Hongbo Qi

Methotrexate-loaded PLGA nanobubbles for ultrasound imaging and Synergistic Targeted therapy of residual tumor during HIFU ablation.

High intensity focused ultrasound (HIFU) has attracted the great attention in tumor ablation due to its non-invasive, efficient and economic features. However, HIFU ablation has its intrinsic limitations for removing the residual tumor cells, thus the tumor recurrence and metastasis cannot be avoided in this case. Herein, we developed a multifunctional targeted poly(lactic-co-glycolic acid) (PLGA) nanobubbles (NBs), which not only function as an efficient ultrasound contrast agent for tumor imaging, but also a targeted anticancer drug carrier and excellent synergistic agent for enhancing the therapeutic efficiency of HIFU ablation. Methotrexate (MTX)-loaded NBs were synthesized and filled with perfluorocarbon gas subsequently using a facile but general double emulsion evaporation method. The active tumor-targeting monoclonal anti-HLA-G antibodies (mAbHLA-G) were further conjugated onto the surface of nanobubbles. The mAbHLA-G/MTX/PLGA NBs could enhance the ultrasound imaging both in vitro and in vivo, and the targeting efficiency to HLA-G overexpressing JEG-3 cells has been demonstrated. The elaborately designed mAbHLA-G/MTX/PLGA NBs can specifically target to the tumor cells both in vitro and in vivo, and their blood circulation time in vivo was much longer than non-targeted MTX/PLGA NBs. Further therapeutic evaluations showed that the targeted NBs as a synergistic agent can significantly improve the efficiency of HIFU ablation by changing the acoustic environment, and the focused ultrasound can promote the on-demand MTX release both in vitro and in vivo. The in vivo histopathology test and immunohistochemical analysis showed that the mAbHLA-G/MTX/PLGA NBs plus HIFU group presented most serious coagulative necrosis, the lowest proliferation index and the highest apoptotic index. Therefore, the successful introduction of targeted mAbHLA-G/MTX/PLGA NBs provides an excellent platform for the highly efficient, imaging-guided and non-invasive HIFU synergistic therapy of cancer with the supplementary functions of killing residual tumor cells and preventing tumor recurrence/metastasis.

Wnt5a inhibited human trophoblast cell line HTR8/SVneo invasion: implications for early placentation and preeclampsia

Abstract Objective: Wnt5a and Wnt signaling play potential roles in human placental and fetal development. The objective of this study is to explore the role of Wnt5a in the invasion of the human trophoblast cell line HTR8/SVneo and the probable mechanism of early placentation and preeclampsia in which Wnt5a is involved. Methods: Human first trimester villous tissues from normal pregnancies and third trimester placentas from pregnancies with or without preeclampsia (PE) were used in the detection of the expression and subcellular location of Wnt5a. The human trophoblast cell line HTR8/SVneo was treated with 0–400 ng/ml recombinant Wnt5a to investigate the role of Wnt5a in human trophoblast invasion. Results: Human first trimester villous is accompanied by the decreased expression of Wnt5a compared with term placenta. Upregulated Wnt5a was detected in PE placenta compared with the normal control. Wnt5a inhibited the migration and invasion of HTR8/SVneo cells with decreased integrin β1, α5 and N-cadherin. Moreover, Wnt5a downregulated β-catenin in HTR8/SVneo cells. Conclusions: These findings strongly suggest that Wnt5a inhibits the invasion of HTR8/SVneo cells. Decreased Wnt5a facilitates early placentation, whereas increased Wnt5a contributes to the pathogenesis of PE with insufficient trophoblast invasion. Aberrant Wnt5a may function by impairing Wnt non-canonical/β-catenin signaling pathway in trophoblasts.

Excessive autophagy induces the failure of trophoblast invasion and vasculature: possible relevance to the pathogenesis of preeclampsia.

Introduction: Preeclampsia affects 5–7% of all healthy pregnancies and is characterized by hypertension and proteinuria. Although the pathogenesis of preeclampsia is still not fully understood, a failure of spiral artery transformation and aberrant placental vasculature are considered to be facets of this disease. Studies have also implicated increased autophagic activity. In this study, we investigated whether oxidative stress could increase autophagic activity and consequently affect trophoblast invasion and the placental vasculature. Methods: Placentas from 18 pregnancies complicated by preeclampsia and from 18 uncomplicated pregnancies, trophoblast HTR8/SVneo cell line (HTR8/SVneo) extravillous trophoblasts, and human umbilical vein endothelial cells (HUVECs) were employed. The levels of autophagy markers LC3, Beclin-1 and autophagosome were quantified by immunohistochemistry, Western blotting and RT-PCR in placental tissue, and in trophoblasts and endothelial cells that had been treated with an oxidative stress inducer glucose oxidase. Trophoblast invasion and endothelial cell tube formation were assessed in HTR8/SVneo cells or HUVECs that had been treated with glucose oxidase. Results: The expression of LC3, Beclin-1 and autophagosome was significantly increased in placentas from pregnancies complicated by early-onset preeclampsia and in HTR8/SVneo cells and HUVECs treated with glucose oxidase. In addition, trophoblast invasion and endothelial cell tube formation were significantly reduced in HTR8/SVneo cells or HUVECs that had been treated with glucose oxidase. Conclusion: Our data suggest that oxidative stress induces increased autophagy in trophoblasts or endothelial cells which affects trophoblast invasion and the placental vasculature. Excessive autophagic activity may be involved in the development of preeclampsia.

The expression of pentraxin 3 and tumor necrosis factor-alpha is increased in preeclamptic placental tissue and maternal serum.

ObjectiveTo evaluate the expression of pentraxin 3 (PTX3) and tumor necrosis factor-alpha (TNF-α) in preeclampsia.MethodsTwenty-two preeclamptic patients, six preeclamptic patients with intrauterine growth restriction (IUGR) and 30 women with uncomplicated pregnancies were included in this study. The expression of PTX3 and TNF-α in placental tissue was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western immunoblotting. Enzyme-linked immunosorbent assay (ELISA) was used to measure the concentration of PTX3 and TNF-α in maternal sera. The localization and immunoreaction of PTX3 and TNF-α in placenta were determined via immunohistochemistry (IHC).ResultsExpression of PTX3 and TNF-α in placental tissues and maternal sera was significantly increased in preeclamptic patients, as well as in those with IUGR. PTX3 was mainly expressed in villous stroma, decidual cells and terminal villi, and TNF-α was mostly localized in trophoblast, vascular endothelial cells, decidual cells and in the stroma of the stem villi. Moreover, PTX3 expression was correlated with TNF-α expression in maternal sera of preeclamptic women.ConclusionsPTX3 and TNF-α are increased in preeclampsia and are likely involved in the pathogenesis of preeclampsia.

Downregulated Krüppel-Like Factor 8 Is Involved in Decreased Trophoblast Invasion Under Hypoxia–Reoxygenation Conditions

Krüppel-like factor 8 (KLF8) is a pivotal transcription factor expressed in the human placenta that can regulate cell invasion. The objective of this study was to assess whether a hypoxia–reoxygenation (H/R) environment affects placental KLF8 expression levels and subcellular localization and to evaluate the relationship between KLF8 levels and trophoblast invasion activity. Human first trimester villous tissues from normal pregnancies and third trimester placentas from pregnancies with or without preeclampsia (PE) were used for the detection of KLF8 expression and correlating its levels with metalloproteinase 9 (MMP-9) expression. In addition, HTR8/SVneo cells were used to mimic the effects of an H/R environment on placentas to study KLF8 expression and trophoblast invasion. The KLF8 levels, MMP-9 levels, and trophoblast invasion were similarly altered; the levels peaked at 8 to 10 weeks of gestation and declined thereafter along with oxygen tension increased from hypoxia to normoxia during early pregnancy, decreased in third trimester placentas from PE pregnancies featured by repeated H/R and HTR8/SVneo cells exposed to H/R compared with the control. Moreover, a visible reduction in KLF8 immunoreactivity was present in the nuclei of cytotrophoblast cells in human villous tissues at 11 weeks, and partial cytoplasmic accumulation of KLF8 was observed in HTR8/SVneo cells treated with H/R. In conclusion, these findings strongly suggest that H/R reduces the expression and nuclear localization of KLF8 to inhibit the trophoblast invasion by downregulating MMP-9 levels. The KLF8 may play a vital role in the pathogenesis of PE as a novel oxygen tension sensor.

Laminin α4 (LAMA4) expression promotes trophoblast cell invasion, migration, and angiogenesis, and is lowered in preeclamptic placentas.

INTRODUCTION The laminin α4 subunit (LAMA4) has been shown to promote migration, proliferation, and survival of various cell types. This study investigated LAMA4s role in trophoblast cells during placental development. METHODS LAMA4 expression was immunohistochemically assessed in the first trimester and term human placentas. LAMA4 siRNA was applied to silence LAMA4 expression in extravillous explants and HTR8/SVneo cells. Hypoxia-reoxygenation (H/R) conditions were applied to mimic preeclampsia. LAMA4 expression and trophoblast cell invasion, migration, and tube formation (a measure of angiogenesis) were assessed in HTR8/SVneo cells. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 was used to study the mechanism underlying LAMA4 activity. LAMA4 promoter methylation was assessed by bisulfite-sequencing polymerase chain reaction (PCR) or methylation-specific PCR. RESULTS LAMA4 levels in preeclamptic placentas were significantly lower than those in controls. LAMA4 silencing significantly inhibited extravillous explant outgrowth as well as HTR8/SVneo cell invasion and migration. H/R conditions significantly lowered LAMA4 expression. Application of either H/R conditions or LAMA4 silencing both significantly decreased HTR8/SVneo cell invasion, migration, and tube formation, decreased MMP2 and MMP9 expression, and increased TIMP2 expression. SB203580 significantly reduced LAMA4 expression. LAMA4 silencing significantly decreased p-p38, p-c-Jun N-terminal kinase (JNK), and p-extracellular signal-regulated kinase (ERK) expressions; by contrast, H/R conditions induced significant upregulation of p-p38 and p-ERK but decreased p-JNK. LAMA4 promoter methylation was not significantly altered in preeclamptic placentas compared to controls. CONCLUSIONS LAMA4 expression is lowered in preeclamptic placentas and promotes trophoblast cell invasion, migration, and angiogenesis. H/R conditions decrease LAMA4 expression and appear to decouple the positive relationship between LAMA4 expression and p38 and ERK activation.

Expression of Gadd45α in human early placenta and its role in trophoblast invasion

OBJECTIVES Well-controlled trophoblast migration and invasion at the maternal-foetal interface are crucial events for normal placentation and successful pregnancy. Growing evidence has revealed that growth arrest and DNA damage-inducible 45 alpha (Gadd45α) participates in tumour migration and invasion as a tumour suppressor. However, the expression and function of Gadd45α in trophoblasts is unknown. This study aimed to determine the Gadd45α expression and function in the human first trimester placenta and identify the underlying mechanisms. METHODS The expression of Gadd45α in human first trimester placenta was determined using immunohistochemistry. HTR8/SVneo cell line was used to investigate the effects of Gadd45α on proliferation, apoptosis, migration/invasion, matrix metalloproteinase (MMP)2/9 activities, and tissue inhibitor of metalloproteinase (TIMP)1/2 expression using cell proliferation assays, flow cytometric analysis, transwell migration/invasion assays, gelatin gel zymography, and western blotting, respectively. Moreover, a placental villous explant model was employed to verify its functions in placentation. RESULTS Gadd45α was strongly expressed in syncytiotrophoblasts and trophoblast columns of human placental villi, extravillous trophoblast cells and glandular epithelium within the maternal decidua. Gadd45α knockdown significantly promoted migration and invasion of HTR8/SVneo cells, whereas it did not affect cell proliferation or apoptosis. Silencing Gadd45α also enhanced trophoblast outgrowth and migration in placental explants. These effects were related to increased activities of MMP2/9 and the decreased expression of TIMP1/2. DISCUSSION AND CONCLUSION Gadd45α may be involved in human trophoblast migration and invasion and may function as an important negative regulator at the foetal-maternal interface during early pregnancy by directly or indirectly regulating MMP2/9 activities.

Oxidative stress-induced C/EBPβ inhibits β-catenin signaling molecule involving in the pathology of preeclampsia.

INTRODUCTION Oxidative stress-induced trophoblast cell dysfunction is a major pathology in preeclampsia (PE). Recently, CCAAT/enhancer binding protein beta (C/EBPβ) has been investigated as a tumor suppressor that participates in tumor invasion. However, the function of C/EBPβ in trophoblast cells remains unknown. Our study was designed to detect the expression of C/EBPβ in the preeclamptic placenta and to identify the underlying mechanisms of oxidative stress. METHODS Human placental tissues with PE were collected. The expression of C/EBPβ and β-catenin were detected. Human first trimester extravillous trophoblast cell (HTR8/SVneo) line exposed to hypoxia/reoxygenation (H/R) was employed as an oxidative stress model in vitro to investigate the effects of C/EBPβ on invasion and the expression of β-catenin. Moreover, first trimester-derived placental villous explants were used to verify the effects of C/EBPβ and β-catenin in placentation. RESULTS In preeclamptic placentas, C/EBPβ was overexpressed and β-catenin was decreased. In addition, C/EBPβ was found to have increased expression in H/R-treated HTR8/SVneo cells and villous explants. C/EBPβ knockdown and β-catenin activation could significantly promote the invasion of HTR8/SVneo cells, enhance the outgrowth and migration in villous explants and inhibit the excessive generation of intracellular ROS. These findings might be related to the increased activities of MMP-2/9 and the decreased expression of TIMP-1/2. Meanwhile, C/EBPβ knockdown remarkably increased the expression of β-catenin. DISCUSSION We hypothesize that the oxidative stress-induced overexpression of C/EBPβ might influence the activity of MMPs by regulating the Wnt/β-catenin signaling pathway to affect the invasion of trophoblast cells, which then participate in the pathogenesis of preeclampsia.

N-acetylglucosaminyltransferase V inhibits the invasion of trophoblast cells by attenuating MMP2/9 activity in early human pregnancy

INTRODUCTION The invasion and migration of trophoblast cells are essential steps of normal placentation and successful pregnancy. The process is well-regulated by many factors at the fetal-maternal surface. Inadequate invasion by trophoblast cells may lead to poor perfusion of the placenta or complications such as preeclampsia (PE). Accumulating evidence suggests that N-acetylglucosaminyltransferase V (MGAT5) is correlated with tumor invasion and metastasis. Our objective was to characterize MGAT5 expression and function during placental development. METHODS The expression of MGAT5 in humans in placental tissue from the first trimester was determined by immunohistochemistry. To investigate whether MGAT5 regulates trophoblast invasion and migration, we investigated invasion/migration of the HTR8/SVneo trophoblast cells and used human villous explants. Cell proliferation and apoptosis were measured by CCK-8 assay and flow cytometry, respectively. The activity of matrix metalloproteinase (MMP) 2/9, and the expression of tissue inhibitors of metalloproteinases (TIMPs) 1/2 were determined by gelatin zymography and Western blot, respectively. RESULTS MGAT5 was specifically localized within the cytotrophoblast, the syncytiotrophoblast and the trophoblast columns of human placental villi, decidual cells and some extravillous cells in the maternal decidua. MGAT5 shRNA significantly enhanced the invasion and migration capability of HTR8/SVneo cells, and increased villous explant outgrowth but did not affect proliferation and apoptosis of the trophoblast. The enhanced effect of MGAT5 shRNA on trophoblast cell invasion was associated with increased gelatinolytic activity of MMP2/9 and decreased expression of TIMP1/2. DISCUSSION AND CONCLUSION Our data support a role for MGAT5 in the inhibition of human trophoblast cell invasion and migration during early pregnancy by direct or indirect regulation of MMP2/9 activity.

IL-27 Activates Human Trophoblasts to Express IP-10 and IL-6: Implications in the Immunopathophysiology of Preeclampsia

Purpose. To investigate the effects of IL-27 on human trophoblasts and the underlying regulatory signaling mechanisms in preeclampsia. Methods. The expression of IL-27 and IL-27 receptor (WSX-1) was studied in the placenta or sera from patients with preeclampsia. In vitro, we investigated the effects of IL-27 alone or in combination with inflammatory cytokine tumor necrosis factor (TNF-α) on the proinflammatory activation of human trophoblast cells (HTR-8/SVneo) and the underlying intracellular signaling molecules. Results. The expression of IL-27 and IL-27 receptor α (WSX-1) was significantly elevated in the trophoblastic cells from the placenta of patients with preeclampsia compared with control specimens. In vitro, IL-27 could induce the expression of inflammatory factors IFN-γ-inducible protein 10 (CXCL10/IP-10) and IL-6 in trophoblasts, and a synergistic effect was observed in the combined treatment of IL-27 and TNF-α on the release of IP-10 and IL-6. Furthermore, the production of IP-10 and IL-6 stimulated by IL-27 was differentially regulated by intracellular activation of phosphatidylinositol 3-OH kinase-AKT, p38MAPK, and JAK/STAT pathways. Conclusions. These results provide a new insight into the IL-27-activated immunopathological effects mediated by distinct intracellular signal transduction molecules in preeclampsia.