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Placenta 29 (2008) 624e630
Novel Homeobox Genes are Differentially Expressed in Placental Microvascular Endothelial Cells Compared with Macrovascular Cells P. Murthi a,b,*, U. Hiden c, G. Rajaraman a,b, H. Liu a,d, A.J. Borg a, F. Coombes a, G. Desoye c, S.P. Brennecke a,b, B. Kalionis a,b a
b
Pregnancy Research Centre, Department of Perinatal Medicine, The Royal Women’s Hospital, Carlton, Victoria 3053, Australia Department of Obstetrics and Gynaecology, The Royal Women’s Hospital and University of Melbourne, Carlton, Victoria 3053, Australia c Department of Obstetrics and Gynecology, Medical University, Graz, Austria d Department of Obstetrics and Gynaecology, Qilu Hospital of Shandong University, Jinan 250012, China Accepted 14 April 2008
Abstract Angiogenesis is fundamental to normal placental development and aberrant angiogenesis contributes substantially to placental pathologies. The complex process of angiogenesis is regulated by transcription factors leading to the formation of endothelial cells that line the microvasculature. Homeobox genes are important transcription factors that regulate vascular development in embryonic and adult tissues. We have recently shown that placental homeobox genes HLX, DLX3, DLX4, MSX2 and GAX are expressed in placental endothelial cells. Hence, the novel homeobox genes TLX1, TLX2, TGIF, HEX, PHOX1, MEIS2, HOXB7, and LIM6 were detected that have not been reported in endothelial cells previously. Importantly, these homeobox genes have not been previously reported in placental endothelial cells and, with the exception of HEX, PHOX1 and HOXB7, have not been described in any other endothelial cell type. Reverse transcriptase PCR was performed on cDNA from freshly isolated placental microvascular endothelial cells (PLEC), and the human placental microvascular endothelial cell line HPEC. cDNAs prepared from control term placentae, human microvascular endothelial cells (HMVEC) and human umbilical vein macrovascular endothelial cells (HUVEC) were used as controls. PCR analyses showed that all novel homeobox genes tested were expressed by all endothelial cells types. Furthermore, real-time PCR analyses revealed that homeobox genes TLX1, TLX2 and PHOX1 relative mRNA expression levels were significantly decreased in HUVEC compared with microvascular endothelial cells, while the relative mRNA expression levels of MEIS2 and TGIF were significantly increased in macrovascular cells compared with microvascular endothelial cells. Thus we have identified novel homeobox genes in microvascular endothelial cells and have shown that homeobox genes are differentially expressed between micro- and macrovascular endothelial cells. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Placenta; Microvascular endothelial cells; Homeobox genes; Macrovascular endothelial cells; Microarray
1. Introduction Angiogenesis is a fundamental aspect of normal placental development that leads to the elaboration of the placental * Corresponding author. Pregnancy Research Centre, Department of Perinatal Medicine, The Royal Women’s Hospital, 132 Grattan St, Carlton, Victoria 3053, Australia. Tel.: þ61 3 9344 2966; fax: þ61 3 9347 2472. E-mail address:
[email protected] (P. Murthi). 0143-4004/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2008.04.006
vasculature [1]. The process of angiogenesis requires the precise and coordinated regulation of cellular proliferation, differentiation, and migration [2]. This is evident in major disorders of pregnancy such as pre-eclampsia and fetal growth restriction where the process of angiogenesis and villous outgrowth are significantly affected [3,4]. Placental angiogenesis involves multiple signaling pathways that control growth and differentiation [5], but relatively few transcription factors are known to mediate these signaling
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pathways in the placenta [6e8]. Homeobox genes, an important family of transcription factors that encode a 60 amino acid DNA-binding motif referred to as the homeodomain, may play essential roles in regulating the function of the placental vascular systems. For example, targeted homeobox gene mutations in the mouse (e.g. Dlx3 and Esx1), have demonstrated a spectrum of placental vascular and angiogenic defects [9,10]. There is evidence in the literature that homeobox genes are essential for vascular development and control many different aspects of cellular growth and differentiation [11]. Gorski and Walsh [12] and Douville and Wigle [13] have extensively reviewed the regulation and function of homeobox genes in embryonic and adult vascular systems. Little is known about how the homeobox genes implicated in angiogenesis and vascular remodeling exert their effects at the cellular level. However, it is clear that at least a subset of homeobox genes appear to function by controlling the differentiation, proliferation, and/or migration of vascular smooth muscle cells or endothelial cells (reviewed by Gorski and Walsh [12]). For example, homeobox genes GAX (MOX2) [14] and HOXB7 [15] control proliferation and migration of vascular smooth muscle cells and HOXB3 promote capillary morphogenesis in vascular endothelial cells [16]. Increasingly, downstream target genes of homeodomain proteins that regulate both pro- and antiangiogenic cardiovascular and lymphatic cell processes are being identified. Clearly, the repertoire of homeobox genes that regulate vascular cellular processes such as migration, invasion, proliferation and tube formation [17] in many organs is not yet known. This is especially true in the placenta where our understanding of the number and role of homeobox genes in these processes is rudimentary. Much of our knowledge on the transcriptional regulation of angiogenesis in endothelial cells in the placenta comes from model systems, particularly from human umbilical vascular endothelial cells (HUVEC). HUVEC have been used as a model for endothelial cells in many studies describing transcriptional regulation of endothelial specific growth factors (for example, see Nagakawa et al. [18]), and although this has proven to be a useful model, HUVEC are macrovascular endothelial cells. We and others have shown that the placental microvascular endothelial cells (PLEC) that comprise the fine fetal capillaries of chorionic villi, differ in phenotype, gene expression and physiology from macrovascular endothelial cells [19,20]. We identified homeobox genes DLX4, MSX2, GAX, DLX3 and HLX (previously called HB24 and HLX1) in the human placenta [21e24] and in placental microvascular PLEC and macrovascular HUVEC [19,20]. However, HLX mRNA levels were significantly lower in macrovascular HUVEC compared with microvascular PLEC [20]. In this study we sought to expand the repertoire of homeobox genes expressed in placental endothelial cells by carrying out microarray expression profiling on endothelial cells and by analysing public microarray expression profile databases created by others. We have employed PCR and real-time PCR methods to corroborate the microarray data and to compare relative expression levels of homeobox genes in freshly
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isolated microvascular PLEC and macrovascular HUVEC. In this study, we also have used primary human microvascular endothelial cells (HMVEC) derived from human foreskin at passage 19 [25]. This control was included to determine whether the novel homeobox genes were expressed generally in primary microvascular endothelial cells or restriction in expression to primary PLEC cells. The human placental microvascular SV-40 transformed endothelial cell line HPEC was included because it is a well studied microvascular cell line that displays phenotypic and genotypic similarities with PLEC [19]. 2. Materials and methods 2.1. Tissue collection Placentae were obtained from uncomplicated, term pregnancies by vaginal delivery or Caesarean section, with informed patient consent and with the approval of The Royal Women’s Hospital’s Research and Human Ethics Committees.
2.2. Endothelial cells Primary human microvascular endothelial cells (HMVEC) were a generous gift from Dr Grant Drummond (Ludwig institute, Melbourne, Australia) and were derived from human foreskin as previously described [25]. Human placental microvascular endothelial cells (HPEC) of SV-40 transformed cell line [19] were a generous gift from Dr Peter Friedl (Darmstadt, Germany). Freshly isolated primary, uncultured HUVEC were prepared as previously described [26], and all cell cultures were maintained at 37 C in an atmosphere of 5% CO2.
2.3. Isolation of PLEC PLEC were isolated as described previously [20]. Briefly, a single isolated placental lobule was perfused via a chorionic cannulation artery and vein pair with filtered, modified Krebs solution (37 C and bubbled with 5% O2, 5% CO2 and 90% N2) at a constant inflow of 5 mL/min. Once the outflow showed no macroscopic evidence of blood contamination, the inflow was exchanged with a sterile 0.05% (w/v) collagenase/dispase solution (Gibco Life Sciences, Australia) at 37 C and an inflow rate of 3 mL/min. The collagenase/dispase solution was in calcium/magnesium-free HBSS with 0.1% (w/v) bovine serum albumin without fatty acid, penicillin (100 U/ml), streptomycin (100 mg/ml) and amphotericin B (0.25 mg/ml). The venous effluent was collected, centrifuged (200 g for 10 min) and the pelleted cells resuspended in HBSS prior to separation on a 65%e15% Percoll density gradient. The fraction containing PLEC at 1.05 g/ml [20] was collected and viable cell counts were determined by the Trypan blue exclusion assay (Invitrogen, Australia). Uncultured fresh PLEC were used for gene expression analysis. All cell preparations were subjected to rigorous immunocytochemical characterization [20]. HPEC were cultured in 1% (v/v) Endothelial growth medium (EGM2, Cambrex, CloneticsÔ, USA) supplemented with the EGMÔMV BulletKit (CloneticsÔ) until 70% confluent.
2.4. RNA isolation and processing for microarray Total RNA from PLEC was isolated using Trizol (MRC, OH) followed by quality assessment using a Bioanalyzer (Agilent, CA). Experimental procedures and data analysis settings followed proposed standards [27]. Total RNA from 10 preparations per cell type that were isolated from different placentae were pooled [28]. From 5 mg pooled RNA, cDNA was synthesized (SuperScript double stranded cDNA Synthesis Kit, Invitrogen, CA), in vitro transcribed (RNA Transcript Labeling Kit, Enzo diagnostics, Farmingdale, NY) and fragmented. To test the quality of the cRNA it was hybridized against
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Test-3 arrays (Affymetrix, CA). As these met the criteria (bioC, bioD and cre were present, the 30 /50 ratio of the polyA controls was 100. Annotations were obtained from NetAffx (http://www.affymetrix.com). Regulated genes selected as above were clustered manually in Microsoft Excel according to the encoded proteins’ putative function using database searches on PubMed and OMIM (http://www.ncbi. nlm.nih.gov).
2.6. RNA isolation for PCR analysis Confluent cultures of HMVEC, HPEC, primary-uncultured PLEC and HUVEC were homogenised using a QIAshredder spin column (Qiagen, Australia), and total RNA extracted using the QIAGEN RNeasy Micro Kit (Qiagen, Australia) as described [20].
2.7. Semi-quantitative reverse transcriptase PCR Primers for novel homeobox genes listed in Table 1 were designed using the public web-page Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer 3_www.cgi). First strand synthesis was performed using Superscript III (Invitrogen, Australia) and approximately 25 ng cDNA was used for PCR amplification as described [20]. PCR was carried out in a GeneAmp PCR System 9700 thermocycler. Amplified products were fractionated by agarose gel electrophoresis.
2.8. Real-time PCR Relative quantitation of novel homeobox genes in HMVEC, HPEC, PLEC and HUVEC was performed on an ABI Prism 7700 Sequence Detector (Perkin-Elmer-Applied Biosystems) using Inventoried gene expression assaysÔ consisting of a 20 mix of unlabelled PCR primers and TaqManÒ MGB FAMÔ dye labeled probes HEX (Hs00242160_m1, Applied Biosystems), TGIF (Hs00820148_g1, Applied Biosystems), LIM6 (Hs00232660_m1, Applied Biosystems), TLX1 (Hs00271457_m1, Applied Biosystems), TLX2 (Hs00818124_g1, Applied Biosystems), PHOX1 (Hs00246567_m1, Applied Biosystems), HOXB7 (Applied Biosystems), MEIS2 (Hs00542636_m1, Applied Biosystems) as described by Murthi et al. [29]. Relative quantitation of novel homeobox gene expression was normalised to b-actin (4326315E, TaqManÒ MGB VICÔ dye labeled probe, Applied Biosystems) in a duplex, co-amplification reaction and calculated according to the 2DDCT method of Livak and Schmittgen [30].
2.9. Data analysis All parameters relating to the isolation and characterisation of PLEC were expressed as mean SEM. The relative quantitation of novel homeobox gene mRNA expression normalized to b-actin in macrovascular HUVEC compared with all three types of microvascular endothelial cells was determined using the Student’s t-test. Results were considered significant at p < 0.05.
3. Results A modified perfusion-based method was used to isolate PLEC and they were enriched by density gradient centrifugation. Red blood cell contamination ranged from 2 to 5% of the total cells, and the mean viability of the cells was 90.8 2.4%, n ¼ 12. All isolated and enriched PLEC and HPEC showed positive staining for CD34 (QBEnd/10), which confirmed the endothelial characteristics of the PLEC [20] (data not shown). Microarray expression data suggested that there were novel homeobox genes expressed in microvascular placental endothelial cells (HPEC and PLEC). These homeobox genes, HEX, PHOX1, LIM6, HOXB7 and TGIF, have not been previously detected in the placenta and were selected because they exhibited the greatest relative expression in the microarray data (Table 2). TLX1 and TLX2 homeobox gene expression data (not shown) was from the GNF Microarray Analysis Data for the Human U95A microarray, Version 2 dataset (http://expression.gnf.org) [31]. As depicted, homeobox genes LIM6, HOXB7, TGIF, PHOX1 and HEX were expressed in endothelial cells of the placenta (Table 2). Expression of these homeobox genes in the placenta or in placental endothelial cells has not been previously reported. HEX [18], PHOX1 [32] and HOXB7 [33] homeobox genes have been previously described in endothelial cells from various tissue sources but have not been described in any placental endothelial cell type and may represent novel endothelial regulatory genes. PCR on endothelial cells, including the microvascular HPEC and HMVEC cell lines, 12 freshly isolated PLEC primary cell preparations, and three freshly isolated macrovascular HUVEC preparations showed expression of homeobox genes TLX1, TLX2, MEIS2, HOXB7, LIM6, PHOX1, HEX and TGIF at their predicted PCR product size. Fig. 1 shows representative PCR products of HMVEC, PLEC, HPEC and macrovascular HUVEC for all the novel homeobox genes tested. Placental cDNA derived from a normal term placenta was used as a control. The housekeeping gene b-actin showed
Table 1 Oligonucleotide sequences of primer pairs Gene
Sense primer
Antisense primer
Size (bp)
HEX HOXB7 PHOX1 TGIF LIM6 PHOX1 TLX1 TLX2 MEISE2
50 -tac tct gga gcc cct tct tg-30 50 -agg tag cga ttg tag tga aa-30 50 -act ata ttc ctt ggc ctt c-30 50 -cag agc aag aaa aag cgt tg-30 50 -cgc tga cat taa cag gag ca-30 50 -tcc ctc ctc aaa tcc tac-30 50 -cgg acg ccc ccc aag aag aag aag-30 50 -ttc gga ggg gtc cgg ttt tg-30 50 -gat gat gca acc tca acc cac-30
50 -ttc aag gtc ttc ctg gga gg-30 50 -tgc gaa tgc ttt att ttc ta-30 50 -tcc ctc ctc aaa tcc tac-30 50 -gac cac tct gtg tat tcg tac ttg-30 50 -atg cta cgg cca cat aat cc-30 50 -act ata ttc ctt ggc ctt c-30 50 -cat cgg tca ttt tga gcg cc-30 50 -ggg tga act tac agg gga cg-30 50 -atc caa cac aaa gct ccc-30
380 477 290 503 203 290 141 259 359
P. Murthi et al. / Placenta 29 (2008) 624e630
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Table 2 Gene expression data from microarray Id No.
Signal to Log ratio
Detection
Genes
219884_at 216973_s_at 203313_s_at 217226_s_at 215933_s_at
667 1154 654 1251 1426
Present Present Present Present Present
LIM6 HOXB7 TGIF PHOX1 HEX
TLX1, TLX2 and MEIS2 homeobox gene expression data (not shown) was from the GNF Microarray Analysis Data for the Human U95A microarray, Version 2 dataset (http://expression.gnf.org).
relatively constant levels of expression for all the samples tested (Fig. 1). Homeobox genes HEX, TGIF and LIM6 showed PCR products corresponding to their predicted size in all the cell types tested. As shown, the PCR product for TLX1 (141 bp), TLX2 (259 bp) and PHOX1 (290 bp) mRNA expression were reduced in macrovascular HUVEC compared with the microvascular HMVEC, PLEC and HPEC. In contrast, mRNA expression for HOXB7 (477 bp) and MEIS2 (359 bp) was more evident in the macrovascular HUVEC and in the placenta than in the microvascular endothelial cell types. The relative quantitation by real-time PCR of the novel homeobox genes in HMVEC, PLEC, cultured HPEC and HUVEC normalised to b-actin is illustrated in Fig. 2. Three independent real-time assays were performed for n ¼ 3 preparations from each endothelial cell type for the Real-Time PCR. TLX1, TLX2 and PHOX1 showed significantly lower levels of expression in macrovascular HUVEC compared with microvascular endothelial cells, and homeobox genes MEIS2, and TGIF mRNA expression levels were significantly increased in macrovascular HUVEC compared to microvascular endothelial cells. It is important note that some of the differences in the band intensity observed for the homeobox genes tested (e.g. HOXB7 in Fig. 1) using the traditional PCR method are not reflected in the real-time PCR results. These differences may reflect the increased level of sensitivity and reliability of the real-time PCR assay. The effect of mode of delivery on the level of novel homeobox genes HEX and TGIF were verified in PLEC. There was no significant difference in the level of homeobox gene expression for HEX [0.93 0.53 vaginal delivery vs 0.84 0.54 Caesarean section, (n ¼ 2), p ¼ 0.22] and TGIF [0.94 0.54 vaginal delivery vs 0.91 0.52 Caesarean section, (n ¼ 2), p ¼ 0.44] suggesting that mode of delivery did not influence novel homeobox gene expression in PLEC.
Fig. 1. Total RNA from HMVEC, PLEC, HPEC, HUVEC and placenta was subjected to RT-PCR using primers for the novel homeobox genes TLX1, TLX2, MEIS2, HOXB7, LIM6, PHOX1, HEX and TGIF as described in Section 2. PCR products were electrophoresed on 2% agarose gel and detected following ethidium bromide staining. b-actin was used as housekeeping gene. A representative RT-PCR is shown. ‘‘Control’’ represents a negative control containing PLEC RNA that was not reverse transcribed. PCR product sizes are shown, as determined by comparison with the 100 bp DNA ladder.
Micro-array expression data suggested that there were novel homeobox genes expressed in microvascular placental endothelial cells. These homeobox genes, HEX, PHOX1, LIM6, HOXB7, MEIS2 and TGIF, have not previously been identified in the placenta and were selected because they
1.50
A number of homeobox genes have already been identified in the cardiovascular system where they carry out important functional roles [12]. Previous studies in our laboratory have shown that the homeobox genes, DLX3, DLX4, MSX2, GAX and HLX, were expressed in placental microvascular endothelial cells and HLX mRNA expression level was significantly decreased in macrovascular HUVEC compared with microvascular PLEC [20].
*
*
1.00
0.50
0.00
4. Discussion
HMVEC PLEC HPEC HUVEC
* * TLX1
* TLX2
MEIS2 HOXB7 LIM6 PHOX1
HEX
TGIF
Fig. 2. Relative quantitation of TLX1, TLX2, MEIS2, HOXB7, LIM6, PHOX1, HEX and TGIF mRNA expression normalised to the house keeping gene bactin in HMVEC, PLEC, HPEC and HUVEC is shown. Real-time PCR using Assays-on-DemandÔ (Gene Expression products from Applied Biosystems), TaqMan and the ABI Prism 7700 Sequence Detection System was performed as described in Section 2. Data was analysed according to the 2DDCT method (Livak and Schmittgen, 2001). Statistical comparisons were performed using the Student’s t-test. The * value denotes significantly decreased levels of TLX1, TLX2 and PHOX1 mRNA and significantly increased levels of MEIS2 and TGIF in macrovascular endothelial cells compared to microvascular endothelial cells ( p < 0.001).
628
P. Murthi et al. / Placenta 29 (2008) 624e630
Fig. 3. A schematic representation of the distinct endothelial cell types in the human placenta. Differential expression of novel homeobox genes TGIF and MEIS2 in macrovascular HUVEC and TLX1, TLX2 and PHOX1 in microvascular HPEC and PLEC are summarised.
showed detectable levels of expression in the micro-array data. TLX1 and TLX2 homeobox gene expression in the placenta was revealed from the GNF Microarray Analysis Data [31] (data not shown). Validation of microarray expression data is important and necessary before further conclusions on novel homeobox gene expression can be drawn. Therefore, the microarray data were corroborated by independent methods such as RT-PCR and real-time PCR on primary uncultured PLEC and cultured microvascular endothelial cell lines (HPEC, HMVEC) and in macrovascular HUVEC endothelial cells. In this study we have demonstrated that the novel homeobox genes TLX1, TLX2, PHOX1, MEIS2 and TGIF are expressed in placental microvascular HPEC and PLEC endothelial cells. In addition, we have also reported differential expression of TLX1, TLX2, PHOX1, MEIS2 and TGIF mRNA levels in macrovascular HUVEC and microvascular HMEC, HPEC and PLEC. HEX (haematopoietically expressed homeobox) also referred to as Prh, is a proline-rich divergent homeobox gene and our study here demonstrate HEX expression in microvascular endothelial cells, and specifically in placental microvascular endothelial cells. Nakagawa et al. [18] demonstrated the expression of HEX in HUVEC, and that it acts as a negative regulator of angiogenesis in endothelial cells in vitro by abrogating the angiogenic properties. HEX has been shown to act as a transcriptional activator increasing the expression of endoglin, which is known to be involved in placental vascular maturation [34]. This makes HEX a likely candidate for a regulatory role in placental angiogenesis. PHOX1 (paired like homeobox) is also referred to as PRRX1 (paired related homeobox gene 1), or Pmx1 (paired mesoderm homeobox) [35]. Although much progress has been made in understanding the behaviour of PHOX1 in tumour cell biology, little information is available in relation to vascular biology. PHOX1 was shown to control pulmonary
vascular endothelial cell differentiation and their subsequent incorporation into functional pulmonary vascular networks [32]. The differential expression of PHOX1 mRNA in microand macrovascular environment suggests a potential role of PHOX1 in placental microvascular endothelial cells. LIM6 also referred to as Lhx6 (LIM-homeobox containing gene) has been shown for the first time to be expressed in endothelial cells, including placental microvascular endothelial cells. The presence of LIM6 at high levels in both the microand macrovascular environment suggests that it has a potential role in vascular biology. LIM6 has been implicated in two major developmental processes that require cell specification and differentiation; the formation of the brain and nervous system of humans and other organisms [36e39] and the development of the mammalian head [40]. Based on the action of LIM6 in these cell types it may prove to play a role in placental endothelial cell differentiation. Studies on neoangiogenesis and tumour invasion have shown that HOXB7 may act as a proangiogeneic stimulus. Care et al. [33] have provided evidence that HOXB7 acts as a key factor in a tumor-associated angiogenic switch in the up-regulation of vascular endothelial growth factor, melanoma growth-stimulatory activity/growth-related oncogene alpha, interleukin-8, and angiopoietin-2. We now report HOXB7 mRNA expression in both microvascular and macrovascular endothelial cell types. MEIS2 belongs to the TALE family (three-amino acid loop extension) homeodomains. In this study we have demonstrated the presence of MEIS2 expression in the human placental microvascular endothelial cells. Meis2 correlates with the Stra (several retinoic acid inducible genes) family of genes in the mouse placenta [41,42], however the role of the human counterpart of these genes in the placental vasculature is unknown. TGIF (TG-Interacting factor) is also a member of the TALE-superclass homeodomains. In this study we have
P. Murthi et al. / Placenta 29 (2008) 624e630
shown that TGIF is expressed in both microvascular endothelial cells (including placental microvascular endothelial cells) and macrovascular endothelial cells. TGIF expression has been implicated in many biological pathways and developmental processes. TGIF was first identified as a competitor of the retinoic acid receptor for binding to retinoic acid response elements and shows restricted expression to only a few tissues where it regulates proliferating and differentiating cell lineages [43,44]. Subsequently, TGIF has been shown to interact with growth factors and other homeobox gene products to exert its action [45,46]. TGIF has not yet been described in endothelial cells, however, based on the ability of TGIF to bind other specific homeobox gene products, these interactions may also be necessary in placental angiogenesis. TLX1 also known as HOX11 is involved in various aspects of embryogenesis and features prominently as an oncogene in human T-cell acute lymphoblastic leukaemia [47]. TLX1 exhibits immortalising activity in a wide variety of blood cell lineages; however, the effect of this gene on endothelial cell differentiation has not been fully investigated. Homeobox gene TLX2 (also known as HOX11L1, Ncx and Enx) is a transcription factor and plays a crucial role in the development of the enteric nervous system, as confirmed by mice models exhibiting intestinal hyperganglionosis and pseudo-obstruction [48]. The role of TLX2 in endothelial cells is yet to be elucidated. In summary, we have used microarray expression analysis to detect homeobox gene expression in placental endothelial cells. We have identified homeobox genes that are novel not only in placental microvascular endothelial cells but also in endothelial cells and in the placenta in general. Fig. 3 summarises the association between the detection of homeobox gene expression and the regions of angiogenic potential in the human placenta. In the microvasculature, where angiogenesis is predominant, homeobox genes TLX1, TLX2 and PHOX1 show increased expression. In the macrovasculature, where there is limited angiogenesis, the level of TGIF and MEIS2 are significantly increased suggesting that the heterogeneity in homeobox gene expression between microvascular PLEC and macrovascular HUVEC that could reflect differences in the angiogenic potential in the two different endothelial environments. Functional studies in cultured endothelial cells are underway in our laboratory to determine the role of novel homeobox genes. Acknowledgements The authors gratefully acknowledge specimen collection by Clinical Research Midwife Ms Susan Nisbet, Pregnancy Research Centre, and Department of Perinatal Medicine at the Royal Women’s Hospital. The authors would like to thank the National Health and Medical Research Council, Australia (509140), Lynne Quayle Charitable Trust Fund (Equity Trustees), the Marian and EH Flack Trust Funds and the Sunshine Foundation for funding support for this project and also the University of Melbourne for the award of a Melbourne
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