Remco Keijser

Expression of Placental FLT1 Transcript Variants Relates to Both Gestational Hypertensive Disease and Fetal Growth

The recent discovery of additional alternative spliced FLT1 transcripts encoding novel soluble (s)FLT1 protein isoforms complicates both the predictive value and functional implications of sFLT1 in preeclampsia. We investigated FLT1 expression levels and splicing patterns in placentas of normotensive and preeclamptic women, and established the tissue specificity of all FLT1 transcript variants. mRNA levels of sFLT1 splice variants were determined by real-time polymerase chain reaction in 21 normal human tissues and placental biopsies from 91 normotensive and 55 preeclamptic women. Cellular localization of placental FLT1 expression was established by RNA in situ hybridization. Of all tissues investigated, placenta has by far the highest FLT1 mRNA expression level, mainly localized in the syncytiotrophoblast layer. More than 80% of placental transcripts correspond to sFLT1_v2. Compared with normotensive placenta, preeclamptic placenta has ≈3-fold higher expression of all FLT1 transcript variants (P<0.001), with a slight shift in favor of sFLT1_v1. Although to a lesser degree, transcript levels are also increased in placenta from normotensive women that deliver a small for gestational age neonate. We conclude that sFLT isoform–specific assays could potentially improve the accuracy of current sFLT1 assays for the prediction of preeclampsia. However, placental FLT1 transcript levels are increased not only in preeclampsia but also in normotensive pregnancy with a small for gestational age fetus. This may indicate a common pathway involved in the development of both conditions but complicates the use of circulating sFLT1 protein levels for the prediction or diagnosis of preeclampsia alone.

Resistance Artery Creatine Kinase mRNA and Blood Pressure in Humans

Hypertension remains the main risk factor for cardiovascular death. Environmental and biological factors are known to contribute to the condition, and circulating creatine kinase was reported to be the main predictor of blood pressure in the general population. This was proposed to be because of high resistance artery creatine kinase-BB rapidly regenerating ATP for vascular contractility. Therefore, we assessed whether creatine kinase isoenzyme mRNA levels in human resistance arteries are associated with blood pressure. We isolated resistance-sized arteries from omental fat donated by consecutive women undergoing uterine fibroid surgery. Blood pressure was measured in the sitting position. Vessels of 13 women were included, 6 normotensive and 7 hypertensive, mean age 42.9 years (SE, 1.6) and mean systolic/diastolic blood pressure, 144.8 (8.0)/86.5 (4.3) mm Hg. Arteriolar creatine kinase isoenzyme mRNA was assessed using quantitative real-time polymerase chain reaction. Normalized creatine kinase B mRNA copy numbers, ranging from 5.2 to 24.4 (mean, 15.0; SE, 1.9), showed a near-perfect correlation with diastolic blood pressure (correlation coefficient, 0.9; 95% confidence interval, 0.6–1.0) and were well correlated with systolic blood pressure, with a 90% relative increase in resistance artery creatine kinase B mRNA in hypertensives compared with normotensives, normalized copy numbers were, respectively, 19.3 (SE, 2.0) versus 10.1 (SE, 2.1), P=0.0045. To our knowledge, this is the first direct evidence suggesting that resistance artery creatine kinase mRNA expression levels concur with blood pressure levels, almost doubling with hypertension. These findings add to the evidence that creatine kinase might be involved in the vasculature’s pressor responses.

Initial characterization of C16orf89, a novel thyroid-specific gene.

BACKGROUND Thyroid hormone is prerequisite for proper fetal and postnatal neurodevelopment, growth, and metabolism. Although much progress has been made in the characterization of genes implicated in thyroid development and function, the majority of genes involved in this process are still unknown. We have previously applied serial analysis of gene expression (SAGE) to identify novel genes preferentially expressed in the thyroid, and this has resulted in the characterization of DUOX2 and IYD (also known as DEHAL1), two genes encoding essential enzymes in the production of thyroid hormone. In the current study we characterize the gene C16orf89, which is linked to another thyroid-specific SAGE tag CCAGCTGCCT. METHODS We establish tissue-specific expression of C16orf89 using novel tissue-specific SAGE libraries and quantitative polymerase chain reaction. In addition, we characterize the C16orf89 gene and protein, and analyze its mRNA expression in response to thyrotropin and during mouse development. RESULTS C16orf89 is predominantly expressed in human thyroid tissue with a specificity intermediate between thyroid transcription factors and proteins involved in thyroid hormone synthesis. C16orf89 shows the same expression pattern as Nkx2-1 (thyroid transcription factor 1) from embryonic day (E) 17.5 onward in the developing mouse thyroid and lung. The developmental timing of C16orf89 mRNA expression is similar to that of the iodide transporter Slc5a5 (also known as Nis). Both transcripts are detected from E17.5 in the developing thyroid. This is clearly later than the onset of Tg mRNA expression (from E14.5), while Nkx2-1 and Iyd mRNA can already be detected in the E12.5 thyroid. In in vitro cell culture C16orf89 expression is stimulated by thyrotropin. The major splice variant encodes a 361 amino acid protein that is well conserved between mammals, contains an N-terminal signal peptide, is secreted in a glycosylated form, and does not contain any known functional domain. CONCLUSIONS We present a novel gene highly expressed in thyroid that encodes a currently enigmatic protein.

Increased glucocerebrosidase expression and activity in preeclamptic placenta

INTRODUCTION Lysosomal glucosidase beta acid (GBA) deficiency is inherent to Gaucher disease, Parkinsonism and Lewy-body dementia. Increased GBA expression has never been associated with human disease. We describe increased GBA expression and activity in placenta from preeclamptic pregnancies. METHODS 112 placenta biopsies were available for qPCR, analysis of GBA gene expression and activity. Microanalysis was performed on 20 placenta samples. Alternatively spliced placental GBA transcripts were cloned, expressed in HEK293 cells and analyzed by Western blot and activity assay. RESULTS GBA is expressed in the syncytiotrophoblast layer of human placenta already at 5 weeks of gestation. We identified five novel GBA transcripts in placenta that enzymatically inactive when expressed in HEK293 cells. Both GBA RNA expression and enzymatic activity are upregulated in preeclamptic placenta. Microarray analysis of 20 placenta tissues identified 158 genes co-regulating with GBA expression and gene enrichment analysis highlights lysosomal function. In our micro-array data GBA expression does not correlate with FLT1 expression, currently the most powerful marker for preeclampsia. There are 89 transcripts that are negatively correlated with GBA expression of which BMP4 and TFEB are interesting as they are essential to early placenta function. DISCUSSION Although very speculative, we hypothesize that increased GBA expression might relate to placentation through decreased BMP4 signaling or vascularization through downregulation of TFEB. Ceramide, the product of hydrolysis of glucosylceramide by GBA and involved in the regulation of cell differentiation, survival and apoptosis, is another putative candidate linking increased GBA activity to preeclampsia. Both pathways merit further investigation.

Total bile acids in the maternal and fetal compartment in relation to placental ABCG2 expression in preeclamptic pregnancies complicated by HELLP syndrome

OBJECTIVE To investigate total bile acid (TBA) levels in maternal (MB) and umbilical cord blood (UCB) in normotensive, preeclamptic (PE), and PE pregnancies complicated by hemolysis elevated liver enzymes and low platelets (HELLP) syndrome in the context of ABCG2 placental gene expression levels, a recently reported placental bile acid transporter. METHODS TBA levels were determined in 83 paired MB and UCB samples of normotensive, PE and PE/HELLP pregnancies and in 22 paired arterial and venous UCB samples from uncomplicated term pregnancies. ABCG2 gene expression was measured in 104 human placentas by reverse transcriptase quantitative polymerase chain reaction. RESULTS Overall, TBA levels in MB are higher compared to levels in UCB (p<0.0001), but this comparison looses statistical significance for the 11 PE/HELLP cases. TBA levels in maternal blood are increased in PE/HELLP compared to PE pregnancies (p=0.016). TBA levels in arterial and venous UCB from 22 normotensive pregnancies are not statistically different. ABCG2 expression is reduced in pregnancies where preeclampsia is further complicated by HELLP syndrome. ABCG2 expression in human placenta is not correlated with TBA levels in either the maternal or fetal compartment. CONCLUSION Increased maternal TBA levels in PE/HELLP pregnancies indicate a relation between bile acids in the maternal circulation and HELLP syndrome. As overall TBA levels in maternal blood are increased compared to UCB, we conclude that the placenta partly protects the fetus from increased maternal TBA levels. This consistent difference in TBA levels between the maternal and fetal compartment is unrelated to the placental expression of ABCG2.

Placental expression of heparan sulfate 3-O-sulfotransferase-3A1 in normotensive and pre-eclamptic pregnancies.

INTRODUCTION The endothelial glycocalyx, consisting of membrane-bound proteoglycans and attached glycosaminoglycans plays an important role in vascular homeostasis. We aimed to assess whether glycocalyx mRNA transcripts are differentially expressed in placental tissue of pre-eclamptic and normotensive women. METHODS We evaluated the expression of transcripts encoding for proteins involved in glycocalyx synthesis and degradation using a microarray analysis of placental mRNA obtained from pre-eclamptic and normotensive women. Participants were recruited from the department of obstetrics at a university hospital in Amsterdam, The Netherlands. The most prominent differentially expressed transcript was validated by qPCR on 112 additional placenta samples. RESULTS Of 78 preselected genes involved in glycocalyx synthesis and degradation, only HS3ST3A1 mRNA was differentially expressed in placental tissue obtained from pre-eclamptic women (N = 12) compared to normotensive women (N = 12, fold change = 0.61, p = 0.02). Validation with qPCR in additional placental samples of 64 normotensive and 48 pre-eclamptic women confirmed that normalized mRNA expression of HS3ST3A1 was decreased by 27% (95% CI 14%-41%) in placental tissue obtained from pre-eclamptic compared to normotensive women (p < 0.001). HS3ST3A1 expression was positively correlated with neonatal birth weight in normotensive women (r = 0.35, p < 0.01) and inversely correlated with mean arterial pressure of women with pre-eclampsia (r = 0.32, p = 0.02). CONCLUSIONS The mRNA expression of HS3ST3A1, which encodes for a 3-O sulfating enzyme of heparan sulfate (3-OST-3A1), is decreased in pre-eclamptic placental tissue. Expression of this glycocalyx synthesis transcript is correlated with maternal blood pressure and neonatal birth weight, suggesting a possible role in pre-eclampsia-associated placental dysfunction.

Higher decidual EBI3 and HLA-G mRNA expression in preeclampsia: Cause or consequence of preeclampsia

The maternal immune system must adapt to tolerate the invasion of the allogeneic feto-placental unit. It is generally accepted that improper adaptation causes pregnancy complications like preeclampsia. The Epstein-Barr virus-induced gene 3 (EBI3) protein is a subunit of immune-modulatory cytokines interleukin 27 (IL-27) and IL-35. EBI3 has been reported to associate with HLA-G. In this small pilot study we find higher decidual EBI3 (p<0.05) and HLA-G (p<0.01) mRNA expression in preeclampsia (n=7) compared to normotensive (n=8) pregnancies. Whether the higher EBI3 and HLA-G mRNA expression is a consequence or cause of preeclampsia remains to be answered. Further research to determine the effects on IL-27 and IL-35 is needed.