Angiogenesis and the placental environment

Placma(1995), 16, 289-296

Angiogenesis Environment

and the Placental

T. WHEELER, C. L. ELCOCK F. W. ANTHONY

&

Obstetrics and Gynaecology, University of Southampton, Princess Anne Hospital, Coxford Road, Southampton, SO16 5YA, UK Paper accepted 12.12.1994

SUMMARY Rapid growth and vascularization of the human placenta are characteristic of early pregnancy and are accomplished in an unusually hypoxic environment. Stimulation of placental growth through hypoxia-induced angiogenesis may therefore be ofparticular importance. We have previously found that several varieties of vascular endothelial growth factor (VEGF) mRNA, including VEGF,,, are present in cultured placentaljbroblasts. We hypothesized that hypoxia would increase the transcription and translation of VEGF by these cells and provide one mechanism linking placental development with its environment. Placentalfibroblasts were grown in aerobic or anaerobic atmospheric conditions for 72 h. By 24 h the oxygen tension of the anaerobic culture media was signajicantly less than that of the aerobic cultures. RNA was extracted from the cells at 24, 48 and 72 h. Following reverse transcription polymerase chain reaction (RT-PCR) stronger signals for VEGF were always found in the anaerobic cultures and this was conj%med by competitive PCR. mRNA for VEGF,,, was represented most strongly but the anaerobic cultures also showed clearly mRNA for VEGF,,,, VEGF,‘,,, and was also measured in the aerobic and anaerobic culture VEGFzo,. The VEGFprotein medium, By 72 h the average concentration of VEGF was sign$cantly higher (p=O.OI) in the anaerobic culture medium. VEGF production is one mechanism through which oxygen supply may injuence placental development. Examples of this may include the compensatory placental hypertrophy associated with maternal anaemia and with reproduction at high altitude.

INTRODUCTION The first 10 weeks of human pregnancy are characterized by rapid trophoblastic growth contrasting with the slower growth of the embyro while organogenesis takes place. The trophoblast forms a shell around the embryo during this period and the maternal spiral arteries, which will ultimately perfuse the placenta, are not patent (Hustin, 1992). Throughout this time the oxygen tension of the trophoblast has been shown to be far lower than that which 01431tOO4/95/030289 + 08 $08.00/O

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prevails when the spiral arteries open and perfusion of the intervillous space is established (Rodesch et al, 1992). The recent demonstration that the transcription of mRNA for the vascular endothelial growth factor (VEGF& is increased in hypoxic conditions (Shweiki et al, 1992) suggested that hypoxia-mediated angiogenesis may have an important role in placenta development. VEGF exists in several forms due to alternative splicing of mRNA (Houck et al, 1991; Tischer et al, 1991). Previous work has shown that the predominant form in the human placenta is VEGFr,, (Houck et al, 1991). Using reverse transcription and the polymerase chain reaction (RT-PCR) we have identified mRNA for VEGF,,, and several other alternatively spliced forms of VEGF (including VEGF,,J in RNA extracts obtained from both the chorionic villi and cultured fibroblasts (Anthony et al, 1994). We hypothesized that the transcription and translation of VEGF would be increased if the placental fibroblasts were grown in an hypoxic environment.

METHODS Cell

AND

MATERIAL

culture

Fresh trophoblastic villi were obtained from five women at the time of therapeutic termination during the first trimester. Separate fibroblast cultures were prepared from the tissue obtained from each individual. The cell culture technique was based upon that of Fant (1991), details being as follows. The villi were dissected from surrounding membranes and digested for 15 min in trypsin/EDTA. The cells obtained were filtered to remove undigested material and purified by density centrifugation over Histopaque 1077. Cells at the interface were seeded into 6-well plates grown in RPM1 1640 medium supplemented with 10 per cent fetal calf serum and antibiotics. The cultures were grown to confluence in air and 5 per cent carbon dioxide over a period of lo-15 days and the cells then trypsinized for subculture. For identification the cells were cultured for 3 days on glass chamber slides. Immunocytochemical staining of these preparations showed that the cells were positive for vimentin and a specific fibroblast antigen (prolyl-4-hydroxylase, SBS, Dakopatts) but negative for cytokeratin, a macrophage specific marker (Ki MS, kindly supplied by Professor H. Radzun, University of Kiel, Germany) and factor 8 related antigen. The subcultures were then grown in aerobic or anaerobic conditions in the following manner: 2 X lo5 fibroblasts were added to three wells of six culture plates; three plates were incubated in 5 per cent carbon dioxide in air (aerobic cultures); the remaining three plates were placed separately into airtight bags with a palladium catalyst to remove oxygen from the surrounding atmosphere (Anaerocult IS, Merck). The effects of the hypoxic environment were assessedat 24,48 and 72 h of culture; the cultures in air and 5 per cent carbon dioxide were assessedat the same times and acted as controls. Initially measurements of the oxygen and carbon dioxide tensions and of the pH in the culture media were made using a Corning 178 pH/blood gas analyser. The cultured cells were then removed, pelleted and prepared for RNA extraction. Using a haemocytometer, the live cells in each well were counted from a small sample obtained before pelleting and following trypan blue staining. The culture media were frozen for subsequent measurements of VEGF protein.

Reverse

transcription

and the PCR

RNA was extracted from cell pellets using RNAzol-B (Biogenesis Ltd). The RNA present in the strands was reverse transcribed to cDNA (Statagene First Strand Kit). Three microlitres

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of each cDNA sample were run for 30 cycles in PCR with primers corresponding to exon 4 and exon 8 of the VEGF gene. The main product of 228 bp was sequenced and corresponded to VEGF,,,. Other products corresponded to VEGF,,,, VEGF,,, and VEGF,,,. Details of our RT-PCR technique have been described previously (Anthony et al, 1994). For standardization, the cDNA was evaluated with beta-actin primers that generated a 610 bp product. The PCR signal for this product was correlated with the number of cells present. The PCR results were substantiated by the use of an internal standard for VEGF and competitive PCR.

VEGF

measurement

(by enzyme-linked

immunosorbent

assay)

The technique was based on that described by Houck et al (1992). Microtitre plates were coated with monoclonal antibody for VEGF (mAb 4.6.1, lot 16288-18, Genetech Inc.). Standards were prepared from recombinant human VEGF (lot no. 14036-16) and diluted in assay buffer. One hundred microlitre aliquots of standards and samples were incubated for 2 h on a plate shaker. A polyclonal antiserum to VEGF (lot no. 11094-70B, Genetech, Inc.) was next incubated for 2 h on the plate and this was followed by a further 2 h incubation with goat anti-rabbit IgG coupled to horse-radish peroxidase (Dako Immunoglobulins). The plates were then incubated with substrate (OPD tablets, Dako Immunoglobulins) and the absorbance measured at 490 nm. We were able to recover 90-100 per cent of VEGF added to our culture medium. The interassay coefficient of variation was 9 per cent for a sample of approximately 1 rig/ml. Parallelism existed between the standard curve and diluted 72 h anaerobic medium.

RESULTS

By 24 h the average oxygen tension in the anaerobic culture media was 10 kPa, significantly less than the oxygen tension (22 kPa) in the aerobic cultures at the same time (Figure 1). The oxygen tension remained significantly lower in anaerobic cultures over the following 2 days (Figure 1). A smaller but significant decrease (PzO.01) in oxygen tension also occurred in the aerobic cultures by 48 h. The pH fell significantly (P=O.O5), but to the same degree in both aerobic and anaerobic cultures over the 3 days of the experiments; there were no significant differences in pH between the aerobic and anaerobic cultures during the experiments (Figure 1). There were no significant trends in carbon dioxide tension during the experiments (Figure 1). The numbers of living cells increased in the aerobic cultures but remained unaltered in the anaerobic cultures (Figure 1). RNA was extracted from the cells at 24, 48 and 72 h. Following RT-PCR stronger signals for messenger VEGF were always indicated in the anaerobic cultures (Figure 2). This was confirmed with an internal standard and competitive PCR. The internal standard yielded a 170 bp product with all reaction mixes under the same conditions including the 24 h samples incubated under aerobic conditions. The ratio of VEGF products to internal standard increased with the incubation and degree of hypoxia. The VEGF,,, message was represented most strongly in the RT-PCR results, but the anaerobic cultures also showed clearly mRNA for VEGF,,t, VEGF,,, and VEGF,,, was present. The VEGF protein was also measured in culture medium. There was considerable variation in the amounts of VEGF protein detected but, individually, greater amounts of VEGF were always found in the anaerobic cultures of the five experiments. By 72 h (Figure 1) the average concentration of VEGF was significantly higher (PzO.01) in the anaerobic cultures (paired non-parametric and parametric t-test).

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1. Average measurements and SEIM of oxygen and carbon dioxide tensions, pH, cell numbers and vascular endothelial growth factor (VEGF) concentration in culture media obtained from placental fibroblasts grown in aerobic and anaerobic conditions. The measurements were made after 24, 48 and 72 h. Data from five experiments, each derived from a different placenta. Mean and SEM for individual groups are displayed on the graph. For statistical comparison the mean and SEM of the differences between matched aerobic and anaerobic cell sets were used in the paired t-test. (0), Aerobic; (O), anaerobic. “*P=
Figure

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Lanes 1

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Figure 2. Electrophoretic pattern of reverse transcription-polymerase chain reaction products for vascular endothelial growth factor (VEGF) mRNA obtained from placental fibroblasts in aerobic and anaerobic culture. Lane 1: 100 bp ladder; lane 2: 24 h in aerobic culture; lane 3: 24 h in anaerobic culture; lane 4: 48 h in aerobic culture; lane 5: 48 h in anaerobic culture; lane 6: 72 h in aerobic culture; lane 7: 24 h in anaerobic culture; lane 8: 100 bp ladder.

DISCUSSION Angiogenic growth factors have only recently been localized within the human placenta. Using immunocytochemistry, Ferriani et al (1994) h ave shown the presence of platelet-derived endothelial cell growth factor and VEGF. The message for VEGF has been found in total RNA extracts prepared from first trimester villi; the mRNA species corresponded to the VEGF isoforms of 189, 165, 145 and 121 amino acids (Sharkey et al, 1993). We have also extracted mRNA from first trimester villi and cultured placental fibroblasts finding messages for similar forms of VEGF but in addition the message for VEGF,,, (Anthony et al, 1994). VEGF,s6 was present in low amounts and we believe that the reason it was not found by Sharkey et al (1993) reflects differences in PCR protocol and in the preparation of the tissue before PCR was undertaken. In our first study (Anthony et al, 1994) it was necessary to perform two rounds of PCR to demonstrate the presence of VEGF,,,; while, in the current study (of placental fibroblasts), we believe that the hypoxic stimulus up-regulated all the forms

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of the VEGF message thus allowing them to be demonstrated in the first round of PCR. The protein isoform for VEGF,s, was also found with western blotting by Jackson et al (1994). Our study shows for the first time that hypoxia can up-regulate several varieties of VEGF and not VEGF,,, alone as was originally demonstrated in glioblastoma cells by Shweiki et al (1992). In addition we carried out one experiment on adult skin fibroblasts, again showing that hypoxia up-regulated mRNA VEGF (data not shown in this paper). Other workers have also found similar effects with the expression of VEGF mRNA in human skin fibroblasts exposed to hypoxia (Minchenko et al, 1994). Earlier investigations of the angiogenic factors present in human placenta and decidua showed that small specimens of human decidua (obtained in the first trimester) produced a strongly angiogenic effect on the chick chorio-allantoic membrane (CAM) whereas placental tissue did not (Fuchs, Lindenbaum and Marcoudas, 1985). Since VEGF has now been shown to be present in the placenta, stimulate blood vessel development in the CAM (Wilting, Christ and Weich, 1992) and the growth of endothelial cells derived from the umbilical cord (Gospodarowicz, Abraham and Schilling, 1989), th ese findings are initially surprising. However, it is possible that VEGF was not present in sufficient concentration to stimulate the CAM in Fuchs et al’s experiments (1985) and that the placental tissue contained other growth factors with inhibitory effects on angiogenesis, which would have antagonized the effect of VEGF. The up-regulation of VEGF by hypoxia, demonstrated in our experiments, may provide an important mechanism through which the vascularization of the placenta develops according to its metabolic requirement. This particular method of regulating the growth of the vascular system has been proposed by Adair, Gray and Montani (1990). The control is homeostatic, thus the neovascularization induced by hypoxia corrects tissue oxygenation and VEGF release will be down-regulated. The regulatiqn of placental vascular growth through oxygen availability may have particular relevance in the first 10 weeks of pregnancy when the oxygen tension of the trophoblastic villi is particularly low (Rodesch et al, 1992). We cannot be certain how VEGF transcription is affected in these circumstances because the oxygen tensions recorded (in vivo) by Rodesch et al (1992) were much lower than those found in our hypoxic cultures. However, there is further evidence that placental function is linked with oxygen availability in early pregnancy. We have found (Wheeler et al, 1994) that the maternal haemoglobin concentrations at 10 weeks of pregnancy is inversely correlated with the levels in the maternal serum of the two placental products, human chorionic gonadotrophin (hCG) and human placental lactogen (hPL). VEGF receptors are present on cytotrophoblast cells (Charnock-Jones et al, 1994) and we speculate that the up-regulation of VEGF through hypoxia increases the growth of the syncytiotrophoblast giving rise to the increased levels of hCG and hPL. There are other examples in pregnancy when a reduction in oxygen availability is associated with changes in placental vasculature, which may be brought about through local VEGF release. Reproduction at high altitude (Kruger and Arias-Stella, 1970) is associated with placental hypertrophy and morphometric studies of such placentae show that there is an increase in oxygen diffusion capacity (Mayhew, Jackson and Haas, 1990; Reshetnikova, Burton and Milovanov, 1993) which is achieved through changes in the fetal placental vasculature (Reshetnikova, Burton and Milovanov, 1993). Experimentally-induced hypoxia in pregnant guinea-pigs results in an increase in the gaseous diffusional capacity of the placenta (Bacon et al, 1994). This is also associated with vascular changes in the form of increased branching and coiling of the capillaries (Scheffen et al, 1990; Bacon et al, 1994). We propose that these changes are the consequence of increased production of VEGF within the placenta. Extreme

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varieties of maternal anaemia, seen in the developing world, are also associated with placental hypertrophy (Be&her et al, 1970) and the stereological changes in such placentae resemble those associated with high altitude (Burton, personal communication). A more recent study from the western world has shown an inverse correlation between maternal haemoglobin concentration and placental weight and that this was present across the entire range of haemoglobin concentration (Godfrey et al, 1991). In a separate study of over 600 women, drawn from the general population, we found an inverse correlation between maternal haemoglobin concentration and placental volume at 18 weeks of pregnancy (Howe and Wheeler, 1993). A follow-up in the same women showed a highly significant correlation between placental volume (at 18 weeks) and placental weight at delivery (Howe, 1994). These observations imply that the environment within which the placenta develops in early pregnancy causes significant changes in its growth trajectory. These changes are likely to be in response to the metabolic demands of the placenta. At present little is known about the integration of the various mechanisms controlling vascularization in the placenta. Other factors than VEGF release are likely to be involved, these include the pressure within the capillaries, the nature of the extracellular matrix and the overall impact of many different growth factors, with either a stimulatory or inhibitory effect on endothelial cells. The in situ hybridization studies of Sharkey et al (1993) showed that the message for VEGF was present in macrophages (Hofbauer cells) and other unspecified cells in the placenta. The placental villous stroma contains high numbers of Hofbauer cells, about 40 per cent, in the first trimester (Goldstein et al, 1988), they are typically circular in shape and are thought to move freely through fluid filled channels in the villous stroma (Jauniaux, Burton and Jones, 1992). Placental fibroblasts, on the other hand, have an elongated shape and lie parallel with the longitudinal axes of the villus trunks and rami (Boyd and Hamilton, 1970). In due course they are surrounded by the extracellular matrix and their position becomes relatively fixed. It seems likely that both Hofbauer cells and fibroblasts contribute to placental angiogenesis but perhaps in different ways. Fibroblasts for example may contribute more of the less soluble forms of VEGF (VEGF,,,, VEGF,,,) that attach to the extracellular matrix. If the release of VEGF reflects local oxygen tension, this would produce gradients of VEGF concentration which may become directional signals influencing capillary growth. ACKNOWLEDGEMENTS Genetech Inc. kindly immunocytochemical was supported by the Karen Creed for the

supplied the antibody and antigen for the VEGF immunoassay. The chamber slide cultures and studies were carried out in our own department by Cheryl Taylor and Pat Englefield. The work Wessex Regional Health Authority and the Wessex Medical Trust. We would like to thank Miss preparation of this manuscript.

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