Deepali P. Sundrani

Differential placental methylation and expression of VEGF, FLT-1 and KDR genes in human term and preterm preeclampsia

BackgroundPreeclampsia, a pregnancy complication of placental origin is associated with altered expression of angiogenic factors and their receptors. Recently, there is considerable interest in understanding the role of adverse intrauterine conditions in placental dysfunction and adverse pregnancy outcomes. Since we have observed changes in placental global DNA methylation levels in preeclampsia, this study was undertaken to examine gene promoter CpG methylation and expression of several angiogenic genes.We recruited 139 women comprising, 46 normotensive women with term delivery (≥37 weeks), 45 women with preeclampsia delivering preterm (<37 weeks) and 48 women with preeclampsia delivering at term. Expression levels and promoter CpG methylation of VEGF, FLT-1 and KDR genes in placentae from respective groups were determined by Taqman-based quantitative real time PCR and by the Sequenom® EpiTYPER™ technology respectively.ResultsWe observed several differentially methylated CpG sites in the promoter regions of VEGF, FLT-1 and KDR between the normotensive and preeclampsia groups. We specifically observed hypomethylated CpGs in the promoter region and an increased expression of VEGF gene between term and preterm preeclampsia. However, mean promoter CpG methylation could not account for the higher expression of FLT-1 and KDR in preterm preeclampsia as compared to normotensive group.ConclusionsOur data indicates altered DNA methylation patterns in the VEGF, FLT-1 and KDR genes in preeclampsia as compared to the normotensive group, which could be involved in the pathophysiology of preeclampsia. Hypomethylation of VEGF promoter and consequent upregulation of VEGF mRNA levels could be a compensatory mechanism to restore normal angiogenesis and blood flow in preterm preeclampsia. This study suggests a role of altered DNA methylation in placental angiogenesis and in determining adverse pregnancy outcomes.

Gestation‐dependent changes in human placental global DNA methylation levels

The placenta, which plays a critical structural and functional role duringpregnancy, is a key link in the chain of events that lead to intrauterine programming of adult health. Placental development involves spatio-temporally programmed epigenetic processes that, if altered, may affect the gene expression programs for different cell types leading to abnormal placentation, thereby affecting birth outcome. The present study, for the first time, examines the global DNA methylation levels as a function of gestation in placentas of normotensivewomendelivering at term and preterm who were matched for age and socioeconomic status. A total number of 98 normotensive pregnant women with singleton pregnancy recruited at the Department of Obstetrics and Gynecology, Bharati Hospital, Pune, during the years 2007--2009, were included in this study. Pregnant women with complications such as preeclampsia, gestational diabetes, anemia, chronic hypertension, and type I or type II diabetes mellitus were excluded from the study. Genomic DNA was isolated from placental tissues and global DNA methylation was measured using the Methylamp Global DNA Methylation Quantification Kit (EpigentekGroup, Inc., NewYork,NY) (Kulkarni et al., 2010). Our results show a positive association (r1⁄4 0.32; P< 0.01) between global DNA methylation and gestational age at birth in the study cohort (Fig. 1). In contrast to our previous study in preeclampsia (Kulkarni et al., 2011), the present study shows decreased DNAmethylation levels in preterm deliveries. The disturbed placental pathophysiology of preeclampsia could account for contrasting trends in DNA methylation levels in normotensive preterm and preeclamptic preterm placentas. Further the observed positive association with gestational age at birth suggests that the gestational age-dependent profile is important to evaluate and control, when considering any methylation change identified as a potential biomarker, particularly in preterm pregnancies. Placental development and function is coordinated by interactions among genetic, epigenetic, and physiological cues that are differentially interpreted as a function of gestational age (Oyama, 2000). It has been shown in animal models that after implantation, the bulk of the genome becomes hypermethylated through active de novo methylation. The dynamic nature ofmethylation patterns during development has also been demonstrated in humans. At every stage of fetal development there is a sequence of de novo methylation and chromatin remodeling that dictates the tissue structure and function through a finely tuned pattern involving the switching on and off of gene expression. There are critical spatiotemporal windows during which these programs must be completed; failure to complete these programs in time may be irrecoverable and have long-term consequences (Hales and Barker, 2001). Our results, therefore, have implications for the consequences of prematurity since premature birth may impede the normal spatio-temporal pattern of gene expression affecting later development of the infant after birth, and for fetal programming of adult diseases since preterm babies are known to be at increased risk of neurodevelopmental and metabolic disorders in later life. However, there is a need to study the methylation patterns of the relevant genes in placenta of women delivering preterm and at term to test this hypothesis.

Gestation dependant changes in angiogenic factors and their associations with fetal growth measures in normotensive pregnancy.

BACKGROUND Earlier studies indicate that altered angiogenesis at birth is associated with poor birth outcome in women with preeclampsia. Now, we hypothesize that the progressive gestation dependant changes in markers of angiogenesis will be more useful to predict birth weight early even in a normotensive pregnancy. This study for the first time examines the association of gestation dependant changes in the levels of maternal angiogenic factors in addition to their levels in cord with birth weight. METHOD Ninety two pregnant women were followed at three different time points: 16-20 weeks, 26-30 weeks and at delivery during pregnancy. Plasma levels of angiogenic and anti angiogenic factors were determined by commercial enzyme-linked immunosorbent assay (ELISA) kits. RESULTS Maternal plasma VEGF levels increased (p<0.01) till the second time point and decreased (p<0.05) up to delivery while plasma sFlt-1 levels increased (p<0.01) at delivery. PlGF levels peaked (p<0.01) at second time point and decreased (p<0.01) at delivery. Cord plasma VEGF levels were higher (p<0.01) and sFlt-1 levels were lower (p<0.01) as compared to maternal values at all time points. Maternal plasma VEGF levels at first time point and PlGF levels at delivery were positively (p<0.05 and p<0.01 respectively), while sFlt-1/PlGF ratio at delivery was negatively associated (p<0.05) with birth weight. CONCLUSION Levels of pro- and anti-angiogenic factors may be differentially regulated across gestation. Maternal VEGF levels at early gestation (16-20 weeks) may be predictive of birth weight in healthy term pregnancies.

A longitudinal study of circulating angiogenic and antiangiogenic factors and AT1-AA levels in preeclampsia

Our earlier studies of preeclampsia (PE) at delivery have demonstrated the alteration of one carbon cycle, reduced placental omega 3 fatty acids, altered circulating levels of angiogenic factors and differential placental gene-specific methylation patterns of angiogenic factors. This study was undertaken to examine changes in the levels of angiogenic factors and angiotensin II type 1 receptor autoantibodies (AT1-AAs) throughout gestation, from early pregnancy until delivery, in women with PE and to examine their association with cord angiogenic factors, blood pressure and infant weight. A total of 81 pregnant women (46 normotensive and 35 with PE) were followed at three different time points during pregnancy: 16–20 weeks (T1), 26–30 weeks (T2) and at the time of delivery (T3). The plasma levels of angiogenic factors and AT1-AAs were determined in the maternal and cord plasma by commercial enzyme-linked immunosorbent assay kits. Maternal plasma levels of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) were lower (P<0.05 for both), whereas soluble fms-like tyrosine kinase-1 (sFlt-1; P<0.05) and the sFlt-1/PlGF ratio (P<0.01) were higher in early pregnancy in the PE group. Maternal plasma AT1-AA levels were higher (P<0.05) at T2 in women with PE. Cord plasma VEGF and soluble kinase insert domain receptor (sKDR) levels were lower (P<0.01 and P<0.05, respectively), whereas AT1-AA levels were higher (P<0.05) in the PE group. Maternal plasma VEGF levels in early pregnancy were positively associated with systolic blood pressure, whereas the sFlt-1/PlGF ratio at T2 was negatively associated with infant weight in the PE group. Low levels of proangiogenic factors (VEGF and PlGF) and high levels of AT1-AAs and antiangiogenic factors (sFlt-1 and sFlt-1/PlGF ratio) are present in the maternal circulation during early gestation in women with PE.

Altered metabolism of maternal micronutrients and omega 3 fatty acids epigenetically regulate matrix metalloproteinases in preterm pregnancy: A novel hypothesis

Preterm birth is an important perinatal health problem. Several possible mechanisms have been proposed but it may be important to have a testable mechanistic hypothesis that can explain the possible common mechanism for preterm births around the globe. Altered metabolism of micronutrients, like folic acid, vitamin B(12), zinc and copper are known to be associated with adverse pregnancy outcomes such as preterm birth. We have recently reported that increased oxidative stress and reduced docosahexaenoic acid levels are associated with preterm delivery. Matrix metalloproteinases and their tissue inhibitors play vital roles in extracellular matrix remodelling/degradation during pregnancy. Expression and the activity of matrix metalloproteinases have been shown to be regulated by oxidative stress and hyperhomocysteinemia. We have recently reported gestation dependant changes in placental global methylation levels. Here, we propose a novel hypothesis that altered maternal micronutrients (folic acid, vitamin B(12)), omega 3 fatty acids, and consequent oxidative stress lead to altered epigenetic mechanisms resulting in altered expression of matrix metalloproteinases and their tissue inhibitors during pregnancy. This may have important implications in the epigenetic programming of adult diseases since preterm infants are known to be at increased risk for neurodevelopmental, metabolic and cardiovascular dysfunctions in later life.

Matrix Metalloproteinase-1 and -9 in Human Placenta during Spontaneous Vaginal Delivery and Caesarean Sectioning in Preterm Pregnancy

Preterm birth is a major public health problem in terms of loss of life, long-term and short term disabilities worldwide. The process of parturition (both term and preterm) involves intensive remodelling of the extracellular matrix (ECM) in the placenta and fetal membranes by matrix metalloproteinases (MMPs). Our previous studies show reduced docosahexaenoic acid (DHA) in women delivering preterm. Further omega 3 fatty acids are reported to regulate MMP levels. This study was undertaken to examine the placental levels of MMPs and their association with placental DHA levels in women delivering preterm. The levels of MMP-1 and MMP-9 in 74 women delivering preterm (52 by spontaneous vaginal delivery and 22 by caesarean sectioning) and 75 women delivering at term (59 by spontaneous vaginal delivery and 16 by caesarean sectioning) were determined by enzyme-linked immunosorbent assay (ELISA) and their association with placental DHA was studied. Placental MMP-1 levels were higher (p<0.05) in women delivering preterm (both by spontaneous vaginal delivery and caesarean sectioning) as compared to those delivering at term. In contrast, placental MMP-9 levels in preterm pregnancies was higher (p<0.05) in women with spontaneous vaginal delivery while lower (p<0.05) in women delivering by caesarean sectioning. Low placental DHA was associated with higher placental MMP-9 levels. Our study suggests a differential effect of mode of delivery on the levels of MMPs from placenta. Further this study suggests a negative association of DHA and the levels of MMP-9 in human placenta although the mechanisms need further study.

Regional differences in the placental levels of oxidative stress markers in pre-eclampsia

To examine placental malondialdehyde (MDA), catalase, and glutathione peroxidase (GPx) levels in four placental regions among women with and without pre‐eclampsia.

Altered Methylation and Expression Patterns of Genes Regulating Placental Angiogenesis in Preterm Pregnancy

Introduction: Altered angiogenesis has been implicated in the pathogenesis of various pregnancy complications, particularly preeclampsia. At present, there is a lack of data on the possible role of angiogenesis and its molecular mechanism in preterm pregnancy. We have previously reported reduced placental global DNA methylation levels in preterm pregnancy. Now, we have extended the study to examine plasma levels of angiogenic factors from maternal and cord blood and correlate them with placental promoter CpG methylation and messenger RNA expression of these angiogenic genes in preterm pregnancies. Methods: We recruited 99 women delivering at term and 90 women delivering preterm. Plasma levels of angiogenic factors, vascular endothelial growth factor (VEGF), placental growth factor (PlGF), fms-related tyrosine kinase 1 (FLT-1), and kinase insert domain receptor (KDR) were analyzed by enzyme-linked immunosorbent assay. Expression levels and promoter CpG methylation of angiogenic genes in placentae were determined by quantitative real-time polymerase chain reaction and by the Sequenom EpiTYPER technology, respectively. Results: Maternal VEGF and PlGF levels (P < .01 for both) were lower but soluble FLT-1 (sFLT-1) levels and sFLT-1–PlGF ratio (P < .05 for both) were higher in the preterm group. Placental VEGF expression (P < .05) was lower, and CpG site 14 in the VEGF promoter was hypermethylated (P < .05) in the preterm group. The KDR expression (P < .05) was higher in women delivering preterm. Conclusions: Our study provides first evidence of differential placental CpG methylation patterns and expression of VEGF, FLT-1, and KDR genes in women delivering preterm. This may explain the possible mechanism for angiogenic imbalance in the pathophysiology of preterm pregnancy.

Maternal nutrition influences angiogenesis in the placenta through peroxisome proliferator activated receptors: A novel hypothesis

Placental angiogenesis is critical to maintain adequate blood flow during gestation, and any alterations in this process can result in an adverse pregnancy. Growing evidence indicates that suboptimal maternal nutrition can alter placental development. Although the underlying mechanisms are not clear, maternal nutrition likely influences the expression of genes involved in placental development through regulation of various transcription factors such as peroxisome proliferator‐activated receptors (PPARs), which can be activated by ligands including long‐chain polyunsaturated fatty acids. Indeed, several studies demonstrated a role for PPAR in implantation, trophoblast differentiation, and angiogenesis. Alterations in maternal nutrition during pregnancy can affect the expression of PPARs via epigenetic mechanisms or through homocysteine, which is known to compete for PPARs. This review discusses the role of maternal nutrition—particularly micronutrients like folate, vitamin B12, and omega‐3 fatty acids—in modulating the activity of PPARs during placentation and angiogenesis, which affects placental and fetal growth. Additional animal and human studies need to be undertaken to elucidate the molecular mechanisms through which maternal nutrition regulates PPARs, specifically to determine whether PPARs affect placental angiogenesis directly through angiogenic factors or indirectly by modulating trophoblast differentiation. Mol. Reprod. Dev. 82: 726–734, 2015.

Matrix metalloproteinases-2, -3 and tissue inhibitors of metalloproteinases-1, -2 in placentas from preterm pregnancies and their association with one-carbon metabolites

Maternal nutrition is an important determinant of one-carbon metabolism and defects in the one-carbon metabolism may lead to poor obstetric outcomes. This study was designed to test the hypothesis that altered intake/metabolism of micronutrients (folic acid and vitamin B12) and docosahexaenoic acid (DHA) contributes to increased homocysteine and oxidative stress leading to altered levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in women delivering preterm. We have earlier reported increased vitamin B12, homocysteine, and oxidative stress along with reduced placental DHA in women delivering preterm. In this study, we further examine the placental levels of MMP2, MMP3, TIMP1, and TIMP2 in 75 women delivering at term and 73 women delivering preterm. Placental levels of MMPs and TIMPs were determined by ELISA. Placental MMP2 and MMP3 levels were higher (P<0.01) in women delivering preterm as compared with term. There was no difference in the placental TIMP1 and TIMP2 levels in women delivering preterm and at term. Further placental MMP2 and MMP3 levels were higher (P<0.01) in women with preterm labor as compared with those in labor at term, suggesting that MMPs may favor degradation of extracellular matrix in the placenta during preterm labor. Our study for the first time suggests a crucial role of micronutrients and MMPs in preterm birth. Future studies need to examine if epigenetic modifications through the one-carbon cycle contribute to increased levels of MMPs leading to preterm deliveries.