Human Reproduction, Vol. 15, No. 2, 293-301,
February 2000
© 2000 European Society of Human Reproduction and Embryology
Isolation, characterization and long-term culture of human myometrial microvascular endothelial cells
Monash University Department of Obstetrics & Gynaecology, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia
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Angiogenesis, defined as the growth of new vessels from pre-existing vessels, involves microvascular rather than large vessel endothelial cells. Accordingly, microvascular endothelial cell (MEC) proliferation assays are an appropriate in-vitro model of angiogenesis. We have developed a method for the isolation and long-term culture of large numbers of MEC from the human myometrium, tissue readily available from hysterectomy specimens. Human myometrial MEC were positively selected from tissue dissociated sequentially with collagenase and trypsin using Ulex europeaus antigen-1 (UEA)-coated dynabeads. Cultured myometrial MEC displayed characteristic endothelial phenotype and function for up to 14 passages: cobblestone morphology, formed capillary-like tubes on Matrigel, expressed CD31, Factor VIII-related antigen, bound UEA lectin, incorporated 1,1'-dioctadecyl-1,3,3,3',3'-tetramethylindocarbocyanine perchlorate-labelled acetylated low density lipoprotein, migrated and proliferated in response to basic fibroblast growth factor (bFGF) and vascular endothlelial growth factor (VEGF), but not epidermal growth factor. Optimal growth of human myometrial MEC occurred in a simple medium comprising M199, 5 ng/ml bFGF, 15% human serum, 5% fetal calf serum (FCS) and heparin. Human serum was essential for growth, although there was a synergistic effect when FCS was included. Almost identical dose–response curves were obtained for bFGF- and VEGF-induced myometrial MEC proliferation in early and late passage cells. Therefore myometrial MEC are a good model for in-vitro studies of uterine angiogenesis, since they have a stable phenotype and proliferative responsiveness to VEGF and bFGF for up to 14 passages.
Key words: angiogenesis/endothelial cell/microvascular/myometrium
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Introduction |
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The microvascular endothelium has a critical role in angiogenesis, the process by which new blood vessels develop from pre-existing vessels. In the adult, angiogenesis rarely occurs under normal circumstances, except in the female reproductive tract during the menstrual cycle and pregnancy (Risau, 1997
). Little is known about the microvasculature of the myometrium and the changes that occur during pregnancy, where angiogenesis undoubtedly occurs as the myometrial smooth muscle hypertrophies. Considerable research effort has been applied to the study of angiogenesis associated with malignant tumours, but there is little information about the role of angiogenesis in benign tumours including fibroids, smooth muscle tumours of the myometrium, which affect up to 50% of women during their reproductive years (Vollenhoven, 1998).
The study of angiogenesis has been significantly advanced by the ability to culture endothelial cells (EC) in vitro. Initially large vessel EC, such as those isolated from the human umbilical vein (HUVEC) were used for these studies (Jaffe et al., 1973), but increasingly it has been recognized that microvascular endothelial cells (MEC) are a more appropriate model since angiogenesis involves MEC rather than large vessel EC (Hewett and Murray, 1993; Bicknell, 1996). It is well known that EC are heterogeneous in phenotype, function, expression of surface molecules and responsiveness to growth factors (Auerbach et al., 1985; McCarthy et al., 1991; Garlanda and Dejana, 1997). Differences exist between large vessel and microvascular EC and the properties of arterial and venous EC are also distinct (Hewett and Murray, 1993). EC express organ-specific antigens and therefore differ between the various organs, but even within an organ, there is heterogeneity of EC from different vascular beds (Auerbach et al., 1985; Kumar et al., 1987).
In comparison to large vessel EC, MEC are difficult to isolate and culture. MEC only comprise 1–5% of the cells in a given tissue, they grow slowly in culture and are contact-inhibited. As a result, MEC cultures are often overgrown by faster growing, non-contact-inhibited fibroblasts, smooth muscle cells and pericytes, usually limiting passage number to five or six (Hewett and Murray, 1993; Bicknell, 1996). A significant improvement in the culture of MEC came with the use of superparamagnetic bead technology (Dynabeads). Initially Dynabeads were coupled with the lectin Ulex europeaus agglutinin-1 (UEA-1) which binds to 
-fucosyl residues on an EC-specific glycoprotein (Jackson et al., 1990). This approach has been extended by using antibodies to specific EC markers such as CD31 (Hewett and Murray, 1993), tumour necrosis factor (TNF)--induced E-selectin (Richard et al., 1998) and CD34 (Iruela-Arispe et al., 1999). These techniques have significantly increased the yields and purity of MEC obtained from a variety of tissues after initial enzyme dissociation.
Microvascular EC have previously been isolated from a number of human tissues including neonatal foreskin, brain, omentum, synovium, retina, endometrium and decidual tissue (Jackson et al., 1990; Su and Gilles, 1992; Grimwood et al., 1995; Chung-Welch et al., 1997; Richard et al., 1998; Iruela-Arispe et al.,1999). Few have attempted to isolate and culture MEC from muscular organs, although there are reports on the isolation of MEC from the human myocardium (Nishida et al., 1993; McDouall et al., 1996). There have been no detailed reports on the isolation and culture of MEC from the human myometrium, a highly vascular tissue that is readily available from hysterectomy specimens. In the present study we report on the isolation and long-term culture of MEC from the human myometrium to serve as a model of MEC derived from sex steroid hormone-responsive tissue and for our studies of angiogenesis in normal myometrium and fibroid tumours. The aims of the present study were to: (i) isolate pure cultures of MEC from human myometrium and (ii) optimize growth conditions for the use of myometrial MEC in in-vitro studies of angiogenesis.
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Materials and methods |
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Human tissues
Human myometrial tissue was obtained from normal ovulating women undergoing hysterectomy (aged 20–40 years) without underlying uterine pathology, usually for prolapse. In some cases, myometrium was obtained from women undergoing hysterectomy for fibroids. Tissues included in this study were from women who had not taken exogenous hormones in the previous 3 months. Informed consent was obtained from each patient and ethical approval from the Monash Medical Centre Human Research and Ethics Committee.
A small portion of each piece of tissue was fixed for 4 h in 10% phosphate-buffered formalin (pH 7.4) for routine paraffin embedding. Sections (5 µm) were used for immunohistochemical analysis. The remaining tissue (2–7 g) was collected in HEPES-buffered M199 culture medium (Gibco BRL, Gaithersburg, MD, USA) containing 10% FCS and antibiotic–antimycotic solution (Gibco BRL) with final concentrations of penicillin 1000 U/ml, streptomycin 1000 U/ml and fungizone 2.5 µg/ml. The tissue was stored overnight at 4°C and then processed.
Microvascular endothelial cell isolation and purification
The endometrial layer of the uterine tissue was identified, removed and discarded together with the first 1 mm of myometrial tissue and the outer third of the myometrium, thus removing any of the serosa and mesothelial layer. The inner 2/3 of the myometrium was then finely chopped for enzyme dissociation, since the inner layers of myometrium show greater responses to sex steroid hormones compared to the outer third (Noe et al., 1999). Chopped tissue was digested with 2 mg/ml collagenase type 2 (Worthington, Biochemical Corporation, Freehold, NJ, USA) and 14.5 µg/ml deoxyribonuclease type I (Boehringer Mannheim GmbH, Mannheim, Germany) in Ca2+- and Mg2+-free phosphate buffered saline (PBS, pH 7.4) containing 0.1% bovine serum albumin (BSA) (Sigma Chemical Co., St Louis, MO, USA) for 2 h at 37°C in a shaking water bath. The cloudy supernatant containing single cells was sequentially removed at 30 min intervals during the dissociation procedure and placed into HEPES-buffered M199 medium containing 50% FCS and stored at 4°C until all the tissue was dissociated. Fresh enzyme solution was added back to the digesting tissue. The cell suspensions were collected, diluted in M199 medium and filtered through 100 µm stainless steel mesh (Sigma), washed several times and incubated with trypsin:EDTA (0.05% trypsin: 0.53 mmol/l EDTA, Gibco BRL) and 24 µg/ml deoxyribonuclease type I for 10 min at 37°C to digest the remaining microvessel fragments and obtain single cell suspensions. The cells were washed and resuspended at 2.5x107/ml in HEPES-buffered M199 medium containing 1% FCS. Cell viability was measured using Trypan Blue dye exclusion and was ~70 and 60% after collagenase and trypsin treatment respectively.
Dynal M-450 magnetic beads (Dynal, Oslo, Norway) were coated with lectin by incubating 2x107 beads with UEA-1 (1 mg/ml in 0.1 mol/l borate buffer, pH 8.5) (Sigma) in a 200 µl volume for 24 h at 4°C, according to the manufacturer's instructions. The beads were collected using a magnetic particle concentrator (MPC; Dynal) and washed three times in PBS/0.1% BSA (one an overnight wash) and resuspended at 2x108/ml in PBS/BSA.
Endothelial cells were positively selected from the dissociated tissue by incubating 1 ml volumes of cell suspension (2.5x107 cells) with 8x107 UEA-1 coated beads for 10 min at 4°C with end-over-end rotation (bead:EC ratio 
5:1). The bead-attached cells were recovered and washed 8–10 times in M199/1% FCS using the MPC.
Microvascular endothelial cell culture
The purified MEC were resuspended in a standard culture medium comprising M199 with Earle's salts containing heat-inactivated 15% pooled male human serum (HS) (obtained from Red Cross Blood Service and male staff volunteers) and 5% FCS (CSL, Melbourne, Australia), 2 mmol/l glutamine (Gibco BRL), 5 ng/ml bFGF (Gibco BRL), 0.1 mg/ml heparin (Gibco BRL), and antibiotic/antimycotic, seeded into culture flasks at 8–10x104 cells/cm2 coated with 10 µg/ml fibronectin (Gibco BRL) and incubated in a humidified atmosphere at 37°C in 5% CO2 in air. To minimize culture variability the same batch of human serum was used for each set of experiments. For some isolations 0.5 µg/ml hydrocortisone (Sigma), 330 µmol/l 3-isobutyl-1-methyl xanthine (IBMX; Sigma), 2 mmol/l MgSO4 (complex culture medium) were also included in the culture medium. Medium was changed every 2–3 days and at 70–80% confluence, MEC were trypsinized (0.025% trypsin: 0.27 mmol/l EDTA) and repurified with UEA-1-coated Dynabeads and subcultured at a split ratio of 1:3 on 0.2% gelatin-coated (Sigma) T175 Falcon tissue culture flasks (Beckton Dickinson, Bedford, MA, USA). On some occasions it was necessary to remove contaminating smooth muscle cells with a further Dynabead purification at a later passage. For freezing, MEC (5x106 to 2x107) were resuspended in 20% FCS/10% DMSO (Sigma)/70% culture medium and slowly cooled to –80°C and then stored in liquid N2. Thawed cells were gently washed in culture medium and seeded at 1.5x104/cm2 on fibronectin-coated flasks.
Immunohistochemistry
MEC were grown on 13 mm gelatin-coated Thermanox coverslips (Nunc, Roskilde, Denmark) and when confluent were rinsed in PBS and fixed in cold acetone for 2 min. Standard immunohistochemistry protocols (Abberton et al., 1999; Goodger (MacPherson) and Rogers, 1994; Gargett et al., 1999) were used after first blocking sections with 0.03% H2O2 for 10 min at room temperature (RT) and protein blocking reagent (PBA) (Lipshaw Immunon, Pittsburgh, PA, USA) for 10 min at RT. The primary antibodies were then incubated for 1 h at 37°C, followed by biotinylated rabbit anti-mouse or goat anti-rabbit secondary antibodies (1/100) for 30 min at RT, streptavidin–HRP conjugate (Zymed, San Francisco, CA, USA) for 30 min at RT and AEC chromogen (Zymed) for 10 min. The following primary antibodies were used: rabbit anti-human Factor VIII-related antigen at 20 µg/ml (Zymed), mouse anti-human CD31, 8.2 µg/ml (Zymed), mouse anti-human CD34, 0.1 µg/ml (QBEND/10 clone; Serotec, Oxford, UK), mouse anti-human smooth muscle actin, 0.18 µg/ml (Dako Ltd, High Wycombe, UK), mouse anti-human cytokeratin, 2 µg/ml (Clone MNF116; Dako). Ulex europeaus A-1 binding antigen was detected on MEC grown on coverslips and in tissue sections of myometrium using 20 µg/ml biotinylated UEA-1 lectin (Sigma) for 30 min at room temperature, followed by streptavidin–HRP conjugate and AEC. For negative controls, primary antibodies were substituted with an isotype matched IgG at the equivalent concentration of primary antibody. Immunohistochemical procedures were performed on six separate MEC cultures at various passages.
Uptake of DiI-Ac-LDL
The presence of scavenger receptors for acetylated low density lipoprotein (ac-LDL) on MEC was detected using 1,1'-dioctadecyl-1,3,3,3',3'-tetramethylindocarbocyanine perchlorate acetylated LDL (DiI-Ac-LDL, Molecular Probes, Eugene, OR, USA). Confluent MEC cultured in 24-well plates on 0.2% gelatin were incubated with 15 µg/ml DiI-Ac-LDL in culture medium for 4 h at 37°C in 5% CO2/air according to a published method (Voyta et al., 1984). LDL uptake was examined using a Zeiss Axiovert 100 epifluorescence microscope, using an optical bandpass filter set with an excitation of 545 nm and emission of 590 nm.
Endothelial cell proliferation assay
The CellTitre 96 MTS tetrazolium-based bioassay (Promega, Madison, WT, USA), where the absorbance of the formazan product is directly proportional to the number of viable cells, was used to indirectly measure cell number after incubation of MEC with various mitogens. It was initially established that the MTS bioassay was linear from 0 to 2x104 MEC/well. MEC in basal M199 medium (M199 medium containing 5% FCS, 2 mmol/l glutamine, antibiotic/antimycotic mixture) were seeded in triplicate onto fibronectin (10 µg/ml)-coated wells of 96-well plates at 2000 cells/well and allowed to attach and quiesce for 36 h at 37°C in 5% CO2/air. Medium was replaced with the standard or complex M199 culture medium (day 0) with or without the following growth factors: 10 ng/ml vascular endothelial growth factor (VEGF121; gift from Dr J.Abrahams, Scios Inc., Sunnyvale, CA, USA), 0.01–20 ng/ml VEGF165 (R&D Systems, Minneapolis, MN, USA), bFGF (0.01–10 ng/ml), 120 µg/ml EC growth supplement (ECGS; Collaborative Research, Bedford, MA, USA), 20 µg/ml EC growth factor (ECGF; Boehringer Mannheim), 50 ng/ml epidermal growth factor (EGF; R&D Systems), depending on the assay conditions being tested. In some experiments different concentrations of HS and FCS and combinations of both were tested and in other experiments various components of the complex medium were omitted. Cells were incubated at 37°C in 5% CO2/air for 6 or 10 days, with medium changes every 2 days. Twenty µl 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulphophenyl)-2H-tetrazolium (MTS) was added to the cells and incubated 2 h at 37°C and absorbance measured at 490 nm using a plate reader (Emax, Molecular Devices, Sunnyvale, CA, USA). Absorbance was also measured on day 0 and this value subtracted from all results to determine MEC growth over 6 days.
MEC migration assay
Early- (P3) and late- (P13) passage MEC were cultured in standard M199 culture medium in 0.2% gelatin-coated wells of a 24-well plate until confluent. The medium was changed to basal medium, the MEC monolayers wounded using a plastic Pasteur pipette and photographed using a Zeiss inverted phase contrast microscope. Duplicate wells were incubated with or without VEGF165 (10 ng/ml) or bFGF (5 ng/ml) in the presence and absence of 40 µg/ml rabbit anti human VEGF antibody (Gargett et al., 1999) or 50 µg/ml goat anti-human bFGF antibody (R&D Systems) respectively for 48 h at 37°C in 5%CO/air and the same field of view was rephotographed.
MEC tube forming assay
Wells of a 24-well plate were coated with 400 µl undiluted Matrigel (growth factor reduced; Collaborative Research, Cat. no. 40230) and allowed to polymerize at 37°C for 1 h. Early (P3) and late (P13) MEC in simplifed M199 medium were seeded onto the Matrigel-coated wells at 6x104/well and incubated 24 h at 37°C in 5% CO2/air, examined for tube formation under an inverted phase contrast microscope and photographed.
Statistical analysis
Data were analysed by Student's t-test using Excel software. P < 0.05 was considered significant.
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Results |
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Isolation and culture of human myometrial microvascular endothelial cells
Human myometrium is a highly vascularized tissue as demonstrated by UEA binding to tissue sections (Figure 1A
). We have adapted the UEA-1-coated Dynabead selection method of (Jackson et al., 1990) to isolate and establish primary cultures of microvascular endothelial cells from human myometrium, which is primarily composed of smooth muscle cells and endothelial cells. Using this method our yields are ~106 MEC per gram tissue and we usually process between 4–7 g. Primary cultures of MEC were readily established, although growth rates varied markedly between isolates, which reached confluence in 5–10 days. We often found that a further UEA-1–Dynabead purification was required during early passage to produce >99.95% pure cultures of MEC (Figures 1B–D,G and 2A,B). The attached dynabeads did not interfere with MEC growth or survival and were diluted out within one or two passages. While all MEC cultures demonstrated typical cobblestone morphology as viewed by phase contrast microscopy, the morphology of primary and P1 MEC was variable with respect to size and shape (Figures 1B, 2A). With increasing passage number, the morphology became very uniform (Figures 2B and 1C,D). At high passages (P14) MEC showed regular morphology but increased in size (Figure 1D) and replicative senescence was reached by P16.
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Characterization of human myometrial MEC
Cultured myometrial MEC displayed characteristic features of endothelial cells. At all passages examined (P1-P14), MEC bound UEA-1 lectin (Figure 1B–D
), expressed Factor VIII-related antigen in Weibel–Palade bodies (Figure 1E), CD31 in the perinuclear region and at sites of cell–cell contact (Figure 1F) and took up DiI-Ac-LDL via the scavenger LDL receptor (Figure 2D). A characteristic feature of EC, which distinguishes them from mesothelial cells (Chung-Welch et al., 1989), is their ability to differentiate into capillary-like tubes when cultured on the basement membrane matrix, Matrigel. Myometrial MEC spontaneously formed capillary-like tubes in the presence and absence of either bFGF or VEGF (Figure 2C) confirming that cultures were indeed MEC and not contaminated by mesothelial cells. Myometrial MEC did not immunostain with cytokeratin antibodies (results not shown) or express the smooth muscle cell marker, -smooth muscle actin, as seen by the lack of staining of the MEC in the background of Figure 1G.
Growth requirements and characteristics of human myometrial MEC
Figure 3 shows that myometrial MEC had an absolute requirement for human serum for growth stimulated by either bFGF (Figure 3A) or VEGF (results not shown), and that this effect was dose–dependent. Even in the absence of growth factor, 40% HS stimulated significant MEC proliferation (P < 0.03) (Figure 3A). While myometrial MEC survived in FCS containing medium, neither bFGF (Figure 3A) nor VEGF (results not shown) induced significant MEC proliferation, even at high FCS concentrations. However, both bFGF (Figure 3B) and VEGF (results not shown) stimulated even greater levels of MEC proliferation when combinations of HS and FCS were present in the medium (Figure 3B). The optimal combination was 15% HS and 5% FCS (Figure 3B).
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, 
) or with 5 ng/ml bFGF (•, 
) for 5 days in M199 culture medium containing human serum (HS) (•, 
), 20% FCS (
), 5% HS/15% FCS (
). Results shown are means ± SEM of triplicates from a single experiment, representative of three.Agents that increase intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) are commonly used in the medium for culture of MEC to stimulate their growth (Karasek, 1989
). The effect of a cAMP phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine (IBMX 330 µmol/l) and a membrane permeant cAMP analogue, dibutyryl cAMP (N-6,2'-O-dibutyryl adenosine 3',5'-cyclic monophosphate, 50 µmol/l, Sigma), was examined on bFGF-stimulated MEC proliferation. Figure 4 shows that bFGF stimulated significantly greater proliferation of myometrial MEC over 10 days in the absence of either IBMX or dibutyryl cAMP (P < 0.05). After 5 days greater growth was observed in the absence of dibutyryl cAMP (P < 0.02) but not IBMX (Figure 4). Hydrocortisone had no significant effect on bFGF-induced myometrial MEC proliferation in the presence or absence of either IBMX or dibuytryl cAMP (results not shown).
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, The responsiveness of human myometrial MEC to a range of growth factors, known to induce proliferation of EC derived from other tissues, was determined by stimulating MEC with maximal concentrations of growth factors for 6 days. Figure 5
shows that ECGS and bFGF were equally effective and produced maximal stimulation of MEC proliferation, followed by the two VEGF isoforms, VEGF121 and VEGF165. ECGF and EGF were weakly mitogenic for myometrial MEC. Both VEGF165 and bFGF stimulated myometrial MEC proliferation in a dose-dependent manner over the concentration range 0.01–20 ng/ml (Figure 6). The dose–response curves obtained for high and low passage MEC were very similar for bFGF and VEGF when reported as the mean percentage of maximal responses (Figure 6). The approximate EC50 for VEGF was 1.5 and 1.6 ng/ml for early and late passage MEC respectively and for bFGF, 0.6 and 0.9 ng/ml. The difference in absorbance between maximal and minimal response measured at day 6 was significantly greater for high passage MEC compared to low passage MEC for bFGF: 0.50 ± 0.07 (n = 3) versus 0.82 (n = 2) respectively (P = 0.044); but not for VEGF: 0.51 ± 0.09 (n = 3) versus 0.70 (n = 2) respectively (P = 0.33). These results indicate that late passage MEC proliferate at a greater rate in response to bFGF compared to early passage MEC.
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Migration responses of myometrial MEC
An integral part of the angiogenic process is the ability of MEC to migrate toward an angiogenic stimulus, such as a source of VEGF (Goodman et al., 1985
; Ferrara and Davis-Smyth, 1997). The migratory capacity of MEC to certain growth factors can be assessed in vitro using Boyden chamber or monolayer wound assays. The migration of early and late passage myometrial MEC in response to bFGF and VEGF was examined using the monolayer wound assay (Goodman et al., 1985) in the presence and absence of neutralizing bFGF and VEGF antibodies. After a 48 h incubation in basal medium in the presence of bFGF or VEGF, both high and low passage MEC migrated into the denuded area to a comparable extent, and this migration was completely inhibited by the respective neutralizing antibodies to each growth factor (Figure 7, Table I).
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Discussion |
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Although MEC have been isolated and cultured from a variety of tissues, this is the first detailed report of the characterization of MEC isolated and cultured from human myometrium. Oestrogen receptor expression in human myometrial MEC has been described in one study, but the details of their culture method were scanty (Tschugguel et al., 1997
). Myometrial tissue is readily available from hysterectomies and from a single sample large numbers of MEC can be isolated and cultured for use in in-vitro studies of angiogenesis. Furthermore, excess myometrial MEC were successfully frozen in dimethylsulphoxide, stored in liquid N2 and thawed for subsequent use. Human myometrial MEC showed typical cobblestone morphology in monolayer culture and spontaneously formed capillary-like tubes on basement membrane matrix, expressed classical endothelial cell markers, CD31, Factor VIIIra, bound UEA-1 lectin and took up DiI-ac-LDL. Futhermore, myometrial MEC migrated and proliferated in response to VEGF and bFGF. Thus myometrial MEC showed typical morphology, phenotype and function of endothelial cells for up to 14 passages.
Certain procedures were important for the successful isolation of MEC, since the presence of too many contaminating smooth muscle cells in the initial stages would result in overgrowth of the slower growing MEC. The success of UEA-1-coated Dynabead purification of the dissociated myometrial tissue relied on the two-step digestion procedure of (Grimwood et al., 1995), where collagenase treatment was followed by a short trypsinization to produce a single cell suspension. Excessive smooth muscle cell and pericyte contamination of primary cultures was a problem if Dynabead purification was attempted on cell suspensions containing small clumps of cells. The 5:1 ratio of Dynabeads to MEC was also an important factor, since we were unable to remove excess Dynabeads by competive binding with fucose (Jackson et al., 1990), and heavily bead-coated cells were unable to attach to fibronectin-coated flasks. However, we found that MEC purified by UEA-1-coated dynabeads attached more readily and produced more reliable cultures than when CD31-coated dynabeads were used (K.Bucak and C.E.Gargett, unpublished data). CD31 or platelet endothelial cell adhesion molecule-1 (PECAM-1) is an adhesion molecule associated with homophilic and heterophilic adhesion (Newman, 1997), and also has a role in angiogenesis (DeLisser et al., 1997). Presumably ligation of CD31 with bead-attached antibodies on the MEC inhibited the cell–cell interactions necessary to establish successful monolayer cultures.
Although primary cultures of MEC were established from almost all samples of myometrium, the growth rate between isolates was very variable and did not relate to age of donor or hormonal status. Different batches of human serum may have contributed to some of the variability between isolates, but our observations would suggest that this effect was minor compared to the differences between isolates. A common feature of all isolates was the absolute requirement of HS to establish and maintain MEC growth. This requirement remained through all passages until senescence was reached. In contrast, Tschugguel et al. (1997) cultured human myometrial MEC in 20% FCS, but provided no further details. We believe that the use of HS is one of the contributing factors in our ability to culture myometrial MEC for a greater number of passages than most other human MEC. Although myometrial MEC proliferated poorly in FCS, when both FCS and HS were used in combination, greater rates of growth were always observed, suggesting that the action of essential component(s) present in HS were enhanced by an unknown constituent of FCS, which alone was ineffectual. Similarly, Abbott et al. (1996) found that a mix of FCS and HS gave optimal growth for human synovial MEC. MEC cultured from a number of human tissues vary in their serum requirements, highlighting the heterogeneity of MEC from vascular beds of different tissues. For example, MEC derived from decidual tissue (Gallery et al., 1991; Grimwood et al., 1995), heart (McDouall et al., 1996), synovium (Abbott et al., 1996) and the pseudointima of vascular stents (Sanyal and Mirshahi, 1998) all failed to proliferate in FCS, but proliferated well in HS, while MEC cultured from neonatal foreskins (Jackson et al., 1990; Kräling and Bischoff, 1998; Richard et al., 1998), omentum (Chung-Welch et al., 1997) and the gastric or intestinal mucosa (Haraldson et al., 1995; Hull et al., 1996) proliferated well in FCS. The ability of these human MEC to proliferate in the presence of FCS is not dependent on the molecular selection of endothelial cell subtypes (i.e. capillary versus venule), since dermal MEC isolated using UEA-1, CD31 or inducible E-selectin markers all grow in FCS. Rather, it appears that the proliferative responses of some human MEC are dependent on a component present in HS, which is not found in FCS.
One of the advantages of using myometrial MEC for angiogenesis assays is their ability to proliferate in a relatively simple culture medium compared to many other human MEC. In particular, agents which raise the intracellular concentration of cAMP were not only unnecessary but actually had an inhibitory effect on myometrial MEC growth. We believe this to be a major advantage, since cAMP is a second messenger signalling molecule which functions as a downstream regulator for many receptor-mediated events in EC. The presence of cAMP promoters in growth medium for MEC therefore complicates any investigation examining receptor-mediated functions that signal through adenylyl cyclase. In addition, cAMP promoters may also interact with other signalling pathways, including growth factor receptors with intrinsic tyrosine kinase activity (e.g. VEGF), given the degree of cross-talk that exists between signalling pathways (Krupinski, 1991). We also found it unnecessary to include corticosteroids in the culture medium of myometrial MEC, since hydrocortisone had no beneficial or detrimental effect on growth. Similarly, Kräling and Bischoff (1998) found that both hydrocortisone and dibutyryl cAMP exerted a small, statistically insignificant, inhibitory effect on bFGF-stimulated proliferation of human dermal MEC.
A second advantage of using human myometrial MEC for in-vitro angiogenesis assays is that reproducible proliferative responses were obtained for a large number of passages (up to P13) with almost identical dose–response curves for the endothelial mitogens, bFGF and VEGF. However, it must be noted that, like most primary cell cultures, early passage myometrial MEC proliferated at a slower rate than those of late passage. However, comparative data can be obtained by allowing an extra day in culture for low passage MEC before measurement of cell number or absorbance. Both VEGF and bFGF stimulate similar mitogenic responses in myometrial MEC, although bFGF produces a greater maximal response and is slightly more potent. MEC derived from a variety of human tissues respond to both bFGF and VEGF (Fawcett et al., 1991; Carley et al., 1992; Hull et al., 1996; Kräling and Bischoff, 1998), although decidual MEC are a notable exception (Grimwood et al., 1995).
In summary, we have isolated and cultured MEC from human myometrium and characterized their phenotype and growth characteristics. We have described a simplified culture medium that is suitable for the study of proliferative responses, which make myometrial MEC a good model for in-vitro angiogenesis assays. Myometrial MEC also have a stable phenotype and proliferative responsiveness to VEGF and bFGF for up to at least 13 passages. Large numbers of MEC are obtainable from a single specimen of myometrial tissue, which is readily available from hysterectomy specimens. Myometrial MEC should prove a useful model of MEC from sex steroid-responsive tissue showing similar characteristics to decidual MEC, only differing in their response to bFGF. We have also used the protocol outlined in this report to isolate and culture MEC from human endometrium where the yield is much higher, but the amount of tissue available for culture much less (K.Bucak and C.E.Gargett, unpublished observations). Furthermore, we have successfully isolated and cultured MEC from fibroids, benign smooth muscle tumours of the myometrium, for our studies examining the role of angiogenesis in the development of these tumours (M.Zaitseva and C.E.Gargett, unpublished data). Fibroid and myometrial MEC should also prove very useful in the study of angiogenesis in the pregnant uterus and in disorders of the myometrium such as fibroids.
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Acknowledgments |
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We with to thank Debbie Plunkett and Fiona Lederman for their technical assistance with the immunohistochemistry, the gynaecologists at Monash Medical Centre for provision of hysterectomy tissue and Sister Nancy Taylor for collection of the tissue, as well as Mal Forsyth and the Australian Red Cross Blood Service, Melbourne for provision of male human serum. This study was supported by a Post-doctoral fellowship from Monash University (C.E.G.) and Grant No. 960041 from the Australian National Health & Medical Research Foundation (P.A.W.R.).
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Notes |
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1 To whom correspondence should be addressed
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References |
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Submitted on August 2, 1999; accepted on November 4, 1999.
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