Jagged1 Research Articles

Alagille syndrome: an uncommon cause of intrahepatic cholestasis in adults

Alagille syndrome (ALGS) is an autosomal-dominant multisystem disorder caused by mutations in Jagged 1 (JAG1) or NOTCH2. The penetrance is low but highly variable. It is almost exclusively diagnosed in children with cholestasis and, more rarely, in their adult relatives. Here, we report the case of a patient diagnosed with ALGS in adulthood. The patient was a 28-year-old male who presented with characteristic facial features, an eye abnormality, chronic cholestasis with bile duct paucity on liver biopsy, atrial defects and stenosis of the left internal carotid artery. A novel frameshift mutation, c.2087_2088insAAAAATGG (p. W697Kfs*49), in JAG1 was identified. To our knowledge, this is the first case of ALGS diagnosed in adulthood in China. ALGS should be considered as a differential diagnosis for intrahepatic cholestasis in adult patients with a wide variety of clinical manifestations, including cardiac disease, skeletal abnormalities, ocular abnormalities and characteristic facial features.

Jagged1 protein processing in the developing mammalian lens.

ABSTRACTNotch signaling regulates a multitude of cellular processes. During ocular lens development this pathway is required for lens progenitor growth, differentiation and maintenance of the transition zone. After ligand-receptor binding, the receptor proteins are processed, first by ADAM proteases, then by γ-secretase cleavage. This results in the release of a Notch intracellular domain (N-ICD), which is recruited into a nuclear transcription factor complex that activates Notch target genes. Previous in vitro studies showed that the Delta-like and Jagged ligand proteins can also be cleaved by the γ-secretase complex, but it remains unknown whether such processing occurs during in vivo vertebrate development. Here we show that mouse and human lens progenitor cells endogenously express multiple Jagged1 protein isoforms, including a Jagged1 intracellular domain. We also found that pharmacologic blockage of γ-secretase activity in vitro resulted in an accumulation of Jagged1 polypeptide intermediates. Finally, overexpression of an epitope-tagged Jagged1 intracellular domain displayed nuclear localization and induced the upregulation of endogenous JAG1 mRNA expression. These findings support the idea that along with its classical role as a Notch pathway ligand, Jagged1 is regulated post-translationally, to produce multiple active protein isoforms.

Molecular signatures identify immature mesenchymal progenitors in early mouse limb buds that respond differentially to morphogen signaling.

ABSTRACTThe key molecular interactions governing vertebrate limb bud development are a paradigm for studying the mechanisms controlling progenitor cell proliferation and specification during vertebrate organogenesis. However, little is known about the cellular heterogeneity of the mesenchymal progenitors in early limb buds that ultimately contribute to the chondrogenic condensations prefiguring the skeleton. We combined flow cytometric and transcriptome analyses to identify the molecular signatures of several distinct mesenchymal progenitor cell populations present in early mouse forelimb buds. In particular, jagged 1 (JAG1)-positive cells located in the posterior-distal mesenchyme were identified as the most immature limb bud mesenchymal progenitors (LMPs), which crucially depend on SHH and FGF signaling in culture. The analysis of gremlin 1 (Grem1)-deficient forelimb buds showed that JAG1-expressing LMPs are protected from apoptosis by GREM1-mediated BMP antagonism. At the same stage, the osteo-chondrogenic progenitors (OCPs) located in the core mesenchyme are already actively responding to BMP signaling. This analysis sheds light on the cellular heterogeneity of the early mouse limb bud mesenchyme and on the distinct response of LMPs and OCPs to morphogen signaling.

Bacoside A Induced Sub-G0 Arrest and Early Apoptosis in Human Glioblastoma Cell Line U-87 MG through Notch Signaling Pathway.

BackgroundGlioblastoma multiforme (GBM) is a highly malignant brain tumor with a worst prognosis of less than one year despite advance treatment facilities. Among various signaling pathway genes displaying genetic modifications, aberrant expression of Notch pathway genes is frequent in GBM offering novel therapeutic targets. Herbal extracts having anticancer properties are used in adjuvant therapy that is safe and affordable as compared to chemotherapeutics. Bacopa monnieri has been used for the development of brain cells because of its neuroprotective properties. Its anticancer properties have shown to be promising in cancer treatment.MethodsThe anticancer properties of Bacoside A, an active and abundant component of Bacopa monnieri was assessed on U-87 MG cell line and its effects on expression of Notch pathway genes were studied. Cell cycle arrest and apoptosis were studied using flow cytometry. Expression of Notch pathway genes comprising of Notch receptors (notch1, notch2, notch3 and notch4), ligands (jagged1 and jagged2), a component of gamma-secretase complex (APH1A) and downstream target (HES1) were evaluated by quantitative real-time PCR.ResultsBacoside A exhibited considerable cytotoxicity on U-87 MG cells inducing cell cycle arrest and apoptosis. Cell cycle analysis revealed a significant arrest of 39.21% cells in sub-G0 phase at 80 µg/mL concentration, increasing to 53.21% at a higher concentration of 100 µg/mL. The fraction of early apoptotic cells in control was low (3.48%) that increased substantially to 31.36% and 41.11% after 80 µg/mL and 100 µg/mL of Bacoside A treatment respectively. Additionally, the expression of notch1 gene decreased after exposure to Bacoside A with a fold change of 0.05, whereas HES1 gene expression was increased by 25 fold.ConclusionThese data indicate that Bacoside A has a possible anticancer activity that could be inducing cell cycle arrest and apoptosis through Notch pathway in GBM in vitro.

M1-Polarized Macrophages Promote Self-Renewing Phenotype of Hepatic Progenitor Cells with Jagged1-Notch Signalling Involved: Relevance in Primary Sclerosing Cholangitis.

The immunologic interaction between parenchyma cells and encircling inflammatory cells is thought to be the most important mechanism of biliary damage and repair in primary sclerosing cholangitis (PSC). Monocytes/macrophages as master regulators of hepatic inflammation have been demonstrated to contribute to PSC pathogenesis. Macrophages coordinate with liver regeneration, and multiple phenotypes have been identified with diverse expressions of surface proteins and cytokine productions. We analyzed the expression of Notch ligand Jagged1 in polarized macrophages and investigated the relevance of Notch signalling activation in liver regeneration. M1 or M2 macrophages were generated from mouse bone marrow-derived macrophages (BMDMs) by classical or alternative activation, respectively. Then, the expression levels of Jagged1 (Jag1) of each phenotype were measured. The effects of polarized BMDMs on the expression of hepatic progenitor cell- (HPC-) specific markers and hairy and enhancer of split-1 (HES1) in HPCs in coculture were also analyzed. Monocyte-macrophage and Notch signalling-associated gene signatures were evaluated in the GEO database (access ID: GSE61260) by gene set enrichment analysis (GSEA). M1 macrophages were found associated with elevated Jag1 expression, which increased the fraction of HPC with self-renewing phenotypes (CD326+CD44+ or CD324+CD44+) and HES1 expression level in cocultured HPC. Blocking Jagged1 by siRNA or antibody in the coculture system attenuates HPC self-renewing phenotypes as well as HES1 expression in HPC. GSEA data show that macrophage activation and Notch signalling-associated gene signatures are enriched in PSC patients. These findings suggest that M1 macrophages promote an HPC self-renewing phenotype which is associated with Notch signalling activation within HPC. In the liver of PSC patients, the prevalence of activated macrophages, with M1 polarized accounting for the main part, is associated with increment of Notch signalling and enhancement of HPC self-renewal.

Invasion of white matter tracts by glioma stem cells is regulated by a NOTCH1-SOX2 positive-feedback loop.

Early invasive growth along specific anatomical structures, especially the white matter tract, is regarded as one of the main causes of poor therapeutic outcome of people with gliomas. We show that some glioma stem cells (GSCs) are preferentially located along white matter tracts, which exhibit a demyelinated phenotype, at the invasive frontier of glioma tissues. These GSCs are CD133+Notch1+, whereas the nerve fibers express the Notch ligand Jagged1. The Notch-induced transcription factor Sox9 promotes the transcription of SOX2 and the methylation level of the NOTCH1 promoter is attenuated by the upregulation of SOX2 to reinforce NOTCH1 expression in GSCs. This positive-feedback loop in a cohort of glioma subjects is correlated with a poor prognosis. Inhibition of Notch signaling attenuates the white-matter-tract tropism of GSCs. These findings provide evidence indicating that the NOTCH1-SOX2 positive-feedback loop controls GSC invasion along white matter tracts.

A non-canonical JAGGED1 signal to JAK2 mediates osteoblast commitment in cranial neural crest cells

During craniofacial development, cranial neural crest (CNC) cells migrate into the developing face and form bone through intramembranous ossification. Loss of JAGGED1 (JAG1) signaling in the CNC cells is associated with maxillary hypoplasia or maxillary bone deficiency (MBD) in mice and recapitulates the MBD seen in humans with Alagille syndrome. JAGGED1, a membrane-bound NOTCH ligand, is required for normal craniofacial development, and Jagged1 mutations in humans are known to cause Alagille Syndrome, which is associated with cardiac, biliary, and bone phenotypes and these children experience increased bony fractures. Previously, we demonstrated deficient maxillary osteogenesis in Wnt1-cre;Jagged1f/f (Jag1CKO) mice by conditional deletion of Jagged1 in maxillary CNC cells. In this study, we investigated the JAG1 signaling pathways in a CNC cell line. Treatment with JAG1 induced osteoblast differentiation and maturation markers, Runx2 and Ocn, respectively, Alkaline Phosphatase (ALP) production, as well as classic NOTCH1 targets, Hes1 and Hey1. While JAG1-induced Hes1 and Hey1 expression levels were predictably decreased after DAPT (NOTCH inhibitor) treatment, JAG1-induced Runx2 and Ocn levels were surprisingly constant in the presence of DAPT, indicating that JAG1 effects in the CNC cells are independent of the canonical NOTCH pathway. JAG1 treatment of CNC cells increased Janus Kinase 2 (JAK2) phosphorylation, which was refractory to DAPT treatment, highlighting the importance of the non-canonical NOTCH pathway during CNC cells osteoblast commitment. Pharmacologic inhibition of JAK2 phosphorylation, with and without DAPT treatment, upon JAG1 induction reduced ALP production and, Runx2 and Ocn gene expression. Collectively, these data suggest that JAK2 is an essential component downstream of a non-canonical JAG1-NOTCH1 pathway through which JAG1 stimulates expression of osteoblast-specific gene targets in CNC cells that contribute to osteoblast differentiation and bone mineralization.

Mismatch repair deficiency and aberrations in the Notch and Hedgehog pathways are of prognostic value in patients with endometrial cancer.

The aim of this study was to investigate the prognostic value of the Hedgehog (Gli, Patched-1, Shh, Smo) and Notch (Jag1, Notch2, Notch3) pathway members, in comparison to a panel of proteins (ER, PgR, HER2/neu, Ki67, p53, p16, PTEN and MMR) previously suggested to be involved in the pathogenesis of endometrial cancer, in association with clinical outcome and standard clinicopathological characteristics. A total of 204 patients with histological diagnosis of endometrial cancer treated from 2004 to 2013 were included. The evaluation of protein expression was assessed by immunohistochemistry. Univariate analysis showed that higher Ki67 labeling, expression of PTEN, p16, Notch2 and Notch3 proteins, as well as MMR proficiency were associated with increased relapse and mortality rate. Additionally, Patched-1 protein expression was associated with worse DFS, while p53 overexpression was associated with worse OS. In multivariate analyses, patients with MMR proficient tumors had more than double risk for death than patients with MMR deficient (MMRd) tumors (adjusted HR = 2.19, 95% CI 1.05–4.58, p = 0.036). Jag1 positivity conferred reduced mortality risk (HR = 0.48, 95% CI 0.23–0.97, p = 0.042). However, as shown by hierarchical clustering, patients fared better when their tumors expressed high Jag1 protein in the absence of Notch2 and Notch3, while they fared worse when all three proteins were highly expressed. Patched-1 positivity conferred higher risk for relapse (HR = 2.04, 95% CI 1.05–3.96, p = 0.036).Aberrant expression of key components of the Notch and Hedgehog signaling pathways, as well as MMRd may serve as independent prognostic factors for recurrence and survival in patients with endometrial cancer.

MIB1 mutations reduce Notch signaling activation and contribute to congenital heart disease.

Congenital heart disease (CHD) is one of the most common birth defects in humans, but its genetic etiology remains largely unknown despite decades of research. The Notch signaling pathway plays critical roles in embryonic cardiogenesis. Mind bomb 1 (Mib1) is a vital protein that activates the Notch signaling pathway through promoting ubiquitination, endocytosis and subsequent activation of Notch ligands. Previous studies show that Mib1 knockout in mice completely abolishes Notch signaling, leading to cardiac deformity. However, the function of MIB1 and its potential disease-causing mutations are poorly studied in human CHD. In this research, we identified four novel non-synonymous heterozygous rare mutations of MIB1 from 417 Han Chinese CHD patients. The following biochemical analyses revealed that mutations p.T312K fs*55 and p.W271G significantly deplete MIB1’s function, resulting in a lower level of JAGGED1 (JAG1) ubiquitination and Notch signaling induction. Our results suggest that pathologic variants in MIB1 may contribute to CHD occurrence, shedding new light on the genetic mechanism of CHD in the context of the Notch signaling pathway.

Notch Signaling Regulates Mitochondrial Metabolism and NF-κB Activity in Triple-Negative Breast Cancer Cells via IKKα-Dependent Non-canonical Pathways.

Triple negative breast cancer (TNBC) patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem-like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Notch signaling is a key pathway regulating TNBC CSC survival. Treatment of TNBC with PI3K or mTORC1/2 inhibitors results in drug-resistant, Notch-dependent CSC. However, downstream mechanisms and potentially druggable Notch effectors in TNBC CSCs are largely unknown. We studied the role of the AKT pathway and mitochondrial metabolism downstream of Notch signaling in TNBC CSC from cell lines representative of different TNBC molecular subtypes as well as a novel patient-derived model. We demonstrate that exposure of TNBC cells to recombinant Notch ligand Jagged1 leads to rapid AKT phosphorylation in a Notch1-dependent but RBP-Jκ independent fashion. This requires mTOR and IKKα. Jagged1 also stimulates mitochondrial respiration and fermentation in an AKT- and IKK-dependent fashion. Notch1 co-localizes with mitochondria in TNBC cells. Pharmacological inhibition of Notch cleavage by gamma secretase inhibitor PF-03084014 in combination with AKT inhibitor MK-2206 or IKK-targeted NF-κB inhibitor Bay11-7082 blocks secondary mammosphere formation from sorted CD90hi or CD44+CD24low (CSCs) cells. A TNBC patient-derived model gave comparable results. Besides mitochondrial oxidative metabolism, Jagged1 also triggers nuclear, NF-κB-dependent transcription of anti-apoptotic gene cIAP-2. This requires recruitment of Notch1, IKKα and NF-κB to the cIAP-2 promoter. Our observations support a model where Jagged1 triggers IKKα-dependent, mitochondrial and nuclear Notch1 signals that stimulate AKT phosphorylation, oxidative metabolism and transcription of survival genes in PTEN wild-type TNBC cells. These data suggest that combination treatments targeting the intersection of the Notch, AKT and NF-κB pathways have potential therapeutic applications against CSCs in TNBC cases with Notch1 and wild-type PTEN expression.

Extravesicular Mir-7977 Released from Leukemic Cells Inhibites the PCBP1-Mediated mRNA Stabilization and Hippo-YAP Signaling Pathway in Bone Marrow Mesenchymal Stromal Cells

[Background] We and others have revealed that various abnormalities of the bone marrow (BM) environment such as aberrant cytokine expression and impaired microRNA (miRNA) biogenesis are observed in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, underlying mechanisms to induce BM stromal dysfunction have not yet been clarified. Recently, we have found that abundant extracellular vesicles including exosomes, were detected in the BM interstitial fluid. Furthermore, we have shown that exosomal miR-7977 was highly released from AML cells (GSE64029) and was taken up by BM mesenchymal stem cells (MSCs). Based on these findings, we hypothesized that exosomal miR-7977 could be involved in the alteration of BM stromal function of MDS/AML.[Materials and Methods] To gain an insight into the function of exosomal miR-7977 in BM, we firstly performed transduction of a miR-7977 mimic into BM MSCs and compared transcriptomes between control-transduced and miR-7977-transduced MSCs (GSE108186). Subsequently, we selected the differentially expressed genes (DEGs) and conduced gene set enrichment analysis (GSEA).[Results] Regarding DEGs, we focused on poly(rC) binding protein 1 (PCBP1), since PCBP1 was predicted to be a target gene of miR-7977 by an online database for miRNA target prediction using miRDB. The reductions in PCBP1 mRNA and protein were confirmed in MSCs after miR-7977 transfer by real time PCR. Because PCBP1 is known to be involved in splicing, and the expression levels of several genes, we analyzed the expression of several hematopoietic factors and found that expression level of stem cell factor (SCF), angiopoietin-1 (ANGPT1) and Jagged-1 (JAG1) were remarkably decreased. Regarding the pathway analysis, GSEA showed that the gene sets of Yes-associated protein 1 (YAP1)_up were significantly enriched (p<0.001, q<0.25), suggesting that miR-7977 modulates the Hippo-YAP signaling pathway. Visualization of pathway and network showed that miR-7977 significantly reduced the expression of Hippo core kinase, STK4. The transfer of miR-7977 mimic inactivated the Hippo-YAP signaling pathway as proven by GFP-tagged YAP nuclear trans localization and TEAD reporter assay. The miR-7977-transduced MSCs showed elevated saturation density and enhanced entry into the cell cycle.[Conclusion] Collectively, these results indicated that miR-7977 is an important factor that inhibits normal hematopoiesis via PCBP1 reduction and up-regulate the growth of functionally-disturbed MSCs via inactivation of Hippo-YAP signaling pathway in MDS/AML. DisclosuresNo relevant conflicts of interest to declare.

Jagged1-expressing adenovirus-infected dendritic cells induce expansion of Foxp3+ regulatory T cells and alleviate T helper type 2-mediated allergic asthma in mice.

Dendritic cells (DCs) are professional antigen-presenting cells that play a key role in directing T-cell responses. Regulatory T (Treg) cells possess an immunosuppressive ability to inhibit effector T-cell responses, and Notch ligand Jagged1 (Jag1) is implicated in Treg cell differentiation. In this study, we evaluated whether bone marrow-derived DCs genetically engineered to express Jag1 (Jag1-DCs) would affect the maturation and function of DCs in vitro and further investigated the immunoregulatory ability of Jag1-DCs to manipulate T helper type 2 (Th2) -mediated allergic asthma in mice. We produced Jag1-DCs by adenoviral transduction. Overexpression of Jag1 by ovalbumin (OVA) -stimulated Jag1-DCs exhibited increased expression of programmed cell death ligand 1 (PD-L1) and OX40L molecules. Subsequently, co-culture of these OVA-pulsed Jag1-DCs with allogeneic or syngeneic CD4+ T cells promoted the generation of Foxp3+ Treg cells, and blocking PD-L1 using specific antibodies partially reduced Treg cell expansion. Furthermore, adoptive transfer of OVA-pulsed Jag1-DCs to mice with OVA-induced asthma reduced allergen-specific immunoglobulin E production, airway hyperresponsiveness, airway inflammation, and secretion of Th2-type cytokines (interleukin-4, interleukin-5, and interleukin-13). Notably, an increased number of Foxp3+ Treg cells associated with enhanced levels of transforming growth factor-β production was observed in Jag1-DC-treated mice. These data indicate that transgenic expression of Jag1 by DCs promotes induction of Foxp3+ Treg cells, which ameliorated Th2-mediated allergic asthma in mice. Our study supports an attractive strategy to artificially generate immunoregulatory DCs and provides a novel approach for manipulating Th2 cell-driven deleterious immune diseases.

Severe Chronic Neutropenia: Primary Immunodeficiency Mutations Are Frequent Causative Agents

Severe Chronic Neutropenia (SChrN) is an heterogeneous group of disorders characterized by persistently low circulating neutrophils (<500/cmm). Within this group, the classical form of Severe Congenital Neutropenia (SCN), once defined Kostman's disease, appears in early infancy, usually shows a marrow block at promyelocyte stage and needs lifetime treatment with G-CSF to prevent severe infections. Mutations in more than 20 genes have been recognized as causative of the disease with a clear prevalence of ELANEgene mutations (70% of cases). However,SChrN may also be an epiphenomena of autoimmune/immune dysregulation disorders (Secondary Neutropenia, SN) which are usually associated to extrahaematological signs and/or positivity of autoimmunity markers, to a normal or “left shifted” bone marrow morphology. In spite of these categorization many cases do not fit either group and share features of both of them. These “Ovelap Neutropenia” (ON) patients represent a challenge for diagnosis and management. AIM OF the study: Investigate the genetic backgroud of a cohort of SChrN subjects screened in two immune-hematology centers in Italy. Material and Methods: Patients SChrN were prospectively seen in participating Centres and diagnosed/followed-up according to published guidelines (3,4). Genetic diagnosis included classical Sanger technique for commonest severe chronic neutropenia genes and an enlarged NGS panel including also genes responsible for PID. Results: From 2008 to 2016, 27 SChrNpatients (52% males)with a median age at last follow of 13y (range 20 mos-53yrs ) entered the study (Table 1).Thirteen/27 subjects (48%) were phenotypically diagnosed as classical SCN. Of them 10 (77%) and 1 (8%) had mutations in ELANEand JAG1 genes respectively and 2 (16%) had no pathogenic gene detectable. Eight/27(30%) patients were phenotypically diagnosed as SN and the remaining 6/27 ( 22%) had the clinical features of ON. A pathogenic mutation in a Primary Immune Deficiency (PID) gene (RAG1, LRBA, CECR1. TACI, CARD11, CD40 and PI3K plus CASP 10) was found in 7/27 patients (26%). Four of them belonged to the group of 8 patients diagnosed with SN (50%) and 3 to that of the 6 ON subjects (50%). In 5/27(19%) patients no pathogenic gene was found. Table 1 shows clinical hematological characteristic of the 3 categories of patients.Conclusions:In our unselected cohort of SChrNa considerable proportion (26%) of subjects bore a genetic defect that qualifies them as PID. These PID genetic defects are located in the SN and ON patients (50% each) that are clinically different from classical ELANE mutated SCN subjects because of the presence of extra-hematological signs, of markers of autoimmunity, of a diverse marrow maturation block and a frequent involvement of more than one hematopoietic lineage. While the use of NGS still leaves a not negligible proportion of SChrN without pathogenic gene, this extensive genetic diagnostic approach enables to identify a relevant portion of subjects with SChrN who indeed carry a PID genetic defect. This has important clinical implications related to specific treatment and monitoring schedules to apply to these patients. The application of the Whole ExomeSequencing technique might fill the gap of the SCHrN patient who are still gene orphan. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cartilage Oligomeric Matrix Protein initiates cancer stem cells through activation of Jagged1-Notch3 signaling

Cancer stem cell populations are important for the initiation, progression and metastasis of tumors. The mechanisms governing cancer stem cell control are only partially understood, but activation of the Notch3 pathway plays a crucial role in the maintenance of breast cancer stem cells. Expression of Cartilage Oligomeric Matrix Protein (COMP) in breast cancer cells is correlated with poor survival and higher recurrence rates in patients. In this study, we provide in vivo and in vitro evidence that COMP expression increases the proportion of cancer stem cells in breast cancer. Thus, MDA-MB-231 and BT-20 cells expressing COMP formed larger tumorspheres in vivo and in vitro and displayed higher ALDH-activity than cells lacking COMP. Additionally, BT-20 COMP-expressing cells displayed higher expression of CD133 compared with the control cells. Furthermore, among the different Notch receptors, Notch3 is specifically activated in COMP-expressing cells. Mechanistically, activation of Notch3 is mediated by secreted, polymeric COMP, which interacts with both Notch3 and its ligand Jagged1, bridging the receptor and ligand together, enhancing Notch3-specific signaling. COMP-dependent Notch3 activation also leads to cross-talk with β-Catenin and AKT pathways. Using the model of MMTV-PyMT mouse breast tumorigenesis, we observed a decrease in the size of tumors and the amount of cancer stem cells as well as reduced Notch3 activation, in COMP knockout mice in comparison to wild type mice. In conclusion, we reveal a novel molecular mechanism whereby COMP regulates the cancer stem cell population through increasing the interaction between Notch3 and Jagged1, leading to increased activation of Notch3 signaling.

Pathogenic factors associated with tetralogy of Fallot

Tetralogy of Fallot(TOF) is the most common cyanotic congenital heart disease with pathological anatomy of ventricular septal defect, overriding aorta, pulmonary stenosis, and right ventricular hypertrophy.At present, the success rate of surgical repair of TOF has been significantly improved, but there is risk of complications after surgery.The study of the pathogenesis of TOF is important for disease prevention and genetic counseling.The etiological mechanism of TOF is multifactorial.It has been reported that NKX2-5, Jagged-1, GATA-4, TBX1 are associated with TOF.Exposure during pregnancy, including harmful environmental exposure, diseases and infections, and lack of key nutritional factors can also increase the risk of morbidity.This article focuses on the progress of research on pathogenic factors associated with TOF. Key words: Tetralogy of Fallot; Pathogenesis; Genetics; Environmental factor

A new method for constructing engineered corneal epithelium based on embryonic stem cells

Objective To construct engineered corneal epithelium from embryonic stem cells (ESCs) using Rock inhibitor combined with hypoxia-normoxia culture condition. Methods Human ESC line H1 was induced to differentiate into epithelial-like cells by addition of retinoic acid (RA) and bone morphogenetic protein 4(BMP4) in the differentiation medium under the adherent culture condition.The ESCs derived epithelial-like cells were expanded in the mixed medium of SHEM and KSFM with the mixture ratio of 1∶2 with or without Rock inhibitor Y27632.The H1 derived epithelial-like cells were seeded on the denuded ammonic membrane to construct engineered corneal epithelium under hypoxia, normoxia and hypoxia-normoxia culture conditions, respectively.The inducted effect of ESCs into epithelial-like cells, the expansion ability of the epithelial-like cells and the characteristics of the constructed engineered corneal epithelium were evaluated by morphological observation, real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) and immunofluorescence technology. Results Compared with the control group, the relative expressions of ESCs marker Oct4 mRNA, Notch signaling pathway related factors Notch1 and Jagged1 mRNA, and Wnt signaling pathways related factors c-myc and Cyclin D1 mRNA were significantly reduced, and the relative expressions of cutaneous ectoderm markers p63 and K18 mRNA were significantly increased at day 8 after induction in the induced group, with significant differences between them (t =14.63, 20.15, 93.50, 11.60, 19.30, 18.44, 22.63; all at P 0.05). The ESCs derived epithelium and constructed under hypoxia-normoxia culture condition showed more obvious stratification and tighter cell arrangement in comparison with those cells cultured in consistent hypoxia culture condition or normoxia culture condition.Epithelial markers Pan-CK and K18 as well as epithelial progenitor cell markers p63 and K14 expressed in the whole cell layers of the ESCs derived epithelium constructed under hypoxia-normoxia culture condition. Conclusions The addition of Y27632 enhances the proliferation ability of H1 derived epithelial cells and actives Notch signaling pathway and inhibits Wnt signaling pathway.The culture and construction in the expansion medium with Y27632 under the hypoxia-normoxia culture condition can promote the stratification of H1 derived engineered corneal epithelium. Key words: Embryonic stem cells; Engineered corneal epithlium; Rock inhibitor; Hypoxia-normoxia culture

Role of miR-214-5p in the migration and invasion of pancreatic cancer cells.

To analyze the role of miR-214-5p in proliferation and metastasis of pancreatic cancer (PC) cells, as well as its underlying mechanism. 30 pairs of PC tissues and adjacent normal tissues were collected in our Department. The expression level of miR-214-5p was detected by quantitative Real-time-polymerase chain reaction (qRT-PCR). Biological information analysis and luciferase report gene assay were used to verify potential target genes of miR-214-5p. Cell counting kit-8 (CCK-8) and transwell methods were applied to observe the interference of miR-214-5p on invasion and migration of PC cells. Western blot (WB) assay was applied to determine the expression changes of Jagged 1 (JAG1) and epithelial-mesenchymal transition (EMT)-related genes in PC cells. QRT-PCR results showed that the expression level of miR-214-5p is significantly down-regulated in PC tissues and cells. Bioinformatics software and luciferase report gene assay identified that JAG1 is a target gene of miR-214-5p. The negative correlation between protein expressions of miR-214-5p and JAG1 was assessed by Western Blot assay. Furthermore, miR-214-5p could suppress cell proliferation, invasion and migration, and it also blocked the EMT in PC cells in vitro. Meanwhile, JAG1 overexpression reversed the inhibitory effects of miR-214-5p on proliferation, invasion and migration of PC cells. Overexpressing miR-214-5p could significantly inhibit malignant behavior of PC cells through targeted regulation of JAG1. Thus, miR-214-5p might be a potential therapeutic target for treatment of PC.

MiR-199b exerts tumor suppressive functions in hepatocellular carcinoma by directly targeting JAG1.

To investigate the regulating roles of miR-199b in hepatocellular carcinoma (HCC). The expression of miR-199b of 45 human HCC tissues and the corresponding para-cancerous tissue samples were detected by quantitative Real-time-polymerase chain reaction (qRT-PCR). Western blot was employed to investigate the level of JAG1. Transwell assay was used to monitor the ability of cell migration and invasion. Cell proliferation was tested by CCK-8 assay and luciferase reporter assay was done to clarify whether JAG1 was a target of mir-199b. miR-199b expression level was decreased in 45 paired tumor tissues in contrast with the corresponding para-carcinoma tissues. The expression level of miR-199b was closely associated with TNM stage, tumor size, and 5-year overall survival. Transwell assay result showed that miR-199b inhibited HCC cell migration and invasion. Cell counting kit-8 (CCK-8) results demonstrated that miR-199b could suppress HCC cell proliferation. Luciferase reporter assay suggested that Jagged1 (JAG1) was a direct target of miR-199b in HCC cells. miR-199b could negatively regulate JAG1 expression by targeting JAG1. miR-199b exerted tumor suppressive functions in HCC by targeting JAG1, and it may be a potential target treatment for hepatocellular carcinoma.

Female Mice Have Higher Angiogenesis in Perigonadal Adipose Tissue Than Males in Response to High-Fat Diet.

Background: Impaired capillary growth (angiogenesis) in skeletal muscle and adipose tissue contributes to the development of metabolic disorders in obese males. This association remains unexplored in females, despite mounting evidence that endothelial cells have sex-specific transcriptional profiles. Therefore, herein we assessed whether males and females show distinct angiogenic capacities in response to diet-induced obesity.Methods: Age-matched male and female mice were fed normal chow or high-fat obesogenic diets for 16 weeks. At the end of diet period, systemic glucose disposal was assessed as well as insulin sensitivity of skeletal muscle and visceral adipose tissue. Capillary content and the expression of angiogenic regulators were also evaluated in these tissues.Results: When placed on a high-fat diet, female mice gained less weight than males and showed a metabolic phenotype similar to NC-fed mice, contrasting with the impaired whole-body glucose metabolism observed in high-fat-fed males. However, high-fat-feeding elevated serum lipid levels similarly in male and female mice. Although skeletal muscle of high-fat–fed female mice had higher insulin sensitivity than male counterparts, no sex difference was detected in muscle capillarization. Metabolic functions of perigonadal white adipose tissue (pgWAT) were retained in high-fat-fed females, as evidenced by smaller adipocytes with preserved insulin sensitivity, greater responsiveness to isoproterenol, higher expression of Adiponectin and a lower ratio of Leptin:Adiponectin mRNA. An enhanced browning phenotype was detected in HF-fed female adipocytes with upregulation of Ucp1 expression. PgWAT from high-fat-fed females also showed augmented capillary number and expression of endothelial cell markers, which was associated with elevated mRNA levels of pro-angiogenic mediators, including vascular endothelial growth factor A (Vegfa) and its receptor (Vegfr2), the Notch ligand Jagged-1 (Jag1) and Angiopoietin-2 (Angpt2).Conclusion: Taken together, our findings provide novel evidence that visceral adipose tissue of female mice display greater levels of pro-angiogenic factors and vascularity than males in response to high-fat diet. This phenotype is associated with preserved metabolic homeostasis at both tissue and systemic levels. Our study discloses that a thus-far-unappreciated sex-specific difference in the regulation of adipose angiogenesis may contribute to an individual’s susceptibility to developing adipose dysfunction and obesity-related metabolic disturbances.

JAG2 signaling induces differentiation of CD14+ monocytes into Langerhans cell histiocytosis-like cells.

Langerhans cell histiocytosis (LCH) is a MAPK pathway-driven disease characterized by the accumulation of CD1a+ langerin+ cells of unknown origin. We have previously reported that the Notch signaling pathway is active in LCH lesions and that the Notch ligand Jagged2 (JAG2) induces CD1a and langerin expression in monocytes in vitro. Here we show that Notch signaling induces monocytes to acquire an LCH gene signature and that Notch inhibition suppresses the LCH phenotype. In contrast, while also CD1c+ dendritic cells or IL-4-stimulated CD14+ monocytes acquire CD1a and langerin positivity in culture, their gene expression profiles and surface phenotypes are more different from primary LCH cells. We propose a model where CD14+ monocytes serve as LCH cell precursor and JAG2-mediated activation of the Notch signaling pathway initiates a differentiation of monocytes toward LCH cells in selected niches and thereby contributes to LCH pathogenesis.