Impaired repair properties of endothelial colony-forming cells in patients with granulomatosis with polyangiitis.

In patients with ANCA-associated vasculitis, interactions between neutrophils and endothelial cells cause endothelial damage and imbalance. Endothelial colony-forming cells (ECFCs) represent a cellular population of the endothelial lineage with proliferative capacity and vasoreparative properties. This study aimed to evaluate the angiogenic capacity of ECFCs of patients with granulomatosis with polyangiitis (GPA). The ECFCs of 13 patients with PR3-positive GPA and 14 healthy controls were isolated and characterized using fluorescence-activated cell sorting, capillary tube formation measurement, scratching assays and migration assays with and without plasma stimulation. Furthermore, three patients with active disease underwent post-treatment recollection of ECFCs for longitudinal evaluation. The ECFCs from the patients and controls showed similar capillary structure formation. However, the ECFCs from the patients with inactive GPA exhibited early losses of angiogenic capacity. Impairments in the migration capacities of the ECFCs were also observed in patients with GPA and controls (12th h, p = 0.05). Incubation of ECFCs from patients with GPA in remission with plasma from healthy controls significantly decreased migration capacity (p = 0.0001). Longitudinal analysis revealed that treatment significantly lowered ECFC migration rates. This study revealed that ECFCs from the patients with PR3-positive GPA in remission demonstrated early losses of tube formation and reduced migration capacity compared to those of the healthy controls, suggesting impairment of endothelial function.

Synergies of Extracellular Vesicles and Microchimerism in Promoting Immunotolerance During Pregnancy.

The concept of biological identity has been traditionally a central issue in immunology. The assumption that entities foreign to a specific organism should be rejected by its immune system, while self-entities do not trigger an immune response is challenged by the expanded immunotolerance observed in pregnancy. To explain this "immunological paradox", as it was first called by Sir Peter Medawar, several mechanisms have been described in the last decades. Among them, the intentional transfer and retention of small amounts of cells between a mother and her child have gained back attention. These microchimeric cells contribute to expanding allotolerance in both organisms and enhancing genetic fitness, but they could also provoke aberrant alloimmune activation. Understanding the mechanisms used by microchimeric cells to exert their function in pregnancy has proven to be challenging as per definition they are extremely rare. Profiting from studies in the field of transplantation and cancer research, a synergistic effect of microchimerism and cellular communication based on the secretion of extracellular vesicles (EVs) has begun to be unveiled. EVs are already known to play a pivotal role in feto-maternal tolerance by transferring cargo from fetal to maternal immune cells to reshape their function. A further aspect of EVs is their function in antigen presentation either directly or on the surface of recipient cells. Here, we review the current understanding of microchimerism in the feto-maternal tolerance during human pregnancy and the potential role of EVs in mediating the allorecognition and tropism of microchimeric cells.

Angiogenesis-Related Genes in Endothelial Progenitor Cells May Be Involved in Sickle Cell Stroke.

Background The clinical aspects of sickle cell anemia (SCA) are heterogeneous, and different patients may present significantly different clinical evolutions. Almost all organs can be affected, particularly the central nervous system. Transient ischemic events, infarcts, and cerebral hemorrhage can be observed and affect ≈25% of the patients with SCA. Differences in the expression of molecules produced by endothelial cells may be associated with the clinical heterogeneity of patients affected by vascular diseases. In this study, we investigated the differential expression of genes involved in endothelial cell biology in SCA patients with and without stroke. Methods and Results Endothelial progenitor cells from 4 SCA patients with stroke and 6 SCA patients without stroke were evaluated through the polymerase chain reaction array technique. The analysis of gene expression profiling identified 29 differentially expressed genes. Eleven of these genes were upregulated, and most were associated with angiogenesis (55%), inflammatory response (18%), and coagulation (18%) pathways. Downregulated expression was observed in 18 genes, with the majority associated with angiogenesis (28%), apoptosis (28%), and cell adhesion (22%) pathways. Remarkable overexpression of the MMP1 (matrix metalloproteinase 1) gene in the endothelial progenitor cells of all SCA patients with stroke (fold change: 204.64; P=0.0004) was observed. Conclusions Our results strongly suggest that angiogenesis is an important process in sickle cell stroke, and differences in the gene expression profile of endothelial cell biology, especially MMP1, may be related to stroke in SCA patients.

Identification of miRNAs and associated pathways regulated by Leukemia Inhibitory Factor in trophoblastic cell lines.

INTRODUCTION: Leukemia Inhibitory Factor (LIF) regulates behavior of trophoblast cells and their interaction with immune and endothelial cells. In vitro, trophoblast cell response to LIF may vary depending on the cell model. Reported differences in the miRNA profile of trophoblastic cells may be responsible for these observations. Therefore, miRNA expression was investigated in four trophoblastic cell lines under LIF stimulation followed by in silico analysis of altered miRNAs and their associated pathways. METHODS: Low density TaqMan miRNA assays were used to quantify levels of 762 mature miRNAs under LIF stimulation in three choriocarcinoma-derived (JEG-3, ACH-3P and AC1-M59) and a trophoblast immortalized (HTR-8/SVneo) cell lines. Expression of selected miRNAs was confirmed in primary trophoblast cells and cell lines by qPCR. Targets and associated pathways of the differentially expressed miRNAs were inferred from the miRTarBase followed by a KEGG Pathway Enrichment Analysis. HTR-8/SVneo and JEG-3 cells were transfected with miR-21-mimics and expression of miR-21 targets was assessed by qPCR. RESULTS: A similar number of miRNAs changed in each tested cell line upon LIF stimulation, however, low coincidence of individual miRNA species was observed and occurred more often among choriocarcinoma-derived cells (complete data set at http://www.ncbi.nlm.nih.gov/geo/ under GEO accession number GSE130489). Altered miRNAs were categorized into pathways involved in human diseases, cellular processes and signal transduction. Six cascades were identified as significantly enriched, including JAK/STAT and TGFB-SMAD. Upregulation of miR-21-3p was validated in all cell lines and primary cells and STAT3 was confirmed as its target. DISCUSSION: Dissimilar miRNA responses may be involved in differences of LIF effects on trophoblastic cell lines.

Involvement of STAT1 in proliferation and invasiveness of trophoblastic cells.

Trophoblast proliferation and invasion are controlled by cytokines and growth factors present at the implantation site. Members of the Interleukin-6 (IL-6) family of cytokines trigger their effects through activation of intracellular cascades including the Janus Kinase/Signal Transducer and Activator of Transcription (JAK-STAT) pathway. Functions of several STAT molecules in trophoblast cells have been described, but the role of STAT1 remained unclear. Here, potential functions of STAT1 and its activation by Oncostatin M (OSM) have been investigated in an in vitro model. STAT1 expression and phosphorylation were analyzed in human term placenta tissue by immunohistochemistry. HTR-8/SVneo cells (immortalized human extravillous trophoblast cells) were stimulated with OSM, IL-6, IL-11, Leukemia Inhibitory Factor (LIF) and Granulocyte Macrophage Colony-Stimulating Factor. Expression and phosphorylation of STAT1 were analyzed by Western blotting and immunocytochemistry. Fludarabine and STAT1 siRNA were employed for STAT1 depletion. STAT1 transcriptional activity was evaluated by DNA-binding capacity assay. Cell viability and invasion were assessed by MTS and Matrigel assays, respectively. STAT1 was expressed in villous and extravillous trophoblast cells. Low phosphorylation was detectable exclusively in extravillous trophoblast cells. Only OSM and LIF induced phosphorylation of STAT1 in the in vitro model. Challenge with OSM increased cell invasion but not proliferation. Inhibition of STAT1 by fludarabine treatment or STAT1 siRNA transfection reduced cell viability and invasiveness in presence and absence of OSM. These results indicate the potential involvement of STAT1 in the regulation of trophoblast behavior. Furthermore, STAT 1 functions are more efficiently inhibited by blocking its expression than its phosphorylation.

MicroRNA-141 is upregulated in preeclamptic placentae and regulates trophoblast invasion and intercellular communication.

Preeclampsia (PE) is one of the leading causes of maternal and perinatal morbidity and mortality worldwide. Abnormal expression of microRNAs (miRNAs) occurs in several pregnancy diseases including PE. Placental trophoblast cells express a specific set of miRNAs which changes during pregnancy. These miRNAs can be released within extracellular vesicles (EVs) and mediate intercellular communication. miR-141 is a pregnancy-related miRNA which is expressed by trophoblast cells at increased levels in maternal plasma in the third trimester. We hypothesize that miR-141 is abnormally expressed in PE placentae, controls trophoblast, and immune cell functions and is involved in the intercellular communication between fetal trophoblast and maternal immune cells. Expression of miR-141 was analyzed by quantitative real-time PCR (qPCR) in normal and preeclamptic placentae and in 2 different trophoblastic cell lines, JEG-3 and HTR-8/SVneo. Changes in JEG-3 and HTR-8/SVneo cell proliferation and invasion were investigated after miR-141 inhibition and overexpression by MTS-, BrdU-, and Matrigel assays. EVs from miR-141 transfected cells were isolated from supernatants and characterized by NanoSight analysis and qPCR. Proliferation of Jurkat T cells and invasion of HTR-8/SVneo cells were investigated after treatment with EVs containing different miR-141 levels. miR-141 expression was higher in placentae from PE patients compared with those from normal pregnancies. miR-141 inhibition in trophoblastic cells resulted in decreased cell viability and reduced invasion capability. After transfection with miR-141-mimic, trophoblastic cells secreted EVs with increased miR-141 content. These vesicles did not exert effects on trophoblastic cell invasion but reduced Jurkat T cell proliferation. In conclusion, miR-141 regulates major functions of trophoblastic and immune cells. Trophoblast cells release EVs whose miRNA content can be modified by transfection of origin cells. Furthermore, elevated levels of miR-141 can be transferred from trophoblast to immune cells by release and internalization of EVs suggesting their role in the immune regulation of normal and pathologic pregnancies.

Oncostatin M and leukaemia inhibitory factor trigger signal transducer and activator of transcription 3 and extracellular signal-regulated kinase 1/2 pathways but result in heterogeneous cellular responses in trophoblast cells.

Leukaemia inhibitory factor (LIF) and oncostatin M (OSM) are pleiotropic cytokines present at the implantation site that are important for the normal development of human pregnancy. These cytokines share the cell membrane receptor subunit gp130, resulting in similar functions. The aim of this study was to compare the response to LIF and OSM in several trophoblast models with particular regard to intracellular mechanisms and invasion. Four trophoblast cell lines with different characteristics were used: HTR-8/SVneo, JEG-3, ACH-3P and AC1-M59 cells. Cells were incubated with LIF, OSM (both at 10ngmL(-1)) and the signal transducer and activator of transcription (STAT) 3 inhibitor S3I-201 (200µM). Expression and phosphorylation of STAT3 (tyr(705)) and extracellular regulated kinase (ERK) 1/2 (thr(202/204)) and the STAT3 DNA-binding capacity were analysed by Western blotting and DNA-binding assays, respectively. Cell viability and invasiveness were assessed by the methylthiazole tetrazolium salt (MTS) and Matrigel assays. Enzymatic activity of matrix metalloproteinase (MMP)-2 and MMP-9 was investigated by zymography. OSM and LIF triggered phosphorylation of STAT3 and ERK1/2, followed by a significant increase in STAT3 DNA-binding activity in all tested cell lines. Stimulation with LIF but not OSM significantly enhanced invasion of ACH-3P and JEG-3 cells, but not HTR-8/SVneo or AC1-M59 cells. Similarly, STAT3 inhibition significantly decreased the invasiveness of only ACH-3P and JEG-3 cells concomitant with decreases in secreted MMP-2 and MMP-9. OSM shares with LIF the capacity to activate ERK1/2 and STAT3 pathways in all cell lines tested, but their resulting effects are dependent on cell type. This suggests that LIF and OSM may partially substitute for each other in case of deficiencies or therapeutic interventions.

Dissimilar microRNA-21 functions and targets in trophoblastic cell lines of different origin.

Trophoblast cells express a singular miRNA expression profile which varies during pregnancy and whose alteration may be associated with pregnancy complications. miR-21, a widely known oncomir, is highly expressed in human placenta but its role in regulating trophoblast cells remains unclear. The aim of this study was to investigate miR-21 functions and targets in HTR-8/SVneo immortalized trophoblast and JEG-3 choriocarcinoma cells, which are trophoblast cell models that differ in their cellular origin. Cells were transfected with miR-21-antagomir, -mimic or their respective controls. Following, cell proliferation (BrdU), migration (Transwell and scratch wound-healing assays), invasion (Matrigel assays) and apoptosis (flow cytometry, TUNEL assay and Western blotting) were assessed. Expression of the potential miR-21 targets phosphatase and tensin homolog (PTEN) and programmed cell death 4 (PDCD4) were analyzed by Western blotting. Inhibition of miR-21 decreased cell proliferation, migration, and invasion in JEG-3 and HTR-8/SVneo cells and additionally, induced apoptosis in JEG-3 cells. Silencing of miR-21 enhanced PDCD4 expression only in JEG-3 cells, and PTEN expression only in HTR-8/SVneo cells. Inhibition of miR-21 significantly increased phosphorylation of AKT in HTR-8/SVneo cells. In conclusion, miR-21 has cell-specific targets depending upon the origin of trophoblastic cells. Furthermore, miR-21 regulates major cellular processes including cell growth, migration, invasion and apoptosis suggesting that its impairment may lead to placental disorders.

STAT5 is Activated by Epidermal Growth Factor and Induces Proliferation and Invasion in Trophoblastic Cells.

Epidermal growth factor (EGF) is expressed by decidual and trophoblast cells and influences manifold cellular functions during embryo implantation. Thus far, signaling of EGF via Signal Transducer and Activator of Transcription 5 (STAT5) has been only partially investigated. STAT5 stimulates proliferation and cell cycle progression in several cell types. Its dysregulation is associated with pregnancy. The aim of this study was to investigate STAT5 activation and function mediated by EGF in 2 trophoblastic cell lines, namely, HTR8/SVneo and JAR. Additionally, expression of STAT5B messenger RNA (mRNA) in trophoblast models has been compared to that of primary cells isolated from term placentas. Our results demonstrate the highest STAT5B mRNA expression in isolated trophoblast cells, lower expression in HTR8/SVneo cells, and the significantly lowest in JAR cells. Moreover, EGF-mediated STAT5 activation increases cell proliferation and viability in both cell lines. The STAT5 knockdown results in significant decrease in cell viability induced by EGF. Only in HTR8/SVneo cells, invasion decreases after STAT5 silencing and this effect cannot be rescued by further addition of EGF. These results demonstrate that STAT5 activated by EGF constitutes an important cascade for the regulation of cell proliferation and invasion in trophoblast cells.

Intranuclear crosstalk between extracellular regulated kinase1/2 and signal transducer and activator of transcription 3 regulates JEG-3 choriocarcinoma cell invasion and proliferation.

Invasiveness of trophoblast and choriocarcinoma cells is in part mediated via leukemia inhibitory factor- (LIF-) induced activation of signal transducer and activator of transcription 3 (STAT3). The regulation of STAT3 phosphorylation at its ser727 binding site, possible crosstalk with intracellular MAPK signaling, and their functional implications are the object of the present investigation. JEG-3 choriocarcinoma cells were cultured in presence/absence of LIF and the specific ERK1/2 inhibitor (U0126). Phosphorylation of signaling molecules (p-STAT3 (ser727 and tyr705) and p-ERK1/2 (thr 202/tyr 204)) was assessed per Western blot. Immunocytochemistry confirmed results, but also pinpointed the location of phosphorylated signaling molecules. STAT3 DNA-binding capacity was studied with a colorimetric ELISA-based assay. Cell viability and invasion capability were assessed by MTS and Matrigel assays. Our results demonstrate that LIF-induced phosphorylation of STAT3 (tyr705 and ser727) is significantly increased after blocking ERK1/2. STAT3 DNA-binding capacity and cell invasiveness are enhanced after LIF stimulation and ERK1/2 blockage. In contrast, proliferation is enhanced by LIF but reduced after ERK1/2 inhibition. The findings herein show that blocking ERK1/2 increases LIF-induced STAT3 phosphorylation and STAT3 DNA-binding capacity by an intranuclear crosstalk, which leads to enhanced invasiveness and reduced proliferation.

Pregnancy-associated miRNA-clusters.

MicroRNAs (miRNAs) are expressed in the placenta and can be detected in maternal plasma. An increasing number of studies have been published on the cellular origin, distribution and function of miRNAs in pregnancy. Specific miRNA profiles have been described for the placenta, maternal plasma and several pregnancy disorders. It has been observed that numerous miRNAs, which are predominantly or exclusively expressed during pregnancy, are clustered in chromosomal regions, may be controlled by the same promoters, may have similar seed regions and targets, and work synergistically. The three most eminent clusters are the chromosome 19 miRNA cluster (C19MC), C14MC and miR-371-3 cluster, which is also localized on chromosome 19. MiRNA members of these clusters are not only detected in the placenta, but also in other compartments, e.g. in serum where they have the potential to become novel biomarkers of pregnancy disorders. Additionally, some members are also expressed in a variety of tumors. Antagonism of selected miRNAs or their targets may lead to novel strategies for the development of new drug classes in pregnancy disorders or other diseases. This review summarizes current knowledge on the pregnancy-related miRNA clusters - the C19MC, C14MC and miR-371-3 cluster - in regard to pregnancy and also other, mostly pathological circumstances.