Department of Obstetrics and Gynaecology, University of Southampton, Southampton, UK
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Abstract |
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Key words: angiogenesis/cell culture/endothelial cells/immunocytochemistry/ovary
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Introduction |
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This process of neovascularization is under the control of heparin-binding growth factors, particularly vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), produced by the granulosa-derived cells of the corpus luteum (Anthony et al., 1994; Asaki and Tamura, 1993
; Kamat et al., 1995
; Shibuya, 1995
; Redmer et al., 1996
). Studies have shown that in the developing corpus luteum up to 98% of the proliferating cells are of endothelial origin. Investigations into the rate of proliferation showed that endothelial proliferation is greatest during the luteal phase (Rodger et al., 1997
). The resulting mature corpus luteum thus contains a considerable percentage of endothelial cells (Bagavandoss and Wilks, 1991
; Redmer and Reynolds, 1996
; Jablonka-Shariff et al., 1993
).
Studies on the control of angiogenesis involving microvascular endothelium would be facilitated by the development of representative, in-vitro physiological models. The use of transformed endothelial cell lines (Hughes, 1996) for studies on angiogenesis is limited by their reduced responsiveness to growth factors. Endothelial cell preparations from large vessels such as the umbilical vein have the advantage of adequate responsiveness to growth factors and have been widely used (Gimbrone et al., 1974
; Cockerill et al., 1994
). However, there are important differences between these preparations and those from microvascular endothelium (Craig et al., 1998
; Fajardo, 1989
). In particular, the morphology and responsiveness of microvascular endothelium varies according to the tissue of origin (Richard et al., 1998
). Ideally, therefore, a representative model for angiogenesis in the corpus luteum would involve the use of endothelial cells from this tissue. In the present study we aimed to: (i) develop a reproducible and convenient method for the isolation and culture of human ovarian microvascular endothelial cells, and (ii) undertake their morphological and functional characterization.
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Materials and methods |
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Human umbilical vein endothelial cells (HUVEC) were harvested from fresh umbilical cords obtained at Caesarean section using the method of Jaffe et al. (1973). Cultures were maintained in supplemented M199 with additional endothelial cell growth supplement (ECGS; 150 µg/ml; Sigma, Poole, UK) using 5% CO2 in air at 37°C. HUVEC cultures were grown to confluence on 1% (w/v) gelatin-coated 50 cm2 flasks. Culture medium was changed every 34 days and cells were used up to and including passage 4.
The ECV304 cell line, a spontaneously transformed HUVEC cell line, was obtained from the European Collection of Animal Cell Cultures (CAMR, Salisbury, UK). Cells were maintained in the supplemented M199 with no additional growth factors in 5% CO2 in air at 37°C. Cultures were grown to confluence on uncoated flasks. Medium was changed every 34 days.
Human foreskin fibroblasts for control studies were kindly supplied by the Department of Medical Oncology, Southampton General Hospital and were maintained in supplemented M199 with no additional growth supplement, in 5% CO2 in air at 37°C. Culture medium was changed every 34 days. Cells were used up to passage 10.
In order to isolate human ovarian microvascular endothelial cells (HOMEC), follicular aspirates were obtained at the time of oocyte recovery for in-vitro fertilization. The treatment protocol, described in detail by Jenkins et al. (1991), involved down-regulation of pituitary function with gonadotrophin releasing hormone analogue and stimulation of multifollicular development with human menopausal gonadotrophin. Follicular aspirates were filtered through 70 µm cell strainers (Becton Dickinson, Cowley, UK) and the tissue pieces retained were resuspended in M199 by backwashing. Centrifugation (100 g, 10 min) yielded a small pellet which was cooled before resuspension in 0.5 ml of Matrigel (Becton Dickinson) at 4°C. Aliquots (50100 µl) of this mixture were dispensed onto the surface of uncoated 25 cm2 flasks and the Matrigel containing the tissue fragments was allowed to set. Cultures were maintained in supplemented M199 with ECGS (150 µg/ml) and VEGF (6.25 ng/ml; R & D Systems, Abingdon, UK) in 5% CO2 in air at 37°C. Medium was changed every 34 days and cultures were monitored for growth daily by phase-contrast microscopy. Cultures not developing by day 12 were discarded. Areas of growth exhibiting a cobblestone morphology were trypsinized and replated in 24-well plates with supplemented M199 and the previously described ECGS/VEGF growth supplements.
Histochemistry
In order to confirm the ovarian origin of the fragments obtained by follicle aspiration, histochemical staining for the steroidogenic marker 3ß-hydroxysteroid dehydrogenase (3ßHSD) was carried out. HOMEC cultures exhibiting cell morphology consistent with endothelial cobblestone growth and the original tissue fragment in Matrigel from which the cells had originally grown were stained according to the method of Aldred and Cooke (1983). Briefly, cultures were washed with HBSS and incubated at 37°C for 2 h with a mixture containing 100 µl 5-androstan-3ß-ol-17-one (2 mg/ml) in dimethyl formamide, 1 ml nitroblue tetrazolium (1.6 mg/ml), 800 µl NAD+-free salt (3 mg/ml) and 4 ml of phosphate-buffered saline (all additions from Sigma). Cultures were then frozen for 1 h at 80°C and thawed at room temperature. The presence of staining for 3ßHSD was observed by light microscopy.
Immunocytochemistry
Cells were characterized by fluorescence immunostaining using a panel of endothelial cell-specific antibodies, listed with dilutions in Table I. Confluent and subconfluent cells were plated on 4-well chamber slides and allowed to settle overnight. Plated cells and capillary-like structures in association with Matrigel were fixed in ice cold methanol for 30 min and washed with PBS containing 0.1% Triton. Slides with primary antibodies were incubated overnight at room temperature. After washing in PBS, biotinylated secondary antibodies (10 µg/ml; Vector, Peterborough, UK) were added and slides incubated for 1.5 h at room temperature. After washing and incubation with fluorescein isothiocyanate-conjugated streptavidin (20 µg/ml; Vector) for 1 h at room temperature and nuclear counterstaining with propidium iodide (1 µg/ml; Sigma) specimens were mounted in Mowiol (Harlow Chemicals, Harlow, UK). Slides were viewed under epifluorescence using a Leica TSC 4D confocal microscope.
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Results |
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RT-PCR analysis showed the presence of the correct size double stranded PCR products for the VEGF receptors KDR and flt-1, in both HOMEC and HUVEC, but not in the transformed ECV304 cells (Figure 3A). Expression of eNOS was also seen in HOMEC and HUVEC as demonstrated by RT-PCR, but an equivalent PCR product was not detectable in ECV304 cells (Figure 3B
).
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Discussion |
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Secondly, immunocytochemical analysis showed that HOMEC possess the endothelial cell specific markers CD31, vWF, E-selectin and UEA-1 which were evident with the same fluorescence intensity as seen in HUVEC cultures. However, in contrast to HUVEC, HOMEC constitutively expressed the adhesion molecule VCAM-1 whose expression is more typically seen in endothelial cells stimulated by cytokines (Haraldsen et al., 1996). Its expression in unstimulated HOMEC in the present study may reflect the special role of these cells in interacting with luteal cells and the tissue matrix during the rapid formation of the corpus luteum in vivo.
Currently available models for the study of ovarian endothelial cells have been based on the isolation of cells directly from the corpus luteum. Some studies have involved the purification of dispersed cells from the primate corpus luteum using immunomagnetic separation (Christenson and Stouffer, 1996b). In our experience, this method proved unsatisfactory for the isolation of endothelial cells from the human corpus luteum because problems associated with the incomplete dispersion of tissue did not allow effective separation of endothelial cells from luteal cells (unpublished data). Ethical difficulty of obtaining human tissue from surgical specimens is also an important limitation of this approach. The method described in the present study allows readily available human tissue obtained at egg collections for IVF to be utilized in a simple technique for the culture of HOMEC. This model also has the advantage of using endothelial outgrowth from the theca layer which would mimic to some extent the processes involved in corpus luteum development in vivo. Cells isolated using this method are at a stage when they might be expected to have the ability to generate the new capillary growth associated with corpus luteum formation after ovulation. Cells isolated from the mature corpus luteum, however, would be at a stage where proliferation has been completed. Further studies will be required to establish the functional properties of isolated endothelial cells using our method, and also to determine whether the hormonal treatment used in the IVF cycle has an influence on their functional activity. Evidence from bovine models suggests that sub-populations of endothelial cells in the corpus luteum may exist, showing differences in cytoskeleton and responsiveness to growth factors (Spanel-Borowski and van der Bosch, 1990
; Fenyves et al., 1993
). Establishing the extent of heterogeneity in the HOMEC cultures derived in the present study would require further work.
VEGF plays an important role in corpus luteum formation in a number of species and the present study shows that its effects could be mediated by either or both of the main VEGF receptors, flt-1 and KDR, which are being expressed by HOMEC. The activation of flt-1 has been shown to mobilize calcium ions which are important for eNOS activity (Ahmed et al., 1997). As eNOS was also shown to be expressed in HOMEC, the constituents for important endothelial mechanisms in relation to VEGF action are in place in these cells. The present observations are qualitative in nature and further studies are required to evaluate the role of these mechanisms, and to establish whether they had been influenced in our study by prior hormonal treatment used in the IVF cycle. Survival of microvascular endothelium under serum-free conditions has been demonstrated and it has been postulated that VEGF may act as a survival factor for microvascular endothelial cells under these conditions (Gupta et al., 1997
). The proliferative activity of microvascular luteal endothelial cells isolated from the primate corpus luteum has been shown to be stimulated by VEGF (Christenson and Stouffer, 1996b
). Taken together, the available evidence thus suggests that VEGF has an important role in controlling the function of microvascular endothelium in the ovary.
A special feature of the ovary is the potential for interaction between steroidogenic and vascular tissues. In a bovine model microvascular endothelial cells were seen to enhance the growth of granulosa cells (Spanel-Borowski et al., 1994). Future studies of HOMEC, luteal cells and matrix interactions will be of great interest, and the present study has shown the feasibility of utilizing an available source of normal human material for such studies.
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Acknowledgments |
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Notes |
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References |
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Aldred, L.F. and Cooke, B.A. (1983) The effect of cell damage on the density and steroidogenic capacity of rat testis leydig cells, using an NADH exclusion for determination of viability. J. Steroid Biochem., 18, 411414.[ISI][Medline]
Anthony, F.W., Watson, R.H., Richardson, M.C. et al. (1994) Reverse transcription and competitive PCR shows increased expression of vascular endothelial growth factor (VEGF) after hCG stimulation of cultured human granulosa cells. J. Reprod. Fertil., 13, 26.
Asaki, R. and Tamura, K. (1993) Basic fibroblast growth-factor (bfgf) receptors decrease with luteal age in rat ovarian luteal cells colocalization of bfgf receptors and bfgf in luteal cells. Endocrinology, 133, 10741084.[Abstract]
Augustin, H.G., Braun, K., Telemenakis, I. et al. (1995) Phenotypic characterization of endothelial cells in a physiological model of blood vessel growth and regression. Am. J. Clin. Pathol., 147, 339351.
Bagavandoss, P. and Wilks, J.W. (1991) Isolation and characterization of microvascular endothelial cells from developing corpus luteum. Biol. Reprod., 44, 11321139.[Abstract]
Charnock-Jones, D.S., Sharkey, A.M., Boocock, C.A, et al. (1994) Vascular endothelial growth factor receptor localization and activation in human trophoblast and choriocarcinoma cells. Biol. Reprod., 51, 524530.[Abstract]
Christenson, L.K. and Stouffer, R.L. (1996a) Proliferation of microvascular endothelial cells in the primate corpus luteum during the menstrual cycle and simulated early pregnancy. Endocrinology, 137, 367374.[Abstract]
Christenson, L.K. and Stouffer, R.L. (1996b) Isolation and culture of microvascular endothelial cells from the primate corpus luteum. Biol. Reprod., 55, 13971404.[Abstract]
Cockerill, G.W., Meyer, G., Noack, L. et al. (1994) Characterization of a spontaneously transformed human endothelial cell line. Lab. Invest., 71, 497509.[ISI][Medline]
Craig, L.E., Spelman, J.P., Strandberg, J.D. and Zink, M.C. (1998) Endothelial cells from diverse tissues exhibit differences in growth and morphology. Microvasc. Res., 55, 6576.[ISI][Medline]
de Vries, C., Escobedo, J.A., Ueno, H. et al. (1992) The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science, 255, 989991.[ISI][Medline]
Fajardo, L.F. (1989) The complexity of endothelial cells. Am. J. Clin. Pathol., 92, 241250.[ISI][Medline]
Fenyves, A.M., Behrens, J. and Spanel-Borowski, K. (1993) Cultured microvascular endothelial cells (mvec) differ in cytoskeleton, expression of cadhedrins and fibronectin matrix a study under the influence of interferon-gamma. J. Cell Sci., 106, 879890.
Ferrara, N. (1996) Vascular endothelial growth factor. Eur. J. Cancer, 32, 2413422.
Ferrara, N., Houck, K., Jakeman, L. and Leung, D.W. (1992) Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocrine Rev., 13, 1832.[ISI][Medline]
Ferrara, N., Chen, H., Davis-Smyth, T. et al. (1998) Vascular endothelial growth factor is essential for corpus luteum angiogenesis. Nature Med., 4, 336340.[ISI][Medline]
Gimbrone, M.A., Cotran, R.S. and Folkman, J. (1974) Human vascular endothelial cells in culture. J. Cell Biol., 60, 673684.
Gupta, K., Ramakrishnan, S., Browne, P.V. et al. (1997) A novel technique for culture of human dermal microvascular endothelial cells under either serum-free or serum-supplemented conditions: isolation by panning and stimulation with vascular endothelial growth factor. Exp. Cell Res., 230, 244251.[ISI][Medline]
Haraldsen, G., Kvale, D., Lien, B. et al. (1996) Cytokine-regulated expression of E-selectin, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in human intestinal microvascular endothelial cells. J. Immunol., 156, 25582565.[Abstract]
Hughes, S.E. (1996) Functional characterization of the spontaneously transformed human umbilical vein endothelial cell line ECV304: Use in an in vitro model of angiogenesis. Exp. Cell. Res., 225, 171185.[ISI][Medline]
Jablonka-Shariff, A., Grazul-Bilska, A.T., Redmer, D.A. and Reynolds, L.P. (1993) Growth and cellular proliferation of ovine corpora lutea throughout the estrous cycle. Endocrinology, 133, 18711879.[Abstract]
Jaffe, E.A., Nachman, R.L., Becker, C.G. and Minidi, C.R. (1973) Culture of human endothelial cells derived from umbilical veins: identification by morphological and immunological criteria. J. Clin. Invest., 52, 27452756.[ISI][Medline]
Janssens, S.P., Shimouchi, A., Quertermous, T. et al. (1992) Cloning and expression of a cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase. J. Biol. Chem., 267, 1451914522.
Jenkins, J.M., Davies, D.W., Devonport, H. et al. (1991) Comparison of poor responders with good responders using a standard buserelin human menopausal gonadotropin regime for in vitro fertilization. Hum. Reprod., 6, 918921.[Abstract]
Kamat, B.R., Brown, L.F., Manseau, E.J. et al. (1995) Expression of vascular permeability growth factor/vascular endothelial growth factor by human granulosa and theca lutein cells. Am. J. Pathol., 146, 157163.[Abstract]
Koolwijk, P., van Erck, M.G.M., de Vree, W.J.A. et al. (1996) Cooperative effect of TNF, bFGF and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity. J. Cell Biol., 132, 11771188.[Abstract]
Kubota, Y., Kawa, Y. and Mizoguchi, M. (1996) Cdw49b/CD29 integrin complex mediates the differentiation of human endothelial cells into capillary-like structures in vitro. J. Dermatol. Sci., 12, 3643.[ISI][Medline]
Nelson, S.E., Mclean, M.P., Jayatilak, P.G. and Gibori, G. (1992) Isolation, characterization and culture of cells forming the pregnant rat corpus luteum. Endocrinology, 130, 954966.[Abstract]
Plendl, J., Neumuller, C., Vollmar, A. et al. (1996) Isolation and characterization of endothelial cells from different organs in fetal pigs. Anat. Embryol. (Berl), 194, 445456.[ISI][Medline]
Redmer, D.A. and Reynolds, L.P. (1996) Angiogenesis in the ovary. Rev. Reprod., 1, 182192.
Redmer, D.A., Dai, Y., Li, J. et al. (1996) Characterization and expression of vascular endothelial growth factor (VEGF) in the ovine corpus luteum. J. Reprod. Fertil., 108, 157165.[Abstract]
Richard, L., Velasco, P. and Detmar, M. (1998) A simple immunomagnetic protocol for the selective isolation and long-term culture of human dermal microvascular endothelial cells. Exp. Cell Res., 240, 16.[ISI][Medline]
Rodger, F.E., Young, F.M., Fraser, H.M. and Moor, R.M. (1997) Endothelial cell proliferation follows the mid-cycle luteinizing hormone surge, but not human chorionic gonadotrophin rescue, in the human corpus luteum. Hum. Reprod., 12, 17231729.[Abstract]
Scott, P.A.E. and Bicknell, R. (1993) The isolation and culture of microvascular endothelium. J. Cell Sci., 105, 269273.
Shibuya, M. (1995) Role of VEGF-FLT receptor system in normal and tumour angiogenesis. Adv. Cancer Res., 67, 281316.[ISI][Medline]
Spanel-Borowski, K. and van der Bosch, J. (1990) Different phenotypes of cultured microvessel endothelial cells obtained from bovine corpus luteum. Cell Tissue Res., 261, 3547.[ISI][Medline]
Spanel-Borowski, K., Ricken, A.M., Saxer, M. and Huber, P.R. (1994) Long-term coculture of bovine granulosa cells with microvascular endothelial cells effect on cell growth and cell death. Mol. Cell. Endocrinol., 104, 1119.[ISI][Medline]
Suzuki, T., Sasano, H., Takaya, R. et al. (1998) Cyclic changes of vasculature and vascular phenotypes in normal human ovaries. Hum. Reprod., 13, 953959.[Abstract]
Submitted on October 6, 1998; accepted on February 2, 1999.