Asmita Joshi

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.

Maternal omega 3 fatty acid supplementation during pregnancy to a micronutrient-imbalanced diet protects postnatal reduction of brain neurotrophins in the rat offspring

An altered one carbon cycle (folic acid, vitamin B(12)) and omega 3 fatty acid metabolism during pregnancy can increase the risk for neurodevelopmental disorders in the offspring. Our earlier studies have shown that a maternal diet imbalanced with micronutrients like folic acid, vitamin B(12) reduces levels of brain docosahexaenoic acid (DHA) and neurotrophins in the offspring at birth. The present study examines whether these effects can be reversed by a postnatal diet. Pregnant female rats were divided into six treatment groups at two levels of folic acid both in the presence and absence of vitamin B(12). Omega 3 fatty acid supplementation was given to the vitamin B(12)-deficient groups. Following delivery, eight dams from each group were randomly shifted back to control and remaining eight continued on the same treatment diet. Plasma homocysteine levels could be normalized by a postnatal control diet. Brain DHA levels were similar in all the groups irrespective of the diet consumed during lactation. Brain-derived nerve growth factor (BDNF) and nerve growth factor (NGF) levels were lower in both the vitamin B(12)-deficient groups even after consuming a diet with normal levels of vitamin B(12) during lactation (p<0.05 for all) indicating that the effects of maternal programing with respect to neurotrophins cannot be reversed by a postnatal diet. Our findings for the first time suggest that omega 3 fatty acid supplementation to a micronutrient-imbalanced diet, during pregnancy and lactation protects the levels of BDNF and NGF. This may have significant implications in the development of psychiatric disorders/cognitive deficits in later life.

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 maternal proportions of long chain polyunsaturated fatty acids and their transport leads to disturbed fetal stores in preeclampsia

Our previous cross-sectional studies have shown altered proportions of long chain polyunsaturated fatty acids (LCPUFA) in preeclampsia (PE) at the end of pregnancy when the pathology has already progressed. The present longitudinal study for the first time reports fatty acid proportions from 16th week of gestation till delivery and placental transport in PE. This is a hospital based study where women were recruited in early pregnancy. Maternal blood was collected at 3 time points i.e. T1=16-20th week, T2=26-30th week and T3=at delivery. Cord blood and placenta were collected at delivery. This study reports data on 140 normotensive control (NC) and 54 PE women. In PE we report lower proportions of DHA in maternal plasma at T1, cord plasma and placenta (p<0.05 for all). The mRNA levels of placental ∆5 desaturase, fatty acid transport proteins -1, -4, were lower (p<0.05 for all) in PE. There was also a positive association between cord and maternal plasma DHA and total omega-3 fatty acids at T1. This study demonstrates that women with PE have lower fatty acids stores at 16-20th week of gestation and lower placental synthesis and transport. It is likely that supplementation of omega-3 fatty acids during the 16-20th week of gestation may help in improving fatty acid status in infants born to mothers with PE.

Maternal micronutrients and omega 3 fatty acids affect placental fatty acid desaturases and transport proteins in Wistar rats.

Adequate supply of LCPUFA from maternal plasma is crucial for fetal normal growth and development. The present study examines the effect of maternal micronutrients (folic acid and vitamin B12) and omega 3 fatty acids on placental mRNA levels of fatty acid desaturases (Δ5 and Δ6) and transport proteins. Pregnant female rats were divided into 6 groups at 2 levels of folic acid both in the presence and absence of vitamin B12. Both the vitamin B12 deficient groups were supplemented with omega 3 fatty acid. Maternal vitamin B12 deficiency reduced placental mRNA and protein levels of Δ5 desaturase, mRNA levels of FATP1 and FATP4 (p<0.05 for all) as compared to control while omega 3 fatty acid supplementation normalized the levels. Our data for the first time indicates that altered maternal micronutrients and omega 3 fatty acids play a key role in regulating fatty acid desaturase and transport protein expression in placenta.

Expression of Genes Encoding Enzymes Involved in the One Carbon Cycle in Rat Placenta is Determined by Maternal Micronutrients (Folic Acid, Vitamin B12) and Omega-3 Fatty Acids

We have reported that folic acid, vitamin B12, and omega-3 fatty acids are interlinked in the one carbon cycle and have implications for fetal programming. Our earlier studies demonstrate that an imbalance in maternal micronutrients influence long chain polyunsaturated fatty acid metabolism and global methylation in rat placenta. We hypothesize that these changes are mediated through micronutrient dependent regulation of enzymes in one carbon cycle. Pregnant dams were assigned to six dietary groups with varying folic acid and vitamin B12 levels. Vitamin B12 deficient groups were supplemented with omega-3 fatty acid. Placental mRNA levels of enzymes, levels of phospholipids, and glutathione were determined. Results suggest that maternal micronutrient imbalance (excess folic acid with vitamin B12 deficiency) leads to lower mRNA levels of methylene tetrahydrofolate reductase (MTHFR) and methionine synthase , but higher cystathionine b-synthase (CBS) and Phosphatidylethanolamine-N-methyltransferase (PEMT) as compared to control. Omega-3 supplementation normalized CBS and MTHFR mRNA levels. Increased placental phosphatidylethanolamine (PE), phosphatidylcholine (PC), in the same group was also observed. Our data suggests that adverse effects of a maternal micronutrient imbalanced diet may be due to differential regulation of key genes encoding enzymes in one carbon cycle and omega-3 supplementation may ameliorate most of these changes.

Maternal micronutrient imbalance alters gene expression of BDNF, NGF, TrkB and CREB in the offspring brain at an adult age

Micronutrients like folate, vitamin B12, and fatty acids which are interlinked in the one carbon cycle play a vital role in mediating epigenetic processes leading to an increased risk for neurodevelopmental disorders in the offspring. Our earlier study demonstrates that a micronutrient imbalanced diet adversely affects docosahexaenoic acid (DHA) and protein levels of neurotrophins like brain‐derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the brain and cognition in the offspring by 3 months of age. In this study we attempt to analyze if these effects are a consequence of a change in gene expression of these molecules. Further, we also examined the effect of either a postnatal control diet or a prenatal omega‐3 fatty acid supplementation on gene expression in the cortex of the offspring. Pregnant rats were divided into control and five treatment groups at two levels of folic acid (normal and excess folate) in the presence and absence of vitamin B12. Omega‐3 fatty acid (eicosapentaenoic acid – EPA + DHA) supplementation was given to vitamin B12 deficient groups. Following delivery, 8 dams from each group were shifted to control diet and remaining continued on the same treatment diet. Our results demonstrate that the imbalanced diet caused a marked reduction in the mRNA levels of BDNF, NGF, TrkB, and cAMP response element‐binding protein (CREB). Prenatal omega‐3 fatty acid supplementation to the maternal imbalanced diet was able to normalize the mRNA levels of all the above genes. This study demonstrates that a maternal diet imbalanced in micronutrients (folic acid, vitamin B12) influences gene expression of neurotrophins and their signalling molecules and thereby adversely affects the brain of the offspring.

Levels of brain derived neurotrophic factors across gestation in women with preeclampsia.

Preeclampsia (PE) is a major pregnancy complication of placental origin which leads to adverse pregnancy outcome. Brain derived neurotrophic factor (BDNF) is suggested to promote trophoblast growth and regulate placental and fetal development. This study for the first time examines the levels of maternal plasma BDNF at various time points during gestation, cord plasma and placental BDNF levels and their association with birth outcome in women with PE. Normotensive control (NC) women (n = 89) and women with PE (n = 61) were followed at three different time points [16–20 weeks (T1), 26–30 weeks (T2) and at delivery (T3)]. Maternal blood at all time points and cord blood was collected. Results indicate that maternal BDNF levels at T1 (p = 0.050) and T3 (p = 0.025) were lower in women with PE than in NC women. Cord BDNF levels at delivery in women with PE were lower (p = 0.032) than those in NC women. Placental BDNF gene expression was also lower (p = 0.0082) in women with PE than in NC women. Our data suggests that BDNF plays an important role in the development of the materno‐fetal–placental unit during pregnancy. Alteration in the levels of BDNF during pregnancy may be associated with an abnormal development of the placenta resulting in PE.

Differential regulation of hepatic transcription factors in the Wistar rat offspring born to dams fed folic acid, vitamin B12 deficient diets and supplemented with omega-3 fatty acids.

Nutritional status of the mother is known to influence various metabolic adaptations required for optimal fetal development. These may be mediated by transcription factors like peroxisome proliferator activated receptors (PPARs), which are activated by long chain polyunsaturated fatty acids. The objective of the current study was to examine the expression of different hepatic transcription factors and the levels of global methylation in the liver of the offspring born to dams fed micronutrient deficient (folic acid and vitamin B12) diets and supplemented with omega-3 fatty acids. Female rats were divided into five groups (n = 8/group) as follows; control, folic acid deficient (FD), vitamin B12 deficient (BD) and omega-3 fatty acid supplemented groups (FDO and BDO). Diets were given starting from pre-conception and continued throughout pregnancy and lactation. Pups were dissected at the end of lactation. Liver tissues were removed; snap frozen and stored at −80°C. Maternal micronutrients deficiency resulted in lower (p<0.05) levels of pup liver docosahexaenoic acid (DHA) and arachidonic acid (ARA) as compared to the control group. Pup liver PPARα and PPARγ expression was lower (p<0.05) in the BD group although there were no differences in the expression of SREBP-1c, LXRα and RXRα expression. Omega-3 fatty acids supplementation to this group normalized (p<0.05) levels of both PPARα and PPARγ but reduced (p<0.05) SREBP-1c, LXRα and RXRα expression. There was no change in any of the transcription factors in the pup liver in the FD group. Omega-3 fatty acids supplementation to this group reduced (p<0.05) PPARα, SREBP-1c and RXRα expression. Pup liver global methylation levels were higher (p<0.01) in both the micronutrients deficient groups and could be normalized (p<0.05) by omega-3 fatty acid supplementation. Our novel findings suggest a role for omega-3 fatty acids in the one carbon cycle in influencing the hepatic expression of transcription factors in the offspring.