Haiyi Liu

Hypoxic trophoblast-derived sFlt-1 may contribute to endothelial dysfunction: implication for the mechanism of trophoblast—endothelial dysfunction in preeclampsia

The maternal systemic disorder of widespread endothelial dysfunction is a primary focus in understanding the development of preeclampsia. sFlt‐1 (soluble fms‐like tyrosine kinase receptor 1), an endogenous inhibitor of VEGF (vascular endothelial growth factor), may play important roles in endothelial dysfunction. The present study aimed to determine whether hypoxic trophoblast‐derived sFlt‐1 could lead to endothelial dysfunction by establishing a cocultured model of anoxic TEV‐1s (human first‐trimester extravillous trophoblasts) and HUVECs (human umbilical vein endothelial cells). The results showed that the hypoxic treatment significantly promoted sFlt‐1 mRNA and protein expression in TEV‐1s in a time‐dependent manner compared with the effect in HUVECs. When HUVECs were cocultured with anoxic TEV‐1s, the endothelial function, which was characterized by NO (nitric oxide) synthesis and monolayer barrier function of HUVECs, were notably decreased, accompanied by increasing sFlt‐1 and decreasing VEGF in cell‐conditioned medium. Moreover, the observed endothelial dysfunction described above was consistent with the dysfunction observed in VEGF siRNA‐treated cultures. The findings presented herein imply that chronically hypoxic trophoblasts may release sufficient sFlt‐1 to cause endothelial dysfunction by depriving cells of VEGF activity.

VEGF deficit is involved in endothelium dysfunction in preeclampsia

SummaryThis study examined the association of expression of vascular endothelial growth factor (VEGF), a promoter of angiogenesis, with endothelium dysfunction in preeclampsia. The level of VEGF protein and mRNA in the placenta and peripheral blood samples of 30 preeclampsia patients and 30 normotensive pregnant women was measured by immunohistochemistry, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. VEGF expression in the human umbilical vein endothelial cells (HUVECs) was blocked by small interfering RNAs (siRNAs). The monolayer barrier function of HUVECs was determined by measuring the fluorescence intensity of BSA that crossed the HUVEC monolayers. The cell proliferation and cell-secreted nitric oxide (NO) level were detected by MTT method and nitrate reductase assay, respectively. The results showed that VEGF was expressed in the syncytiotrophoblasts and endothelial cells of vessels and capillaries in the placenta tissue. The serum level of VEGF in the preeclampsia patients was significantly decreased as compared with that in normal pregnant subjects, although VEGF mRNA expression in the placenta tissue of preeclampsia patients remained still high. Moreover, VEGF deficit could lead to endothelium cell dysfunction, and the administration of VEGF could protect endothelium cells from injury. It was concluded that lack of VEGF contributes to endothelium dysfunction, which may lead to the occurrence and development of preeclampsia.This study examined the association of expression of vascular endothelial growth factor (VEGF), a promoter of angiogenesis, with endothelium dysfunction in preeclampsia. The level of VEGF protein and mRNA in the placenta and peripheral blood samples of 30 preeclampsia patients and 30 normotensive pregnant women was measured by immunohistochemistry, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. VEGF expression in the human umbilical vein endothelial cells (HUVECs) was blocked by small interfering RNAs (siRNAs). The monolayer barrier function of HUVECs was determined by measuring the fluorescence intensity of BSA that crossed the HUVEC monolayers. The cell proliferation and cell-secreted nitric oxide (NO) level were detected by MTT method and nitrate reductase assay, respectively. The results showed that VEGF was expressed in the syncytiotrophoblasts and endothelial cells of vessels and capillaries in the placenta tissue. The serum level of VEGF in the preeclampsia patients was significantly decreased as compared with that in normal pregnant subjects, although VEGF mRNA expression in the placenta tissue of preeclampsia patients remained still high. Moreover, VEGF deficit could lead to endothelium cell dysfunction, and the administration of VEGF could protect endothelium cells from injury. It was concluded that lack of VEGF contributes to endothelium dysfunction, which may lead to the occurrence and development of preeclampsia.

Effect of epigenetic modification of maspin on extravillous trophoblastic function.

SummaryThis study investigated the effect of epigenetic modification of maspin on extravillous trophoblastic function. The mRNA expression of maspin in placentae from normotensive and preeclamptic pregnant women was detected by RT-PCR. TEV-1 cells, a human first-trimester extravillous trophoblast cell line, were cultured and treated with CoCl2 (300 μmol/L) to induce chemical hypoxia and with 5-aza (500 nmol/L) to induce demethylation. The mRNA expression of maspin in TEV-1 cells subjected to different treatments was determined by RT-PCR, and the proliferative and migratory abilities of TEV-1 cells were assessed by cell counting kit-8 (CCK-8) and Transwell assays. Our results showed that the maspin mRNA expression level in placentae from preeclamptic women was much higher than that from normotensive women. CoCl2 or 5-aza could up-regulate the mRNA expression of maspin and significantly suppress the proliferation and migration of TEV-1 cells. It was concluded that the epigenetic modification in promoter region of maspin contributes to incomplete trophoblast invasion, which offers a novel approach for predicting and treating placental dysfunction.This study investigated the effect of epigenetic modification of maspin on extravillous trophoblastic function. The mRNA expression of maspin in placentae from normotensive and preeclamptic pregnant women was detected by RT-PCR. TEV-1 cells, a human first-trimester extravillous trophoblast cell line, were cultured and treated with CoCl2 (300 μmol/L) to induce chemical hypoxia and with 5-aza (500 nmol/L) to induce demethylation. The mRNA expression of maspin in TEV-1 cells subjected to different treatments was determined by RT-PCR, and the proliferative and migratory abilities of TEV-1 cells were assessed by cell counting kit-8 (CCK-8) and Transwell assays. Our results showed that the maspin mRNA expression level in placentae from preeclamptic women was much higher than that from normotensive women. CoCl2 or 5-aza could up-regulate the mRNA expression of maspin and significantly suppress the proliferation and migration of TEV-1 cells. It was concluded that the epigenetic modification in promoter region of maspin contributes to incomplete trophoblast invasion, which offers a novel approach for predicting and treating placental dysfunction.

Promoter hypomethylation and increased maspin expression in preeclamptic placentas in a Chinese population

OBJECTIVE Preeclampsia is thought to begin with shallow trophoblast invasion and inadequate spiral artery remodeling. Maspin, a tumor-suppressor gene, plays a regulatory role in trophoblast invasion and motility. The tissue-specific methylation of the maspin promoter can regulate maspin gene expression in various cancers. We sought to detect maspin gene expression and assess the degrees of methylation of maspin promoter regions in preeclamptic placentas in the Han Chinese population and to investigate the potential role of maspin in the pathophysiology of preeclampsia. METHODS We conducted RT-PCR, immunohistochemistry and western blotting to characterize maspin gene expression and protein levels in the placentas from normal and preeclamptic pregnancies. Finally, using methylation-specific PCR and bisulfite sequencing PCR, we detected the degrees of methylation of the promoter regions of maspin in each of the two studied groups. RESULTS Maspin expression was increased at the mRNA and protein levels in the preeclamptic placentas compared to the control group. Maspin immunohistochemical staining revealed positive staining in the syncytio-cytotrophoblast layers and more diffuse staining in the preeclamptic group. The mean methylation level of the analyzed promoter region was significantly hypomethylated in the preeclamptic placentas compared to the control placentas, pointing to a negative relationship between maspin promoter methylation and gene expression. DISCUSSION Hypomethylation of the maspin promoter results in increased expression of maspin in preeclamptic placentas, which suggests a negative relationship between maspin methylation and maspin expression in this Han Chinese population. Thus, maspin is likely involved in the etiology of preeclampsia.

Implication of expression of osteopontin and its receptor integrin ανβ3 in the placenta in the development of preeclampsia

To investigate the expression of osteopontin (OPN) and its receptor integrin ανβ3 in the placental tissue from pregnant women complicated with preeclampsia, the expression of OPN and ανβ3 in the placenta of the pregnant women with preeclampsia and healthy pregnant women was detected by immunohistochemistry, Western blotting and RT-PCR. Our results showed that OPN and ανβ3 protein were expressed in the placenta from normal pregnant woman and those with preeclampsia. OPN was located in the placental syncytiotrophoblasts and the cytoplasm of capillary endothelial cells and integrin ανβ3 was mainly expressed on the surface of trophoblast cells. Expression of OPN and integrin ανβ3 in the placental tissue from preeclampsia subjects was significantly lower than that from the control group (P<0.05). Compared with the control group, expression of OPN in the placental tissue from preeclampsia group was significantly lower (P<0.05) but there was no significant difference in the expression of αν and β3 between the preeclampsia group and the controls. It is concluded that OPN and its receptor integrin ανβ3 may be involved in the pathogenesis of preeclampsia.SummaryTo investigate the expression of osteopontin (OPN) and its receptor integrin ανβ3 in the placental tissue from pregnant women complicated with preeclampsia, the expression of OPN and ανβ3 in the placenta of the pregnant women with preeclampsia and healthy pregnant women was detected by immunohistochemistry, Western blotting and RT-PCR. Our results showed that OPN and ανβ3 protein were expressed in the placenta from normal pregnant woman and those with preeclampsia. OPN was located in the placental syncytiotrophoblasts and the cytoplasm of capillary endothelial cells and integrin ανβ3 was mainly expressed on the surface of trophoblast cells. Expression of OPN and integrin ανβ3 in the placental tissue from preeclampsia subjects was significantly lower than that from the control group (P<0.05). Compared with the control group, expression of OPN in the placental tissue from preeclampsia group was significantly lower (P<0.05) but there was no significant difference in the expression of αν and β3 between the preeclampsia group and the controls. It is concluded that OPN and its receptor integrin ανβ3 may be involved in the pathogenesis of preeclampsia.

Effect of leptin on cytotrophoblast proliferation and invasion

The effects of leptin on cytotrophoblast proliferation and invasion activity were investigated. Immunohistochemistry was used to determine the placental expression of leptin in first-trimester pregnancy. By using RT-PCR and quantitative real-time PCR, the expression of leptin in cytotrophoblast and the effect of leptin on cytotrophoblast secretion were detected. The potential of cell proliferation, invasiveness and migration was assessed by MTT, Transwell invasion assay and migration assay respectively when the cytotrophoblast was cultured with different concentrations of leptin. The results showed that: (1) Leptin was distributed diffusely around cell membrane, in cytoplasma, and on nuclear membrane of cytotrophoblast; (2) Leptin mRNA was expressed in cytotrophoblast. Ten ng/mL leptin could promote the secretion of cytotrophoblast significantly (P<0.01); (3) After culture with different concentrations of leptin for 24 h or longer, the proliferation of cytotrophoblast was inhibited, while in 24 h leptin could promote cytotrophoblast invasion and migration. Leptin at a concentration of 500 ng/mL could promote cytotrophoblast invasiveness and migration significantly as compared with controls (P<0.05). It was suggested that leptin could inhibit cytotrophoblast proliferation, and promote cytotrophoblast invasion and migration activity.SummaryThe effects of leptin on cytotrophoblast proliferation and invasion activity were investigated. Immunohistochemistry was used to determine the placental expression of leptin in first-trimester pregnancy. By using RT-PCR and quantitative real-time PCR, the expression of leptin in cytotrophoblast and the effect of leptin on cytotrophoblast secretion were detected. The potential of cell proliferation, invasiveness and migration was assessed by MTT, Transwell invasion assay and migration assay respectively when the cytotrophoblast was cultured with different concentrations of leptin. The results showed that: (1) Leptin was distributed diffusely around cell membrane, in cytoplasma, and on nuclear membrane of cytotrophoblast; (2) Leptin mRNA was expressed in cytotrophoblast. Ten ng/mL leptin could promote the secretion of cytotrophoblast significantly (P<0.01); (3) After culture with different concentrations of leptin for 24 h or longer, the proliferation of cytotrophoblast was inhibited, while in 24 h leptin could promote cytotrophoblast invasion and migration. Leptin at a concentration of 500 ng/mL could promote cytotrophoblast invasiveness and migration significantly as compared with controls (P<0.05). It was suggested that leptin could inhibit cytotrophoblast proliferation, and promote cytotrophoblast invasion and migration activity.

Promoter Hypomethylation of Maspin Inhibits Migration and Invasion of Extravillous Trophoblast Cells during Placentation

Objective Extravillous trophoblast (EVT) cells invade the endometrium and the maternal spiral arterioles during the first trimester. Mammary Serine Protease Inhibitor (Maspin, SERPINB5) plays a putative role in regulating the invasive activity of cytotrophoblasts. The maspin gene is silenced in various cancers by an epigenetic mechanism that involves aberrant cytosine methylation. We investigated the effect of the methylation status of the maspin promoter on the maspin expression and the aggressiveness of EVT cells. Methods Western blotting was used to detect the maspin protein expression in EVT cells upon hypoxia. The proliferative ability, the apoptosis rate and the migration and invasiveness were measured with Cell Counting Kit-8 assay, Flow Cytometry technology and Transwell methods. Subsequently, we treated cells with recombinant maspin protein. The methylation degree of maspin promoter region upon hypoxia/ decitabine was detected by bisulfite sequencing PCR and methylation-specific PCR. Finally, we explored the effects of decitabine on maspin protein expression and the aggressiveness of EVT cells. Results Hypoxia effectively increased maspin protein expression in EVT cells and significantly inhibited their aggressiveness. The addition of recombinant maspin protein inhibited this aggressiveness. Decitabine reduced the methylation in the maspin promoter region and effectively increased the maspin protein expression, which significantly weakened the migration and invasiveness of EVT cells. Discussion The methylation status of the maspin promoter is an important factor that affects the migration and invasion of EVT cells during early pregnancy. A decrease in the methylation status can inhibit the migration and invasion of EVT cells to affect placentation and can result in the ischemia and hypoxia of placenta.

Low molecular weight heparin improves proteinuria in rats with L-NAME induced preeclampsia by decreasing the expression of nephrin, but not podocin

Objective: We investigated the relationship between proteinuria in L-NAME induced preeclampsia and the expression of nephrin and podocin, and the effect of low-molecular-weight-heparin (LMWH) on proteinuria in rats. Methods: We detected nephrin and podocin expression of kidneys of pregnant rats after L-NAME and after LMWH intervening pregnant rats. Results: Glomerular nephrin expression in L-NAME induced preeclampsia significantly decreased, but not podocin. Nephrin was relatively increased after LMWH intervention and this was accompanied by a decrease in proteinuria. Conclusion: We demonstrate that down-regulation of nephrin is involved in L-NAME induced proteinuria, and that LMWH reduces proteinuria by up-regulation of neprhin.

Proapoptotic effect of metalloproteinase 9 secreted by trophoblasts on endothelial cells.

Aim:  During normal placentation, the uterine spiral arteries are remodeled to form high‐flow, low‐resistance vessels, which is necessary to meet the increasing demands of the fetus for blood and oxygen. Remodeling of uterine arteries is mediated by extravillous trophoblasts, which penetrate the arterial walls and induce the endothelial apoptosis. But the mechanism of endothelial apoptosis is not yet fully understood. In this study, we investigate the involvement of matrix metalloproteinase 9 (MMP‐9) in trophoblast‐induced endothelial apoptosis.

Decitabine Improves the Clinical Manifestations of Rats With l-NAME-Induced Pre-eclampsia: A Potential Approach to Studying Pre-eclampsia

Objective: Pre-eclampsia is a major cause of maternal mortality and morbidity. Conditions with low oxygen tension are regarded as a key factor. Decitabine can partly attenuate the effects of hypoxia. This research was designed to investigate the effects of decitabine in rats with NG-Nitro-L-arginine Methyl Eater (L-NAME) induced pre-eclampsia and to explore the molecular mechanisms. Methods: A Wistar rat model of pre-eclampsia was established by intraperitoneal injection of L-NAME, and the intervention reagent was decitabine. Blood pressure (BP) and 24-h urinary protein were monitored. The expression of Mammary Serine Protease Inhibitor (SERPINB5, maspin) in the placenta was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting. Results: Systolic BP in the tail artery of pregnant rats was increased by more than 30 mm Hg, and 24-h urinary protein was significantly increased after L-NAME was added. After decitabine treatment, blood pressure and 24-h urinary protein were significantly decreased. The expression of SERPINB5 in the placenta significantly increased after L-NAME was added. Decitabine significantly elevated the expression of SERPINB5 in the placenta of rats with L-NAME-induced preeclampsia. Conclusion: Decitabine reduced 24-h urinary protein and partly decreased blood pressure of pre-eclampsia in late pregnancy in rats with L-NAME-induced pre-eclampsia and increased the expression of SERPINB5, but the molecular mechanism of decitabines effect remains unknown. This research provided a potential approach to studying the pathogenesis, treatment and prevention of pre-eclampsia.