Increased levels of macrophage colony-stimulating factor in the placenta and blood in preeclampsia

PROBLEM: Macrophage colony-stimulating factor (M-CSF) is considered an essential cytokine for placental growth and maintenance. We evaluated whether M-CSF levels in the placenta and blood in preeclampsia differed from those in normal pregnancies. METHOD OF STUDY: The subjects were 37 pregnant women carrying single fetuses, of whom 19 were women with normal pregnancies and 18 were women with preeclampsia. Their average gestational age at entry was 38 weeks of gestation. Blood was collected before the onset of labor, and separated serum was obtained after centrifugation. A tissue segment of the placenta was cut immediately after delivery. The frozen placental tissue was placed in a plastic tube containing phosphate-buffered saline. The tissue was fully homogenized and then centrifuged. Separated supernatant was used for subsequent determination. M-CSF levels in separated serum were measured, and M-CSF and total protein (TP) levels in separated supernatant were also measured. RESULTS: Both M-CSF/TP levels in the placenta and M-CSF levels in blood were significantly higher (P < 0.05-0.01) in preeclampsia than in normal pregnancies. CONCLUSIONS: This is the first report concerning high placenta levels of M-CSF/ TP in preeclampsia. Increased M-CSF in the placenta supports the hypothesis that immunological abnormalities contribute to the etiology of preeclampsia.     

脂蛋白脂酶基因在子痫前期胎盘中的变化及意义

目的研究脂蛋白脂酶(lipoproteinlipase,LPL)mRNA在于痫前期患者胎盘组织中的表达与定位,探讨其在子痫前期病理生理过程中的作用。方法利用cDNA表达谱芯片检查子痫前期胎盘组织与正常胎盘组织之间的差异表达基因;根据筛选结果,采用半定量RT-PCR检测子痫前期患者胎盘组织(研究组)和正常孕妇胎盘组织(对照组)中LPLmRNA的表达;以原位杂交方法进行定位。结果在4轮杂交过程中,共筛选出22条有差异表达的基因,其中LPL基因为表达降低基因之一;正常胎盘组织和子痫前期胎盘组织中均存在LPLmRNA,子痫前期胎盘组织中LPLmRNA表达明显低于正常胎盘组织(0.208±0.067vs0.524±0.139,P<0.05);LPLmRNA分布在胎盘绒毛滋养细胞胞浆。结论胎盘组织中LPL的低表达可能参与子痫前期的发病过程。     

Increased cystatin C expression in the pre-eclamptic placenta

Trophoblast invasion is regulated by proteinases and their inhibitors. Cystatin C inhibits cysteine proteinases. The serum concentration of cystatin C is increased in late pregnancy and pre-eclampsia. We aimed to investigate whether the expression of cystatin C is increased in the pre-eclamptic placenta and to investigate the expression pattern of cystatin C mRNA and protein in placental tissue. Tissue samples from the central part of the placenta from 13 normal and 22 pre-eclamptic pregnancies were included. We used real-time polymerase chain reaction (RT-PCR) and in situ hybridization for mRNA expression analysis and immunohistochemistry and Western blotting for protein expression analysis. RT-PCR showed a significantly higher expression of cystatin C mRNA in pre-eclampsia than in normal pregnancy, with the highest expression in cases with severe pre-eclampsia. In situ hybridization revealed a distinct pattern of high expression in the extravillous trophoblast cells of the basal plate and low expression in the syncytiotrophoblast covering villi. The cystatin C protein distribution matched the mRNA expression pattern. Western blot analysis revealed an increased protein expression in cases with severe pre-eclampsia and confirmed the presence of cystatin C in amniotic fluid samples. The high expression of cystatin C mRNA in the extravillous trophoblast cells of the basal plate suggests a role for cystatin C in the regulation of proteases in placentation. Placental expression and secretion of cystatin C could contribute to the elevated maternal plasma levels seen in pre-eclampsia.     

Activation of the novel prothrombinase, fg12, as a basis for the pregnancy complications spontaneous abortion and pre-eclampsia

PROBLEM: Impaired trophoblast invasion during the first trimester of pregnancy is linked to spontaneous abortion, and defective invasion in the second trimester to hypertension + proteinuria (pre-eclampsia). Hypertension developing during the third trimester of human pregnancy represents, in part, a corrective response in the mother to provide adequate placental perfusion for fetal growth when trophoblast has not to invaded and converted the myometrial porprtion of maternal spiral arteries into to low resistance-high capacitance conduits. Deportation of vesicles from hypoxemic trophoblast is thought to cause hypertension plus proteinuria, vascular damage and a systemic coagulopathy. Trophoblast invasion may be inhibited by local cytokines, such as TGF-betas but Thl-type cytokines associated with pre-eclapmsia and spontaneous abortions (e.g., IL-1, TNF-alpha, IFN-gamma) are not known to inhibit migration at in situ concentrations. Trophoblast invasion is also inhibited by the binding of surface integrins to fibronectin and fibrin, and fibrin production is stimulated by these Th1 cytokines via up-regulation of prothrombinases(s) such as fg12 which directly and via TNF-alpha-facilitated inflamation compromise trophoblast cell integrity. We, therefore, asked if fg12 expression and TNF-alpha are increased in first trimester human miscarriage and in third trimester pre-eclampsia. METHODS: fg12 mRNA was detected using in situ hybridization and fg12 protein by immunohistochemistry. TNF-alpha mRNA and protein were similarly tested. The techniques were validated using uterine sections from day 8.5 of CBA x DBA/2 pregnancies, and then were applied to sections of placentae from normal and pre-eclamptic pregnancies with and without intrauterine fetal growth restriction (IUGR). Fibrin was detectectd by immunohistochemistry. RESULTS: Expression of fg12 protein correlated with fg12 mRNA expression in mouse uteri and in placentae from normal human pregnancies. Increased expression of fg12 and TNF-alpha mRNA and protein, and increased fibrin deposition was detected in placental trophoblast. CONCLUSIONS: Activation of fg12 prothrombinase by Th1-type cytokines in pregnancy may lead to spontaneous abortion, or in ongoing pregnancy, to pre-eclampsia and/or IUGR.     

Expression profile of trophoblast invasion-associated genes in the pre-eclamptic placenta

The primary pathology of pre-eclampsia is thought be a defect in placentation due to failure of trophoblast invasion. Here, we aim to identify the expression profile of invasion-associated genes in the pre-eclamptic placenta. Messenger RNA (mRNA) expression levels of extracellular matrix molecule-related genes in five pre-eclamptic placentas and in five strictly matched normal placentas were assayed using complementary DNA (cDNA) microarrays representing over 220 human cytokine-associated or hormone-associated genes. Results demonstrated greater than two-fold higher expression of 18 extracellular matrix molecule genes, including cadherin, collagen, integrin and selectin, in the pre-eclamptic placenta. Extracellular matrix molecule degradation-related genes, including matrix metalloproteinase (MMP)-10, MMP-13, MMP-15, tissue inhibitor of metalloproteinase (TIMP)-2, TIMP-3, plasminogen and plasminogen activator, were also highly expressed in the pre-eclamptic placenta, compared to the normal placenta. Results suggest that the abnormal expression profiles of extracellular matrix molecules and degrading proteinases might be associated with the pathogenesis of pre-eclampsia.     

The etiological role of allogeneic fetal rejection in pre-eclampsia

PROBLEM: It has been demonstrated that allogeneic fetal rejection in normal pregnancy is prevented by placental indoleamine 2,3-dioxygenase (IDO). Further, an immunological etiology has been implicated in pre-eclampsia. METHOD OF STUDY: We examined the differences in placental IDO activity between normal and pre-eclamptic pregnancies. RESULTS: IDO mRNA expression and enzyme activity levels in the placenta were low in patients with severe pre-eclampsia. The enzyme activity also inversely correlates with the blood pressure of the patients. In the placentas from severe pre-eclampsia, IDO immunoreactivity was low, whereas regional T-cell infiltration was observed reciprocally proportional to the IDO activity. CONCLUSION: Our findings implicate a potential role for IDO activity and a maternal immunological reaction against an allogeneic fetus in the etiology of pre-eclampsia.     

细胞命运决定因子numb在先兆子痫胎盘组织中的表达及作用

目的探讨先兆子痫胎盘组织中细胞命运决定因子numbs表达情况及意义。方法获取正常妊娠胎盘组织以及先兆子痫胎盘组织,分别运用实时荧光定量PCR法、免疫印迹法、免疫组织化学法检测numbmRNA、蛋白分布及其表达水平。结果numb在以上两种胎盘组织中均有表达,且主要集中在细胞膜上表达。numb在先兆子痫组（实验组）的表达水平明显高于正常胎盘组（对照组）（P〈0．05）。结论numb参与的滋养细胞凋亡失调可能是先兆子痫的发病机制之一。     

Aberrant uterine natural killer (NK)-cell expression and altered placental and serum levels of the NK-cell promoting cytokine interleukin-12 in pre-eclampsia

PROBLEM: Natural killer (NK) cells are the most abundant lymphocyte population at the maternal-fetal interface. They are suggested to be important during placentation by controlling trophoblast invasion. If placentation is suboptimal, pre-eclampsia can occur. METHOD OF STUDY: Decidual NK (dNK) cells were examined at delivery in 46 women, 22 pre-eclamptic women and 24 healthy controls, by staining for CD56 and CD94 with immunohistochemistry (IHC). Furthermore, we investigated the placental expression and the serum levels of the NK-cell activating cytokines interleukin(IL)-12, IL-15, IL-18 and the anti-inflammatory cytokine IL-10 by IHC and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: Pre-eclamptic women had higher number of CD56+ and CD94+ cells in the decidua, indicating an altered receptor expression of dNK cells. We also demonstrate for the first time that the villous trophoblasts show strong immunostaining for IL-12 in placentae from healthy controls, while women suffering from pre-eclampsia have significantly less IL-12. However, pre-eclamptic women had significantly elevated IL-12 and IL-15 levels in serum. CONCLUSION: Results show increased numbers and altered phenotype of dNK cells in pre-eclampsia, supporting the importance of these cells for a healthy pregnancy. The altered receptor expression of dNK cells together with diminished placental IL-12 expression could implicate an altered NK cell-regulation in pre-eclampsia.     

Increased levels of pregnancy-associated plasma protein-A2 in the serum of pre-eclamptic patients

Pregnancy-associated plasma protein-A and -A2 (PAPP-A and -A2) are proteases that cleave insulin-like growth factor-binding proteins (IGFBPs), resulting in local activation of IGF signaling pathways. Here, we examined PAPP-A and -A2 mRNA and protein levels in placenta and maternal sera from women with pre-eclampsia and compared them with samples from uncomplicated pregnancy. PAPP-A2 but not PAPP-A mRNA and protein were elevated in pre-eclamptic placenta (P < 0.01). PAPP-A2 is normally produced in placental syncytiotrophoblast cells and maternal decidua. PAPP-A2 in syncytiotrophoblast cells was dramatically increased in pre-eclampsia. Maternal serum concentrations of PAPP-A2 but not PAPP-A were also significantly elevated in pre-eclampsia as compared with uncomplicated pregnancy. mRNA levels of IGFBP5, a specific substrate for PAPP-A2 protease activity, were also significantly increased, suggesting a potential role for IGFBP5 in fetal and placental growth suppression during pre-eclampsia. However, IGFBP5 protein levels were not increased in placenta from pre-eclampsia, possibly due to cleavage by up-regulated PAPP-A2. These data might imply that PAPP-A2 may be up-regulated in pre-eclamptic pregnancy to compensate for IGFBP5-mediated suppression of the IGF pathway, although final birthweights are still low in pre-eclamptic pregnancy.     

Lack of human leukocyte antigen-G expression in extravillous trophoblasts is associated with pre-eclampsia

Pre-eclampsia, a common complication of first pregnancies, is thought to result from a poorly perfused placenta and may reflect an abnormal maternal immune reaction to the hemiallogenic fetus. Human leukocyte antigen (HLA)-G, a major histocompatibility tissue-specific antigen expressed in extravillous trophoblast cells (fetal-derived), may protect trophoblasts from maternal-fetal immune intolerance and allow these cells to invade the uterus. Through RNA in-situ hybridization analysis, we studied the expression pattern of HLA-G in normal placentae and placentae from pregnancies complicated by severe pre-eclampsia. In normal placenta we found HLA-G expression in the anchoring extravillous trophoblasts with an increasing gradient of expression in the more invasive cells. However, in nine out of 10 pre-eclamptic placentae HLA-G expression was absent or reduced. We conclude that HLA-G is normally expressed in invasive trophoblasts and HLA-G expression is defective in most pre-eclamptic placentae. We propose that trophoblasts lacking HLA-G are vulnerable to attack by the maternal immune system. These defective trophoblasts will be unable to invade the maternal spiral arteries effectively, thereby developing vessels which cannot adequately nourish the developing placenta. This poorly perfused placenta may initiate the systemic cascade of events associated with pre-eclampsia.     

Endothelial nitric oxide synthase in placental villous tissue from normal, pre-eclamptic and intrauterine growth restricted pregnancies

Nitric oxide (NO) regulates blood flow in the human placenta. As increased resistance to blood flow is seen in the fetal-placental vasculature in pregnancies complicated by pre-eclampsia and/or intrauterine growth restriction (IUGR), we examined expression of endothelial nitric oxide synthase (eNOS) in these placentas. Placental villous tissue sections were obtained from normotensive control (n = 5), IUGR alone (n = 5) or pre-eclamptic (with or without IUGR (n = 9) patients, immunostained for eNOS and scored for localization, type (punctate or diffuse) and intensity of eNOS staining in syncytiotrophoblast and placental vessels. The significance of differences was calculated using the Mann-Whitney U-test. No differences in intensity or type of immunostaining in syncytiotrophoblast were seen. Placentas from patients with pre- eclampsia with or without IUGR had a significantly more basal distribution of eNOS in syncytiotrophoblast. eNOS immunostaining was absent in terminal villous capillary and faint in stem villous vessel endothelium of normal placentas, but was intense in the endothelium of both of these types of vessels in the IUGR and pre-eclampsia groups, with significantly greater staining seen in stem vessels of patients with IUGR alone. This increased eNOS expression and hence increased NO production in the fetal-placental vasculature may be an adaptive response to the increased resistance and poor perfusion in these pathological pregnancies.      

Defective endovascular trophoblast invasion in the first trimester is associated with increased maternal serum ischemia-modified albumin

BACKGROUND: Ischemia-modified albumin (IMA), a protein elevated in cardiac ischemia, is also increased to supra-physiological levels in early normal pregnancy. This finding supports the hypothesis that normal trophoblast development is stimulated by a hypoxic intrauterine environment. The aim of this study was to examine whether first trimester IMA levels are further elevated with defective trophoblast development. METHODS: Prospective study of healthy women with singleton pregnancies undergoing nuchal translucency assessment at 11-14 weeks. First trimester maternal serum IMA concentrations in those subsequently developing pre-term pre-eclampsia (n = 19) were compared to randomly chosen controls with normal pregnancy outcome (n = 69). RESULTS: Median first trimester serum IMA concentrations were significantly higher in women who subsequently developed pre-eclampsia (median 126.5 kU/L, interquartile range (IQR) 114.33-134.36 kU/L) when compared to those with normal pregnancy outcome (median 115.01 kU/L, IQR 102.29-124.81 kU/L, P = 0.02). CONCLUSIONS: Maternal serum IMA levels are elevated in the first trimester in women with pre-eclampsia, a clinical manifestation of defective endovascular trophoblast development. This suggests that abnormally high intrauterine hypoxia and subsequent reperfusion oxidative damage may be associated with defective trophoblast development. First trimester serum IMA may be a potential biomarker for abnormal placental development.     

Heme oxygenases in pregnancy II: HO-2 is downregulated in human pathologic pregnancies

PROBLEM: We previously reported a diminished expression of the heme-degrading enzymes heme oxygenases (HO)-1 and HO-2 in decidua and placenta from mice undergoing Th1-mediated abortion, strongly indicating the protective effect of HO in murine pregnancy maintenance. Here we investigated whether the expression of HO-1 and HO-2 is also reduced at the feto-maternal interface of pathologic human pregnancies. METHOD OF STUDY: Immunohistochemistry was used to detect HOs expression in placental and decidual first-trimester tissue from patients with: spontaneous abortion (n = 14), choriocarcinoma (n = 14), hydatidiform mole (H-mole) (n = 12), compared with normally progressing pregnancies (n = 15). Further, we investigated early third-trimester decidual and placental tissue from patients with pre-eclampsia (n = 13) compared with fetal growth retardation (n = 14) as age-matched controls. RESULTS: In first trimester tissue, we observed a significant reduction of HO-2 expression in invasive trophoblast cells, endothelial cells, and syncytiotrophoblasts in samples from patients with spontaneous abortion compared with normal pregnancy. H-mole samples showed a diminished expression of HO-2 in invasive trophoblast cells and endothelial cells in comparison with NP, whereas choriocarcinoma samples showed no significant differences compared with the control. In third trimester tissue, HO-2 was also reduced in syncytiotrophoblasts and invasive trophoblast cells from pre-eclampsia compared with samples from fetal growth retardation. HO-1 expression was diminished in all pathologies investigated; however, the differences did not reach levels of significance. CONCLUSIONS: Our data indicate that HOs play a crucial role in pregnancy and low expression of HO-2, as observed in pathologic pregnancies, may lead to enhanced levels of free heme at the feto-maternal interface, with subsequent upregulation of adhesion molecules, allowing enhanced inflammatory cells migration to the feto-maternal interface.     

Expression of vascular endothelial growth factor receptors 1, 2 and 3 in placentas from normal and complicated pregnancies

Extensive angiogenesis and invasion of the maternal decidua by trophoblasts are essential for the development and function of the placenta. Vascular endothelial growth factors (VEGF), placenta growth factor (PlGF) and their receptors VEGFR-1/Flt-1, VEGFR-2/KDR and VEGFR-3/Flt4 have important roles in vasculogenesis and angiogenesis. We have studied the localization of these proteins by immunohistochemistry and Western blotting in the placenta and of PlGF in maternal serum, and their association with diabetes, pre-eclampsia, fetal growth restriction (FGR) and fetal alcohol syndrome (FAS). VEGFR-1 and VEGFR-3 were detected mainly in the syncytiotrophoblastic layer whereas VEGFR-2 was detected in the vascular endothelial cells of the placenta. VEGFR-1, but not the other receptors, showed increased expression in placental syncytiotrophoblasts from 50% of patients with severe pre-eclampsia and FGR when compared with normal placentas. PlGF was undetectable in 38 of 44 samples of amniotic fluid of mothers with normal and complicated pregnancies. However, maternal serum PlGF concentrations were significantly lower in pre-eclamptic patients and in those with FGR when compared to diabetic women or healthy controls. These results suggest that low maternal serum PlGF and increased placental expression of its receptor VEGFR-1 are associated with pre-eclampsia and FGR.     

Immunolocalization of alphaV,alpha3 and beta1 integrins in the human placenta with pre-eclampsia

Signs of pre-eclampsia are considered to be caused by maternal endothelial dysfunction due to circulating factors of placental origin. Integrins are a large family of cell surface, proteins that serve as receptors involved in cell-cell and cell-matrix interactions during placentation. Therefore, low expression of integrins or the lack of it may be encountered during pre-eclampsia. In the present study, we investigated the immunolocalisation of integrins alphaV, alpha3 and beta1 in placentas of normal and pre-eclamptic women. Thirty-two placentas from pre-eclamptic (n = 14) and normotensive (n = 18) women were used. Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue specimens, using anti-alphaV, anti-alpha3 and anti-beta1 antibodies and the indirect immunoperoxidase technique. A semi-quantitative grading system (HSCORE) was used to compare immunohistochemical staining intensities. Distribution patterns of alphaV, alpha3 and beta1 integrins were detected in cytotrophoblasts and Hofbauer cells in normal and pre-eclamptic placentas. Immunostaining of alphaV and beta1 integrins was slightly decreased in pre-eclamptic samples but alpha3 integrin immunostaining was similar in pre-eclamptic and normal placentas. Decreased immunostaining of integrins in the cytotrophoblasts may considered to be a structural basis for decreased placental perfusion in pre-eclampsia.     

Immunohistochemical adrenomedullin expression is decreased in the placenta from pregnancies with pre-eclampsia

The purpose of the present study was to investigate whether placental immunohistochemical adrenomedullin expression in normal normotensive pregnancies is different from that in pregnancies with pre-eclampsia. Placental tissues were obtained from seven normal normotensive pregnancies and 12 pregnancies with pre-eclampsia. The intensity of adrenomedullin staining in syncytiotrophoblasts was evaluated by means of immunohistochemistry and the ratio of the number of intact tertiary villi to that of total tertiary villi (intact/total villi ratio) was determined. The intensity of adrenomedullin expression in the placenta obtained from pregnancies with pre-eclampsia was significantly decreased compared with expression in placentas from uncomplicated normotensive pregnancies (P < 0.005). The intact/total villi ratio in placentas obtained from pregnancies with pre-eclampsia was significantly lower than that in placentas from normal normotensive pregnancies (P < 0.0001). In the amnion and extravillous trophoblast cells in both groups, no difference for the intensity of adrenomedullin expression was noted. These results suggest that adrenomedullin synthesis in the villous syncytiotrophoblasts is decreased in pregnancies with pre-eclampsia.     

Decreased expression of the angiogenic regulators CYR61 (CCN1) and NOV (CCN3) in human placenta is associated with pre-eclampsia

The pregnancy disorder pre-eclampsia (PE) is thought to be caused in part by shallow invasion of the extravillous trophoblast (EVT) leading to uteroplacental insufficiency and hypoxia. Here, we focused on the expressions of cysteine-rich 61 (CYR61, CCN1) and nephroblastoma overexpressed (NOV, CCN3), members of the CCN family of angiogenic regulators, in human placenta during normal pregnancy compared with pre-eclamptic and HELLP placentae using quantitative RT-PCR, western blotting and immunocytochemistry. During normal pregnancy, both proteins showed increasing expression levels and were strongly coexpressed in endothelial cells of vessels, stromal cells and interstitial EVT giant cells. However, NOV showed an earlier onset of expression in villous endothelial cells during gestation compared with CYR61, which may signify distinct roles of these proteins in placental angiogenesis. In early-onset pre-eclamptic placentae, both CYR61 and NOV were expressed at a significantly lower level compared with normal matched controls. This decrease of CYR61 and NOV in pre-eclamptic placentae is not associated with a decrease of the endothelial marker CD34 or vimentin. No obvious changes in the localization of CYR61 and NOV in pre-eclamptic placentae were detected but a change in the intracellular distribution in trophoblast giant cells. Our data point to a potential role of both molecules in the pathogenesis of early-onset PE.     

Placental adaptive responses and fetal programming

Fetal programming occurs when the normal pattern of fetal development is disrupted by an abnormal stimulus or 'insult' applied at a critical point in in utero development. This then leads to an effect, for example diabetes or hypertension, which manifests itself in adult life. As the placenta is the regulator of nutrient composition and supply from mother to fetus and the source of hormonal signals that affect maternal and fetal metabolism, appropriate development of the placenta is crucial to normal fetal development. Placental function evolves in a carefully orchestrated developmental cascade throughout gestation. Disruption of this cascade can lead to abnormal development of the placental vasculature or of the trophoblast. Timing of a developmental 'insult' will be critical in consequent placental function and hence programming of the fetus. The 'insults' that alter placental development include hypoxia and abnormal maternal nutrient status, to which the placenta may adapt by alterations in transporter expression and activity to maintain fetal growth or by epigenetic regulation of placental gene expression. Hypoxia is physiological for organogenesis and placental tissue normally exists in a relatively hypoxic environment, but intrauterine growth restriction (IUGR) and pre-eclampsia are associated with a greater degree of trophoblast hypoxia. The metabolic activity of placental mitochondria leads to oxidative stress even in normal pregnancy which is exacerbated further in IUGR, diabetic and pre-eclamptic pregnancies and may also give nitrative stress known to lead to covalent modification and hence altered activity of proteins. Hypoxia, oxidative and nitrative stress all alter placenta development and may be a general underlying mechanism that links altered placental function to fetal programming.