% confluent. After culture, cells were washed twice in PBS and fixed in 10% neutral buffered formalin for 1 h at room temperature. Whole follicles were dissected from slaughterhouse ovaries and fixed in 10% neutral buffered formalin overnight at 4 C and processed according to standard paraffin-embedded histological procedures. For double-label immunofluorescence, sections were incubated overnight at 4 C with a combination of the following primary antibodies: anti-VEGF (rabbit polyclonal; 1:600 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-Flk-1 (mouse monoclonal; 1:500 dilution; Santa Cruz Biotechnology). Cultured granulosa, aortic endothelial, and fibroblast cells were incubated overnight at 4 C with an antibody specific for active caspase-3 (mouse monoclonal; 1:200 dilution; Oncogene Research Products, Boston, MA). fluorescein isothiocyanate- or Texas Red-conjugated antimouse and antirabbit secondary antibodies (all 1:100 dilution; Vector Laboratories, Burlingame, CA) were used for detection. Nuclei were counterstained with propidium iodide or 4',6'-diamino-2-phenylindole (DAPI) and specimens were imaged with a BX-61 fluorescent microscope (Olympus, Melville, NY).

Apoptosis detection
Cultured cells and histological sections of follicles were processed and fixed as above. Detection of apoptotic cells was performed with a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) kit (Roche Applied Science, Laval, Quebec, Canada) according the manufacturer’s protocol. Briefly, after fixation tissues and cells were permeabilized in 0.1% Triton X-100 (Sigma, St. Louis, MO), washed in PBS, and incubated with the fluorescein isothiocyanate-conjugated TUNEL enzyme for 60 min to detect DNA fragmentation. Nuclei were counterstained with propidium iodide and imaged with an Olympus BX-61 microscope. For analysis, 10 fields of view at x250 magnification were quantified in each experiment (n = 3).

Immunoblot analysis
GC lysate protein was isolated from transvaginal aspirations of small (<0.5 cm), medium (0.5–1.0 cm), and large (>1.0 cm) follicles after estrous synchronization and from bovine follicles obtained at slaughter. Western blotting was performed according to standard protocols. Granulosa, aortic endothelial, and fibroblast cells were cultured as described above. Proteins were detected using the following antibodies: anti-VEGF (Santa Cruz; 1:500 dilution), anti-Flk-1 (Santa Cruz; 1:500 dilution); antirabbit POD conjugated (Sigma; 1:1000 dilution), and antimouse POD conjugated (Sigma; 1:1000 dilution). Immunoreactive bands were visualized by enzyme chemiluminescence (Roche Applied Science) and quantified against commercially available VEGF and Flk-1/KDR protein standards (Santa Cruz Biotechnology). After enzyme chemiluminescence detection, membranes were stained with Coomassie Blue to ensure equal protein load. Equal protein loading was present within all membranes.

Expression of VEGF and VEGF receptor DNA
Primer sequences specific for bovine VEGF and Flk-1/KDR have been validated previously by others (34). Sequences were: VEGF forward, 5'-TGT AAT GAC GAA AGT CTG CAG-3'; VEGF reverse, 5'-TCA CCG CCT CGG CTT GTC ACA-3'; Flk-1 forward, 5'-AGA CTG GTT CTG GCC CAA C-3'; and Flk-1 reverse, 5'-GAA GCC TTT CTG GCT GTC-3'. The VEGF primer allowed detection of all four isoforms (186, 318, 390, and 441 bp) representing VEGF121, VEGF165, VEGF189, and VEGF206 splice variants, respectively. PCR was performed with 35 cycles of denaturation at 94 C for 1 min, annealing at 55 C for 1 min, and primer extension at 72 C for 1 min. Incubation for 5 min at 72 C followed to complete extension. PCR products were run on a 1% agarose gel, and visible bands were excised and subjected to DNA sequencing (Molecular Supercenter, University of Guelph).

Data analysis
In Western blot experiments, levels of VEGF and Flk-1 protein were normalized to commercially available protein standards for these molecules. For apoptosis detection and active caspase-3 expression in whole follicle sections, six fields of view at x250 magnification were quantified and averaged as percent immunopositive cells. A minimum of six follicles (from different animals) for each group was used for comparison. For in vitro culture data collection, experiments were performed on three separate occasions, with each treatment replicated three times. Logit transformation was used and analyzed in a two-level nested ANOVA to evaluate whether group differences were present, taking into account treatment or follicle classification, experimental error, and subsampling error. Pairwise, multiple comparisons among the means were performed, using Tukey’s test, to assess statistical differences among treatment groups in in vitro culture experiments, immunostaining experiments, and Western blot graphs. Statistical analyses were conducted using a statistical software package (version 8.2, SAS Institute, Cary, NC), and any P < 0.05 was considered significant. Results are expressed as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GCs express VEGF and Flk-1/KDR
RT-PCR experiments analyzed the expression of transcripts for VEGF and its receptor Flk-1/KDR in bovine endothelial, fibroblast, and granulosa cells. BAECs were used as positive controls for PCR experiments because these cells are widely recognized to express both VEGF receptor types (35, 36). Fibroblasts derived from explant cultures of fetal bovine skin were used as negative controls for Flk-1/KDR mRNA because expression of this gene is not expected in these cells (37). Analysis of the VEGF transcripts by RT-PCR revealed that cultured bovine endothelial, fibroblast, and granulosa cells predominantly expressed the smallest VEGF isoforms (VEGF121 and VEGF165) and weakly expressed the large VEGF189 isoform (Fig. 1AGo). RT-PCR analysis revealed Flk-1/KDR gene expression in cultured endothelial and granulosa cells but not in fibroblasts (Fig. 1BGo). Each PCR product showed 100% homology to the corresponding regions of the bovine VEGF and Flk-1/KDR genes after sequencing (results not shown).



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  		FIG. 1. RT-PCR analysis of VEGF and Flk-1/KDR transcripts in BAECs, bFs, and bGCs. A, Three VEGF transcripts (186, 318, and 390 bp) were expressed in BAECs, bFs, and bGCs. Bands corresponded to 121, 165, and 189 amino acid isoforms of the VEGF-A gene. B, A 379-bp band corresponding to the bovine Flk-1/KDR gene was observed in BAECs and bGCs. No gene expression was observed in bFs. Sequencing verified 100% homology to bovine VEGF and Flk-1/KDR genes. The larger bands in bGCs were sequenced and were not homologous to known bovine genes.

 
Detection of VEGF protein levels in GC lysates revealed that expression of the growth factor was varied in follicles of different sizes, and early and late corpora lutea. VEGF protein was detected by Western blot analysis as a 20-kDa band under reducing conditions (Fig. 2AGo). Histological data are presented as percent intensity of a commercially available VEGF protein standard (Fig. 2AGo, lane 1). Expression of VEGF in small (<0.5 cm) follicles was very low (Fig. 2AGo, lane 2). Medium follicles (0.5–1.0 cm) exhibited significantly (P < 0.05) higher VEGF protein levels, and this was followed by a greater than 2-fold higher (P < 0.05) VEGF expression in large (>1.0 cm) follicles (Fig. 2AGo, lane 4). Lysates from freshly isolated TCs were also analyzed for VEGF (Fig. 2AGo, lane 5) and demonstrated VEGF protein expression at levels greater than small and medium follicles and similar to large follicles and early corpora lutea. Corpora lutea were classified as early or late based on established morphological criteria (38). VEGF protein level in early corpora lutea tissue was similar to that seen in GC lysates from large follicles and TCs (Fig. 2AGo, lane 6). VEGF immunoreactivity was significantly (P < 0.05) increased in the late CL, compared with GCs from small and medium follicles and TCs but was not different from GCs from large follicles or early CL (Fig. 2AGo, lane 7). Immunohistochemical detection revealed that the majority of immunopositive cells in the CL were large luteal cells (data not shown).



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  		FIG. 2. VEGF and Flk/KDR protein expression increases during follicular development. Standard immunoblot analysis was performed for VEGF (A) with 40 µg GC lysate protein from small (<0.5 cm, lane 2), medium (0.5–1.0 cm, lane 3), and large (>1.0 cm, lane 4) bovine follicles; TCs (lane 5) and homogenized tissue of early (lane 6) and late (lane 7) corpora lutea. Lane 1 (A), purified VEGF protein (Santa Cruz Biotechnology) as positive control. Immunoblot analysis was performed for Flk-1/KDR (B) with 40 µg GC lysate protein from small, medium, and large follicles and compared against a purified Flk-1 protein standard (Santa Cruz Biotechnology). Graphs of densitometry measurements show data from four blots of different lysate samples. Groups within each graph that contain the same letters are not significantly different; those with different letters (A) a–d and (B) a–c are statistically different (P < 0.05).

 
Flk-1/KDR protein levels were measured by Western blot in GC lysates collected from small (<0.5 cm), medium (0.5–1.0 cm) and large (>1.0 cm) bovine follicles to determine changes in expression during follicular development. After ensuring equal protein loading, densitometric analysis was performed, and band intensity was calculated as a ratio to intensity of purified Flk-1 protein (Santa Cruz; Fig. 2BGo, lane 1). There was a 1.8-fold higher (P < 0.05) expression of Flk-1/KDR protein in medium compared with small follicles. Large follicles exhibited significantly higher (P < 0.05) Flk-1 protein levels, compared with small or medium follicles (Fig. 2BGo).

VEGF and Flk-1/KDR are coordinately expressed in the bovine follicle
Dual-labeling immunofluorescence was used to determine localization of VEGF and Flk-1/KDR in follicles and in vitro cultured cells. In experiments with tissue sections, VEGF ligand was localized to the GCs and TCs of preovulatory bovine follicles (Fig. 3Go, A and B, green staining). Flk-1/KDR colocalized with VEGF primarily in the GC layer, with some immunopositive colocalization in TCs (Fig. 3Go, A and B, Flk-1/KDR, red staining; colocalization, yellow staining). In vitro cultured GCs also exhibited colocalization of VEGF and its receptor, Flk-1/KDR (Fig. 3Go, C and D; VEGF, green, Flk-1/KDR, red, DAPI, blue). Cultured BAECs demonstrated colocalization of VEGF and Flk-1/KDR in a similar fashion to GCs, whereas bFs were only immunopositive for VEGF (data not shown). Omission of either primary or secondary antibodies or incubation with preimmune serum abolished immunoreactivity.



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  		FIG. 3. VEGF and Flk-1/KDR are colocalized in bovine follicle tissue and in vitro cultured GCs. Immunofluorescent detection of VEGF (green) and Flk-1/KDR (red) in bovine follicles (A and B) revealed that ligand and receptor were colocalized (yellow) in the GC layer, whereas the TC layer expressed predominantly VEGF alone. In vitro cultured bGCs (C–F) demonstrated expression of VEGF (green; C) and Flk-1/KDR (red; D). Nuclei were stained with DAPI (blue; E). Overlay of C-E shows colocalization (yellow; F) of ligand and receptor. Magnification, x400 (A); x1000 (B–F).

 
VEGF is cytoprotective for GCs
Because VEGF is known to have cytoprotective effects in some extravascular cells, we evaluated the effect of VEGF and Flk-1/KDR on spontaneous and induced apoptosis in BAECs, bovine GCs (bGCs), bF cells, and SIRGCs. Cells cultured in DMEM/F12 in the presence of 10% fetal bovine serum exhibited very low incidence of apoptosis (1.8 ± 1.3 to 3.2 ± 1.25%) (Fig. 4AGo). Switching BAECs, bGCs, and SIRGCs to serum-free media resulted in an increase (P < 0.05) in apoptotic cell death (Fig. 4CGo). When serum-free cultures were treated with 50 ng/ml rhVEGF (R&D Systems), there was a significant (P < 0.05) reduction in apoptosis in bGCs from 48.6 ± 9.2% to 19.8 ± 3.9% (Fig. 4CGo), in BAECs from 39.1 ± 9.6% to 11.1 ± 3% and in SIRGCs from 26.2 ± 5.4% to 16.1 ± 1.8%. Addition of 30 ng/ml of the Flk-1/KDR kinase inhibitor SU1498 (39) abrogated the cytoprotective effect of VEGF in serum-free culture (Fig. 4Go).



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  		FIG. 4. VEGF and Flk-1/KDR interact to protect granulosa and endothelial cells from apoptosis. A commercial TUNEL apoptosis kit was used to detect apoptotic cells after culture with 10% serum (A); serum starvation (B); treatment with VEGF (C); exposure to VEGF and the Flk-1/KDR inhibitor SU1498 (D); TNF{alpha} challenge (E); TNF{alpha} and VEGF (F); TNF{alpha} and IGF-I (G); TNF{alpha} and both VEGF and IGF-I (H); and TNF{alpha}, VEGF, and SU1498 (I) for 24 h. Images are of bGCs, and results are representative of experiments with BAECs and SIRGCs as well. Graph represents mean ± SEM; n = 3 experiments, each in triplicate. Groups within each graph that contain the same letters are not significantly different; those with different letters are statistically different (P < 0.05).

 
In separate experiments, apoptosis was induced by adding 100 ng/ml TNF{alpha} to serum-free cultures for 24 h. Addition of the cytokine resulted in almost universal cell death in the different cell types (Fig. 4EGo). When 50 ng/ml VEGF were added in conjunction with 100 ng/ml TNF{alpha} (in serum-free media), there was a dramatic reduction in apoptotic cell death (P < 0.05) (Fig. 4FGo). Because IGF-I has well-characterized cytoprotective effects in GCs, we used this growth factor as a positive control for cytoprotection. Thirty nanograms per milliliter IGF-I were added to serum-free cultures in the presence of 100 ng/ml TNF{alpha}. In bovine and immortalized rat GCs, IGF-I treatment resulted in a 5-fold reduction in apoptosis, compared with TNF{alpha} alone, which was similar to the cytoprotection provided with VEGF treatment (Fig. 4GGo). In endothelial cells, however, treatment with IGF-I resulted in only a 2.2-fold decrease in apoptosis (Fig. 4Go). When TNF{alpha}-challenged cells were treated with a combination of 50 ng/ml VEGF and 30 ng/ml IGF-I, there was a variable effect, with no difference in bGCs or BAECs and slightly reduced (P < 0.05) apoptotic rate in SIRGCs, compared with VEGF or IGF-I treatment alone (Fig. 4HGo). To determine whether the cytoprotective influence of exogenous VEGF was due to Flk-1/KDR, TNF{alpha}-challenged cells were treated with 50 ng/ml VEGF and 30 ng/ml SU 1498. Compared with combined TNF{alpha} and VEGF treatments, addition of SU1498 resulted in a significant (P < 0.05) increase in granulosa and endothelial cell apoptosis (Fig. 4IGo). The rate of apoptosis, however, did not reach levels observed in cells treated with TNF{alpha} alone. The effect of VEGF on granulosa cell proliferation was also evaluated. Serum-deprived GCs were cultured in the presence of 50 ng/ml VEGF for 24 h. [3H]thymidine incorporation was measured at 1, 3, 6, 12, and 24 h. There was no difference in the proliferation index of treated or untreated bGCs (data not shown). This experiment was repeated with a higher dose of VEGF (100 ng/ml), and there was still no difference in proliferation between treated and untreated cells or in treated cells at any time point (data not shown).

VEGF inhibits caspase activation in GCs
Numerous reports have indicated that apoptosis in the follicle occurs through activation of different caspase cascades (40, 41). The activation of latent caspase-3 to its active form is one such important determinant of apoptosis within the ovarian follicle. We therefore determined whether the apoptosis observed in our in vitro experiments was mediated by caspase-3 activation. Culture experiments were identical to those performed in the evaluation of in vitro apoptosis. The results of the in vitro assay for active caspase-3 closely replicated those described above for apoptosis. Serum starvation of bGCs, BAECs, and SIRGCs resulted in 6-fold, 25-fold, and 14-fold increases in active caspase-3 immunoreactivity, respectively (Fig. 5Go). Addition of 50 ng/ml rhVEGF (R&D Systems) to the serum-free culture significantly (P < 0.05) reduced expression of the active form of caspase-3 in all three cell types. Inhibition of Flk-1/KDR signaling by treatment with 30 ng/ml SU1498 resulted in a significant (P < 0.05) increase in active caspase-3 staining in bGCs (3.2-fold), BAECs (5.3-fold), and SIRGCs (2.7-fold) (Fig. 5Go). Treatment of TNF{alpha} (100 ng/ml)-challenged cells with 50 ng/ml rhVEGF significantly (P < 0.01) reduced the presence of active caspase-3 in bGCs, BAECs, and SIRGCs. Once again, IGF-I was used as a positive control for cytoprotection because it has previously been shown to inhibit caspase-3 activation (42). Addition of 30 ng/ml IGF-I to TNF{alpha} (100 ng/ml)-challenged cells resulted in a decrease in the percentage of GCs expressing active caspase-3 (bGCs 16 ± 2.5%; SIRGCs 14 ± 5.1%), similar to that seen in VEGF-treated GCs. IGF-I treatment of TNF{alpha}-challenged BAECs resulted in a more modest protection, with 42.6 ± 11.3% immunopositive. Treatment with both 50 ng/ml VEGF and 30 ng/ml IGF-I resulted in an incidence of caspase-3 expression of 10 ± 2.2% in bGCs, 16 ± 3.8% in BAECs, and 9 ± 4.1% in SIRGCs. Concurrent treatment with 100 ng/ml TNF{alpha}, 50 ng/ml VEGF, and 30 ng/ml SU1498 ameliorated the cytoprotective effects of VEGF and resulted in 3.3-fold (bGCs), 3.2-fold (BAECs), and 2.6-fold increases (P < 0.05) in active caspase-3 expression, compared with treatment with only TNF{alpha} and VEGF (Fig. 5Go).



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  		FIG. 5. VEGF and Flk-1/KDR reduce expression of active caspase-3. Treated cells were subjected to immunofluorescence for the active form of caspase-3 (green). Nuclei were stained with propidium iodide (red). Staining was performed after culture with serum (A), serum starvation (B), treatment with VEGF (C), exposure to VEGF and the Flk-1/KDR inhibitor SU1498 (D), TNF{alpha} challenge (E), TNF{alpha} and VEGF (F), TNF{alpha} and IGF-I (G), TNF{alpha} and both VEGF and IGF-I (H) and TNF{alpha}, VEGF and SU1498 (I) for 24 h. Images are of bGCs and are representative of BAEC and SIRGC experiments. Graph represents mean ± SEM; n = 3. Significantly different groups within each graph (P < 0.05) are represented by different letters (a–f).

 
Because follicular atresia is characterized by apoptotic death of the GCs (41), we compared the incidence of apoptosis and the expression of Flk-1/KDR between healthy and atretic follicles to determine whether receptor expression may act as a functional survival factor in the follicle. Histological sections of bovine follicles were evaluated morphologically for evidence of atresia. Indicators of follicle atresia included disruption of the architecture of the GC layer and condensation of GC nuclei (43). Follicles were classified as healthy or in the early or late stages of atresia. Healthy follicles exhibited very low levels of GC apoptosis, with only 4 ± 6% of cells being TUNEL positive (Fig. 6Go). The majority (88 ± 14%) of GCs from healthy follicles expressed Flk-1/KDR. Conversely, in the early stages of follicle atresia, there was a significant (P < 0.05) increase in the incidence of GC apoptosis. Inversely, early atretic follicles exhibited significantly (P < 0.05) reduced Flk-1/KDR expression, with only 42 ± 10% of cells positive. More dramatically, late atretic follicles had a very disrupted GC layer, with a significant (P < 0.01) increase in apoptosis, resulting in 90 ± 18% cells with fragmented DNA. Late atretic follicles revealed the lowest immunostaining for Flk-1/KDR with a significant (P < 0.01) reduction to only 9 ± 8% of GCs staining positive (Fig. 6Go). Six follicles from each of the healthy, early atretic, and late atretic groups were imaged (six fields of view at x250 magnification from each follicle) for quantification.



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  		FIG. 6. Flk-1/KDR expression is inversely associated with follicle atresia and GC apoptosis. Healthy, early atretic, and late atretic bovine follicles were subjected to TUNEL analysis for apoptosis and immunofluorescence for Flk-1/KDR localization. Immunopositive cells (apoptosis, Flk-1/KDR) stained green, and nuclei were counterstained with propidium iodide (red). Graph represents mean ± SEM of percent labeling from six visual fields at x250 magnification of six ovaries from each group. Groups with different letters (a–c) within each graph are statistically different (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we demonstrated for the first time that VEGF and its receptor, Flk-1/KDR, are coexpressed in ovarian GCs in which they interact to protect these cells against apoptotic cell death and follicle atresia. These results reveal a novel extravascular role for VEGF in the follicle. VEGF is produced by most cell types, whereas its receptors, Flt-1 and Flk-1/KDR, have previously been thought to be expressed exclusively by vascular cells (for review, see Ref. 44). The effects of VEGF on endothelial cells have long been known and include proliferation, chemotaxis, and protection against apoptosis. However, the role of VEGF in extravascular cells and tissues is poorly understood.

This study revealed coexpression of VEGF and Flk-1/KDR by ovarian GCs in vivo and in vitro. Immunofluorescence experiments demonstrated that expression of ligand and receptor was restricted primarily to the granulosa layer of healthy follicles. VEGF and Flk-1/KDR protein increased as follicle size increased and were important in protecting in vitro cultured GCs against serum starvation and cytokine-induced apoptosis. Inhibition of Flk-1/KDR abrogated the cytoprotective effects of VEGF. Examination of healthy and atretic bovine follicles revealed an inverse pattern of expression between apoptosis and Flk-1/KDR in the different classes of follicles. Healthy follicles exhibited a very low incidence of apoptosis and high Flk-1/KDR expression in the GCs. Early atretic follicles, however, had significantly more apoptotic GCs and reduced Flk-1/KDR staining. Apoptosis was further increased and Flk-1/KDR presence was more reduced in GCs of late atretic follicles. These in vivo results suggest a functional protective role for VEGF and its kinase receptor within the follicle.

In the present study, VEGF protein increased during development of healthy follicles. Similar findings have been reported by others, and the increased ligand expression has been shown to result in enhanced capillary proliferation (45). The present study also demonstrated a concomitant increase in GC Flk-1/KDR protein during development and growth of the follicle. The mechanisms responsible for regulating VEGF and VEGF receptor expression during follicular development are not well understood. Exposure of rats to elevated levels of gonadotropins increased expression of VEGF and Flk-1/KDR in the ovary, which resulted in vascular hyperpermeability (25). The VEGF receptor-ligand system in extravascular GCs may respond in a similar fashion, with increased ligand and receptor expression in GCs occurring in response to altered gonadotropic profile during the ovarian cycle. In vitro and in vivo studies are currently underway in our laboratory to determine whether gonadotropins modulate Flk-1/KDR expression in this system.

VEGF is known to be important in regulating development of the follicle. Inhibition of VEGF with a VEGF Trap antibody in the primate results in decreased follicle angiogenesis, reduced recruitment and growth of antral follicles, and decreased Flt and Flk-1/KDR expression (22). These data support the findings of the present study in which Flk-1/KDR expression was inversely related to GC apoptosis and the incidence of follicle atresia. Therefore, functional interaction between VEGF and its receptor may be crucial in protecting follicular cells against apoptosis or determining dominant vs. atretic follicles.

One particularly interesting and potentially important finding of the present study involves the role of VEGF in the regulation of GC apoptosis. VEGF treatment reduced the incidence of apoptosis and active caspase-3 expression in endothelial and granulosa cells but not fibroblasts, which lack VEGF receptors. The caspases are a family of intracellular cysteine proteases that are involved in mediating apoptotic cell death in all cells from vertebrates. Currently, there are 14 members of the vertebrate caspase family (46), and some have been implicated in apoptosis in the ovary. GCs of healthy follicles express the inactive form of caspase-3, whereas cells of atretic follicles express elevated levels of active caspase-3 (47, 48). The results of this study indicate that cultured granulosa and endothelial cells undergo caspase-3-mediated apoptosis in the absence of serum and in the presence of the cytotoxic cytokine, TNF{alpha}. Cells deprived of serum or those treated with 100 ng/ml TNF{alpha} exhibited significantly higher rates of apoptosis, and the cell death was mirrored by increases in cellular expression of active caspase-3. In our in vitro granulosa cell culture, induced apoptotic cell death occurs in a caspase-dependent fashion, which is consistent with other reports of apoptosis in the ovary (49, 50). In this study, addition of VEGF significantly reduced both the incidence of apoptosis, and the expression of active caspase-3 in cultured endothelial and granulosa cells. The reduction of endothelial cell caspase-3 expression in the present study is similar to that shown by others (51). Although caspase-3 activation is an important mediator of apoptosis in the follicle, there are other pathways that are important in mediating GC death, and the effects of VEGF on these systems remains to be explored.

The protective effect of VEGF in the GCs, as evidenced by reduced expression of active caspase-3 and decreased apoptosis, appears to occur via interaction with Flk-1/KDR. Inhibition of Flk-1/KDR by the receptor-tyrosine kinase inhibitor SU1498 significantly inhibited the ability of cells to respond to endogenous and exogenous VEGF, reducing protection against caspase-3 activation and apoptosis. Addition of SU1498 to cells treated with TNF{alpha} and VEGF resulted in an increase in apoptosis but not to the extent observed when the cells were treated with TNF{alpha} alone. Exogenous VEGF appears to be able to provide some protection against cytokine- and serum deprivation-induced cell death after inhibition of Flk-1. VEGF may be protecting against cell death through other signaling mechanisms, such as via the Flt-1 receptor. Although there is debate about the signaling and functional characteristics of the type 1 (flt-1) receptor, others have claimed that VEGF can stimulate cell proliferation and protect against organ damage through this receptor (11). VEGF may also stimulate expression of other survival factors such as survivin, which is known to be associated with VEGF-induced protection in other cell types (52, 53). Conversely, the addition of SU1498 may not result in complete inhibition of Flk-1/KDR, allowing for some functional interaction with VEGF. SU1498 has been shown to reduce Flk-1-mediated proliferation in another extravascular cell line, neuronal precursor cells (33). The Flk-1/KDR receptor is known to be critical in VEGF-induced endothelial cell survival (4, 54), and it appears, with the results of the present study, that a similar system exists in ovarian GCs. Selective pharmacological inhibition of Flk-1/KDR expression is difficult because these inhibitors may affect other angiogenically relevant molecules such as TGFß (55).

The net effect of VEGF and receptor expression in the GCs of healthy, growing follicles may be to protect the cells against apoptotic death in an avascular environment until ovulation occurs. Upon ovulation, there is a disruption of the basement membrane, and rapid angiogenesis occurs during the formation of the CL (56). Having VEGF and Flk-1/KDR present in the GCs may be advantageous in placing the ovulated follicle in a permissive, proangiogenic state to facilitate the extensive angiogenesis that occurs at this time (57).

The expression of the type 2 VEGF receptor in ovarian GCs is interesting in light of recent studies demonstrating that this cell population functions as a specific subtype of endothelial-like cells (58, 59). These studies show that granulosa cells can respond to endothelial cell-like stimuli such as hypoxia, which may assist in regulating follicular development, follicle steroidogenesis, and other follicular functions. Our studies suggest that this may extend to responsiveness to growth factors formerly thought to regulate endothelial cells exclusively.

The results of this study may facilitate understanding of the role of VEGF in some reproductive disorders common in females. VEGF is implicated in the etiology of serious reproductive disorders such as the polycystic ovary syndrome (60, 61), and OHSS (39). In polycystic ovary syndrome, elevated VEGF may interact with Flk-1/KDR in the affected ovaries, preventing GC apoptosis and the resultant follicle atresia, thus contributing to the growth and persistence of a large number of follicles. Excessive ovarian expression of VEGF contributes to the development of OHSS (39, 62), but its exact role in this disease is not well understood. The results of this study may provide insight into the mechanisms by which VEGF contributes to these and other ovarian disorders and thereby contribute to the development of new therapeutic strategies.

In conclusion, this study demonstrates for the first time that VEGF and Flk-1/KDR are expressed in a coordinated fashion in ovarian GCs in which they function to protect these cells from apoptosis. Flk-1/KDR expression is significantly reduced in early and late atretic follicles, suggesting a physiological role for this receptor in the follicle. The results demonstrate a novel role for VEGF and its receptor in the ovarian follicle.


   Acknowledgments
 
We thank Dr. R. Burghardt (Texas A&M University, College Station, TX) for generously providing the SIRGCs and Michelle Ross for technical support.


   Footnotes
 
This work was supported by supporting grants from the Natural Sciences and Engineering Research Council of Canada and Ontario Ministry of Agriculture and Rural Affairs.

Abbreviations: BAEC, Bovine aortic endothelial cell; bF, bovine fibroblast; bGC, bovine GC; CL, corpus luteum; DAPI, 4',6'-diamino-2-phenylindole; GC, granulosa cell; OHSS, ovarian hyperstimulation syndrome; rh, recombinant human; SIRGC, spontaneously immortalized rat granulosa cell; TC, theca cell; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling; VEGF, vascular endothelial growth factor.

Received December 1, 2003.

Accepted for publication February 18, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals