Is the atherosclerotic phenotype of preeclamptic placentas due to altered lipoprotein concentrations and placental lipoprotein receptors? Role of a small-for-gestational-age phenotype

Atherosis of spiral arteries in uteroplacental beds from preeclamptic women resemble those of atherosclerosis, characterized by increased plasma lipids and lipoproteins. We hypothesized that: 1) lipoprotein receptors/transporters in the placenta would be upregulated in preeclampsia, associated with increased maternal and fetal lipoprotein concentrations; and 2) expression of these would be reduced in preeclamptic placentae from women delivering small-for-gestational-age (SGA) infants. Placental biopsies and maternal and umbilical serum samples were taken from 27 normotensive and 24 preeclamptic women. Maternal/umbilical cord serum LDL, HDL, total cholesterol, and triglycerides were measured. Placental mRNA expression of lipoprotein receptors/transporters were quantified using quantitative RT-PCR. Protein localization/expression of LDL receptor-related protein 1 (LRP-1) in the preeclamptic placentae with/without SGA was measured by immunohistochemistry. Placental mRNA expression of all genes except paraoxonase-1 (PON-1), microsomal triglyceride transfer protein (MTTP), and protein disulfide isomerase family A member 2 (PDIA2) were observed. No differences for any lipoprotein receptors/transporters were found between groups; however, in the preeclamptic group placental LRP-1 expression was lower in SGA delivering mothers (n = 7; P = 0.036). LRP-1 protein was localized around fetal vessels and Hofbauer cells. This is the first detailed study of maternal/fetal lipoprotein concentrations and placental lipoprotein receptor mRNA expression in normotensive and preeclamptic pregnancies. These findings do not support a role of altered lipid metabolism in preeclampsia, but may be involved in fetal growth.     

Endothelia of term human placentae display diminished expression of tight junction proteins during preeclampsia

This study explores the molecular composition of the tight junction (TJ) in human term placenta from normal women and from patients with preeclampsia, a hypertensive disorder of pregnancy. Maternal endothelial dysfunction is a critical characteristic of preeclampsia; hence, we have analyzed its impact on placental vessels. The study concentrates on the TJ because this structure regulates the sealing of the paracellular route. We have found that, in placental endothelial vessels, TJ components include the peripheral protein ZO–1 and the integral proteins occludin and claudins 1, 3, and 5. During preeclampsia, the amounts of occludin and ZO–1 exhibit no significant variation, whereas those of claudins 1, 3, and 5 diminish, suggesting the presence of leakier TJs in the endothelia of the preeclamptic placenta, possibly in response to the decreased perfusion of this organ during preeclampsia. We have unexpectedly found that, in normal placentae, the multinucleated syncytiotrophoblast layer displays claudin 4 at the basal surface of the plasma membrane, and claudin 16 along the apical and basolateral surfaces. The presence of membrane-lined channels that cross the syncytiotrophoblast constituting a paracellular pathway has been determined by transmission electron microscopy and by the co-immunolocalization of claudin 16 with the plasma membrane proteins Na⁺K⁺-ATPase and GP135. Since claudin 16 functions as a paracellular channel for Mg²⁺, its diffuse pattern in preeclamptic placentae suggests the altered paracellular transport of Mg²⁺ between the maternal blood and the placental tissue.This work was supported by grants 45691-Q from the Mexican Council for Science and Technology (CONACYT) and 2005/1/I/012 from the Research Promotion Fund of the Mexican Institute of Social Security (IMSS/FOFOI).     

Immunohistochemical expression of von Willebrand factor in the preeclamptic placenta

Preeclampsia is a high-prevalence systemic pregnancy disorder associated with maternal and foetal mortality. Its pathogenesis is unknown, but it is thought that oxidative stress and endothelial dysfunction may play a fundamental role. Von Willebrand factor (vWF), a marker of endothelial cell injury, can be found in different cells and zones of the placenta. To determine the differential immunoexpression of vWF at different tissue types of preeclamptic placenta and endothelial dysfunction markers at maternal serum of preeclamptic pregnancies. A case–control study was performed on a population of pregnant women with preeclampsia (n = 14), and normal pregnancies (n = 8). Placental and blood plasma samples were withdrawn at delivery. Immunohistochemical vWF expression in the placental tissue was determined. Endothelial dysfunction was assessed through plasminogen activator inhibitor (PAI) 1 and 2 ratio and vWF concentration in maternal plasma. P values less than 0.05 were considered statistically significant. Preeclamptic women showed increased plasma PAI-1/PAI-2 ratio (P < 0.05). There was diminished placental vWF expression in syncytiotrophoblast and increased in the intervillous space of preeclamptic placentas (P < 0.05). No significant differences in vWF expression were found in the villous endothelium and stroma, but it was significantly higher in maternal plasma (P < 0.05). In preeclampsia occurs endothelial damage and placental cell injury. Cell damage in syncytiotrophoblast that occurs in preeclampsia could liberate vWF from syncytiotrophoblast to the placental intervillous space, and this may have pathogenic implications.     

Distribution of Notch protein members in normal and preeclampsia-complicated placentas

Notch proteins are a transmembrane receptor family that is structurally and functionally conserved from worms to humans. The mammalian family of Notch proteins consists of several genes encoding Notch receptors and related Notch ligands. Notch signaling is involved in different aspects of the cell-fate decision tree: differentiation, proliferation, and apoptosis. These three processes are finely regulated in human placenta in order to allow a successful pregnancy and correct fetal growth. Notch and its ligands also participate in vascular remodeling and stabilization. Vasculogenesis and blood regulation are of importance in the human placenta for normal fetal development and growth; any disorder of these systems leads to preeclampsia. Drawing on this background, we have investigated the expression of Notch-1, Notch-4, and Jagged-1, together with two members related to the Notch pathway in angiogenesis: VEGF and p21. Normal and preeclamptic human placentas have been evaluated by immunohistochemistry. In preeclamptic samples, a down-regulation of Notch pathway members occurs with a weak/moderate expression of the Notch protein members in all components of placenta compared with physiological placentas that, at term, exhibit the strong expression of Jagged-1 and a moderate expression of both Notch-1 and Notch-4 in all compartments of the placental villi. Moreover, preeclamptic samples also reveal a down-regulation of VEGF expression, together with a moderate nuclear expression of p21^Cip1 in the syncytiotrophoblast, cytotrophoblast, and endothelial cells. This down-regulation of VEGF in preeclamptic placentas, in turn, probably decreases Notch protein expression in placental compartments and in endothelial cells and could offer an ethiopathogenetic explanation for the onset of this pathology.     

Reduced placental docosahexaenoic acid levels associated with increased levels of sFlt-1 in preeclampsia

Our earlier studies, in preeclamptic women have shown altered levels of long chain polyunsaturated fatty acids (LCPUFA), essential constituents of the cell membrane lipids responsible for membrane stability as one of the key factors contributing to the pathophysiology of preeclampsia. We have also reported elevated levels of sFlt-1 in preeclampsia. The present study examines the levels of LCPUFA and their association with sFlt-1 levels in 69 pre-eclamptic women and 40 normotensive women. DHA and omega 3 fatty acid levels were lower (p<0.001) while arachidonic acid and omega 6 fatty acid levels were higher (p<0.05) in preeclamptic women as compared to normotensive women. Maternal plasma sFlt-1 levels were higher (p<0.05) in preeclamptic women and were negatively associated with DHA (p=0.008) and omega 3 fatty acids concentrations (p=0.031). Our results suggest that altered placental LCPUFA may result in altered membrane lipid fatty acid composition leading to increased release of sFlt-1 in circulation.     

Placental Aromatase Is Deficient in Placental Ischemia and Preeclampsia

IntroductionPreeclampsia is a maternal hypertensive disorder with uncertain etiology and a leading cause of maternal and fetal mortality worldwide, causing nearly 40% of premature births delivered before 35 weeks of gestation. The first stage of preeclampsia is characterized by reduction of utero-placental blood flow which is reflected in high blood pressure and proteinuria during the second half of pregnancy. In human placenta androgens derived from the maternal and fetal adrenal glands are converted into estrogens by the enzymatic action of placental aromatase. This implies that alterations in placental steroidogenesis and, subsequently, in the functionality or bioavailability of placental aromatase may be mechanistically involved in the pathophysiology of PE.MethodsSerum samples were collected at 32–36 weeks of gestation and placenta biopsies were collected at time of delivery from PE patients (n = 16) and pregnant controls (n = 32). The effect of oxygen tension on placental cells was assessed by incubation JEG–3 cells under 1% and 8% O₂ for different time periods, Timed-mated, pregnant New Zealand white rabbits (n = 6) were used to establish an in vivo model of placental ischemia (achieved by ligature of uteroplacental vessels). Aromatase content and estrogens and androgens concentrations were measured.ResultsThe protein and mRNA content of placental aromatase significantly diminished in placentae obtained from preeclamptic patients compared to controls. Similarly, the circulating concentrations of 17-β-estradiol/testosterone and estrone/androstenedione were reduced in preeclamptic patients vs. controls. These data are consistent with a concomitant decrease in aromatase activity. Aromatase content was reduced in response to low oxygen tension in the choriocarcinoma JEG–3 cell line and in rabbit placentae in response to partial ligation of uterine spiral arteries, suggesting that reduced placental aromatase activity in preeclamptic patients may be associated with chronic placental ischemia and hypoxia later in gestation.ConclusionsPlacental aromatase expression and functionality are diminished in pregnancies complicated by preeclampsia in comparison with healthy pregnant controls.     

Increased levels of cell-free hemoglobin,oxidation markers,and the antioxidative heme scavenger α1-microglobulin in preeclampsia

Preeclampsia is a major cause of morbidity and mortality during pregnancy. To date, the pathogenesis of the disease is not fully understood. Recent studies show that preeclampsia is associated with overexpression of the hemoglobin genes α2 and γ and accumulation of the protein in the vascular lumen of the placenta. Hypothesizing that cell-free hemoglobin leaks from the placenta into the maternal circulation and contributes to the endothelial damage and symptoms by inducing oxidative stress, we analyzed fetal and adult hemoglobin (HbF, HbA), haptoglobin, oxidation markers, and the heme scavenger and antioxidant α₁-microglobulin in plasma, urine, and placenta in preeclamptic women (n = 28) and women with normal pregnancy (n = 27). The mean plasma concentrations of HbF, HbA, protein carbonyl groups, membrane peroxidation capacity, and α₁-microglobulin were significantly increased in preeclamptic women. The levels of total plasma Hb correlated strongly with the systolic blood pressure. The plasma haptoglobin concentrations of women with preeclampsia were significantly depressed. Increased amounts of α₁-microglobulin mRNA and protein were found in placenta from preeclamptic women, and the levels of plasma and placenta α₁-microglobulin correlated with the plasma Hb concentrations. The heme-degrading form t-α₁-microglobulin was significantly increased in urine in preeclampsia. These results support the idea that hemoglobin-induced oxidative stress is a pathogenic factor in preeclampsia.     

Proliferation of villous trophoblast of the human placenta in normal and abnormal pregnancies.

The proliferation of villous trophoblast in the human placenta was estimated throughout normal gestation and in term placentae from preeclamptic and smoking mothers by two different methods. These were: 1) labeling of DNA producing cells by bromodeoxyuridine (BrdU) followed by immunohistochemistry using a monoclonal anti-BrdU antibody, and 2) immunohistochemical identification of all proliferating cells by the monoclonal antibody Ki67. Both methods revealed comparable results. In uncomplicated pregnancies there was a remarkable decrease in the labeling indices from early gestation to term. This was the result of a diminution of the number of Langhans' cells, although the cell division rate within the Langhans' cell layer remained nearly constant throughout gestation. A prolongation of the cell cycle in the cytotrophoblast cells at term was indicated by an increase in the Ki67/BrdU ratio. Compared with normal term placentae, there was an increase in the trophoblast proliferation rate in preeclampsia, but not in placentae from smoking mothers. Moreover, the number of Langhans' cells was diminished in placentae from smokers. The results indicate that there are different pathogenetic mechanisms of placental impairment in preeclampsia and in maternal smoking. In preeclampsia an injury to the syncytiotrophoblast seems to lead to a repair hyperplasia of the cytotrophoblast, whereas in maternal smoking, there seems to be a direct toxic effect on the cytotrophoblastic cells.     

Oxidative stress and apoptosis in preeclampsia

We aimed to determine the oxidative stress and antioxidant status in preeclamptic placenta. Also, we investigated the apoptotic index of villous trophoblast and proliferation index of cytotrophoblasts. The study included 32 pregnant with preeclampsia and 31 normotensive healthy pregnant women. Malondialdehyde (MDA) and total antioxidant status (TAS) levels were measured in the placenta. For detection of apoptosis and proliferation in trophoblast, apoptosis protease activating factor 1 (APAF-1) and Ki-67 were used. Placental MDA levels in preeclamptic women were significantly higher than normal pregnancies (p = 0.002). There was no significant difference between the groups in the TAS levels of placenta (p = 0.773). Also, the apoptotic index in villous trophoblasts increased (p < 0.001), but proliferation index did not change in preeclampsia (p = 0.850). Increased oxidative stress and apoptosis in pathological placenta are not balanced by antioxidant systems and proliferation mechanisms.     

Newborns of Preeclamptic Women Show Evidence of Sex-Specific Disparity in Fetal Growth

BackgroundEvidence suggests that in response to in utero insults, male versus female infants have greater disadvantages in pregnancy outcome. In addition, there is a growing body of evidence suggesting that there is a sex-specific fetal response to maternal disease during pregnancy. We considered that a sex-specific relationship may exist between preeclampsia and reduced fetal growth.ObjectiveWe investigated if the relationship between preeclampsia and fetal growth was modified by fetal sex.MethodsWe limited the study population to singleton pregnancies of black and white normotensive and preeeclamptic women enrolled in the Collaborative Perinatal Project (1959–1965). The patients were offspring of 516 preeclamptic and 8801 normotensive women. After adjustment for confounders, interaction terms between preeclamptic status and fetal sex were evaluated to determine if the influence of preeclampsia on fetal growth varied with fetal sex. Separate linear and logistic regression models were then fitted for males and females to report the estimate of the relationship between preeclampsia and fetal growth by fetal sex. The results were stratified by preterm status (<37 vs ≥37 completed weeks of gestation). The mean head and chest circumferences, birthweight, ponderal index, and frequency of small for gestational age were examined. A 2-sided P value of <0.05 was considered statistically significant.ResultsThe results were stratified by preterm status. Male preterm offspring of preeclamptic mothers had greater reductions in chest circumference, head circumference, and birthweight than preterm female offspring of preeclamptic women (P = 0.01, P = 0.02, and P = 0.05, respectively, for interaction). Female versus male preterm offspring exposed to preeclampsia were less susceptible to being small for gestational age (synergy index 0.38; 95% CI, 0.00–0.84). The influence of preeclampsia on the growth of term offspring was more modest, and the influence of sex was opposite that in preterm infants. Compared with term offspring of normotensive women, the reduction in mean ponderal index was greater for female versus term male offspring of preeclamptic women (P = 0.02, interaction).ConclusionFetal growth was more impaired among male versus female preterm infants born to preeclamptic women. Our study underlined the importance of incorporating sex differences in the study of biological mechanisms for immediate- and long-term consequences of suboptimal fetal growth.     

Modulation of apelin and APJ receptor in normal and preeclampsia-complicated placentas

Apelin is an endogenous ligand of the human orphan receptor APJ. This peptide is produced through processing from the C-terminal portion in the pre-pro-protein consisting of 77 amino acid residues and exists in multiple molecular forms. Although the main physiological functions of apelin have not yet been clarified, it is known that apelin is involved in the regulation of blood pressure, blood flow and central control of body fluid homeostasis in different organs. Since human placenta is a tissue where vasculogenesis, blood pressure and flow are dramatically important to allow a normal embryonic and fetal growth and development, the aim of the present study was to investigate the immunohistochemical distribution of apelin and APJ in normal placentas throughout pregnancy and in preeclampsia-complicated placentas. Specifically, we observed that in normal placentas the expression levels of apelin decreased from the first to the third trimester of gestation in both cytotrophoblast and syncytiotrophoblast cells and in the stroma of placental villi, in contrast with increased expression levels of APJ in the cytoplasm of cytotrophoblast cells and in the cytoplasm of endothelial cells of normal placenta samples. In contrast, in preeclampsia-complicated pregnancies, we observed a very strong increase of expression levels of both apelin and APJ receptor in all the placental compartments, cytotrophoblast, syncytiotrophoblast and stroma with a particular increase in endothelial cells inside preeclamptic placental villi. Our data seem to indicate an important role of apelin and APJ in the regulation of fetal development through a correct regulation of human placenta formation during pregnancy. Moreover, the strong expression levels of apelin and APJ in preeclamptic placentas, suggest their possible involvement in the onset of this pathology.     

Increased immunohistochemical expression of thrombomodulin at placental perivascular myofibroblast in severe preeclampsia (PE)

The presence of pro-coagulant and anti-coagulant components of the placental vascular endothelium and syncytiotrophoblast are essential for homeostasis. Vascular endothelium prevents blood clot formation in vivo by involving a cell surface thrombin-binding glycoprotein, thrombomodulin (TM), that activates plasma anti-coagulant protein C. The TM levels increase during pregnancy, but the fibrinolytic capacity diminishes. Since vascular lesions with placental coagulation disorders can be associated with preeclampsia (PE), we hypothesized that TM expression in the stem villous vasculature and syncytiotrophoblast of the placenta are impaired in PE. Plasma and placental tissue samples were collected from PE (n=12) and normotensive pregnant patients (n=11). Patient's gestational age was 35.7+/-1.2 (normotensive) and 30.6+/-1.5 weeks (PE). Blood samples were drawn 30 min before delivery. Serum PAI-1 and PAI-2 antigens were determined by enzyme-linked immunoabsorbent assay (ELISA). A monoclonal antibody specific for TM was used for immunohistochemical tissue staining (ABC) and the staining was quantified by semi quantitative scores. Results show no intensity differences at the apical syncytiotrophoblast between the two groups. However, in preeclamptic placenta, TM expression diminished in the endothelium of the stem villi arteries and increased in the perivascular and stromal myofibroblats in cases of severe PE. TM changes were associated with an increased PAI-1/PAI-2 ratio. It is suggested that in severe PE, the decreased placental blood flow may be due to structural and functional impairment of the endothelium of the stem villi vessels and the surrounding perivascular and stromal myofibroblast, by increasing TM expression which may modulate fetal blow flow in the villous tree.     

Relationship between tissue damage and heme oxygenase expression in chorionic villi of term human placenta

Heme oxygenase (HO) catalyzes the oxidation of heme to carbon monoxide (CO), biliverdin, and iron and is thought to play a role in protecting tissues from oxidative damage. There are three isoforms of HO: HO-1 (inducible), HO-2 (constitutive), and HO-3 (unknown function). Preeclampsia is characterized by an inadequately perfused placenta and areas of tissue damage. We hypothesized that damaged areas of placentas from women with PE and uncomplicated pregnancies are associated with an alteration in HO expression. Compared with microsomes isolated from morphologically normal and peri-infarct chorionic villi of pathological placentas, microsomes from infarcted chorionic villi from the same placentas had decreased HO activity measured under optimized assay conditions. There was no correlation between microsomal HO levels and activity and tissue damage in uncomplicated pregnancies. Whereas there was no significant difference in HO-1 protein levels across all regions of uncomplicated and mildly preeclamptic pregnancies, HO-2 protein levels were decreased (P < 0.05) in peri-infarct regions and infarcted chorionic villi of mildly preeclamptic pregnancies. Immunohistochemical analysis revealed an apparent decrease in both HO-1 and HO-2 protein expression in damaged tissues. HO-1 and HO-2 were immunolocalized in the syncytiotrophoblast layer of the chorionic villi, the underlying cytotrophoblast, and in the vascular endothelium. This study suggests that the ability of the chorionic villi to oxidize heme to CO, biliverdin, and iron may be compromised in areas of tissue damage in the placenta of women with preeclampsia.