Exosome enriched leucine-rich alpha-2-glycoprotein-1 and extracellular matrix protein 1 proteins induce abnormal placental angiogenesis in pregnant mice.

INTRODUCTION: Gestational Diabetes Mellitus (GDM) is characterized by a high risk of fetal macrosomia and placenta hypervascularization. Exosomes has been known participating in various physiological and pathological processes, including pro-angiogenic function. However, the effects of umbilical cord blood derived exosomes from cases of GDM (GDM-exo) on placental vascular network formation remain unclear. METHODS: In the current study, we isolated and identified exosomes in umbilical cord blood from both normal (N-exo) and GDM pregnancies. Meanwhile, we investigated the effects of umbilical cord blood derived exosomes on placental angiogenesis both in vitro and in vivo. RESULTS: Our data indicated that in a mouse model, the placenta and fetus weight were significantly higher in the ones administrated with GDM-exo when compared with N-exo. Meanwhile, GDM-exo significantly enhanced placental endothelial cells functions in both HUVEC and HPMEC endothelial cell models. Importantly, we explored two up-regulated proteins in GDM-exo, namely leucine-rich alpha-2-glycoprotein-1 (LRG1) and extracellular matrix protein 1 (ECM1) by proteome analysis, which performed largely pro-angiogenic function and probably resulted in hypervascularization in GDM placenta. DISCUSSION: Thus, we proposed that abundant LRG1 and ECM1 enriched GDM-exo may take important roles in regulating pathological placental angiogenesis.

S100-A9 protein in exosomes derived from follicular fluid promotes inflammation via activation of NF-κB pathway in polycystic ovary syndrome.

Exosomes have recently emerged as key mediators of different physiological and pathological processes. However, there has been few report about proteomic analysis of exosomes derived from human follicular fluid and their association with the occurrence of PCOS. Herein, we used TMT-tagged quantitative proteomic approach to identify proteomic profiles in exosomes derived from follicular fluid of PCOS patients and healthy controls. We identified 662 proteins in exosomes derived from human ovarian follicular fluid. Eighty-six differently expressed proteins (P < .05) were found between PCOS and healthy women. The alterations in the proteomic profile were related to the inflammation process, reactive oxygen species metabolic process, cell migration and proliferation. Importantly, we observed that follicular fluid exosomes contain S100 calcium-binding protein A9 (S100-A9) protein. Exosome-enriched S100-A9 significantly enhanced inflammation and disrupted steroidogenesis via activation of nuclear factor kappa B (NF-κB) signalling pathway. These data demonstrate that exosomal proteins are differentially expressed in follicular fluid during disease process, and some proteins may play important roles in the regulation of granulosa cell function. These results highlight the importance of exosomes as extracellular communicators in ovarian follicular development.

Exosomes From Women With Preeclampsia Induced Vascular Dysfunction by Delivering sFlt (Soluble Fms-Like Tyrosine Kinase)-1 and sEng (Soluble Endoglin) to Endothelial Cells.

Preeclampsia is a unique multiple system disorder that affects 5% to 8% of pregnancies. Exosomes, membrane-encapsulated vesicles that are released into the extracellular environment by many cell types, can carry signals to the recipient cells to affect inflammation, apoptosis, and angiogenesis. We hypothesize that exosomes from women with preeclampsia complications impair vascular development by delivering antiangiogenic factors to endothelial cells. In the current study, plasma samples from gestational age-matched preeclampsia and normal pregnancies were used to isolate circulating exosomes by commercial kits. Next, application of transwell and matrigel tube formation assays showed that exosomes from preeclampsia patients impaired angiogenesis of human umbilical vein endothelial cells. We found that exosomes from preeclampsia expressed abundant sFlt-1 (soluble fms-like tyrosine kinase-1) and sEng (soluble endoglin). Considering the possibility that extracellular sFlt and sEng were horizontally transferred to human umbilical vein endothelial cells, we successfully collected exosomes containing high levels of sFlt-1 and sEng by overexpressing them in human embryonic kidney 293 cells. Furthermore, we demonstrated that these exosomes can attenuate the proliferation, migration, and tube formation of human umbilical vein endothelial cells in vitro. In a mouse model, exosomes from preeclampsia patients caused vascular dysfunction directly resulted in adverse preeclampsia-like birth outcomes. Thus, we proposed that exosomes mediated efficient transfer of sFlt-1 and sEng to endothelial cells to damage vascular functions and induce complications in preeclampsia patients.

Maternal and umbilical cord serum-derived exosomes enhance endothelial cell proliferation and migration.

We investigated the role of exosomes derived from maternal and umbilical cord blood in the regulation of angiogenesis. We report here that both maternal exosomes (MEs) and umbilical exosomes (UEs) significantly enhance HUVEC proliferation, migration, and tube formation. Importantly, ME-treated HUVECs (MEXs) displayed significantly increased migration, but not proliferation or tube formation, compared with UE-treated HUVECs (UEXs). We found that the expression of a subset of migration-related microRNAs (miRNAs), including miR-210-3p, miR-376c-3p, miR-151a-5p, miR-296-5p, miR-122-5p, and miR-550a-5p, among others, were significantly increased or decreased in UEs, and this altered expression was likely correlated with the differential regulation of HUVEC migration. We also found that the mRNA expression of hepatocyte growth factor (HGF) was up-regulated in MEXs and UEXs and, moreover, that inhibiting HGF partially abolished the enhanced cell migration induced by UEs. Our results suggest that both MEs and UEs greatly enhanced endothelial cell (EC) functions and differentially regulated EC migration, which was mostly attributed to the different expression profiles of exosomal miRNA. These findings highlight the importance of exosomes in the regulation of angiogenesis during pregnancy. Exosomal miRNAs, in particular, may be of great significance for the regulation of angiogenesis in maintaining normal pregnancy.-Jia, L., Zhou, X., Huang, X., Xu, X., Jia, Y., Wu, Y., Yao, J., Wu, Y., Wang, K. Maternal and umbilical cord serum-derived exosomes enhance endothelial cell proliferation and migration.

RhoA/MLC signaling pathway is involved in Δ9-tetrahydrocannabinol-impaired placental angiogenesis.

Cannabis is a widely used illicit drug and its abuse during pregnancy has been related to adverse reproductive outcomes. In addition, placental angiogenesis is considered to be responsible for the transport of nutrients critical for placental development and fetal growth. The purpose of this study is to determine the effects of Δ9-tetrahydrocannabinol (THC), the major component of cannabis, on placental angiogenesis, involving endothelial cell (EC) proliferation, migration and tube formation. Here, we observe dramatic alterations in placental vascular network of cannabis users correlated with an impaired HUVE cell proliferation, migration and tube formation after treated with THC. Mechanistically, the activity of RhoA/MLC is involved in the THC-impaired EC migration and angiogenesis. To further analyze the role of cannabis in mice placental and embryonic development, we inject pregnant mice with THC daily. This treatment results in an altered placental microvasculature, accompanied by the decreased expression of CD31 and activity of RhoA/MLC. Taken together, these findings identify THC plays a pivotal role in impairing placental angiogenesis potentially via RhoA/MLC signaling nexus.

LIFR increases the release of soluble endoglin via the upregulation of MMP14 expression in preeclampsia.

Preeclampsia (PE) is a pregnancy-specific disorder that is the main cause of maternal and perinatal morbidity and mortality worldwide. Inadequate trophoblastic invasion and endothelial dysfunction in the placenta are considered the foundation of the pathogenesis of preeclampsia in which soluble endoglin (sENG) plays an antiangiogenic role in the development of PE. The leukemia inhibitory factor receptor (LIFR) has been widely studied and is highly involved in arterial injury in vivo and in the migration of cancer cells in vitro Here, we tested the hypothesis that LIFR may be correlated with preeclampsia through its regulation of the release of sENG. Our data showed that LIFR protein, the expression of which significantly decreased with the progression of pregnancy, was located in the syncytiotrophoblast and cytotrophoblast. The LIFR protein level was increased in pregnancies with preeclampsia compared with normotensive full-term pregnancies. After the overexpression of LIFR in HTR8/SVneo cells, the release of sENG as well as the migration and invasion were significantly enhanced. Moreover, we also observed that LIFR induced the expression of matrix metalloproteinase14 (MMP14) and that the knockdown or inhibition of MMP14 decreased the release of sENG, as well as increased the LIFR-induced migration and invasion of HTR8/SVneo cells. These studies demonstrated that LIFR promoted the release of sENG through MMP14 in vitro, which indicates that LIFR may be involved in the development of preeclampsia.

Hypoxia-induced TET1 facilitates trophoblast cell migration and invasion through HIF1α signaling pathway.

Low oxygen is a typical extrinsic factor for the regulation of trophoblast biological function, including cell migration, invasion and proliferation. Ten-eleven translocation methylcytosine dioxygenase 1 (TET1), an enzyme converting 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), is transcriptionally activated by hypoxia in cancer cells. Therefore, we focus on the role of TET1 on trophoblast function in a physiologically hypoxic environment (3% oxygen), which is related to early placentation. Here, we found that TET1 was highly expressed in first trimester villi compared with normal term placentas. In vitro, both TET1 mRNA and protein expression levels in JEG3 cells were increased following exposure to 3% oxygen, and the migration and invasion capacities of JEG3 cells were up-regulated. Furthermore, TET1 knockdown decreased the migration, invasion and proliferation of JEG3 cells exposed to 3% oxygen, and the expression of HIF1α and its downstream target genes was also decreased, which was related to hyper-methylation of the HIF1α promoter. Finally, increased HIF1α protein expression reversed the inhibitory effect of TET1 knockdown on the migration and invasion of JEG3 cells exposed to 3% oxygen. These data show that hypoxia-induced TET1 expression facilitates trophoblast cell migration and invasion through the HIF1α signaling pathway, which plays an important role during placentation.

Suppression of STAT3 Signaling by Δ9-Tetrahydrocannabinol (THC) Induces Trophoblast Dysfunction.

AIMS: Marijuana is a widely used illicit drug and its consumption during pregnancy has been associated with adverse reproductive outcomes. The purpose of this study was to determine the effects of chronic intake of Δ9-tetrahydrocannabinol (THC), the major component of marijuana, on trophoblast function, placental development, and birth outcomes. METHODS: The pathological characteristics and distribution of cannabinoid receptors in placenta were observed by immunohistochemical (IHC) staining. Cell migration in response to THC was measured by transwell assays. The levels of cannabinoid receptors and Signal Transducer and Activator of Transcription 3 (STAT3) were detected by western blot. RESULTS: We found the placenta expressed two main cannabinoid receptors, suggesting that THC induced biological responses in placental cells. Supporting this hypothesis, we observed dramatic alterations of placental morphology in marijuana users. Using THC and inhibitors of cannabinoid receptors, we demonstrated that THC impaired trophoblast cell migration and invasion partly via cannabinoid receptors. Additionally, pregnant mice injected with THC showed adverse reproductive events including reduced number of fetuses, lower maternal and placental weights. Mechanistically, STAT3 signaling pathway was involved in the THC-induced suppression of trophoblast cell motility and pregnancy outcomes. CONCLUSION: Our study indicates that the STAT3 signaling pathway plays a critical role in THC-induced trophoblast dysfunction.

Potential Role of Hyperglycemia in Fetoplacental Endothelial Dysfunction in Gestational Diabetes Mellitus.

BACKGROUND: Gestational diabetes mellitus (GDM) is associated with structural and functional alterations in various tissues including endothelial dysfunction. The aim of this study was to explore the effects of hyperglycemia on fibroblast growth factor 2 (FGF2)- and vascular endothelial growth factor (VEGF)-stimulated placental angiogenesis and the underlying molecular signaling mechanisms. METHODS: The density of fetal placental capillaries was examined using immunohistochemistry. Human umbilical vein endothelial cells (HUVECs) derived from GDM (dHUVECs) and normal healthy patients (nHUVECs) were used as cell models in this study. Cell proliferation, migration and tube formation were measured with an MTS assay, a transwell system and a matrigel assay, respectively. The activation of ERK1/2 was analyzed with Western blot. The specific inhibitor of extracellular signal-regulated kinases 1/2 (ERK1/2) PD98059 was used to elucidate the involved signaling pathway. RESULTS: GDM did not alter the capillary density of the fetus-placenta. Both the GDM and hyperglycemic conditions inhibited the proliferation of the FGF2- but not the VEGF-stimulated HUVECs and the basal migratory capacity. Hyperglycemic condition significantly inhibited tube formation and ex vivo angiogenesis. Moreover, hyperglycemia inhibited the FGF2- but not the VEGF-induced activation of ERK1/2. PD98059 significantly inhibited the FGF2-activated ERK1/2 phosphorylation and the FGF2-stimulated cell proliferation in HUVECs. CONCLUSION: Both GDM and hyperglycemia may impair placental angiogenesis by reducing HUVEC proliferation, migration and tube formation. Hyperglycemia-inhibited cell proliferation stimulated by FGF2 probably contributed to the suppression of the MEK1/2/ERK1/2 pathways in the HUVECs.