SEARCH

SEARCH BY CITATION

Keywords:

  • Early growth response factor-1;
  • endothelial cells;
  • fibroblast growth factor receptor-1;
  • fibroblast growth factor-2;
  • placental vascular disease

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Objective  To seek evidence of early vascular injury in the placental villous microcirculation in placental insufficiency identified by a high-resistance umbilical Doppler study by examining for expression of fibroblast growth factor receptor-1 (FGFR-1), its transcription factor, early growth response factor-1 (Egr-1) and plasma fibroblast growth factor-2 (FGF-2).

Design  Case–control study.

Setting  University teaching hospital.

Sample  Placentas and umbilical vein blood were collected at delivery from 12 women with normal pregnancy delivered at term and 14 with placental vascular disease defined by an abnormal umbilical artery Doppler study.

Methods  Microvascular endothelial cells were isolated from fresh human placentas using collagenase digestion and Dynabeads coated with monoclonal antibody against CD31. RNA was extracted from the isolated endothelial cells. The messenger RNA (mRNA) expression of FGFR-1 and Egr-1 production were assessed by reverse transcription polymerase chain reaction and factored relative to 18S ribosomal RNA. To confirm that FGF-2 was playing a significant role in this microvascular endothelial cell injury in the placenta, we also measured the soluble fraction of FGF-2 in fetal plasma from same groups of pregnancies using an enzyme-linked immunosorbent assay.

Main outcome measures  Microvascular endothelial cells expression of Egr-1mRNA, FGFR-1 mRNA and presence of soluble FGF-2 in fetal plasma.

Results  The soluble level of FGF-2 in the fetal placental circulation from pregnancy with placental vascular disease was increased when compared with normal pregnancy (median 10.15 pg/ml and interquartile range 5.34–21.83 pg/ml versus 4.46 pg/ml and 3.69–5.66 pg/ml; P < 0.05). Microvascular endothelial cells from the placental villi with placental vascular disease showed upregulation of both FGFR-1 mRNA expression (median 0.72 and interquartile range 0.40–1.64 versus 0.34 and 0.19–0.71; P < 0.05) and Egr-1 expression (median 0.79 and interquartile range 0.27–1.86 versus 0.23 and 0.17–0.67; P < 0.05) in comparison with normal pregnancy.

Conclusions  Endothelial cells from the placental villi are upregulated for expression of Egr-1 transcription factor gene in placental vascular disease. The FGFR-1 activation and increase in FGF-2 in the fetal circulation are known to be very early features of the response of endothelium to injury. Egr-1 is a promoter of many key pathophysiologically relevant target genes, which influence the development of subsequent vascular lesions. This change may occur before the pathological features recognised on microscopy.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

This study is focused on the molecular events that lead to the cellular movements and proliferative changes in vessel walls following injury. The early growth response factor-1 (Egr-1) gene is linked to the host response to vascular and inflammatory stress.1 When endothelium is exposed to injury, it releases fibroblast growth factor-2 (FGF-2) from cell walls and this binds to the fibroblast growth factor receptor-1 (FGFR-1) and induces the expression of the Egr-1 transcription factor.2 Egr-1 is not present in unperturbed endothelium but is immediately and rapidly induced by injury. It has been likened to a master switch. Thus, it is dramatically present in endothelium and vascular smooth muscle cells and tissue phagocytes after injury. It is important in regulation of genes mediating inflammatory and procoagulant processes. It is also important in angiogenesis. It has been linked to both the initiation and the progression of atherosclerotic vascular lesions.3–5

In recent work, we have described endothelial cell activation6 and proinflammatory cytokine production7 in the umbilical vascular tree of the placenta villi in thrombotic placental vasculopathy. We studied pregnant women identified to have vascular disease in the placenta by a high-resistance umbilical artery Doppler flow velocity waveform pattern.8 We demonstrated that endothelial cells isolated from the placental villi were activated and producing proinflammatory cytokines. We also showed a factor in fetal plasma from these pregnancies, capable of inducing this change in endothelial cells in culture.9,10

In this study, we sought to explore further the origin of the vascular pathology identified by the high-resistance umbilical artery Doppler flow velocity waveform. We hypothesised that injury to the endothelium in placental vascular disease would be evidenced by upregulation of the Egr-1 transcription factor. We therefore measured Egr-1 gene expression in placental villous endothelium. We also studied the release of FGF-2 by vascular trees and measured FGF-2 in fetal plasma and upregulation of the receptor FGFR-1 by measuring gene expression of the FGFR-1 by the endothelium.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Study groups

We studied two groups of pregnant women. In the control group, placentas were collected from 12 normal pregnancies delivered at term. In the normal group, all pregnancies were uncomplicated, with no identifiable medical or obstetric diseases, and delivered by spontaneous vaginal delivery (n= 9) or elective caesarean section (n= 3) at term (for reasons not associated with fetal compromise). The study group comprised 14 complicated pregnancies in which an abnormal umbilical artery Doppler study indicated umbilical placental vascular disease. The umbilical placental vascular disease group was identified by an abnormal umbilical artery Doppler study carried out within 1–4 days prior to delivery. The systolic–diastolic ratio was greater than the 95th centile using our previously reported normal range.11 All cases in this group were delivered by elective caesarean section. This group was subdivided according to the absence (n= 5) or presence (n= 9) of maternal pre-eclampsia. Maternal pre-eclampsia was defined as a blood pressure ≥140/90 mmHg on at least two occasions occurring after the 20th week of gestation accompanied by proteinuria (>300 mg/24 hours). This study was conducted with the approval of the Western Sydney Area Health Service Ethics Committee.

Preparation of placental villi, isolation and purification of endothelial cells

The placentas from our normal group of term pregnancies and from our study group were collected at delivery and immediately processed. Three to four placental lobes were immediately excised and cleared of their main vascular trunks and stored in cold phosphate buffer (phosphate-buffered saline; Gibco BRL, Gaithersburg, MD, USA). The method for isolation and purification of endothelial cells has been previously described.6 These microvascular endothelial cells were confirmed by special studies. A small sample was taken and grown to confluence over 48 hours. Fluorescence immunohistochemistry was then performed to confirm that the cells would stain with von Willebrand’s factor. We also showed that the cells incorporated Dil (a red fluorophore) fluorescence labelled-acetylated low-density lipoprotein. Under phase-contrast microscopy, these cells had a typical elongated spindle form.

Reverse transcription polymerase chain reaction

Total RNA was isolated from these purified endothelial cells extracted from the placental microvessels using RNAzolTM B (Tel-Test Inc., Friendswood, TX, USA). The first-strand complementary DNA (cDNA) synthesis reaction was performed using Perkin-Elmer GeneAmp PCR Systems’ Reverse Transcription kit (Roche, Branchburg, NJ, USA). Semiquantitative polymerase chain reaction (PCR) was used to examine the expression of Egr-1 and FGFR-1 messenger RNA (mRNA) in microvascular endothelial cells. Primers for Egr-1, FGFR-1 and 18S ribosomal RNA (rRNA) were chosen from the published sequences. Egr-1: 5′-CCT TCC CCA CGC CGA ACA C-3′ and 5′-CCT GGG AGC CCG ACT GAG TG-3′; FGFR-1: 5′-TGC CCG CCA ACA AAA CAG T-3′ and 5′-GGG CTT CCA GAA CGG TCA AC-3′ and 18S rRNA: 5′-AGC TTC CGG GAA ACC AAA GT-3′and 5′-CAA TCT CGG GTG GCT GAA C-3′. Amplification reactions were performed using a thermostable DNA polymerase kit (Advanced Biotechnologies, Surrey, UK). Housekeeping gene 18S rRNA was used as an internal control. Preliminary experiments were performed to optimise the amount of cDNA and the reaction conditions. The conditions for each PCR were optimised with respect to the linear range of the amplification reaction. The Egr-1, FGFR-1 and 18S rRNA products were then respectively loaded into 2% agarose gel. Separation of DNA fragments was achieved by electrophoresis with ethidium bromide staining. The gels were scanned and analysed by Fluor-STM MultiImager using Bio-Rad Multi-Analyst Version 1.0.2 software (Bio-Rad Laboratories, Hercules, CA, USA).

Statistical analysis

The quantitative data about clinical characteristics between the placental vascular disease group and the normal pregnancy group were analysed by independent Student’s t test. The differences in expression of Egr-1 mRNA, FGFR-1 mRNA and the soluble level of FGF-2 between the study pregnancy group and the normal control group were assessed using Mann–Whitney U test. The categorical data about parity and low centile birthweight among the groups were analysed by Fisher’s exact test. A P value less than 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Clinical outcome of the pregnancies studied

In all the pregnancies, the last Doppler study and delivery occurred in the third trimester. The group with umbilical placental vascular disease delivered earlier and the infant birthweight was reduced in comparison with the normal control group. The centile birthweight for gestational age and the placental weight were also lower in this group. The clinical characteristics of our investigation groups are summarised in Table 1.

Table 1.  Clinical characteristics*
 Normal pregnancy group (n= 12)Placental vascular disease group (n= 14)P value
  • NS, not significant.

  • *

    Values are expressed as mean (SD).

Primigravida (n)36NS
Maternal age (years)30.1 (3.9)29.8 (5.6)NS
Gestational age (weeks)39.9 (1.4)32.0 (2.1)≤0.001
Infant birthweight (g)3472.9 (578.1)1316.6 (359.8)≤0.001
Placenta weight (g)686.0 (133.3)322.9 (101.0)≤0.001
Centile weight (%)49.1 (32.8)7.4 (5.8)≤0.001
≤10th centile (n)010≤0.001

mRNA expression for Egr-1 and FGFR-1 in endothelial cells

The expression of Egr-1 and FGFR-1 mRNA by endothelial cells from placental villi in the study group with placental vascular disease and in the normal pregnancy group are shown in Figure 1. In the study group, the expression of Egr-1 mRNA by the endothelial cells was upregulated (median 0.79 and interquartile range 0.27–1.86) in comparison with the normal control group (median 0.23 and interquartile range 0.17–0.67; P < 0.05) (Figure 1A). The expression of FGFR-1 mRNA was also enhanced in the placental vascular disease group (median 0.72 and interquartile range 0.40–1.64) when compared with the normal control group (median 0.34 and interquartile range 0.19–0.71; P < 0.05) (Figure 1B).

image

Figure 1. Box and whiskers plot to show median and interquartile range for expression of Egr-1 and FGFR-1 mRNA in microvascular endothelial cells from placental villi with umbilical placental vascular disease and normal pregnancy. The expression of Egr-1 mRNA (A) and FGFR-1 mRNA (B) in these endothelial cells was upregulated in the placental vascular disease in comparison with the normal pregnancy (P < 0.05).

Download figure to PowerPoint

The impact of maternal pre-eclampsia

In the group with placental vascular disease, we examined for differences in the subgroups with and without maternal pre-eclampsia. The differences were not significant and there was much overlap of the data range. Egr-1 mRNA expression was greater in the group with pre-eclampsia (median 0.29 and interquartile range 0.19–1.95 versus 0.84 and 0.40–1.73; P > 0.05) (Figure 2A). There was no difference in expression of FGFR-1 mRNA in the subgroups without maternal pre-eclampsia or with maternal pre-eclampsia (median 0.81 and interquartile range 0.39–1.85 versus 0.72 and 0.39–1.69; P > 0.05) (Figure 2B).

image

Figure 2. Box and whiskers plot to show median and interquartile range for expression of Egr-1 and FGFR-1 mRNA in microvascular endothelial cells from placental villi in the whole group of placental vascular disease and the subgroup without and with maternal pre-eclampsia. In the whole group of placental vascular disease, the expression of Egr-1 mRNA (A) and FGFR-1 mRNA (B) was not significant (NS) in the subgroup without maternal pre-eclampsia in comparison with the subgroup with maternal pre-eclampsia (P > 0.05).

Download figure to PowerPoint

The soluble level of FGF-2 in the fetal plasma

The soluble fraction of FGF-2 in the fetal plasma in the study group with placental vascular disease and in the normal pregnancy group is shown in Figure 3. The level of FGF-2 in the fetal circulation in pregnancies with placental vascular disease (median 10.15 pg/ml and interquartile range 5.34–21.83 pg/ml) was increased in comparison with the normal pregnancies (median 4.46 pg/ml and interquartile range 3.69–5.66 pg/ml; P < 0.05) (Figure 3A).

image

Figure 3. Box and whiskers plot to show median and interquartile range for soluble fraction of FGF-2 in fetal plasma in the study group with placental vascular disease and the normal pregnancy group. The soluble level of FGF-2 was increased in the study group when compared with the normal control group (A) (P < 0.05). However, in the whole group of placental vascular disease, there was no difference in the subgroup without maternal pre-eclampsia or in the subgroup with maternal pre-eclampsia (B) (P > 0.05). NS, not significant.

Download figure to PowerPoint

In the whole group with placental vascular disease, the difference in the soluble level of FGF-2 in the subgroups with maternal pre-eclampsia and without pre-eclampsia was not significant (median 10.53 pg/ml and interquartile range 8.70–66.27 pg/ml versus 8.79 pg/ml and 4.75–26.54 pg/ml; P > 0.05) (Figure 3B).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Our study demonstrates that thrombotic placental vasculopathy is associated with endothelial cell injury. These findings are novel. The increase in gene expression for the Egr-1 transcription factor and FGFR-1 in the endothelial cells is associated with an increase in soluble FGF-2 in fetal plasma. These findings indicate injury to vascular endothelium and release of FGF-2. The FGF-2 binds to the FGFR-1 in a paracrine fashion and leads to activation of the specific Egr-1 transcriptional factor. We have therefore demonstrated a change in endothelial cell message together with a change in the relevant plasma signal. These findings indicate that injury to endothelium is present, and they support our previously suggested hypothesis that this may lead to endothelial cell activation and proinflammmatory change. We have displayed the results of these three measures in Figures 1A, B and 3A using a box and whiskers plot. We choose this because the results were not normally distributed. For all three measures, the median value of the control group is below the interquartile range (25th–75th centile) of the study group, which means that 75% of the study group had a value above the median of the normal group. It was the purpose of this study to investigate whether endothelial cell injury was a feature of placental vascular pathology. The pathways we studied are the earliest change observed when endothelium is perturbed. Our results therefore indicate continuing injury in these cases.

Normal unperturbed endothelium does not express Egr-1. It is a response to injury. Our results also show this in the control group (Figure 1A) in that the median value is very close to the lowest value observed. Expression of Egr-1 is a response to injury and, as stated above, is present in our study group. It is very likely that Egr-1 transcription activation is a common pathway recruited by a range of biochemical and biological agents. This is seen in atherosclerosis. The precise mechanisms that evoke the chronic inflammatory process is uncertain. In a recent study, we have demonstrated that endothelium from the placental villi displays an increase in gene expression for toll-like receptor 4,12 opening the possibility of infection as the mechanism for vascular injury. The findings of our study are important in confirming the presence of vascular injury. There is need now for research directed at finding the cause of this injury.

There are limitations to our study. We have studied pregnancy with established evidence of placental vascular pathology revealed by the Doppler high-resistance flow velocity waveform pattern in the umbilical artery. Our study group delivered early in the third trimester. We used a group with normal pregnancy delivered at term as a control. Gestational age is therefore different. Many premature deliveries have unrecognised pathology as a cause, so we did not feel that this group was suitable. We have previously commented upon the evidence of vascular pathology in that group.12 Placental vascular pathology is present in a group with premature rupture of membranes,13 and fetal plasma from such cases will cause endothelial cell stimulation.14

In this study, we did perform a subgroup analysis in our study group with placental vascular disease according to the presence and absence of maternal pre-eclampsia. We found no significant difference (Figures 2A, B and 3B). Small numbers do limit the power of this finding. However, this has been a finding in our other studies of placental villous endothelium6,7,9,10,12 and is supporting evidence for our hypothesis that the placental vascular pathology and its fetal syndrome are independent of the presence of maternal pre-eclampsia.

The management of the thrombotic placental vasculopathy in current obstetric practice is focused on defining fetal condition to determine the optimal time for delivery. This is a balance of risk of oxygen and nutrient deprivation to the fetus in utero against risk of prematurity. Of the many tests of fetal welfare, only umbilical artery Doppler to identify placental vascular disease has been shown to reduce perinatal mortality. Meta-analysis of randomised controlled trails has shown that clinical identification of the placental vascular disease with umbilical artery Doppler study reduces prenatal mortality by 32% in high-risk pregnancy.15 However, there has been a little progress in understanding the cause of vasculopathy. Our initial reports associating abnormal umbilical artery Doppler and adverse fetal condition were first made 25 years ago,16 and we described the placental pathology 20 years ago,17 and published the first randomised trial 18 years ago.18 Large multicentric trials are now being carried out focussing on delivery timing, with long-term survival (2 years) free of neurological handicap as the primary end point.19 The authors suggest a better approach to manage the fetus that is a victim of thrombotic placental vasculopathy, which is to treat the placental pathology. This study is important because Egr-1 transcription controls many of the effector genes whose products influence the development of the vascular lesions. Defining the cause of the injury will open up the possibility of strategies that selectively target and inhibit these pathophysiologically relevant genes. This raises the possibility of a therapeutic approach to reverse the vasculopathy and so allow the pregnancy to continue.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  • 1
    Rupp J, Maass M. Egr-1, a major link between infection and atherosclerosis? Circ Res 2003;92:e78.
  • 2
    Fahmy RG, Dass CR, Sun LQ, Chesterman CN, Khachigian LM. Transcription factor Egr-1 supports FGF-dependent angiogenesis during neovascularization and tumor growth. Nat Med 2003;9:102632.
  • 3
    Tarnawski AS, Jones MK. Inhibition of angiogenesis by NSAIDs: molecular mechanisms and clinical implications. J Mol Med 2003;81:62736.
  • 4
    Mcmahon SB, Monroe JG. The role of early growth response gene 1 (egr-1) in regulation of the immune response. J Leukoc Biol 1996;60:15966.
  • 5
    Ha E, Bucciarelli LG, Lu Y, Stern DM, Zou YS, Schmidt AM, et al. Early growth response-1 promotes atherogenesis. Circ Res 2004;94:3339.
  • 6
    Wang X, Athayde N, Trudinger B. Microvascular endothelial cell activation is present in the umbilical placental microcirculation in fetal placental vascular disease. Am J Obstet Gynecol 2004;190:596601.
  • 7
    Wang X, Athayde N, Trudinger B. A proinflammatory cytokine response is present in the fetal placental vasculature in placental insufficiency. Am J Obstet Gynecol 2003;189:144551.
  • 8
    Trudinger BJ, Giles WB, Cook CM, Bombadieri J, Collins L. Fetal umbilical artery flow velocity waveforms and placental resistance: clinical significance. Br J Obstet Gynaecol 1985;92:2330.
  • 9
    Wang X, Athayde N, Trudinger B. Endothelial cell expression of cell adhesion molecules is stimulated by fetal plasma from pregnancy with umbilical placental vascular disease. BJOG 2002;109:7707.
  • 10
    Wang X, Athayde N, Trudinger B. Fetal plasma stimulates endothelial cell production of cytokines and the family of suppressor of cytokine signaling in umbilical placental vascular disease. Am J Obstet Gynecol 2003;188:51016.
  • 11
    Thompson RS, Trudinger BJ, Cook CM. Doppler ultrasound waveform indices: A/B ratio, pulsatility index, and Pourcelot ratio. Br J Obstet Gynaecol 1988;95:5818.
  • 12
    Wang X, Athayde N, Trudinger B. Placental vascular disease and toll-like receptor 4 gene expression. Am J Obstet Gynecol 2005;192:9616.
  • 13
    Kim YM, Chaiworapongsa T, Gomez R, Bujold E, Yoon BH, Rotmensch S, et al. Failure of physiologic transformation of the spiral arteries in the placental bed in preterm premature rupture of membranes. Am J Obstet Gynecol 2002;187:113742.
  • 14
    Athayde N, Wang J, Wang X, Trudinger B. Fetuses delivered following preterm prelabor rupture of the membranes are capable of stimulating a proinflammatory response in endothelial cells. J Soc Gynecol Investig 2005;12:11822.
  • 15
    Alfirevic Z, Neilson JP. Doppler ultrasonography in high risk pregnancies: systematic review with meta-analysis. Am J Obstet Gynecol 1995;172:137987.
  • 16
    Trudinger BJ, Cook CM. Fetal umbilical artery velocity waveforms. Ultrasound Med Biol 1982;8(Suppl 1):197.
  • 17
    Giles WB, Trudinger BJ, Baird PJ. Fetal umbilical artery flow velocity waveforms and placental resistance: pathological correlation. Br J Obstet Gynaecol 1985;92:318.
  • 18
    Trudinger BJ, Cook CM, Giles WB, Connelly A, Thompson RS. Umbilical artery flow velocity waveforms in high-risk pregnancy. Randomised controlled trial. Lancet 1987;1:18890.
  • 19
    Thornton JG, Hornbuckle J, Vail A, Spiegelhalter DJ, Levene M., GRIT study group. Infant wellbeing at 2 years of age in the growth restricted intervention trial (GRIT): multicentred randomised controlled trial. Lancet 2004;364:4834.