Preeclampsia (PE) originates in the placenta and involves inadequate cytotrophoblast invasion, maternal endothelial dysfunction and altered expression of angiogenic and anti-angiogenic factors which ultimately leads to the clinical manifestations (Noori et al, 2010). Vasculogenesis and angiogenesis are considered to be central processes in the development of the placenta. Vascular endothelial growth factor (VEGF) is the key factor promoting vasculogenesis and angiogenesis and altered levels of VEGF and its receptors can disrupt angiogenesis, leading to placental insufficiency and endothelial dysfunction seen in preeclampsia (Zhou et al., 2002). VEGF exerts its biologic effects through two high-affinity tyrosine kinase receptors i.e. vascular endothelial growth factor receptor-1 (VEGFR-1) / fms-like tyrosine kinase-1 (FLT-1) and vascular endothelial growth factor receptor-2 / kinase insert domain containing receptor (KDR). FLT-1 interactions with VEGF are critical for invasion and pseudovasculogenesis while KDR is a major mediator of mitogenic, angiogenic processes, enhancing permeability and endothelial survival (Holash et al., 2005).
A number of studies have examined the mRNA levels of different angiogenesis-regulating factors in preeclamptic placenta although results are inconsistent. Some studies report increased VEGF expression (Kweider et al., 2011; Lee et al., 2010; Munaut et al., 2008; Kumazaki et al., 2002) while others report reduced expression (Kim et al., 2012; Cooper et al., 1996; Lyall et al., 1997) in preeclamptic women. Still other studies found no difference (Toft et al., 2008; Ranheim et al., 2001; Sgambati et al., 2004). Expression of FLT-1 (Lee et al., 2010; Munaut et al., 2008; Shibata et al., 2005; Rajakumar et al., 2009; Gu et al., 2007; Jarvenpaa et al., 2007) and KDR (Munaut et al., 2008; Toft et al., 2008; Tripathi et al., 2008) have also been examined. Very few studies have simultaneously examined the expression of VEGF and both receptors, which are essential for embryonic development (Horta et al., 2009), simultaneously in human placenta under pathologic conditions (Lyall et al., 1997; Trollmann et al., 2003) or in human endothelial cells (Munaut et al., 2008). Further, most of the reported studies have been carried out on smaller sample size and are limited by the broad range of gestational ages.
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The placenta serves as the interface between the mother and fetus and placental gene expression and methylation in humans and animals has been shown to be influenced by a number of environmental factors such as diet (Kim et al, 2009; Novakovic et al, 2009; Gallou-Kabani, 2010), life style (Bruchova et al, 2010) etc. In our previous studies we have observed increased homocysteine and oxidative stress levels in preeclampsia. Further we have also reported alterations in placental global DNA methylation levels in preeclampsia and their association with blood pressure and homocysteine levels. Healthy placental development involves spatio-temporally programmed gene expression patterns and any alteration in this process compromises placental function. Under suboptimal uterine conditions, normal methylation of DNA, a process that is important for regulation of gene expression and DNA stability, altering gene expression and subsequently preventing normal growth (Lins and Murray, 2008).
The objective of this study was to examine the gene promoter CpG methylation of the angiogenic factors VEGF, FLT-1 and KDR and their mRNA levels in placentas from women diagnosed with preeclampsia as compared to normal pregnancies.
Materials and Methods
This study was conducted at the Department of Obstetrics and Gynaecology, Bharati Hospital, Pune during the year 2007-2009. This study was conducted with the understanding and consent of each subject and was approved by the Bharati Vidyapeeth Medical College Institutional Ethical Committee. A total number of 135 pregnant women with singleton pregnancy were recruited for this study. 47 women had normotensive pregnancies and delivered at term, 90 women had preeclampsia during pregnancy, of which 42 delivered preterm (â‰¤ 37 weeks), while 46 delivered at term (â‰¥ 37 weeks).
Women were excluded from the study if there was evidence of other pregnancy complications, such as multiple gestation, chronic hypertension, type I or type II diabetes mellitus, seizure disorder and renal or liver disease. Pregnant women with alcohol or drug abuse were also excluded from the study.
The normotensive group consisted of pregnant women with no medical or obstetrical complications. Preeclampsia was defined by systolic and diastolic blood pressures greater than 140 and 90 mm Hg, respectively, with the presence of proteinuria (>1+ or 300mg /24 hrs) on a dipstick test. Edema was present in some cases. Blood pressure was measured in the left arm with a mercury sphygmomanometer. Preeclampsia was confirmed by repeated recording of the blood pressure starting at enrollment and at every follow-up visit, which occurred approximately once a month until delivery. The data given here are the blood pressures at the time of delivery, i.e., just before going to the labor room, to ensure that a similar time point was used for both groups to rule out the effect of stress due to labor on blood pressure. Gestational age was based on day of last menstrual period and then confirmed by ultrasound. All women were routinely given iron and folic acid supplements as per the National Prophylaxis programme. All recruited women were from a similar socioeconomic background and well matched for dietary and lifestyle patterns.
Tissue collection and processing
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Placental tissues: Fresh placental tissues were obtained from normal and preterm pregnancies immediately after delivery. Fetal membranes were trimmed off and the placenta was weighed. Small pieces (approximately 1 cm (l) x 1cm (w) x 0.5-1 cm (h)) were randomly cut out from different regions of the placental cotyledon. The tissue pieces were individually rinsed in phosphate buffered saline (PBS) to wash off maternal and fetal blood. Tissue pieces were dipped in liquid nitrogen and stored at -80°C until assayed.
Gene promoter methylation assay
Genomic DNA was isolated from placental samples using the Qiagen DNA Blood and Tissue kit (Qiagen, Germany). The purified genomic DNA was bisulfite treated using the EZ DNA Methylationâ„¢ Kit (Zymo Research) as per the manufactirer's instructions. This step converts all non-methylated cytosine bases to uracil while all methylated cytosine bases remain unchanged. The bisulfite modified DNA was used for Sequenom MassARRAY EpiTYPING for gene specific methylation analysis of VEGF, FLT-1, and KDR. This technique employs base-specific cleavage followed by MALDI-TOF mass spectrometry in which the size ratio of the cleaved products provides quantitative methylation estimates for CpG sites within a target region. Genomic sequences for assay design were extracted from the UCSC genome browser (http://www.genome.ucsc.edu/). Primer pairs for amplification were designed using EpiDesigner web tool (http://www.epidesigner.com/). The promoter sequences used in this study for CpG methylation analysis of VEGF (Chromosome 6, 43737274-43737739), FLT-1(Chromosome 13, 29067752-29068196) and KDR ( Chromosome 4, 55991374-55991777) genes were selected using the UCSC genome browser and are given in Fig 1. For PCR amplification, a T7-promoter tag was added to the reverse primer, and a 10-mer tag sequence was added to the forward primer to balance the PCR primer length. The primers are listed in Table.1.
Bisulfite treated genomic DNA was amplified using the designed primers. The thermal cycling conditions were as follows: For VEGF- 1 cycle: 95Â°C for 15 min; 5 cycles : 95Â°C for 1 min, 62Â°C for 2 min, 72Â°C for 2 min; 32 cycles: 95Â°C for 1 min, 62Â°C for 1 min, 72Â°C for 1 min then 72Â°C for 7 min. For FLT-1 and KDR-1 cycle : 95Â°C for 15 min; 5 cycles : 95Â°C for 1 min, 60Â°C for 2 min, 72Â°C for 2 min; 35 cycles: 95Â°C for 1 min, 60Â°C for 1 min, 72Â°C for 1 min then 72Â°C for 7 min.
Following PCR amplification, in vitro transcription and T-cleavage assay was performed using MassCLEAVEâ„¢ Reagent Kit (Sequenom). Unincorporated dinucleotide triphosphates (dNTPs) were removed by shrimp alkaline phosphatase (SAP) treatment. Typically, 2 Âµl of the PCR product was directly used as template for the in vitro transcription reaction. T7 RNA & DNA polymerase was used to incorporate thymidine triphosphate in the transcripts. In the same step, RNase-A was added to cleave the in vitro transcripts (T-cleavage assay). Samples were diluted with 20 Âµl of water. Conditioning of the phosphate backbone was achieved by adding 6 mg of Clean Resin before performing MALDI-TOF MS analysis. For Mass Spectrometry analysis RNase-A treated product was robotically dispensed onto silicon matrix preloaded chips (SpectroCHIP; Sequenom), the mass spectra were collected using a MassARRAY Compact MALDI-TOF (Sequenom), and spectra's methylation ratios were generated by the EpiTYPER software v1.0 (Sequenom).
Samples that yielded data in greater than 70% for all CpG units within a promoter were passed for that sample/promoter pair. For each sample the methylation analysis was done in duplicates and sites showing more than 10% difference in methylation were excluded. Sites that were tagged as low mass or high mass by Epityper software were excluded.
Extraction of total RNA, cDNA synthesis and qRT-PCR Assays
Total RNA from placenta samples was isolated using Trizol method and quantified by Nanodrop (ND1000 v3.5.2) spectrophotometer. 1Î¼g of total RNA was transcribed to cDNA with the High-Capacity cDNA reverse transcription Kit (Applied biosystems, Foster city, USA).
Real-time quantitative PCR for VEGF, FLT-1, KDR mRNAs, and 18S rRNA were performed using the Applied biosystems 7500 FAST system. The relative quantitation of our data has been performed using the standard curve method according to the manufacturer's recommendation (PE Applied Biosystems in User Bulletin #2). The relative expression level of the gene of interest was computed with respect to 18S rRNA to normalize for variation in the quality of RNA and the amount of input cDNA. PCR was performed with the TaqMan Universal PCR Master Mix (PE Applied Biosystems, Foster city, USA) using cDNA equivalent to 10ng total RNA. Ct-values were set in the exponential range of the amplification plots using the 7500 Fast System Sequence Detection Software v1.4.0. Î”Î”Ct-values corresponded to the difference between the Ct-values of the genes examined and those of the 18sRNA (internal control) gene. Relative expression levels of genes were calculated and expressed as 2-Î”Î”Ct. The following TaqManÂ® assays (Applied Biosystems, Foster city, USA) were used in this study: 18S RNA (Hs99999901_s1); VEGF (Hs00900058_m1); FLT-1 (Hs01052936_m1); KDR (Hs00176676_m1).
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The data were analyzed using SPSS/PC+ package (Version 11.0, Chicago, IL). Values are reported as mean Â± SD (demographic characters) or mean Â± SE (methylation studies). Mean values of the estimates were compared using one-way ANOVA at conventional levels of significance (p<0.05). Skewed variables were transformed to normality using the following transformations: log to the base 10. Correlation between variables was studied using Pearson's correlation analysis after adjusting for gestation and BMI.
Maternal and neonatal characteristics
The maternal and neonatal characteristics are given in Table 2. All the women recruited in the study had similar age, income, education and parity. The body weight and gestational age at delivery were significantly lower (p<0.05) in women with preeclampsia who delivered preterm. The maternal systolic and diastolic blood pressures were significantly higher in the term and preterm preeclampsia groups as compared to the control group. Baby weight was significantly reduced (p<0.01) in term PE group as compared to control. The baby weight, height, head and chest circumference were significantly lower (p<0.01) in the preterm preeclampsia group as compared to the control and term preeclampsia groups.
Promoter CpG methylation of VEGF, FLT-1 and KDR genes
We analysed the cytosine methylation at 23 CpG sites in the VEGF gene promoter region, 30 CpG sites in FLT-1 gene promoter region and 37 CpG sites in the KDR gene promoter. The mean percent methylation at each CpG site in the VEGF, FLT-1 and KDR promoter are given in Table.3a, 3b and 3c. In the VEGF gene promoter, the mean methylation at CpG site 6.7 (p<0.05) and CpG site 8 (p<0.01) was significantly reduced whereas that at CpG site 14 was significantly higher (p<0.05) in preterm PE group as compared to normotensive group.
The mean methylation of VEGF promoter was significantly lower (p<0.05) in the preterm preeclampsia group as compared to the control group.
In the FLT-1 promoter region, the mean methylation at CpG site 16 was significantly reduced (p<0.01) in term PE group as compared to the control group while mean methylation at CpG site 17 was significantly reduced (p<0.05) in preterm PE group as compared to the control group. Further mean methylation at CpG site 24 was significantly reduced (p<0.05) in both term PE and preterm PE group as compared to normotensive group. Mean methylation of the FLT-1 gene promoter was similar between the groups.
In the KDR gene promoter region, the mean methylation at CpG site 12.13 was significantly higher in term PE (p<0.05) and preterm PE (p<0.01) group as compared to the normotensive group. Mean methylation of the FLT-1 gene promoter was not different between the groups.
VEGF, Flt-1, KDR mRNA levels in placenta
There was a 10 fold increase (p<0.05) in placental VEGF mRNA levels in preterm preeclamptic group compared to term preeclamptic as well as normotensive group, while the levels were significantly reduced (2 Fold decrease) (significance) in term preeclamptic placenta as compared to normotensive. Flt-1 and KDR mRNA levels were comparable in normotensive and term preeclamptic groups. Flt-1 and KDR mRNA levels were higher in preterm preeclamptic group as compared to term preeclamptic and normotensive groups (significance and fold change?) (Figure 2).
Associations between CpG methylation and gestation
Mean promoter methylation of VEGF gene was negatively (n=24, r=-0.461, p=0.018) associated with gestation in the term PE group but not in the control and preterm PE groups. Further CpG site 6.7 in the VEGF promoter region was negatively associated with gestation in both the term PE (n=24, r=-0.399, p=0.044) and preterm PE group (n=19, r=-0.463, p=0.034).
Mean promoter methylation in the FLT-1 gene was not associated with gestation. However, CpG site 16 showed a positive association with gestation in the preterm PE group (n=30, r=0.420, p=0.020). There were no associations between methylation and gestation in the KDR gene promoter region.
In this study we examined the CpG methylation of VEGF, FLT-1 and KDR gene promoters and their expression in human placentas. These genes encode important factors that determine placental angiogenesis. This study shows some interesting findings 1) some CpG sites showed differential methylation between the control, term and preterm preeclampsia groups 2) mean promoter methylation in the VEGF gene was significantly lower in the preterm PE group as compared to control, while it was comparable to control in the term PE group 3) VEGF expression was significantly (10 fold) higher in the preterm PE group as compared to control, while it was 2 fold lower in the term PE group. 4) Although mean methylation in the FLT-1 and KDR promoters was similar between the three groups, FLT-1 and KDR gene expression was significantly higher in the preterm PE group as compared to term PE and control group.5) Mean methylation at the VEGF promoter and methylation at some differentially methylated CpG sites in the VEGF and FLT-1 promoters was associated with gestation in the term and preterm PE groups.
DNA methylation is an important epigenetic mechanism of gene regulation. In the VEGF promoter CpG site 8 was hypomethylated while CpG site 14 was hypermethylated in the preterm PE group as compared to the control group. In the FLT-1 promoter CpG sites 16 and 17 were hypomethylated in the term PE and preterm PE group respectively as compared to control. CpG site 24 of the FLT-1 promoter was hypomethylated in both the term and preterm PE groups as compared to control. In the KDR promoter region CpG site 12.13 was hypermethylated in both term PE and preterm PE groups as compared to the control group. This differential methylation between the normotensive and preeclampsia groups indicates aberrant DNA methylation patterns in the VEGF, FLT-1 and KDR genes in preeclampsia which may be involved in the pathophysiology of preeclampsia. In humans, DNA methylation is mediated by DNA methyltransferases (DNMTs) that are responsible for de novo methylation and maintenance of methylation patterns during replication. There is abundant evidence that suggests that DNA methylation patterns can be altered as a component of disease pathogenesis (van Vliet J et al., 2007; Abdolmaleky et al., 2006). However, further studies are needed to determine the potential predictive and therapeutic value of these findings in PE.
Alterations in methylation status within promoter regions can affect gene expression and hence the phenotype (Ball et al., 2009). Our results show a 10 fold increase in VEGF mRNA in preterm preeclampsia as compared to term preeclamptic and normotensive placentas. This may be due to the fact that in severe preeclampsia, higher levels of placental hypoxia inducible transcription factors up-regulate VEGF expression (Torry and Torry, 1997). Up-regulation of VEGF due to hypoxia in preterm preeclamptic placenta may be a compensatory mechanism in attempting to restore the blood flow toward normal. Further, as seen from our data, the VEGF promoter was significantly hypomethylated in the preterm PE group and this may be responsible for the increased expression of VEGF observed in this group. These results are consistent with other reports that suggest an inverse relation between promoter methylation and gene expression and suggest epigenetic control of VEGF expression in preterm PE. It is also possible that the CpG sites 6.7, 8 and 14 which are differentially methylated in the preterm PE group as compared to the control group, may be involved in the upregulation of VEGF mRNA levels in the preterm PE group.
Although the mean methylation of the VEGF promoter was comparable between the control and term PE group, the mRNA levels were significantly lower in the term PE group as compared to the control group. Generally, the pathology of preterm PE is regarded as more severe and different as compared to term PE. Previous studies have suggested the existence of different subsets of preeclampsia and that pathophysiologic mechanisms may contribute differently to the development of preterm versus term preeclampsia (Roberts and Catov, 2008; Phillips et al, 2010).In our data, the significant differences in the term and preterm preeclamptic groups with respect to the maternal weight, duration of gestation and birth outcome parameters further support the existence of these two different subsets within the preeclamptic group and also indicate differences in severity of the condition. It is likely that since term PE is less severe as compared to preterm PE, the compensatory increase in VEGF mRNA levels is not observed, neither is there a difference in the promoter methylation levels. The observed decrease in VEGF expression in the term PE may be due to alternative pathways of gene expression regulation such as alterations in transcription factor expression or histone modifications as a consequence of the pathology. Some previous studies have also shown reduced placental VEGF mRNA levels at term in preeclampsia compared to control (Andraweera et al, 2012; Cooper et al, 1996).
Mean methylation of the FLT-1 and KDR promoters was comparable between groups yet the mRNA levels of FLT-1 and KDR were significantly higher in the preterm PE group as compared to the other two groups. Although there was no difference in the mean promoter methylation in the FLT-1 gene promoter, CpG site 17 was significantly hypomethylated in the preterm PE group as compared to the control and term PE group. This site may be important in influencing the FLT-1 expression in preterm PE, however further studies are needed to determine the exact role of this site in regulation of FLT-1 expression. Further KDR expression in preterm PE may not be mediated through DNA methylation changes but through other factors affecting gene expression such as transcription factors, mRNA stability and histone modifications. Modulation of factors affecting gene expression by intracellular signals in different physiological states is a well established. Further, the opposite trends in VEGF, FLT-1 and KDR mRNA levels and the difference in epigenetic patterns between these two groups provide support for the existence of differences in pathology between term and preterm preeclampsia.
Our results show that mean methylation of the VEGF promoter and some differentially methylated sites in the VEGF and FLT-1 promoters was associated with gestation in the term and preterm PE groups. CpG 6.7 methylation in the VEGF promoter was negatively associated with gestation in the term and preterm PE groups. Methylation of CpG 16 in the FLT-1 promoter was positively associated with gestation in the preterm PE group. Human pregnancy comprises a complex series of differentiation and growth processes that are spatio-temporally regulated (Lins and Mitchell, 2008). We have previously reported gestation dependant changes in placental global DNA methylation (Chavan-Gautam et al, 200). Novakovic et al (2009) have reported changes in promoter CpG methylation (both increase and decrease in methylation) with gestation in the placenta. In addition to CpG sites that consistently change over gestation, they also report the existence of CpG sites that show inter-individual variability within each gestational age and suggest that such variability could be attributed to cumulative differences in environmental exposure. In our study these results could be attributed to changes in the intrauterine environment as a result of the pathology.
As seen from our data the CpG methylation patterns and expression of the angiogenic factors differ in the preterm PE and term PE groups and could be associated with the differences in pathology. However, it is unclear whether the observed differences are a cause or effect of the underlying pathophysiology. This study reiterates that CpG methylation is dynamic and influenced by the intrauterine environment. DNA methylation changes could account for the alterations in gene expression, and the ability to induce compensatory mechanisms to circumvent adverse pregnancy outcome. This study also highlights that DNA methylation may not explain all gene expression changes, and other mechanisms of gene expression regulation also come into play. Further, the role of CpG sites, in other regions in the gene cannot be ruled out. There are several techniques for evaluation of CpG methylation, but most can analyze only a few CpG sites in a target region. The methodology used in this study overcomes this limitation. Nevertheless, this is the first account of CpG methylation changes in the VEGF, FLT-1 and KDR gene promoters in pregnancies complicated by preeclampsia.