Xiao-Yong Zhu

CXCL8 enhances proliferation and growth and reduces apoptosis in endometrial stromal cells in an autocrine manner via a CXCR1-triggered PTEN/AKT signal pathway

BACKGROUND Chemokine CXCL8 (also known as IL-8) has been identified as a potential regulator of endometrial stromal cells (ESCs), but it is unclear how CXCL8 regulates the survival of ESCs in the pathogenesis of endometriosis. METHODS We assessed the secretion of CXCL8 by enzyme-linked immunosorbent assays and the expression of its receptors, CXCR1 and CXCR2, by in-cell Western assay and immunohistochemistry. The effects of CXCL8 on the activation or expression of various cell mediators were also investigated by in-cell Western assay. The effects of CXCL8 on the proliferation, growth and apoptosis of ESCs in vitro were assessed by BrdU assays, cell counts and annexin V labeling, respectively. RESULTS Secretion of CXCL8 and expression of CXCR1 in the eutopic ESCs from women with endometriosis were significantly higher than that in control ESCs, but the expression of CXCR2 showed no significant difference between these two cell types. CXCL8 stimulated proliferation and growth and reduced apoptosis of ESCs in an autocrine manner, and these effects were abolished by anti-human CXCL8 and CXCR1 neutralizing antibodies and by a PI3K/Akt inhibitor. Moreover, CXCL8 up-regulated the expression of the anti-apoptotic proteins, survivin and Bcl-2, inhibited the expression of the Phosphatase and tensin homolog (PTEN) and activated the phosphorylation of Akt. CONCLUSIONS This study suggests that CXCL8 and CXCR1 are involved in the pathogenesis of endometriosis by up-regulating proliferation and growth and restricting apoptosis in ESCs by activating the PTEN/Akt pathway and mediating the expression of survivin and Bcl-2.

IL-33 enhances proliferation and invasiveness of decidual stromal cells by up-regulation of CCL2/CCR2 via NF-κB and ERK1/2 signaling

Interleukin (IL)-33, a newly described member of the IL-1 family, has been reported to facilitate primary tumor progression and metastatic dissemination. However, its biological function on decidual stromal cells (DSCs) remains unclear. In this study, we tested the hypothesis whether IL-33 promotes proliferation and invasion of DSCs, and the possible mechanism. IL-33 and its orphan receptor ST2 was found to be co-expressed by DSCs in human first-trimester pregnancy. Addition of IL-33, enhanced the proliferation and invasion of DSCs in a dosage-dependent manner, concomitantly with increasing expression of proliferation relative gene (PCNA, survivin) and invasion relative gene (titin, MMP2). Blocking IL-33/ST2 signaling by soluble sST2 apparently abolished the stimulatory effect on the proliferation, invasiveness and related gene expression in DSCs. We also demonstrated that chemokines CCL2/CCR2 was significantly increased with IL-33 administration. Moreover, inhibition of CCL2/CCR2 activation using CCL2 neutralizing antibody or CCR2 blocker prevented IL-33-stimulated proliferation and invasiveness capacity of DSCs. Increasing phosphorylation of nuclear factor NF-κB p65 and extracellular signal-regulated kinases ERK1/2 after treatment with IL-33 was confirmed by western blotting. And the IL-33-induced CCL2/CCR2 expression was abrogated by treatment with the NF-κB inhibitor BAY 11-7082 or ERK1/2 inhibitor U0126. Finally, we showed that decreased IL-33/ST2 expression was observed in DSCs from spontaneous abortion compared with normal pregnancy at both gene and protein levels. This study provides evidence for the molecular mechanism of IL-33 in promoting proliferation and invasiveness of DSCs by up-regulation of CCL2/CCR2 via NF-κB and ERK1/2 signal pathways and thus contributes insight to the potential of IL-33 involved in successful pregnancy via inducing DSCs mitosis and invasion.

Cyclosporin A Promotes Growth and Invasiveness In Vitro of Human First-Trimester Trophoblast Cells Via MAPK3/MAPK1-Mediated AP1 and Ca2+/Calcineurin/NFAT Signaling Pathways

Cyclosporin A (CsA) has provided the pharmacologic foundation for organ transplantation as a calcineurin inhibitor blocking T-cell activation. We have demonstrated that CsA promoted trophoblast viability/proliferation and invasion in vitro. In the present study, we further investigated the intracellular signalling pathways involved in enhancing cell viability/proliferation and invasiveness of the human trophoblast induced by CsA. We showed that blocking mitogen-activated protein kinase 3 (MAPK3)/MAPK1 signaling by U0126 attenuated CsA-increased cell viability and invasiveness of trophoblasts. Cyclosprin A inhibited ionomycin-stimulated nuclear factor of activated T-cells (NFAT) transactivation in JAR cells and reversed the ionomycin-inhibited trophoblast invasiveness. However, either activating calcineurin by ionomycin, resulting in NFAT transactivation, or inhibiting NFAT using an NFAT inhibitor had no effect on trophoblast cell viability/proliferation and apoptosis in vitro. Hence, the CsA-induced promotion of trophoblast growth and invasion occurred by overlapping but independent pathways. The MAPK3/MAPK1 pathway was essential for both trophoblast growth and invasion, whereas the Ca(2+)/calcineurin/NFAT pathway was only involved in the CsA-promoted trophoblast invasiveness. Finally, potential cross-talk between MAPK3/MAPK1 and Ca(2+)/calcineurin/NFAT and its relationship to activator protein 1 activation was investigated. Our findings explored possible signal transduction pathways modulated by CsA, which may lead to the expansion of the clinical applications of this drug.

Cyclosporin A improves murine pregnancy outcome in abortion-prone matings: involvement of CD80/86 and CD28/CTLA-4

Immune regulation during pregnancy is complex, and thus an optimal therapy for pregnancy complications is always a big challenge to reproductive medicine. Cyclosporin A (CsA), a potent immunosuppressant, prevents rejection of allografts by hosts, but little is known about the modulating effect of CsA on the materno-fetal relationship. Here, pregnant CBA/J females mated with DBA/2 males as an abortion-prone model were administered with CsA on day 4.5 of gestation, and the pregnant CBA/J females mated with BALB/c males were established as successful pregnancy control. It was demonstrated that administration of CsA at the window of implantation significantly up-regulated the expression of CTLA-4, while down-regulating the levels of CD80, CD86, and CD28 at the materno-fetal interface in the CBA/J x DBA/2 abortion-prone matings, and the embryo resorption rate of the abortion-prone matings reduced significantly after CsA treatment, implying that modulation of costimulatory molecule expression by CsA might contribute to preventing the fetus from maternal immune attack. In addition, treatment with CsA induced enhanced growth and reduced cell apoptosis of the murine trophoblast cells. Together, these findings indicate that CsA has a beneficial effect on the materno-fetal interface in abortion-prone matings, leading to a pregnancy outcome improvement, which might provide new therapeutics for spontaneous pregnancy wastage.

Programmed cell death-1 (PD-1) and T-cell immunoglobulin mucin-3 (Tim-3) regulate CD4+ T cells to induce Type 2 helper T cell (Th2) bias at the maternal–fetal interface

STUDY QUESTION Are the immune regulatory molecules programmed cell death-1 (PD-1) and T-cell immunoglobulin mucin-3 (Tim-3) involved in regulating CD4+ T cell function during pregnancy? SUMMARY ANSWER PD-1 and Tim-3 promote Type 2 helper T cell (Th2) bias and pregnancy maintenance by regulating CD4+ T cell function at the maternal-fetal interface. WHAT IS KNOWN ALREADY The maternal CD4+ T cell response to fetal antigens is thought to be an important component of maternal-fetal tolerance during pregnancy. PD-1 and Tim-3 are important for limiting immunopathology. The co-expression of PD-1 and Tim-3 on T cells identifies a T cell subset with impaired proliferation and cytokine production. Combined blockade of Tim-3 and PD-1 could restore T cell function to the greatest degree. STUDY DESIGN, SIZE, DURATION The expression of PD-1 and Tim-3 on CD4+ T cells was analyzed by flow cytometry, and in vitro and in vivo analyses were used to investigate the role of PD-1/Tim-3 signal in the regulation of CD4+ T cells function and pregnancy outcome. PARTICIPANTS/ MATERIALS, SETTING, METHODS A total of 88 normal pregnant women, 37 women with recurrent spontaneous abortion, 36 normal pregnant mice and 45 abortion-prone mice were included. We measure the expression of PD-1 and Tim-3 on CD4+ T cells and their relationship to the function of CD4+ T cells and pregnancy outcome, as well as the effects of blocking PD-1 and Tim-3 pathways on decidual CD4+ T (dCD4+ T) cells during early pregnancy. MAIN RESULTS AND THE ROLE OF CHANCE PD-1 and Tim-3, by virtue of their up-regulation on dCD4+ T cells during pregnancy, define a specific effector/memory subset of CD4+ T cells and promote Th2 bias at the maternal-fetal interface. Using in vitro and in vivo experiments, we also found that combined targeting of PD-1 and Tim-3 pathways results in decreased production of Th2-type cytokines by dCD4+ T cells and increased fetal resorption of normal pregnant murine models. Moreover, decreased PD-1 and Tim-3 on dCD4+ T cells may be associated with miscarriage. LIMITATIONS AND LIMITS OF CAUTION Further study is required to examine the mechanism of PD-1 and Tim-3 effects on Th2 cytokine production by CD4+ T cells during pregnancy. WIDER IMPLICATIONS OF THE FINDINGS These results have important implications for understanding the physiological mechanisms that promote maternal-fetal tolerance. Our study also indicates that targeting Tim-3 and PD-1 pathways may represent novel therapeutic strategies to prevent pregnancy loss. STUDY FUNDING/COMPETING INTERESTS This study was supported by the National Basic Research Program of China (2015CB943300); National Nature Science Foundation of China (81490744, 91542116, 31570920, 81070537, 31171437, 81370770, 31270969, 31570920, 91542116); the Key Project of Shanghai Municipal Education Commission (14ZZ013) and the Key Project of Shanghai Basic Research from Shanghai Municipal Science and Technology Commission (12JC1401600). None of the authors have any conflict of interest to declare.

Thymic stromal lymphopoietin downregulates NME1 expression and promotes invasion in human trophoblasts via the activation of STAT3 signaling pathway

Our previous study has demonstrated that TSLP induces the invasion of human trophoblasts and plays a role in embryo implantation. The present study is undertaken to explore the mechanism by which TSLP modulates trophoblast invasion. It was revealed that TSLP treatment significantly downregulated NME1 and TIMP1 expression in both human trophoblasts and JEG-3 cells. The stimulatory effect of TSLP on trophoblast invasion was partially inhibited by either STAT3 inhibitor or STAT5 inhibitor. The downregulation of NME1 expression by TSLP was completely abrogated by STAT3 inhibitor. TIMP1 expression was attenuated by NME1 siRNA interference and enhanced by NME1 overexpression in JEG-3 cells. TSLP-stimulated increase in trophoblast invasion was partially attenuated by NME1 overexpression. Our data suggest that TSLP might downregulate NME1 expression via STAT3 signaling pathway, affecting TIMP1 expression in influencing trophoblast invasion. Our studies suggest that further studies on TSLP as a potential therapeutic for recurrent spontaneous abortion are warranted.

A retrospective review of 10 cases of villoglandular papillary adenocarcinoma of the uterine cervix including one with successful pregnancy

Objective: Villoglandular papillary adenocarcinoma (VGPA) of the uterine cervix is a subtype of cervical adenocarcinoma. In the present study, we summarized the clinical features of VGPA of the uterine cervix and discussed the potential indications for a conservative treatment. Methods: A retrospective review of clinical characteristics and treatment aspects of 10 patients with VGPA at the Obstetrics and Gynecology Hospital of Fudan University was conducted between January 2007 and December 2016. Almost all of the existing 40 English papers on “villoglandular papillary adenocarcinoma [title/abstract]” identified from PubMed were obtained. Clinical data from these papers were analyzed in terms of age, International Federation of Gynecology and Obstetrics (FIGO) stage, recurrence rate, mortality, and conservation treatment aspects. Results: The median age of 10 patients with VGPA was 40 years. All cases had Stage IB 1 disease. Seven patients underwent human papillomavirus examinations, which revealed 6 positive and 1 negative case(s) of infection. Six patients underwent ThinPrep cytologic tests, which revealed 4 patients with atypical glandular cells, 1 with a high-grade squamous intraepithelial lesion, and 1 who tested negative for intraepithelial malignancy. None of the patients had lymph node metastases. During the 6–114 months of follow-up, no disease recurrence or death occurred. Of note, one patient who received conservative treatment successfully became pregnant. Conclusions: VGPA can be detected at an early FIGO stage with excellent prognosis. For young patients who do not exhibit poor prognosis factors, conservative treatment may be the first treatment choice based on overall assessment of clinical conditions.

High Glucose Promotes Epithelial-Mesenchymal Transition of Uterus Endometrial Cancer Cells by Increasing ER/GLUT4-Mediated VEGF Secretion

Background/Aims: Uterus endometrial cancer (UEC) is the common malignancy among gynecologic cancers, and most of them are type I estrogen-dependent UEC. Diabetes is well-known risk factor for the development of UEC. However, the underlying link between high glucose (HG) and the estrogen receptor in UEC remains unclear. Epithelial-mesenchymal transition (EMT) has also been shown to occur during the initiation of metastasis in cancer progression. The aim of this study was to determine the relationships and roles of HG, estrogen receptor and EMT in the growth and migration of UEC. Methods: The expression of glucose transport protein 4 (GLUT4) in the control endometrium and UEC tissues was detected by immunohistochemistry (IHC); the cell viability and invasion were analyzed through CCK-8 and Matrigel invasion assays; the transcriptional level of EMT-related genes was evaluated through real-time PCR; and the effect of HG and / or GLUT4 on estrogen receptors, vascular endothelial growth factor (VEGF) and its receptor VEGFR was analyzed through western blotting, ELISA and flow cytometry (FCM) assay, respectively. In addition, Ishikawa-xenografted nude mice were constructed and were used to analyze the effect of estrogen and GLUT4 on the growth of UEC in vivo. Results: Here, we found that exposure to HG led to a high level of viability and invasion of UEC cell lines (UECC, Ishikawa and RL95-2 cells). Compared with the normal endometrium, a higher level of GLUT4 was observed in UEC tissues. Silencing GLUT4 obviously inhibited the HG-promoted viability, invasion and expression of EMT-related genes (TWIST, SNAIL and CTNNB1) of UECC promoted by HG. Further analysis showed that HG and GLUT4 promoted the secretion of VEGF and expression of VEGFR in UECC. Treatment with HG led to the increase of estrogen receptor α (ERα) and β (ERβ) in UECC, blocking ERα or ERβ resulted in the decreases in GLUT4 expression, TWIST, SNAIL and CTNNB1 transcription, and VEGF and VEGFR expression in UECC. Treatment with anti-human VEGF neutralizing antibody restricted the viability and invasion of UECC that was induced by HG and estrogen. Exposure to estrogen accelerated growth, VEGF production, and TWIST and CTNNB1 expression in UEC in Ishikawa-xenografted nude mice, and silencing GLUT4 restricted these effects. Conclusion: These data suggest that HG increases GLUT4 and VEGF/VEGFR expression, further promotes EMT process and accelerates the development of UEC by up-regulating ER