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RESEARCH |
1 , School of Veterinary Medicine and Science and2 School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics LE12 5RD, UK
Correspondence should be addressed to R S Robinson; Email: bob.robinson{at}nottingham.ac.uk
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Introduction |
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The critical importance of angiogenesis in luteal development was shown by the immunoneutralization of vascular endothelial growth factor A (VEGFA) at ovulation in marmosets, which blocked the development of normal capillary bed. Moreover, there was a marked suppression in post-ovulatory progesterone concentrations (Fraser et al. 2000, Zimmermann et al. 2001). Preliminary experiments in the cow also found that VEGFA antibody administration adversely affected luteal growth, and the post-ovulatory rise in progesterone was delayed (Kamada et al. 2004). This emphasizes the link between angiogenesis and progesterone concentrations and subsequently embryo development. This is particularly important in the cow, where as little as a day's delay in the rise of post-ovulatory concentrations has been associated with retarded embryo development, reduced interferon-
production and a failure to establish pregnancy (Larson et al. 1997, Mann & Lamming 2001). This also represents a major reproductive problem for a number of other species.
While there is a plethora of angiogenic growth factors, the principal ones are considered to be VEGFA and fibroblast growth factor 2 (FGF2) with other factors playing lesser roles. VEGFA and FGF2 are potent stimulators of both endothelial cell proliferation and migration (Carmeliet 2003, Ferrara et al. 2003). Both VEGFA and FGF2 mediate their action through their high affinity tyrosine kinase receptors, namely VEGF receptor (VEGFR)-1 and VEGFR-2 and FGF receptor (FGFR)-1 and FGFR-2 respectively (Ferrara et al. 2003, Wiedlocha & Sorenson 2004). In the pre-ovulatory follicle, granulosa cells are the major source of VEGFA, while FGF2 is mainly theca derived (Berisha et al. 2000a, Greenaway et al. 2005). Contrary to expectation, we have recently showed that FGF2 production, rather than VEGFA, is the dynamic driver during the follicle–luteal transition and early CL development in the cow (Robinson et al. 2007). Specifically, follicular fluid concentrations of FGF2 increased dramatically post-LH surge and were maintained at high concentrations in the collapsed follicle but declined thereafter. Conversely, there was no such up-regulation of VEGFA during the follicle–luteal transition. This suggested that FGF2 may be the initiator of luteal angiogenesis in the cow.
While it has been known that VEGFA and FGF2 are present and active in the ovary there are still a number of questions that remain unanswered. For example, what are the exact roles of FGF2, VEGFA and their different isoforms? Are they both required for angiogenesis? Furthermore, there are numerous other angiogenic controlling factors expressed during early CL development that have a potential roles in either stimulating or inhibiting luteal angiogenesis. However, currently there is no in vitro method that contains all the cell types in the CL to evaluate the exact roles of these different angiogenic factors and their interactions. Hence, the objective of this study was to develop a physiologically relevant culture system which mimicked luteal angiogenesis, was regulated by angiogenic factors and produced progesterone.
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Discussion |
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While a number of cell culture systems have been developed to investigate angiogenesis, the vast majority use a combination of cell lines such as human umbilical vein endothelial cells (HUVECs) or bovine aortic endothelial cells (BAEC) with human fibroblasts (Bishop et al. 1999, Kunz-Schughart et al. 2006) rather than primary-derived cells. While these systems provide an invaluable tool to screen potential pro- and anti-angiogenic compounds for use in tissue repair and treatment of diseases such as cancer, they are less relevant to study the physiological mechanisms controlling angiogenesis. Other in vitro angiogenesis systems have used either primary endothelial cells alone (e.g. Bagavandoss & Wilks 1991, Davis et al. 2003, Hirschberg et al. 2005) or endothelial cell derived from embryonic stem cells (Hermant et al. 2007). In these systems, endothelial cell network and tubule structures are formed but generally only after long-term culture. Furthermore, angiogenesis in vivo involves the communication between numerous different cell types (e.g. pericytes, fibroblasts, large and small luteal cells), hence the physiological relevance of these systems is limited. This probably explains why long-term culture is required for tubule formation in solely endothelial cultures (Hirschberg et al. 2005). For example, pericytes in the developing CL are closely associated with the leading tip of capillary sprouts (Amselgruber et al. 1999) and steroidogenic cells are a source of VEGFA (Berisha et al. 2000b) and FGF2 (Berisha et al. 2006). Hence, an important feature of our culture system was the co-culturing of primary endothelial with all other luteal cell types derived from the CL. Hence, the endothelial cells will be exposed to a similar environment to the in vivo situation (e.g. similar cell types such as small and large luteal cells and pericytes all at similar ratios). Furthermore, steroidogenic cells were present and, more importantly, are functional as determined by progesterone production, which increased with time in culture and responded to LH. In fact, these in vitro effects showed great temporal similarity to those observed in vivo, namely the rise in plasma progesterone and LH responsiveness first occur 4–5 days after ovulation (Niswender et al. 2000, Mann & Lamming 2001, Robinson et al. 2006). The characteristics of the endothelial cells themselves are an important feature since, despite common morphological and functional properties, there is much heterogeneity both between tissues and within a particular tissue (Bagavandoss & Wilks 1991, Davis et al. 2003). For example, Plendl et al. (1996) showed that endothelial cells derived from a pregnant CL formed connected ring-like structure after prolonged culture, while no such structures were observed from those isolated from the cyclic CL. Hence, we have used primary endothelial-derived cells from very early CL, which were able to form tubule-like structures with branch points, both in a similar manner to other cell line systems (Bishop et al. 1999, Donovan et al. 2001) and reproducibly. A further refinement to this system would be to make it truly three dimensional with the use of collagen gels as recently described (Kunz-Schughardt et al. 2006, Zhao et al. 2007).
The importance of the extra-cellular matrix components on vasculature development was demonstrated by the formation of endothelial networks being greatly increased by fibronectin coating, while collagen I coating had little or no effect. This is in strong agreement with Christenson & Stouffer (1996) who demonstrated that fibronectin but not collagen I increased luteal endothelial cell proliferation in response to VEGFA. This is also supported by the in situ observations that fibronectin abundance is particularly high during the early growth phase of the CL while collagen I is relatively low, which again emphasizes its physiological relevance. Then as the CL develops, the fibronectin:collagen ratio decreases, enabling a firm structure to be formed (Silvester & Luck 1999, Irving-Rodgers et al. 2006). Moreover, Amselsgruber et al. (1999) reported that initial endothelial sprouts were characterized by a fibrillary network of fibronectin. This is similar to changes in extra-cellular matrix during wound repair (Silvester & Luck 1999). These suggest that fibronectin rather than collagen I plays a crucial role in the structural backbone for the development of vasculature. Fibronectin has been shown to stimulate endothelial cell adhesion, migration and growth (Milner 2007). Moreover, this suggests that disposition of fibronectin and its cell source are the drivers of luteal angiogenesis.
It is widely accepted that LH is crucial for luteal development and function; however, less is known about its direct role in angiogenesis. We have previously shown that follicular FGF2 was increased by the LH surge, suggesting that LH initiates the angiogenesis (Robinson et al. 2007). Moreover, LH is known to stimulate FGF2 (Robinson et al. 2007) and VEGFA (Schams et al. 2001) production in luteal and granulosa cells respectively. The present study confirmed the ability of LH to promote the development of endothelial networks and strengthens its role in angiogenesis. This builds on in vivo observations where GnRH antagonist inhibited the post-ovulatory peak in proliferation by 90% in primates (Dickson & Fraser 2000). However, hCG, but not LH, stimulated sprout formation in endothelial–granulosa spheroids (Walz et al. 2005). In the current study, it is unclear whether LH acted on endothelial cells directly or by acting on luteal cells to stimulate the production of angiogenic factors. The latter is more likely since no LH receptors have been detected on luteal endothelial cells (Mamluk et al. 1998). Furthermore, LH increased FGF2 concentrations 50% in bovine-dispersed luteal cells (Robinson et al. 2007). The effect of LH in the presence FGF2 and VEGFA antagonists during the culture warrants further investigation.
It is well recognized that FGF2 and VEGFA are critical angiogenic growth factors in the CL; however, their precise roles remain to be elucidated. Both FGF2 and VEGFA alone and in combination stimulated the development of endothelial networks and thus provided further validation of the culture system. Moreover, the image analysis of these networks revealed a particularly interesting observation that the pre-dominate effect of FGF2 was to increase the size of the endothelial cluster rather than their numbers. VEGFA, however, had a lesser effect on cluster size but increased the number of clusters. The development of endothelial networks occurred in stages in the present study, similar to previous observations in isolated endothelial cells (Hirschberg et al. 2005). Specifically, the stages are migration and clustering, then sprouting, cellular alignment and tubule initiation followed by network establishment and finally three-dimensional organization (Hirshberg et al. 2005). Our results suggest that VEGFA increases endothelial clusters by promoting endothelial cell survival, migration and proliferation of the initial angiogenic clusters, while pre-dominant action of FGF2 was involved in the development of these clusters by inducing sprouting and tubule initiation. This is in agreement with other reports of distinct FGF2 and VEGFA actions in various systems (Parsons-Wingerter et al. 2000, Castellon et al. 2002). For example, Castellon et al. (2002) showed that only platelet-derived growth factor BB and FGF2 were capable alone of inducing secondary sprouting in bovine retinal endothelial cells. While VEGFA increased migration, FGF2 was ineffective. Testing of these hypotheses with targeted use of FGF2 and VEGFA receptor inhibitors certainly warrants further investigation. Furthermore, there were no interactions between the effects of VEGFA and FGF2 that both were required for maximal angiogenic response. This indicates that their effects were additive and that they have independent and different actions on the angiogenic process itself.
In conclusion, the successful development of endothelial cell networks in vitro in a physiologically relevant manner heralds a new era in elucidating the physiological control of the angiogenic process in the developing CL. Future studies will be conducted that mimic the in vivo dynamics of FGF2 and VEGFA expression during the follicle–luteal transition, i.e. a transient high dose of FGF2 and elucidation of the exact roles of FGF2 and VEGFA at the different stages of angiogenesis. Also, we are planning to adapt the culture system to study follicular angiogenesis. Finally, future work will involve determining the role of the other cell types and in particular the potential interactions of endothelial cells and pericytes.
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Materials and Methods |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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Establishment of luteal angiogenesis culture system
Coating of coverslips
Coverslips (circular, 19 mm diameterx0.25 mm thick, SLS, Nottingham, UK) were soaked in 70% (v/v) industrial methylated spirits (IMS) for 30 min, air-dried for 1 h and then transferred to a 12-well plate. Fibronectin (1 ml of 10 µg/ml; Sigma; optimized in Expt 1a, see below) was added to each well and incubated in humidified incubator at 39 °C. After 4 h, the fibronectin was removed and the wells were left to dry at 39 °C overnight. On the following day, the wells were washed once with sterile distilled water and allowed to equilibrate with endothelial cell medium (Cambrex, Wokingham, UK) until cell plating.
Endothelial cell medium (EC medium)
The medium used for the luteal angiogenesis culture was a specialized endothelial cell medium (EBM-2; Cambrex) to which gentamycin, heparin, human epidermal growth factor (EGF), LR3-insulin-like growth factor-I (IGF-I), hydrocortisone and ascorbic acid were added as per the manufacturer's instructions. In addition, the medium was supplemented with 5 ng/ml LH (AFP11743B, biopotency 1.06xoLH NIDDK-I-2; a gift from Dr A F Parlow, NIDDK, Torrance, CA, USA), 100 units/ml penicillin, 10 µg/ml streptomycin, 10 µg/ml insulin, 5.5 µg/ml transferrin, 5 ng/ml selenium (all Sigma) and 2% (v/v) foetal bovine serum (Cambrex).
Tissue collection and luteal angiogenesis culture system
Ovaries were collected from a local abattoir and transported back to the laboratory in 1xPBS at room temperature. In all experiments, only early CL (days 1–4) were used and these were selected based on criteria outlined by Ireland et al. (1980). The CLs were removed from the ovary with all connective tissue trimmed away and then sliced up using scissors. All cells (including luteal, endothelial, pericytes and fibroblasts) were dispersed by incubating the luteal tissue in DMEM/F12 medium (Invitrogen) containing 2 mg/ml collagenase I type 1A and 25 µg/ml DNase I (both Sigma) for 2x45 min in a shaking water bath at 37 °C. The dispersed cells from each incubation were pooled together and then filtered through 70 µm cell strainer (BD Biosciences, Oxford, UK) and washed thrice in endothelial cell medium. The cells were then plated out at 2x105 viable cells (as determined by trypan blue exclusion; Tennant 1964) per well in a 12-well plate (total volume: 2 ml) at 39 °C in a humidified incubator in 5% CO2/95% air. The cells were allowed to attach for 24 h and thereafter every 2 days the medium was changed. After 2, 5 and 9 days, the medium was collected and the cells fixed in acetone:methanol (1:1) at room temperature for 5 min. The endothelial cells were then immunostained using VWF as an established endothelial cell marker in order to assess the formation of tubule-like structures over time (Al-zi'abi et al. 2003, Martelli et al. 2006).
Immunostaining of endothelial cell with VWF
After fixation and each step, the cells were washed twice with 1xPBS for 5 min each wash. The first step was to block endogenous peroxidase activity with 3% (v/v) hydrogen peroxide in methanol. Non-specific binding was blocked by incubating sections with 20% normal goat serum (Sigma) for 30 min and with no washing step, the cells were incubated with 4 µg/ml polyclonal rabbit anti-human VWF (Dako, High Wycombe, UK) in 2% (v/v) normal goat serum. For the control sections, 4 µg/ml rabbit IgG was used. The slides were incubated in a humidified chamber at 4 °C overnight. On the following day, the primary antibody was detected using the Vectorstain ABC method as per instructions (Vector, Peterborough, UK) and visualization was performed with diaminobenzidine (Vector). The cells were counterstained with haematoxylin and dehydrated in graded IMS. The coverslips were then removed from the well and further dehydrated in xylene before mounting in DPX mountant.
Immunostaining of steroidogenic cells with 3β-HSD
The presence of steroidogenic cells was determined by immunostaining for 3β-HSD. The procedure performed was the same as for VWF except that the primary antibody used was polyclonal rabbit anti-human placental 3β-HSD (kindly provided by Dr Richard Parker, University of Alabama-Birmingham, AL, USA; diluted 1 in 400 in PBS).
Quantification of VWF immunostaining by image analysis
The quantification method was developed, based on a protocol previously used to quantify area of VWF staining in bovine CL sections (Robinson et al. 2006). All the image analysis was performed using Image Pro-Plus 5.0 (Media Cybernetics, Wokingham, UK). The sections were visualized under a 6x objective lens and the area of brown (VWF) staining was highlighted. The highlighted area was then smoothed, any holes in the staining removed and any area of VWF staining <150 µm2 excluded (in order to eliminate very small cluster of isolated endothelial cells of <3 cells). Only areas stained positively for VWF with tubule-like appearance were included in the analysis. The number, area and perimeter of each cluster were recorded. This was repeated for a total of 10 fields of view across the whole well. The total area and perimeter of VWF staining were then calculated by multiplying the number of clusters by the mean area or perimeter. Both wells were examined for each treatment and from this the mean values recorded for each treatment.
Experiment 1: effect of extra-cellular matrix on formation of endothelial networks
In order to optimize the in vitro luteal angiogenesis culture system, the effect of the extra-cellular matrix coating of the coverslips was investigated. The coverslips were prepared as above and coated as follows: (1) plastic alone, (2) 20 µg per well calf skin collagen I (Sigma) or (3) 10 µg per well bovine fibronectin (Sigma; n=3, early CL for treatment). The cells were dispersed and prepared as described above and were plated out at 2x105 cells per well, with 2 wells per each coating. The cells were cultured in endothelial cell medium supplemented with 1 ng/ml FGF2 and 1 ng/ml VEGFA165 (Cambrex) throughout the culture. After 9 days in culture, the cells were fixed for 5 min in acetone:methanol (1:1) at room temperature, then immunostained for VWF.
Experiment 2: effect of LH on progesterone production and formation of endothelial networks
Cultures were prepared from early CL (days 1–4; n=5) as described above and the dispersed cells were plated out at 2x105 per well and cultured in endothelial cell medium. A 3x2 factorial design was set-up with effects of LH (at 0, 5 and 100 ng/ml) determined in the presence and absence of 1 ng/ml FGF2+1 ng/ml VEGFA165. Each treatment was performed in duplicate. The medium was collected for progesterone analysis and changed after 1, 4, 6 and 8 days. After 9 days, the cells were fixed in acetone:methanol 1:1 at room temperature (RT) for 5 min, then immunostained for VWF.
Progesterone analysis
The medium was collected and stored at –20 °C for progesterone analysis. The progesterone concentration in spent medium was determined in a single assay using RIA as previously validated by Corrie et al. (1981). The samples were diluted 300-fold into RIA buffer and the intra-assay coefficient of variation was 13.7%.
Experiment 3: effect of FGF2 and VEGFA development of endothelial networks
Luteal cells were dispersed from early CL (days 1–4, n=4) and then plated out at 2x105 cells per well and were cultured in endothelial cell medium. The cells were then treated with the following: (a) control, (b) 1 ng/ml FGF2, (c) 1 ng/ml VEGFA165 or (d) 1 ng/ml FGF2 and 1 ng/ml VEGFA165 with each treatment performed in duplicate. The medium was changed after 1, 3, 5 and 7 days. After 9 days in culture, the cells were fixed in acetone:methanol (1:1) at RT for 5 min, then immunostained for VWF. The number, area and perimeter of each cluster as well as the total area and perimeter of the VWF staining were analysed.
Statistical analyses
All the data were checked for normality and heterogeneity of variance and were log transformed where appropriate.
Experiment 1
The effect of coating was analysed by one-way randomized block ANOVA with no coating, collagen and fibronectin as factors and blocked for each culture. Bonferroni's multiple comparisons were performed to determine where any significant difference lay.
Experiment 2
The effect of LH and FGF+VEGFA on the production of progesterone was determined using repeated measurements ANOVA with progesterone production per day as the variable and LH and FGF2+VEGFA as the factors. A randomized block two-way ANOVA was performed to analyse these factors on endothelial development parameters. Bonferroni's multiple comparisons were performed to determine where any significant difference lay.
Experiment 3
The effects of FGF2 and VEGFA on endothelial tubule parameters were determined using two-way randomized block ANOVA with presence of FGF2 and VEGFA as factors and culture as the block. If there was a significant effect, then one-way randomized block ANOVA were performed to elucidate where the differences lay. For all experiments, P<0.05 was considered as significant and all data are quoted as mean±S.E.M. unless stated otherwise.
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Received 10 August 2007
First decision 7 September 2007
Accepted 30 October 2007
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...), 5 (–
–) or 100 (—
—) ng/ml LH (n=5 per treatment). There was a significant day (P<0.001), LH (P<0.001) and addition of FGF2 and VEGFA effect (P<0.05). The data are mean±S.E.M.
