缺氧调控滋养细胞和内皮细胞sFlt-1差异表达的研究

目的:探讨缺氧条件下,人早孕绒毛滋养细胞(TEV-1)及人脐静脉内皮细胞(EVC-304)中可溶性血管内皮生长因子受体-1(sFlt-1)差异表达的特点。方法:CoCl2诱导TEV-1及EVC-304化学缺氧,并于0,6,12,24,48,72,96,120h用ELISA法分别检测上清中sFlt-1蛋白表达,RealtimeRT-PCR法检测各组sFlt-1mRNA表达。结果:缺氧72h后滋养细胞sFlt-1mRNA及蛋白表达随时间延长明显升高(P<0.05),而内皮细胞sFlt-1mRNA及蛋白表达差异无统计学意义(P>0.05)。结论:正常情况下滋养细胞及脐静脉内皮细胞中均有sFlt-1表达;缺氧诱导滋养细胞sFlt-1表达明显增加,而内皮细胞sFlt-1表达对缺氧刺激无明显反应。     

Placental growth factor and soluble fms-like tyrosine kinase-1 are useful markers for the prediction of preeclampsia but not for small for gestational age neonates: a longitudinal study

Objective

To determine maternal serum concentrations of placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFlt-1) longitudinally in normal pregnancies, pregnancies that developed preeclampsia and pregnancies that deliver a small for gestational age (SGA) infant, in order to evaluate them as markers for the prediction of preeclampsia.

Study design

In this case–control study we included 12 singleton pregnancies that developed preeclampsia and 104 randomly selected singleton normal pregnancies. Fourteen of the normal pregnancies gave birth to an SGA infant. Blood samples and ultrasonographic data were collected during the 1st, 2nd and 3rd trimesters of pregnancy.

Results

In preeclamptic pregnancies, PlGF (pg/mL) (median; inter-quartile range) was significantly lower in the 2nd (208; 84–339) (p = 0.035) and in the 3rd trimester (202; 109–284) (p = 0.002) while sFlt-1 was significantly higher only in the 3rd trimester (2521; 2101–3041) (p = 0.011) compared to normal pregnancies (PlGF 2nd: 311; 243–440, PlGF 3rd: 780; 472–1037, sFlt-1 3rd: 1616; 1186–2220). In pregnancies with SGA infants, PlGF and sFlt-1 did not differ significantly from normal pregnancies in any trimester. The sFlt-1 to PlGF ratio was significantly higher in preeclamptic pregnancies than in normal pregnancies, in both the 2nd and 3rd trimesters. The relative difference and the slope of PlGF concentration between 1st and 2nd trimester were significantly reduced in preeclampsia compared to normal pregnancies. A logistic regression model with predictors BMI, 2nd trimester Doppler PI and relative difference of PlGF from the 1st to the 2nd trimester gave 46% sensitivity and 99% specificity for the prediction of preeclampsia, with a very high negative predictive value of 98.3%.

Conclusions

Our study confirms that maternal serum PlGF concentration is significantly lower, at least after 20th week, while sFlt-1 concentration is significantly higher in 3rd trimester, in pregnancies destined to develop preeclampsia. Pregnancies that gave birth to SGA infants do not have altered angiogenic factor concentrations throughout pregnancy. The relative difference of PlGF from the 1st to the 2nd trimester, uterine artery Doppler PI in the 2nd trimester and BMI are the most powerful markers for the prediction of preeclampsia.     

脂氧素对脂多糖诱导的脐静脉内皮细胞通透性的影响

目的 研究脂氧素对脂多糖诱导的脐静脉内皮细胞通透性的影响,并进一步探讨其机制.方法 取华中科技大学同济医学院附属同济医院产科因社会因素行剖宫产术的健康产妇分娩的新生儿脐带,分离原代脐静脉内皮细胞后进行传代培养并分组:(1)对照组.(2)脂多糖组:加入10 mg/L脂多糖培养24 h.(3)脂多糖+脂氧素A4组:加入100 nmol/L脂氧素A4和10 mg/L脂多糖共同培养24 h.(4)脂多糖+脂氧素A4+BOC-2组:BOC-2为脂氧素受体拮抗剂,共同培养24 h.计算各组内皮细胞通透系数;逆转录(RT)PCR技术检测内皮细胞中肿瘤坏死因子α(TNF-α)mRNA表达水平;观察不同浓度脂多糖(0、0.1、1、10 mg/L)对对照组内皮细胞功能的影响(以TNF-α mRNA水平表示);蛋白印迹法检测内皮细胞中核因子κB蛋白表达水平;ELISA法检测内皮细胞培养液中TNF-α水平.结果 (1)内皮细胞通透系数:脂多糖组内皮细胞通透系数为(183.1±1.7)%,脂多糖+脂氧素A4组为(103.1±2.2)%,脂多糖+脂氧素A4+BOC-2组为(162.2±2.8)%,对照组为100%,脂多糖+脂氧素A4组通透系数较脂多糖组减小了(80.0±2.2)%,两组比较,差异有统计学意义(P<0.05).脂多糖+脂氧素A4+BOC-2组通透系数与脂多糖+脂氧素A4组相比增加了(59.1±2.8)%,两组比较,差异有统计学意义(P<0.01).脂多糖+脂氧素A4+BOC-2组与脂多糖组比较,差异无统计学意义(P>0.05).(2)TNF-α mRNA表达水平:脂多糖浓度为0、0.1、1、10 mg/L时,对照组脐静脉内皮细胞中的TNF-α mRNA表达水平分别为1.11±0.11、1.27±0.03、1.60±0.06、1.82±0.04,其中,脂多糖浓度为1、10 mg/L时,分别与0浓度比较,差异均有统计学意义(P<0.05).(3)核因子κB蛋白及TNF-αmRNA表达水平:核因子κB蛋白及TNF-αmRNA表达水平,对照组分别为0.24±0.06及0.25±0.05,脂多糖组分别为0.53±0.06及0.81±0.09,脂多糖+脂氧素A4组分别为0.19±0.05及0.41±0.07,脂多糖组核因子κB蛋白及TNF-α mRNA表达水平明显高于对照组,差异有统计学意义(P<0.05);脂多糖+脂氧素A4组核因子κB蛋白及TNF-αmRNA表达水平明显低于脂多糖组,差异有统计学意义(P<0.05).(4)细胞培养液中TNF-α水平:脂多糖组TNF-α水平[(31.94±0.01)ng/L]明显高于对照组[(18.17±0.03)ng/L],两组比较,差异有统计学意义(P<0.05);脂多糖+脂氧素A4组[(15.72±0.07)ng/L]明显低于脂多糖组,两组比较,差异有统计学意义(P<0.05).结论 脂氧素A4可以抑制脂多糖诱导的脐静脉内皮细胞高通透性,其作用可能是首先与其受体结合然后通过抑制核因子κB及其下游细胞因子TNF-α的表达而实现的.

Abstract：

Objective To explore whether lipoxin A4 (LXA4)could prevent lipopolysaccharide (LPS)-induced human umbilical vein endothelial cells (HUVEC) monolayer hyperpermeability and its possible mechanism. Methods Human umbilical cords were obtained from women with normal pregnancy immediately after delivery from Tongji Hospital Affiliated of Tongji Medical College. Primary HUVEC were isolated from umbilical veins and subcultured, then, HUVEC were divided into four groups:control group;LPS group (10 mg/L of LPS); LPS + LXA4 group(10 mg/L of LPS and 100 nmol/L of LXA4); LPS +LXA4 + BOC-2 group [10 μmol/L of BOC-2, an effective antagonist of formyl peptide receptor like 1 (FPRL-1)]. All expriments were performed after cells were treated for 24 hours. Endothelial permeability was measured by fluorescein isothiocyan-ate labelled bovine serum albumin (FITC-BSA) clearance across the monolayer; tumor necrosis factor α(TNF-o) mRNA and secretion were detected by reverse transcriplase (RT) -PCR and ELISA assay respectively, and nuclear factor κB(NF-κB) protein change was determined by western blot. Results (1) LPS induced a significant increase in the permeability [Pa value of LPS group was (183.1 ±1.7)%], while co-administrating with LXA4 obviously attenuated this LPS-induced hyperpermeability, Pa value of LPS + LXA4 group was (103.1 ±2.2)%, LPS + LXA4 + BOC-2 group was (162.2 ± 2.8)%, control group was 100%, the permeability of HUVEC monolayer was significantly increased by LPS which was (83.1 ± 1.7)% of control (P <0.01), however, it was notably inhibited by LXA4 (P<0.05); the blockade of FPRL-1 could attenuate the effect of LXA4, that is, there was no difference between the LPS + LXA4 + BOC-2 group and the LPS group. (2) After treatment with different concentration of LPS(0,0.1, 1,10 mg/L), the mRNA expressions of TNF-α were increased (1.11 ±0.11,1.27 ± 0.03, 1.60 ± 0.06, 1.82 ± 0. 04, respectively), compared with the control group, at the concentration of 1,10 mg/L LPS, the difference was statistically significant (P<0. 05). (3) The increased levels of NF-κB and inflammatory mediator TNF-α in the LPS group were both inhibited by LXA4. Levels of NF-κB protein and TNF-o mRNA secretion in LPS treated group (0.53 ±0.06 and 0.81 ±0.09 ,respectively)were both inhibited by LXA4 (0.19 ± 0.05 and 0.41 ± 0.07, respectively, and both had significant difference, P<0.05). (4) Levels of TNF-α in HUVEC culture medium of LPS group [(31.94 ±0.01)ng/L] was significantly higher than the control group [(18.17 ± 0.03) ng/L, P<0.05], LPS + LXA4 group [(15.72 ± 0.07) ng/L] was significantly lower than the LPS group (P<0.05). Conclusion Our findings demonstrated that LXA4 could prevent the endothelial cell hyperpermeability induced by LPS in HUVEC under which the possible mechanism was through inhibiting the expression of NF-κB and its related cytokines through receptor-dependent.     

Decrease and dysfunction of endothelial progenitor cells in umbilical cord blood with maternal pre-eclampsia

BACKGROUND: The pre-eclampsia is characterized by placental defective angiogenesis and maternal vascular/endothelial dysfunction. Recently, the decrease and senescence of endothelial progenitor cells (EPC) has been observed in maternal circulation with pre-eclampsia. Given the essential involvement of EPC in neovascularization and reendothelialization, we investigate whether or not the depletion of EPC is existent in placental/fetal circulation with maternal pre-eclampsia. METHODS: Samples of venous cord blood were collected during the labor of preeclamptic mothers (n = 14) and normotensive controls (n = 10). Circulating EPC were enumerated as AC133+/KDR+ cells via fluorescence-activated cell sorting (FACS) analysis. Additionally, EPC were expanded in vitro and identified by DiI-acLDL uptake and lectin staining by direct fluorescent staining under a laser scanning confocal microscope. EPC proliferation, migration and vasculogenesis activities were determined by MTT, modified Boyden chamber assay and in vitro vasculogenensis assay. RESULT: The placental/fetal circulating EPC numbers were significantly decreased in the pre-eclampsia group compared with the control (median, 200; range, 100-440 cells/mL vs 390; 270-440 cells/mL, P < 0.001), and after in vitro cultivation the numbers of EPC also decreased in pre-eclampsia group (19.5; 5.0-32.0 vs 39.5; 31.2-52.0 EPC/x200 field; P < 0.001). Both circulating EPC and cultivated EPC were inversely correlated with cord blood level of soluble fms-like tyrosine kinase 1 (sFlt-1). In addition, the EPC from patients with pre-eclampsia were significantly impaired in their proliferation, migration and vasculogenesis capacities. CONCLUSION: The present study documented the decrease and dysfunction of placental/fetal circulating EPC in patients with pre-eclampsia. The alteration is probably associated with the increased sFlt-1 levels in the umbilical cord blood.     

Nitric oxide generation affects pro- and anti-angiogenic growth factor expression in primary human trophoblast

Objectives

Preeclampsia is associated with reduced trophoblast placenta growth factor (PGF) expression, elevated soluble fms-like tyrosine kinase-1 (sFlt-1) and decreased bioactivity of nitric oxide (NO). Elevated sFlt-1 reduces bio-availability of PGF and vascular endothelial growth factor (VEGF) leading to maternal endothelial dysfunction. Although NO can regulate gene expression, its ability to regulate trophoblast expression of angiogenic growth factors is not known.

Study design

Human primary term trophoblast and JEG-3 choriocarcinoma cells were cultured under 21%O₂ or 1%O₂ conditions in the presence or absence of NO donor (SNP) or inhibitor (L-NAME). Effects on PGF, VEGF and Flt-1 isoform mRNA expression were determined by quantitative real-time PCR. Changes in expression of soluble protein isoforms of FLT-1 was monitored by ELISA.

Results

Hypoxia decreased PGF mRNA but increased VEGF, sFlt-1 and Flt-1 mRNA expression in trophoblast. Generation of NO in trophoblast under 1%O₂ culture conditions significantly reversed sFlt-1 mRNA and protein expression, independent of mFlt-1. Conversely NO generation in hypoxic trophoblast increased VEGF and PGF mRNA expression.

Conclusions

NO production in primary human trophoblast cultures had divergent effects on pro-angiogenic (PGF, VEGF) versus anti-angiogenic (sFlt-1) mRNA expression, resulting in an enhanced pro-angiogenic gene expression environment in vitro.     

CDX1 restricts the invasion of HTR-8/SVneo trophoblast cells by inhibiting MMP-9 expression

Introduction

Pathogenesis of early-onset preeclampsia (PE) is generally recognized by impaired trophoblast invasion of the myometrial arteries, which results in placental insufficiency. Recently, we reported that CDX1 is hypermethylated in the human preeclampsia placenta. However, whether CDX1 participates in trophoblast invasion has not been clearly elucidated.

Methods

We investigated the function of CDX1 in the extravillous trophoblast cell line HTR-8/SVneo using stable transfection of CDX1. Using a CDX1 stable transfected cell line, we determined the cell invasion using a QCM ECMatrix 24-well kit. The cell viability was detected using an MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. Quantitative RT-PCR and western blotting analyses were performed to examine the changes in the expression of downstream target genes and proteins. To disrupt PI3K/AKT signaling, we used the PI3K inhibitor perifosine.

Results

Cell invasion assays demonstrated that CDX1 restricts trophoblast cell invasiveness. In contrast, quantification of cumulative cell numbers revealed that CDX1 did not affect cell proliferation. Western blotting analysis and quantitative real time PCR demonstrated that MMP-9 expression was reduced, whereas TIMP-1 expression was increased in CDX1-overexpressed cells. However, overexpression of CDX1 did not affect PI3K/AKT signaling in HTR-8/SVneo trophoblast cells. In contrast, CDX1 was regulated by the PI3K/AKT signaling pathway.

Conclusions

Altogether, we found that in trophoblast cells, CDX1 reduced invasion independently of the PI3K/AKT signaling pathway. Furthermore, CDX1 functions in concert with PI3K/AKT signaling to regulate trophoblast invasion. We concluded that CDX1 restricts the invasion of HTR-8/SVneo trophoblast cells by inhibiting MMP-9 expression independently of the PI3K/AKT pathway.     

Effects of sFlt-1 and alpha 2-macroglobulin on vascular endothelial growth factor-induced endothelin-1 upregulation in human microvascular endothelial cells

Introduction

Soluble fms-like tyrosine kinase-1 (sFlt-1) is a vascular endothelial growth factor (VEGF) binding protein and potent antagonist of VEGF. Alpha 2 macroglobulin (α₂M) is another major binding protein for circulating VEGF, which is present in human plasma at higher concentration (2–4 mg/mL) than sFlt-1. This study investigated the effects of sFlt-1 and α₂M on VEGF-induced endothelin-1 (ET-1) upregulation in human microvascular endothelial cell-1 (HMEC-1).

Methods

HMEC-1 was cultured and incubated with varying concentrations of sFlt-1 and α₂M in combination with VEGF. ET-1 mRNA expression in the cells was measured by real time RT-PCR and ET-1 protein by western blot analysis.

Results

ET-1 expression in HMEC-1 incubated with VEGF significantly increased in time- and dose-dependent manners. Next, HMEC-1 was treated with the sFlt-1 (10–1000 ng/mL) or α₂M (10–10000 ng/mL) in the presence of VEGF (10 ng/mL). We found that sFlt-1 induced a significant decrease of ET-1 expression upregulated by VEGF, while α₂M did not affect the VEGF-induced ET-1 expression.

Conclusions

sFLT-1 suppressed the VEGF-induced the ET-1 expression of HMEC-1. However, α₂M did not show a significant effect on the ET-1 expression that was induced by VEGF. The results suggest that a certain proportion of the bound form α₂M-VEGF have a biological action involved in the pathophysiology of preeclampsia.     

Activation of adenosine A2B receptor impairs properties of trophoblast cells and involves mitogen-activated protein (MAP) kinase signaling

Introduction

Shallow trophoblast invasion of the maternal spiral arteries contributes to impaired placental perfusion and is hypothesized to be involved in the pathophysiology of preeclampsia. Hypoxia is a potent stimulus for the release of adenosine.

Methods

We investigated the effects of hypoxia and A_2B adenosine receptor signaling on migration, invasion, proteolytic activity of matrix metalloproteinase (MMP)-2, expression of MMP-2 and vascular endothelial growth factor (VEGF) mRNA, and production of human chorionic gonadotropin (hCG) in trophoblast cells (HTR-8/SVneo, BeWo).

Results

The adenosine A_2B receptor agonist 5-N-ethylcarboxamidoadenosine (NECA) reduced trophoblast (HTR-8/SVneo and BeWo) migration at 2%, 8% and 21% O₂ compared to untreated control cells. A_2B adenosine receptor stimulation decreased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) at all three O₂ concentrations. ProMMP-2 activity, MMP-2 mRNA levels and hCG levels were markedly decreased after A_2B adenosine receptor activation in trophoblast cells. Adenosine receptor A_2B stimulation decreased VEGF expression at 2% and 8% O₂ but led to increased levels at 21% O₂.

Conclusions

These data indicate A_2B receptor activation blunts trophoblast migration possibly as a result of reduced activation of the MAPK signaling pathway and lower proMMP-2 levels. These data suggest a role for adenosine receptor A_2B in placental development and possibly in the pathophysiology of preeclampsia.     

Extracellular ATP decreases trophoblast invasion,spiral artery remodeling and immune cells in the mesometrial triangle in pregnant rats

Introduction

Preeclampsia is characterized by deficient trophoblast invasion and spiral artery remodeling, a process governed by inflammatory cells. High levels of the danger signal extracellular adenosine triphosphate (ATP) have been found in women with preeclampsia and infusion of ATP in pregnant rats induced preeclampsia-like symptoms such as albuminuria and placental ischemia. We hypothesized that ATP inhibits trophoblast invasion and spiral artery remodeling and affects macrophages and natural killer (NK) cells present in the rat mesometrial triangle.

Methods

Pregnant rats were infused with ATP or saline (control) on day 14 of pregnancy. Rats were sacrificed on day 15, 17 or 20 of pregnancy and placentas with mesometrial triangle were collected. Sections were stained for trophoblast cells, α-smooth muscle actin (spiral artery remodeling), NK cells and various macrophage populations. Expression of various cytokines in the mesometrial triangle was analyzed using real-time RT-PCR.

Results

ATP infusion decreased interstitial trophoblast invasion on day 17 and spiral artery remodeling on day 17 and 20, increased activated tartrate resistant acid phosphatase (TRAP)-positive macrophages on day 15, decreased NK cells on day 17 and 20, and decreased inducible nitric oxide synthase (iNOS)-positive and CD206-positive macrophages and TNF-α and IL-33 expression at the end of pregnancy (day 20).

Discussion

Interstitial trophoblast invasion and spiral artery remodeling in the rat mesometrial triangle were decreased by infusion of ATP. These ATP-induced modifications were preceded by an increase in activated TRAP-positive macrophages and coincided with NK cell numbers, suggesting that they are involved.

Conclusion

Trophoblast invasion and spiral artery remodeling may be inhibited by ATP-induced activated macrophages and decreased NK cells in the mesometrial triangle in rat pregnancy.     

Production of vasoactive substances by human umbilical vein endothelial cells after incubation with serum from preeclamptic patients.

OBJECTIVE: To determine the in vitro effect of serum from preeclamptic patients on the proliferation, viability and secretion of vasoactive substances by human umbilical vein endothelial cells (HUVEC). STUDY DESIGN: HUVEC were incubated for 24h with sera from 16 preeclamptic, 19 healthy pregnant and 8 healthy nonpregnant women. Proliferation rates were determined by cell counting and vitality by trypan blue staining. The vasoactive substances, 6-keto-prostaglandin F(1alpha), nitrite and nitrate, and endothelin-1 (ET-1) were measured in endothelial cell supernatants. RESULTS: The preeclamptic serum had no effect on cell proliferation or vitality compared with control sera. It induced more HUVEC production of ET-1, but not of prostacyclin (PGI2) or nitric oxide compared with control serum. CONCLUSION: Preeclamptic serum appears to contain a factor(s) that specifically stimulates ET-1 secretion from HUVEC without altering cell growth or vitality.     

Using Doppler ultrasound of the uterine and umbilical arteries and disordered angiogenesis markers (sFlt-1/PlGF) in unified monitoring of ischemic placental syndrome patients

Objective: The shared pathogenesis of placental ischemia entitles us to create a single treatment model. We attempted to develop a unified method for monitoring ischemic placental syndrome patients using Doppler ultrasound of the uterine and umbilical arteries and disordered angiogenesis markers sFlt-1 and PlGF. Material and methods: 182 pregnant women suffering from the ischemic placental syndrome were divided into four groups depending on the severity of their lesions revealed in the Doppler ultrasound examination and weeks of pregnancy. We analyzed the behavior of clinical and biochemical parameters in these groups and the correlations between the ultrasound examination and the disordered angiogenesis markers. Results: In the group of patients demonstrating more severe Doppler ultrasound lesions, the clinical and biochemical parameters were significantly more expressed, whereas unfavorable obstetric events occurred either earlier or more frequently. Lesions revealed in Doppler occur more commonly in groups before 34th week of pregnancy. Disordered angiogenesis markers are significantly correlated with ultrasound examination results. Conclusions: A unified method for monitoring the ischemic placental syndrome based on pathogenetic, biophysical (Doppler ultrasound), and biochemical (sFlt-1/PlGF) parameters is feasible and constitutes a valuable supplement to the existing standards, while the high correlations between Doppler ultrasound examinations and both sFlt-1 and PlGF point to a shared pathogenesis of the lesions. Intensity of Doppler changes is connected with time of testing and pregnancy duration.     

Trophoblast debris extruded from preeclamptic placentae activates endothelial cells: A mechanism by which the placenta communicates with the maternal endothelium

Introduction

Preeclampsia is characterized by maternal endothelial dysfunction. While the mechanisms leading to preeclampsia are unclear, a factor(s) from the placenta is responsible for triggering the disease. One placental factor implicated in triggering preeclampsia is trophoblast debris which may transmit pathogenic signals from the placenta to endothelial cells. In this study, we investigated whether trophoblast debris from preeclamptic placentae triggered endothelial cell activation.

Methods

Trophoblast debris from preeclamptic or normotensive placentae, or trophoblast debris from normal placental explants that had been cultured with preeclamptic (n = 14) or normotensive sera (n = 14) was exposed to endothelial cells. Activation of the endothelial cells was quantified by cell surface ICAM-1 and U937 adhesion to endothelial cells. The levels of IL-1β, pro-caspase-1 and active caspase-1 in the trophoblast debris were measured.

Results

Compared to controls, the levels of ICAM-1 and U937 adhesion to endothelial cells were significantly increased following exposure of the endothelial cells to trophoblast debris from preeclamptic placentae or placentae treated with preeclamptic sera. The levels IL-1β, pro-caspase-1 and active caspase-1 were significantly increased in both trophoblast debris from preeclamptic placentae and placentae treated with preeclamptic sera.

Discussion

These results provide the first direct evidence that trophoblast debris produced from preeclamptic placentae or placentae treated with preeclamptic sera can activate the endothelium.

Conclusions

Trophoblast debris from preeclamptic but not normotensive placentae can induce endothelial cell activation. This may be one mechanism by which the preeclamptic placenta communicates with the maternal endothelium to induce activation of the endothelium.     

Correlation between uterine artery Doppler and the sFlt-1/PlGF ratio in different phenotypes of placental dysfunction

Objective: To explore correlations between the sFlt-1/PlGF ratio and uterine arteries (UtA) Doppler indexes in placental dysfunction-related disorders (PDD).

Methods: We prospectively included women with a singleton pregnancy with preeclampsia (PE) only (n = 22), preeclampsia with fetal growth restriction (FGR) (n = 32), FGR only (n = 12), or normal pregnancy (n = 29).

Results: In PDDs, significantly positive correlations between the sFlt-1/PlGF ratio and the mean UtA pulsatility (mPI-UtA), as well as the resistance index (mRI-UtA) were found (p = 0.015, p = 0.019, respectively), but not in normal pregnancies. PDD with signs of impaired placentation, evidenced by the increased sFlt-1/PlGF ratio and mPI-UtA, was found in 50.0%, and, by the increased sFlt-1/PlGF ratio and mRI-UtA, in 65.2%. PDD without signs of impaired placentation, evidenced by the increased sFlt-1/PlGF ratio but normal mPI-UtA, was found in 24.2%, and, by the increased sFlt-1/PlGF ratio but normal mRI-UtA, in 7.6%. A substantial proportion of women with signs of impaired placentation were diagnosed with FGR with or without PE.

Conclusion: In PDD, the sFlt-1/PlGF ratio and UtA Doppler indexes increase proportionally. Correlations between the sFlt-1/PlGF ratio and UtA Doppler indexes might help to distinguish between PDDs with and without impaired placentation. However, further studies are needed to explore the correlations in different phenotypes of PDD.     

Regulation by hypoxia of adrenomedullin output and expression in human trophoblast cells

Objectives

Plasma adrenomedullin concentrations are increased in the fetal circulation in acute and chronic hypoxic conditions. The effect of hypoxia in regulating adrenomedullin synthesis and secretion was investigated in human placental trophoblast cells.

Study design

Human trophoblast cells obtained from term placentas (n = 7) were cultured in hypoxic condition (3% oxygen). Cytotrophoblast cells were cultured for up to 48 h and syncytiotrophoblasts for 2, 8 and 24 h. Changes in adrenomedullin output compared to normoxic conditions were measured by radioimmunoassay. Protein expression was evaluated with Western blot and immunocytochemistry.

Results

Hypoxia induced a time-dependent increase in adrenomedullin output and protein expression by placental trophoblast cells.

Conclusions

Hypoxia regulates adrenomedullin secretion and expression by human placenta, thereby promoting increased adrenomedullin concentration in the fetal circulation in clinical circumstances characterized by reduced oxygen levels.     

Trophoblast deportation part I: review of the evidence demonstrating trophoblast shedding and deportation during human pregnancy

Trophoblast deportation was first described before the turn of the 20th Century by the German Scientist Schmorl and is now considered a normal physiological process during human pregnancy. Increased shedding and deportation of placental trophoblast is well documented in preeclampsia, one of the most common diseases of pregnancy. This review summarises the seminal historical and contemporary publications that have contributed to our knowledge of trophoblast deportation to the maternal lungs, their presence and quantity in the maternal circulation during normal pregnancy and during preeclampsia, and the range of morphologies deported trophoblasts display. Finally, the contentious nature of the deported multinucleated trophoblasts’ current nomenclature (syncytial knots vs. sprouts) is considered.     

合体滋养细胞微粒对人脐静脉内皮细胞增殖和凋亡的影响

目的:检测合体滋养细胞微粒(STBM)对人脐静脉内皮细胞(HUVECs)增殖和凋亡的影响,探讨子痫前期可能的病因和发病机制。方法:选取2009年6月至10月在上海交通大学附属第六人民医院住院行选择性剖宫产的4例正常健康孕妇的胎盘,机械法体外制备STBM。STBM与HUVECs共培养,MTT法和流式细胞仪检测HUVECs的增殖和凋亡情况。结果:(1)STBM作用12h后,HUVECs悬浮细胞数增加,48h后可见大量悬浮细胞,细胞生存受到明显抑制;(2)30、90、150mg/L STBM作用48h后,内皮细胞增殖抑制率分别为(55.58±2.40)%、(67.94±1.93)%、(86.37±1.59)%,组间差异显著(P<0.01),呈剂量依赖性。(2)30、90、150mg/L STBM作用24h后,内皮细胞凋亡率分别为(17.39±1.11)%、(22.13±1.02)%、(30.42±1.98)%,组间差异显著(P<0.01),呈剂量依赖性。结论:STBM可抑制HUVECs细胞增殖,诱导细胞凋亡,提示其可能是子痫前期内皮细胞功能紊乱的原因。     

脂氧素A4对缺氧损伤的人脐静脉内皮细胞的保护作用

目的 探讨脂氧素A4(LXA4)对缺氧损伤的人脐静脉内皮细胞(HUVEC)的保护作用及其机制.方法 体外培养HUVEC,培养后的细胞分别以单纯M199培养基培养(对照组)、氯化钴(CoCl2)诱导细胞缺氧(缺氧组)、不同浓度LXA4(1、10、100 nmol/L)加入缺氧组细胞培养液中(药物干预组);倒置相差显微镜下观察各组HUVEC的形态学变化;四甲基偶氮唑蓝(MTT)比色法检测不同浓度LXA4培养24 h和100 nmol/L的LXA4作用不同时间(4、8、12、24 h)后缺氧HUVEC存活率;免疫细胞化学法检测细胞胞质内第Ⅷ因子相关抗原抗体(vWF)水平变化(以灰度值表示),细胞质内出现棕黄色颗粒为阳性;激光共聚焦显微镜观察细胞质内游离Ca2+荧光强度变化.结果 (1)细胞形态:缺氧组HUVEC明显失去原有正常细胞形态,细胞变圆,核固缩;药物干预组正常细胞数量增多,加入100 nmol/L浓度的LXA4后,大部分细胞形态与正常细胞形态基本相似.(2)细胞存活率:缺氧组细胞存活率为(40.1±3.9)%,药物干预组细胞培养液中加入1、10、100 nmol/L浓度的LXA4后,细胞存活率分别为(52.9±1.4)%、(64.1±3.3)%、(76.6±1.6)%,分别与缺氧组比较,差异均有统计学意义(P<0.05).药物干预组细胞培养液中LXA4浓度为100 nmol/L时,作用4、8、12、24 h,细胞存活率分别为(68.2±2.3)%、(82.5±1.4)%、(82.9±1.4)%和(72.2±8.5)%,且在12 h时达峰值;药物干预组各时间点细胞存活率分别与缺氧组比较,差异均有统计学意义(P<0.05).(3)vWF水平:随着药物干预组细胞培养液中LXA4浓度的增加,其胞质内vWF水平逐步降低,LXA4浓度为1、10、100 nmol/L时.vWF水平分别为203.9±0.7、204.6±0.9、191.8±0.5,分别与缺氧组比较,差异均有统计学意义(P<0.05).(4)Ca2+荧光强度:激光共聚焦显微镜观察结果显示,LXA4可提高缺氧HUVEC胞质内Ca2+荧光强度.结论 LXA4对缺氧HUVEC维持正常的细胞形态起重要作用,并可提高其细胞存活率及降低细胞质内vWF水平,其保护机制可能是通过降低细胞内钙超载和转移细胞核内Ca2+,使细胞质内Ca2+增加.     

凋亡、坏死合体滋养细胞微粒对内皮细胞增殖与凋亡的影响

目的:探讨凋亡、坏死合体滋养细胞微粒对人脐静脉内皮细胞(HUVECs)增殖、凋亡的影响。方法:培养原代HUVECs,物理方法诱导滋养细胞凋亡、坏死。采用三步超速离心法制备凋亡合体滋养细胞微粒(a STBM)、坏死合体滋养细胞微粒(n STBM)(实验组),同时制备RBC膜(对照组),并检测胎盘碱性磷酸酶(PLAP)含量。将不同浓度(20、80、320μg/ml)a STBM、n STBM和RBC膜分别与HUVECs共同孵育4、16、32h。采用MTT法检测细胞增殖抑制率,采用流式细胞仪AV-PI双标记观察细胞凋亡情况。结果:(1)80μg/ml a STBM组作用4、16、32h时的细胞增殖抑制率分别为29.19±0.47、44.73±0.69、54.01±1.61;80μg/ml n STBM组作用4、16、32h的细胞增殖抑制率分别为57.15±0.70、64.35±0.89、71.67±0.63。a STBM、n STBM均能明显抑制HUVEC增殖,抑制作用呈浓度时间依赖性;n STBM对HUVECs的增殖抑制性明显高于a STBM,差异有统计学意义(P0.001)。RBC膜处理组对HUVECs增殖无明显影响,与空白组比较差异无统计学意义(P0.05)。(2)80μg/ml a STBM组的细胞早期凋亡率、晚期凋亡-坏死率分别为29.05%和7.46%。80μg/ml n STBM组分别为15.93%和30.31%。RBC膜处理组对HUVECs的凋亡无明显影响,与空白对照组相比,差异无统计学意义(P0.05)。a STBM、n STBM组均能引起HUVECs凋亡,n STBM作用HUVECs的晚期凋亡率高于a STBM组,两者有统计学意义(P0.001)。a STBM主要诱导HUVECs早期凋亡。结论:a STBM、n STBM对内皮细胞有抑制细胞增殖作用,a STBM主要以促进内皮细胞早期凋亡为主,而n STBM以内皮细胞晚期凋亡为主,n STBM对内皮细胞造成的损害强于a STBM。     

慢病毒介导干扰TM4SF1基因表达对血管内皮细胞体外生物学行为的影响

目的:探讨RNAi下调4跨膜区的L6超家族成员1(TM4SF1)基因表达对人脐静脉血管内皮细胞(HUVEC)的增殖、周期、迁移及血管生成的影响。方法:构建靶向沉默TM4SF1基因表达的慢病毒载体(LV-RNAi),感染高表达TM4SF1的HUVEC。实验分组:干扰组(LV-TM4SF1-RNAi)、阴性对照组(LV-CON-RNAi)、空白对照组(HUVEC),经流式细胞仪分选出稳定感染的细胞株。实时荧光定量PCR(RT-q PCR)检测细胞TM4SF1 mRNA表达水平;免疫荧光细胞化学及Western blot法检测蛋白定位及表达;CCK-8法检测细胞增殖能力;FCM检测细胞周期;Transwell小室检测细胞迁移能力;基质胶成管实验检测细胞血管生成能力。结果:成功构建的慢病毒载体高效稳定地感染了HUVEC。干扰组中TM4SF1 mRNA和蛋白表达明显低于阴性对照组和空白对照组,差异均有统计学意义(P0.05)。接种72h时,干扰组细胞的增殖能力(0.59±0.06)显著低于阴性对照组(0.94±0.04)和空白对照组(1.04±0.05),差异均有统计学意义(P0.05)。干扰组细胞G0/G1期的比例[(69.30±5.48)%]显著多于阴性对照组[(54.40±4.09)%]和空白对照组[(51.69±4.16)%],差异均有统计学意义(P0.05)。干扰组细胞的迁移能力、细胞成管能力均显著低于阴性对照组和空白对照组,差异均有统计学意义(P0.05)。结论:下调TM4SF1基因表达可使HUVEC的增殖、迁移及血管生成能力受抑制,有可能成为抗血管生成的一个潜在靶点。