To determine placental microRNA (miRNA) expression at different gestational age, total RNA from six first and six third trimester placentas was isolated. miRNA expression was analyzed by Affymetrix miRNA microarray, and miRNA clusters were identified by web-based programs MirClust and miRGen Cluster. qRT-PCR was carried out to validate miRNA expression, and in situ hybridization (ISH) was performed to determine compartmental localization of miRNAs within villous tissue. A total of 208 miRNA transcripts, which represent 191 mature miRNAs, were found differently expressed between first and third trimester placentas. miRNAs within the miR-17-92 cluster, C14MC, miR-371 cluster, and C19MC were significantly upregulated in the first trimester placentas. In contrast, miRNAs of the let-7 family, miR-34 family, miR-29a cluster, miR-195 cluster, and miR-181c cluster were significantly upregulated in the third trimester placentas. Increased miR-371-5p, miR-17-3p, and miR-708 -5p expression and decreased miR125b-5p and miR-139 -5p expression in the first trimester placentas were confirmed by qRT-PCR. Different expression pattern for miR-371-5p and miR-125b-5p within villous tissue was demonstrated by ISH. Distinct miRNA cluster expression profiles between the first and third trimester placentas were identified. miRNAs that regulate innate/ adaptive immune responses are strongly expressed in both first and third trimester placentas. miRNAs that exert oncogenic, angiogenic, and antiapoptotic properties are dominantly expressed in the first trimester placentas, whereas miRNAs that promote cell differentiation and function as tumor suppressors are strongly expressed in the third trimester placentas. These results indicate that miRNAs play critical roles in placental development. miRNA; placenta; pregnancy; development MICRORNAS (MIRNAS) HAVE EMERGED as important posttranscriptional regulators of gene expression. miRNAs induce translational repression, target degradation, and gene silencing by binding to complementary sequences on target messenger RNA (mRNA) transcripts. It has been estimated that ϳ30 -60% of mRNAs are regulated by miRNAs, which virtually control almost every aspect of cellular events from stem cell differentiation (4, 14) and organ development and formation (2, 6), and aging (15) to physiological/pathophysiological processes in cancer cell growth and metastasis (19, 39), genetic and cardiovascular diseases (13,20,38), and metabolic disorders (34, 36).The placenta is the first organ formed during pregnancy. It connects the developing fetus to the mother's uterine wall. Normal placental development is critical for a healthy pregnancy outcome to both the mother and the fetus. Although the cellular and molecular mechanisms in the regulation of placental development are largely unknown, recent studies suggest that deregulation of miRNA expression in placental trophoblasts may contribute to pregnancy complications that are associated with placental insufficiency including preeclampsia and intrauterine growth restrict...

Vitamin D insufficiency/deficiency during pregnancy has been linked to increased risk of preeclampsia. Placenta dysfunction plays an important role in the pathogenesis of this pregnancy disorder. In this study, we tested the hypothesis that disturbed vitamin D metabolism takes place in preeclamptic placentas. Protein expressions of vitamin D binding protein (VDBP), 25-hydroxylase (CYP2R1), 1␣-hydroxylase (CYP27B1), 24-hydroxylase (CYP24A1), and vitamin D receptor (VDR) were examined in placentas from normotensive and preeclamptic pregnancies. By immunostaining we found that in normal placenta VDBP, CYP24A1, and VDR expressions are localized mainly in trophoblasts, whereas CYP2R1 and CYP27B1 expressions are localized mainly in villous core fetal vessel endothelium. Protein expressions of CYP2R1 and VDR are reduced, but CYP27B1 and CYP24A1 expressions are elevated, in preeclamptic compared with normotensive placentas. Because increased oxidative stress is an underlying pathophysiology in placental trophoblasts in preeclampsia, we further determined whether oxidative stress contributes to altered vitamin D metabolic system in placental trophoblasts. Trophoblasts isolated from normal-term placentas were treated with hypoxic-inducing agent CoCl 2, and protein expressions of VDBP, CYP2R1, CYP27B1, CYP24A1, and VDR were determined. We found that hypoxia-induced downregulation of VDBP, CYP2R1, and VDR and upregulation of CYP27B1 and CYP24A1 expressions were consistent with that seen in preeclamptic placentas. CuZnSOD expression was also downregulated in trophoblasts treated with CoCl 2. These results provide direct evidence of disrupted vitamin D metabolic homeostasis in the preeclamptic placenta and suggest that increased oxidative stress could be a causative factor of altered vitamin D metabolism in preeclamptic placentas. Vitamin D status during pregnancy has been drawing great attention (19,22,32,39). Studies have shown that sufficient vitamin D intake during pregnancy reduces the risk of complications, including gestational diabetes, preterm birth, and infection (8, 36). Conversely, vitamin D deficiency during pregnancy has been linked to several adverse pregnancy outcomes (19,22,32,39), including those associated with placental insufficiency such as preeclampsia and low birth weight (8,36). Maternal 25(OH)D 3 levels are lower in women with preeclampsia than in normotensive pregnant women (1, 7). Moreover, a nested case control study revealed that maternal vitamin D deficiency at less than 22 wk of gestation is a strong, independent risk factor for preeclampsia (7). The finding of the association of maternal vitamin D deficiency and increased risk of preeclampsia (3,7,28,29) further emphasizes the importance of adequate vitamin D levels and proper vitamin D metabolism during pregnancy.Normal placental development and function ensure a healthy pregnancy outcome. It is believed that during pregnancy, 1,25(OH) 2 D 3 may be produced not only by kidneys but also by placenta trophoblasts. Human placenta and decidua are c...

Renal injury is a common pathophysiological feature in women with preeclampsia as evidenced by increased protein leakage (proteinuria) and glomerular injury (glomerular endotheliosis). Recently, podocyturia was found in preeclampsia, suggesting podocyte shedding occurs in this pregnancy disorder. However, podocyte function in preeclampsia is poorly understood. In this study, the authors have examined podocyte-specific protein expressions for nephrin, glomerular epithelial protein 1 (GLEPP-1), and ezrin in kidney biopsy tissue sections from women with preeclampsia. Expressions for vascular endothelial growth factor (VEGF) and its receptor Flt-1 and oxidative stress marker nitrotyrosine and antioxidant CuZn-superoxide dismutase (CuZn-SOD) were also examined. Kidney tissue sections from nonhypertensive and chronic hypertensive participants were stained as controls. The findings were (1) nephrin and GLEPP-1 were mainly expressed in glomerular podocytes; (2) ezrin was expressed in both glomerular podocytes and tubular epithelial cells; (3) compared to tissue sections from nonhypertensive and chronic hypertensive participants, nephrin and GLEPP-1 expressions were much reduced in tissue sections from preeclampsia and ezrin expression was reduced in podocytes; (4) enhanced VEGF, Flt-1, and nitrotyrosine, but reduced CuZn-SOD, expressions were observed in both glomerular podocytes and endothelial cells in tissue sections from preeclampsia; and (5) the expression pattern for nephrin, GLEPP-1, ezrin, VEGF, Flt-1, and CuZn-SOD were similar between tissue sections from nonhypertensive and chronic hypertensive participants. Although the authors could not conclude from this biopsy study whether the podocyte injury is the cause or effect of the preeclampsia phenotype, the data provide compelling evidence that podocyte injury accompanied by altered angiogenesis process and increased oxidative stress occurs in kidney of patients with preeclampsia.

We evaluated the leukocyte differentials in women with normal pregnancies and in pregnancies complicated by preeclampsia (PE). A retrospective study was performed in 240 women who were delivered at Louisiana State University Health Sciences Center– Shreveport, Louisiana, from January 1, 2002, to July 31, 2003. A total of 80 patients were studied in each group: normal pregnancy, mild PE, or severe PE. Leukocyte total and neutrophil, lymphocyte, monocyte, eosinophil, basophil, hemoglobin, and platelet counts were analyzed by analysis of variance and pairwise comparison. Data are presented as mean ± standard deviation. A p value <0.05 was set as statistically different. The total leukocyte count was significantly increased in women with severe PE compared with women with mild PE and normal pregnant controls: 10.66 ± 3.70 (p < 0.0001) versus 9.47 ± 2.59 and 8.55 ± 1.93 (1 × 103/µL), respectively. The increased total leukocyte count was mainly due to the increase in neutrophil numbers: 8.05 ± 4.01 (severe; p < 0.0001) versus 6.69 ± 2.23 (mild) and 5.90 ± 1.79 (controls), respectively. The total neutrophil count was further increased 48 hours after delivery in the group with severe PE. No statistical differences for monocyte and lymphocyte counts were observed between normal and PE groups. Increased neutrophil numbers account for the leukocytosis in women with PE.

Habituation is a measure of the ability to inhibit responding and is a more mature form of behavior than is persistent responding. We examined the developmental trend in habituation of the fetal startle response to repeated vibroacoustic stimulation in 90 normal human fetuses between 28 and 40 weeks of gestation. Fetal movement was graded according to the nature of the behavioral response: general startle (3), fast limb movement (2), slow rolling movement (1), and no movement (0). A significant developmental difference (p < 0.0004) in the rate of habituation was found, with response decrement occurring faster in fetuses of more than 32 weeks of gestation. Furthermore, by dividing the patients into three gestational age groups, it was determined that the greatest change in the rate of habituation occurred between 28 and 32 weeks and 32 and 36 weeks. We conclude that the rate of fetal habituation may be determined by the degree of maturation of the neural circuitry governing this form of nonassociative learning.

Wang Y, Zhao S, Loyd S, Groome LJ. Increased urinary excretion of nephrin, podocalyxin, and ␤ig-h3 in women with preeclampsia. Am J Physiol Renal Physiol 302: F1084 -F1089, 2012. First published February 1, 2012 doi:10.1152/ajprenal.00597.2011Emerging evidence has shown that podocyte injury and reduced specific podocyte protein expressions contribute to proteinuria in preeclampsia. We collected urine specimens from women with preeclampsia to study whether podocyte-specific protein shedding is associated with renal barrier dysfunction. Urine specimens from women with normal pregnancies and from pregnant women complicated by chronic hypertension were used for comparison. We determined soluble podocyte slit protein nephrin levels in the urine specimens. Podocalyxin, ␤ig-h3, and VEGF concentrations were also measured. We found that nephrin and podocalyxin were barely detectable in the urine specimens from normal pregnant women and from women with chronic hypertension. In preeclampsia, urinary nephrin and podocalyxin concentrations were significantly increased and highly correlated to each other, r 2 ϭ 0.595. Nephrin and podocalyxin were also correlated with urine protein concentrations. ␤ig-h3 was detected in the urine specimens from women with preeclampsia, and it is highly correlated with nephrin and podocalyxin concentrations in preeclampsia. ␤ig-h3 was undetectable in normal pregnancy and pregnancy complicated by chronic hypertension. Elevated VEGF levels were also found in women with preeclampsia compared with those of normal pregnancy and pregnancy complicated by chronic hypertension. These results provide strong evidence that podocyte protein shedding occurs in preeclampsia, and their levels are associated with proteinuria. The finding of urinary ␤ig-h3 excretion in preeclampsia suggests that increased transforming growth factor activity might also be involved in the kidney lesion in this pregnancy disorder. kidney lesion; glycoprotein shedding; TGF-␤; proteinuria; pregnancy complication NEWLY DEVELOPED HYPERTENSION and proteinuria after 20 wk of gestation are the two major clinical diagnostic criteria for preeclampsia, a hypertensive and multiple-system disorder unique to human pregnancy. Plasma protein leakage into the urine signifies renal/kidney lesions in this pregnancy disorder. Pathophysiologically, renal lesions are attributed to the glomerular endotheliosis characterized by glomerular endothelial cell swelling and subendothelial deposits of protein materials (5). Renal vasoconstriction is another contributing factor as part of the global systemic vasoconstriction in preeclampsia (9). Emerging evidence has revealed that other than glomerular endotheliosis, podocyte injury also plays a significant role in glomerular barrier dysfunction in preeclampsia (7,8,12,27,28). Supporting evidence includes 1) reduced podocyte-specific protein nephrin and synaptopodin expressions in kidney autopsy or biopsy specimens from women who had preeclampsia (7, 27); 2) detection of viable podocytes in the urine specimens ...

Endothelial dysfunction associated with vitamin D deficiency has been linked to many chronic vascular diseases. Vitamin D elicits its bioactive actions by binding to its receptor, vitamin D receptor (VDR), on target cells and organs. In the present study, we investigated the role of VDR in response to 1,25(OH)2D3 stimulation and oxidative stress challenge in endothelial cells. We found that 1,25(OH)2D3 not only induced a dose- and time-dependent increase in VDR expression, but also induced up-regulation of vascular endothelial growth factor (VEGF) and its receptors (Flt-1 and KDR), as well as antioxidant CuZn-superoxide dismutase (CuZn-SOD) expression in endothelial cells. We demonstrated that inhibition of VDR by VDR siRNA blocked 1,25(OH)2D3 induced increased VEGF and KDR expression and prevented 1,25(OH)2D3 induced endothelial proliferation/migration. Using CoCl2, a hypoxic mimicking agent, we found that hypoxia/oxidative stress not only reduced CuZn-SOD expression, but also down-regulated VDR expression in endothelial cells, which could be prevented by addition of 1,25(OH)2D3 in culture. These findings are important indicating that VDR expression is inducible in endothelial cells and oxidative stress down-regulates VDR expression in endothelial cells. We conclude that sufficient vitamin D levels and proper VDR expression are fundamental for angiogenic and oxidative defense function in endothelial cells.

Increased placental release of soluble VEGF receptor-1 (sFlt-1) is believed to play an important role in the pathogenesis of preeclampsia (PE). Although the reason for increased placental sFlt-1 release in PE is unknown, proteolytic effect has been proposed as one of the mechanisms that mediate sFlt-1 release in the placenta. In this study, using various protease inhibitors, we tested the possible role of proteases in sFlt-1 release by human placenta. Villous explants from normal term placentas were incubated with various protease inhibitors including serine protease inhibitors (PMSF, aprotini, and specific chymotrypsin inhibitor (CI)), cysteine protease inhibitor E-64, metalloendopeptidase inhibitor PAD, and universal metalloprotease (ADAM) inhibitor PTM. Culture medium was collected and measured for sFlt-1 by ELISA. Our results showed that villous tissue treated with CI and PTM produced significantly less sFlt-1 than those of controls. PMSF, aprotini, E-64, and PAD had no effect on sFlt-1 release. We further examined chymotrypsin-like protease/chymase and ADAM10 expressions in tissue sections from normal and PE placentas by immunohistochymistry. We found that immunostaining for chymase and ADAM10 was significantly increased in the layer of syncytiotrophoblasts in PE placentas compared to normal placentas. These results suggest chymotrypsin-like serine protease and ADAM10, but not cysteine protease and metalloendopeptidase, may play a role in inducing sFlt-1 release in PE placentas.