Molecular Human Reproduction, Vol. 8, No. 9, 855-863,
September 2002
© 2002 European Society of Human Reproduction and Embryology
Uterine physiology |
Microarray analysis of VEGF-responsive genes in myometrial endothelial cells
1 Centre for Women’s Health Research, Department of Obstetrics and Gynaecology, Monash University, 246 Clayton Rd, Clayton, Victoria 3168 and 2 Peter MacCallum Cancer Institute, Melbourne, Australia
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There is evidence that the vasculature of different organs display different functional characteristics in response to cytokines and growth factors. The aim of this study was to use cDNA gene expression microarray to analyse changes in gene expression following stimulation of myometrial microvascular endothelial cells (MMECs) with vascular endothelial growth factor (VEGF). Primary isolates of MMECs were obtained from fresh hysterectomy specimens and purified with magnetic beads. Cells were stimulated with 15 ng/ml VEGF for 3, 6 and 12 h, and two unstimulated experiments served as controls. A total of six arrays was performed over these time-points. A total of 110 genes were identified as up-regulated by VEGF, 19% of which (21 genes) have previously been reported as up-regulated by VEGF or by angiogenesis. Among the novel genes to be up-regulated by VEGF were brain-derived growth factor, oxytocin receptor and estrogen sulphotransferase. The significance of the genes identified in the physiological and pathological functioning of the myometrial vasculature is discussed.
angiogenesis/endothelial cell/fibroids/myometrium/vascular endothelial growth factor (VEGF)
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Introduction |
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There is evidence to suggest that endothelial cells display considerable heterogeneity, both between tissue beds of different organs and between normal and pathological tissue (Garlanda and Dejana, 1997
). Organ-specific antigens have been demonstrated on capillary endothelial cells, and may influence cell adhesion as well as interactions with circulating leukocytes (Auerbach et al., 1985).
The myometrium possesses an abundant and adaptive vasculature. Myometrial blood vessels undergo major growth and remodelling during pregnancy. During pregnancy, the wet weight of the uterus increases 10-fold (Yen et al., 1999), accompanied by radical growth and remodelling of its blood vessels to meet the increasing demands of the developing fetus. Following implantation, the trophoblast invades and displaces maternal endothelial cells in the myometrial layer. In pre-eclampsia this process is abnormal, with inadequate replacement of the maternal endothelium and a consequent higher flow resistance in the placental bed.
Most pathology occurring in the myometrium involves alterations in the myometrial vasculature. Fibroids are the most common benign tumour to occur in women, contributing to almost half of the hysterectomies performed due to menorrhagia. Differences in vascularity in fibroids compared with normal myometrium have been reported (Casey et al., 2000). Adenomyosis, another common cause of menorrhagia, is defined as implanted endometrial glands and stroma within the myometrium. It has an increased microvascular density compared with adjacent endometrium (Schindl et al., 2001).
Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis both in fetal development and in the adult (Ferrara, 2001). It is a potent endothelial cell mitogen, found in a wide range of human tissues including myometrium (Harrison-Woolrych et al., 1995). There is a growing family of VEGF growth factors, including five isoforms of VEGF A, but also VEGFs B to E and placental growth factor (PlGF) (Neufeld et al., 1999). VEGF has been the focus of intense study due to the critical role played by angiogenesis in many human diseases, including growth of solid tumours. Pharmacological manipulation of angiogenesis has yet to be successful, in part because of the number and complexity of endogenous factors with a role in angiogenesis (Epstein et al., 2001). Studies based on angiogenesis in solid tumours may not reflect the angiogenesis seen in other tissues. A major rationale for our study was to establish baseline molecular responses to VEGF in the myometrium to allow comparisons with other studies.
There is little doubt that VEGF will have some common effects on all endothelial cells. Endothelial cell proliferation, migration and modification of the extracellular matrix (ECM) and basement membrane are well known steps of sprouting angiogenesis, one of the major mechanisms by which new blood vessels form. Other well known effects include an increase in vascular permeability as well as alterations in the extrinsic coagulation cascade. However, we hypothesize that endothelial cell heterogeneity will dictate subtle variations in the effects of VEGF in different organs.
In recent years, cDNA microarrays have emerged as a powerful tool to rapidly screen up to several thousand genes at once for changes in gene expression (Lander, 1999). This technology has been applied to many tissues including ovarian cancer (Welsh et al., 2001). It has also been used to improve our understanding of key physiological events such as decidualization of the endometrial stroma (Popovici et al., 2000) and embryo implantation (Reese et al., 2001). The aim of this experiment was to identify early response genes to a VEGF stimulus in myometrial endothelial cells, using microarray technology.
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Materials and methods |
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Cell culture
Ethical approval for the study was obtained from the institutional ethics committee at Monash Medical Centre and Melbourn Southern Health Human Research Ethics Committee B. Informed consent was obtained from patients undergoing hysterectomy at the pre-admission clinic.
Myometrial microvascular endothelial cells (MMECs) were isolated and cultured according to a previously described method (Gargett et al., 2000) from 5–10 g of myometrial tissue excised from the mucosal half of the myometrium of hysterectomy specimens. All hysterectomy specimens were from premenopausal women having operations for benign pathology, and tissue was only taken from women who had not received exogenous hormones for the previous 3 months. The purity of MMEC cultures was determined by flow cytometry using 5.4 µg/ml CD31 mouse anti-human antibodies (Clone JC/70A; Dako Ltd, Glostrup, Denmark) and mouse IgG1 (Chemicon Inc., Temecula, CA, USA) of equivalent concentration as a negative control. Experiments were performed on >97% pure cultures, at passage 3–4.
Treatment conditions
MMEC were grown to 70–80% confluence at passage 3–4. A minimum of three 175 cm2 culture flasks was required for each experiment. Quiescence was achieved by incubation for 48 h in M199 culture medium (Gibco BRL, Gaithersburg, MD, USA) with 5% fetal calf serum (FCS; CSL, Melbourne, Australia) and 0.1 mg/ml heparin. VEGF165 (Minneapolis, MN, USA) 15 ng/ml was added to M199/5% FCS with 0.1 mg/ml heparin (Gibco) for 3, 6 or 12 h. For 0 h time-points, the cells were harvested after 48 h quiescence.
Preparation of total RNA
Total RNA was isolated from cells using the RNeasy total RNA kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Total RNA was resuspended in RNase-free water, and precipitated by storing overnight at –20°C in 0.1 volume 3 mol/l sodium acetate and 2 volumes 100% ethanol. RNA precipitate was centrifuged at 15 000 g for 30 min at 4°C, and the pellet was washed twice with 70% ethanol prior to resuspension in RNase-free water. Resuspension was performed in a volume for a predicted concentration of 5 µg/µl total RNA. Quantification and purity of the total RNA was assessed by A260/A280 absorption and by electrophoresis in 1% TBE agarose gels.
A reference population of RNA (Alizadeh, et al., 2000) was created from a mixture of large and small vessel endothelial cells [human umbilical vein endothelial cells (HUVECs) and MMECs]. The cell isolates for the reference population were from passages 3–8 in a variety of activation states—some in a quiesced state (confluent in 5% FCS for 48 h) and some activated with an endothelial cell growth factor (either b-fibroblast growth factor or VEGF). The reference RNA went through the same quality checks as the experimental RNA.
5K glass slide cDNA microarray
The total RNA samples were used to prepare fluorescently labelled cDNA for probing with glass microarray slides spotted with 5000 cDNA sequences (prepared in-house at the Peter MacCallum Microarray Facility, Melbourne, Australia). A total of 60–90 µg of experimental total RNA was used for each microarray, with an equal amount of reference total RNA used as the control. A direct-labelling procedure was used for creating fluorescently labelled cDNA. Both RNA samples for each microarray were centrifuged at 15000g for 3 min prior to use, made up to 19.7 µl with RNase free water, then incubated for 10 min at 65°C with 2 µl oligo dT anchor primer (catalogue no. 11146–016; Gibco; 2 µg/µl) and 0.5 µl RNasin (cat. no. N2511; Promega, Madison, WI, USA; 40 IU/µl). Next were added 8 µl 5x first strand buffer (cat. no. 11146016; Gibco), 4 µl 0.1 mol/l dithiothreitol (cat. no. 11146–016; Gibco), 0.8 µl low C dNTP mix (cat. no. 11146–016; Gibco), and 2 µl of either 1 mmol/l Cy3-labelled dCTP (cat. no. PA53021; Amersham Bioscience, Cardiff, UK) for reference control RNA or 1 mmol/l Cy5-labelled dCTP (cat. no. PA55021; Amersham) for experimental RNA. The mix was brought to 42°C for 5 min before addition of 2 µl Superscript II (cat. no. 18064–014; Gibco).
The RT reaction was left at 42°C for 2.5 h, and the RNA was hydrolysed by incubation at 65°C for 20 min with 10 µl 0.25 mol/l NaOH and 5 µl 0.5 mol/l EDTA. Acetic acid (1.5 µl, 2 mol/l) was added to stop the reaction prior to passing the two separately labelled cDNA populations through a purification column (Qiagen PCR purification kit) according to the manufacturer’s instructions. The resulting mixed-labelled cDNA was combined with a combination of blockers to reduce cross-hybridization [3 µl yeast tRNA 4 mg/ml, 3 µl human cot-1 DNA (cat. no. 15279–011; Gibco) 10 mg/ml, 0.75 µl poly dA 8 mg/ml, 0.75 µl 50x Denharts herring sperm DNA (cat. no. 14430–029; Gibco), and 2 µl Cy3 and Cy5-labelled luciferase mRNA control] and dried in a heated vacuum. The pellet was resuspended in 8 µl 6.25x standard saline citrate (SSC), and heated to 100°C for 3 min with an equal volume of formamide before being placed on ice. Sodium dodecyl sulphate (SDS) (0.2 µl, 10%) was added before applying the labelled cDNA to the glass cDNA microarray slide for hybridization under a coverslip. Throughout, the cDNA was kept out of bright light, and all solutions used were filtered prior to use. Hybridization was in a Hybaid (Ashford, Middlesex, UK) hybridization oven at 42°C overnight. The next day, slide washes were 1 min with 0.5xSSC and 0.01% SDS, 3 min with 0.5xSSC, and 3 min with 0.06xSSC. The slides were dried by centrifugation for 5 min at 300 g and stored in a dark dry place until scanning.
Scanning and analysis
Slides were scanned using a dual UV-laser GSI Luminomics scanner (Hewlett Packard, USA) and fluorescent intensity was analysed using the Quantarray program (Hewlett Packard Bioscience). Images were formed by superimposing the Cy3 and Cy5 images for each slide using Scanalyze software (Eisen lab, Stanford, USA). Images were only included in the analysis if all of the spots on the array were visible (i.e. no displacement of the coverslip causing rows to be missing on scanning). Images included in the analysis were also required to have a low background fluorescent intensity (i.e. not obscuring any of the lowest intensity spots on the array on scanning). Lists of all the genes on the slide were analysed in Microsoft Excel and normalized by log transformation. Once normalized, direct ratios of the 3, 6 and 12 h time-points of VEGF stimulation compared with no VEGF stimulation could be calculated by dividing each of the 3, 6 and 12 h experiments by the average of the two 0 h experiments. This was made possible by the use of the common reference pool RNA which was used as a control in all of the experiments. A total of six microarrays were performed: one 3 h VEGF-stimulated MMECs versus the reference, one 6 h VEGF-stimulated MMECs versus the reference, two arrays of unstimulated MMECs versus the reference, and two arrays with 12 h VEGF-stimulated MMECs versus the reference. Each of the arrays was performed using a separate primary isolate of MMECs.
All cDNA spots on the array were duplicated next to each other, so for each gene, two image intensities were obtained for each slide. Furthermore, some genes had more than one representative cDNA on the slide. The ratios obtained as described were fed into an updated gene database using the Genespring software package (Silicon Genetics, USA), and only genes showing an average up-regulation of 
3-fold over the 0 h experiments were considered up-regulated. Genes were rejected from the list if the range of the ratios obtained was <2. The cut-offs for up-regulation were arbitrary, but have been used in other microarray experiments (DeRisi et al., 1997; Perou et al., 1999).
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Results |
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Six glass cDNA microarray slides were used in this experiment to quantitate relative changes in transcript abundance resulting from VEGF stimulation. A total of 4608 genes were screened (complete list available at http://www.CCGPM.org). A representative scanned image of the 5K array is shown in Figure 1
. The overall transcript abundance in the experiment relative to the reference control can be determined by the total brightness of each spot on the array, while the amount of transcript can be determined by the colour of each spot. By convention, the scanning software shows an increase in transcript abundance in the experimental RNA relative to the control as red, and a decrease as green. A yellow spot indicates a gene with equal amounts of transcript in both the experiment and the control.
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Both the 0 and 12 h time-points consisted of two repeats. Despite the biological variability expected from using separate primary isolates, correlation coefficients of all 4608 data-points for the relative image intensity were 0.537 for the two 0 h experiments, and 0.575 for the two 12 h experiments (see Figure 2
for the scatterplots of the repeated time-points). Due to the use of reference RNA as the control for all six microarrays, direct comparisons could be made between slides.
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A total of 110 genes passed our criteria for up-regulation by VEGF in at least one of the three time-points studied. Of these, 76 genes were up-regulated at 3 h (47 of them unique to the 3 h time-point; Figure 3
), 59 were up-regulated at 6 h (18 of them unique to 6 h) and 35 were up-regulated at 12 h (one unique, while the other 34 genes were shared with either the 3 or 6 h time-point).
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Of the genes up-regulated in MMECs by VEGF in our experiments, 19.1% (21 genes) have previously been reported to be up-regulated in endothelial cells either after VEGF stimulation or in an angiogenic state. Within the group of genes previously identified, most encoded cytokines, receptors or proteins involved in ECM formation/ remodelling or in inflammation; all of which are areas of endothelial cell function that have been major foci of research efforts.
Examining the data by graphing the gene expression ratios against the 0 h results over time, a distinct pattern emerged. The majority of the genes encoding transcription factors were up-regulated at 3 h, seven of them uniquely so (Figure 4a). A second wave of gene transcription appeared in the genes involved in the synthesis of ECM, with most of these genes not being up-regulated until 6–12 h (Figure 4b). The genes which were found to be up-regulated by VEGF are shown in Table I.
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Transcription factors
The transcription factors found to be responsive to VEGF included Sp3, ISL1 and cardiac ankyrin repeat protein (CARP).
ECM remodelling
VEGF up-regulated four of the matrix metalloproteases (MMP-1, -2, -3 and -10), all of which have already been reported in endothelial cells (see Table I
for references). Various types of collagen, as well as matrix Gla protein, syndecan-4 and nidogen were all up-regulated. Other ECM components with increased gene expression following VEGF treatment included biglycan, sarcoglycan epsilon and chondroitin sulphate proteoglycan 2. Many of the genes involved in ECM remodelling/degradation, including the MMPs, were up-regulated at 3 h. However, many of the ECM components, such as the collagens, were not up-regulated until at least 6 h.
Secreted cytokines and growth factors
VEGF induced several genes for secreted cytokines, growth factors and their receptors. MCP-1, a well characterized chemotactic protein known to be up-regulated in microvascular endothelial cells by VEGF stimulation (Marumo et al., 1999), was up-regulated in our microarrays. Insulin-like growth factor-binding protein 3 (IGF-BP3), proteinase-activated receptor (PAR)-1, 5-hydroxytryptamine (serotonin) receptor, PlGF, oxytocin receptor, estrogen receptor, brain-derived neurotrophic factor (BDNF) and CD34 have not previously been reported to be up-regulated by VEGF. VEGF receptor 2, tie-1 receptor and angiopoietin 2 were up-regulated by VEGF, and have been previously reported as such.
Intracellular signalling and metabolism
VEGF stimulation resulted in up-regulation of several cell signalling pathways known to be active after mitogenic stimuli; these included Ran GTPase binding protein 1, Gem GTPase and N-Shc. Another two genes up-regulated were the Ras-related protein RAL-A and Ras homologue B.
With the VEGF stimulus, enzymes for many metabolic pathways were up-regulated. Of particular interest in a steroid responsive tissue such as the myometrium was the up-regulation of 11ß-hydroxysteroid dehydrogenase and estrogen sulphotransferase. GTP-cyclohydrolase 1, an enzyme that catalyses the formation of tetrahydrobiopterin, a co-factor for NO synthesis, was also up-regulated.
Cell–cell interactions
Connexin 37 was up-regulated by VEGF. Cell adhesion molecules (VCAM-1, ICAM-2) were also up-regulated, as has been previously reported (see references in Table I).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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In the set of gene expression microarrays we have performed, >80 genes not previously reported to be up-regulated by VEGF stimulation have been identified. Many of the genes, such as nucleoside phosphorylase, protein kinase and diubiquitin, are involved in general metabolism. They are likely to be common pathways of action for many cytokine stimuli to the endothelial cell. However, some of the genes identified are more likely to regulate specific functions of VEGF by promoting endothelial cell survival and mitosis, increasing vessel permeability and affecting vasomotor function. The fact that many of the genes with an increased expression have been previously reported gives greater confidence in the results.
Of the transcription factors up-regulated by VEGF in our experiment, Sp3 is a nuclear protein known to affect KDR/VEFGR2 expression by attenuating the activation of a KDR promoter by Sp1 (Hata et al., 1998). ISL-1, or islet-1, is a transcriptional regulator known to interact with the estrogen receptor to modulate its transcriptional activity (Gay et al., 2000). This has important implications in an estrogen-responsive tissue such as the myometrium. Another transcriptional regulator up-regulated in the microarrays was CARP. CARP has been shown to be expressed not only in the heart, but also in the vasculature (Kanai et al., 2001). Although it has not been shown to be up-regulated by VEGF, both CARP and VEGF have been shown to be up-regulated in coronary vessels following balloon angioplasty (Tsurumi et al., 1997; Kanai et al., 2001).
One of the largest groups of genes up-regulated in the VEGF-stimulated MMECs was that of those involved in ECM production and remodelling. This was not surprising, as degradation of the ECM to allow endothelial cell migration and subsequent laying down of ECM to assist in stabilization of the new blood vessel are important parts of the angiogenic process which VEGF promotes. Collagen VI
1, collagen VI3 and collagen I2 have been shown to be expressed in angiogenic blood vessels (see references in Table I). Matrix Gla protein has been identified as an endothelial cell marker (Croix et al., 2000). Syndecan 4 has been reported to play a major role in wound healing, where angiogenesis and VEGF also play a key part (Subramanian et al., 1997).
The up-regulation of IGF-BP3, which had been previously reported to be down-regulated by VEGF (Dahlfors and Arnquist, 2000), was surprising. It may be that there were a larger number of confluent patches of endothelial cells in the VEGF-stimulated flasks, since IGF-BP3 is up-regulated in endothelial cells after achieving confluence (Delafontaine et al., 1996). Many of the cytokines and receptors up-regulated in the arrays have a well characterized role in angiogenesis; these include PlGF, angiopoietin-2 and VEGF receptor 2. The GRO-1 oncogene up-regulated by VEGF at 3 h has been shown to promote angiogenesis, making it a possible secondary mediator of VEGF action in the myometrium (Loukinova et al., 2000). BDNF has not previously been reported in the human uterus, although it is secreted by HUVECs (Nakabashi et al., 2000) and is an endothelial cell survival factor (Donovan et al., 2000). N-Shc has previously been identified in neurons, and is an important mediator of BDNF signalling (Nakamura et al., 1996). BDNF and N-Shc were up-regulated at the same time-points in our experiment, demonstrating a possible role for this pathway in mediating BDNF action in myometrial MECs.
Several genes with a potential role in modulating the vasomotor function of the myometrial vessels were detected in our experiments. 11-ß hydroxysteroid dehydrogenase type 2 is an enzyme which converts cortisol, a vasoconstrictor, to cortisone, its inactive metabolite. As this enzyme was up-regulated by VEGF, it could be a mediator of VEGF action on vasomotor function. RGS-5, a signalling protein which modulates the function of G proteins, has been shown to inhibit angiotensin and endothelin-induced vasoconstriction by suppressing Ca2+ signalling (Zhou et al., 2001). It was also up-regulated by VEGF. Another up-regulated gene with vasoactive functions was 5-hydroxytryptamine receptor.
Some of the up-regulated genes have possible tissue-specific roles within the myometrial vasculature. One example of this is estrogen sulphotransferase, an enzyme that catalyses the conversion of estrogen to inactive metabolites. VEGF may play a role in modulating the availability of estrogen by acting on the myometrial vasculature via this enzyme. Another gene with a possible tissue-specific role is the oxytocin receptor. The expression of oxytocin receptor was up-regulated in our VEGF-treated cultures at 6 and 12 h. Oxytocin receptors have been reported to be expressed by a variety of endothelial cell types (Thibonnier et al., 1999), but it is possible that some of the actions of oxytocin on myometrial contractility could be mediated via its actions on the endothelial cell. Although endothelial cell purity was very high (>97%), and previous work has established successful microarray data using cell cultures of 98% purity (Alizadeh et al., 2000), we cannot entirely rule out the possibility of uterine smooth muscle cell contamination. Smooth muscle cells are known to express VEGF receptor 2 (Ishida et al., 2001) and therefore could respond in some way to VEGF stimulation. However, we have confirmed that MMECs do express oxytocin receptor by alternative methodology (unpublished data), making it likely that the microarray result is from the endothelial cells, which made up at least 97% of the cells in the culture.
It must be noted that the results presented here are from in-vitro cultured cells. Although the endothelial cells are obtained from myometrial primary isolates, in-vitro results must always be interpreted with caution when extrapolating to the in-vivo situation. It is possible that lengthy periods of time in culture may cause the loss of specialization (Garlanda and Dejana, 1997). For example, estrogen receptor disappears from in-vitro cultures of myometrial microvascular endothelial cells within two passages (C.Gargett, personal communication), and even more rapidly from human leiomyoma and myometrial explant cultures (Severino et al., 1996). In the experiments we performed, the 0 h time-points had less time in culture than the VEGF-stimulated cultures. Although the cells were in culture for 3–4 weeks on average prior to the experiments, the VEGF-stimulated cells were in culture for 3, 6 or 12 h longer than the 0 h time-points. Although unlikely, it is possible that some of the gene expression changes between the time-points were due to the slightly longer culture period for the VEGF-stimulated cells. Any genes identified in our experiment will require in-vivo verification before their ultimate role in human myometrial vascular biology can be appreciated. Technical limitations due to the amount of total RNA currently required for the glass slide arrays (between 60–100 µg per sample), as well as the need for multiple passages with repeated positive selection to ensure cell purity (>97%), meant that the cells needed to be grown through three or four passages before the experiment could be performed. Linear amplification using a bacteriophage promoter (Wang, E. et al., 2000) may reduce the amount of RNA template needed to make fluorescently labelled cDNA, but may also alter the transcript ratios due to differential efficiency of the amplification.
There are technical limitations to microarrays, as is to be expected with such new technology. A microarray will only detect a gene if it has a probe spotted in the array. Although our arrays screen 4806 genes, there are estimated to be 
30 000 genes in the human genome, meaning that information on >25 000 of them is absent from our experiments. Another problem currently being encountered with microarray experiments is the need to regularly update gene databases being used in analysis software (such as GenespringTM) due to the increasing speed at which new information on gene sequence data is being added to public databases with the completion of the Human Genome Project. Our own database is updated on a monthly basis, and we have found that over time, gene sequences listed as expressed sequence tags often turn out to be sequences of known genes following an update.
The data presented here are the first microarray data on myometrial endothelial cells to be reported. The data identify a large number of downstream molecular responses to the stimulation of purified cells with VEGF.
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Acknowledgements |
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We would like to acknowledge the technical assistance of the staff of the microarray facility at the Peter MacCallum Cancer Institute (D.Bowtell, S.Katsambamis and M.Murphy), and P.Hutchinson from the Department of Immunology at Monash University for assistance with flow cytometry. The salary of P.A.W.R. is paid by the National Health and Medical Research Council of Australia under project grant no. 124331. G.W. is supported by the National Health and Medical Research Council of Australia clinical postgraduate scholarship no. 008202. The Australian Red Cross Blood Service, Melbourne provided male human serum.
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3 To whom correspondence should be addressed. E-mail: gareth.weston{at}med.monash.edu.au
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Submitted on February 18, 2002; accepted on June 20, 2002.
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