A cochlear progenitor pool influences patterning of the mammalian sensory epithelium via MYBL2.

During embryonic development, Wnt signaling influences both proliferation and sensory formation in the cochlea. How this dual nature of Wnt signaling is coordinated is unknown. In this study, we define a novel role for a Wnt-regulated gene, Mybl2, which was already known to be important for proliferation, in determining the size and patterning of the sensory epithelium in the murine cochlea. Using a quantitative spatial analysis approach and analyzing Mybl2 loss-of-function, we show that Mybl2 promoted proliferation in the inner sulcus domain but limited the size of the sensory domain by influencing their adjoining boundary position via Jag1 regulation during development. Mybl2 loss-of-function simultaneously decreased proliferation in the inner sulcus and increased the size of the sensory domain, resulting in a wider sensory epithelium with ectopic inner hair cell formation during late embryonic stages. These data suggest that progenitor cells in the inner sulcus determine boundary formation and pattern the sensory epithelium via MYBL2.

Jagged-1+ skin Tregs modulate cutaneous wound healing.

Skin-resident regulatory T cells (Tregs) play an irreplaceable role in orchestrating cutaneous immune homeostasis and repair, including the promotion of hair regeneration via the Notch signaling ligand Jagged-1 (Jag1). While skin Tregs are indispensable for facilitating tissue repair post-wounding, it remains unknown if Jag1-expressing skin Tregs impact wound healing. Using a tamoxifen inducible Foxp3creERT2Jag1fl/fl model, we show that loss of functional Jag1 in Tregs significantly delays the rate of full-thickness wound closure. Unlike in hair regeneration, skin Tregs do not utilize Jag1 to impact epithelial stem cells during wound healing. Instead, mice with Treg-specific Jag1 ablation exhibit a significant reduction in Ly6G + neutrophil accumulation at the wound site. However, during both homeostasis and wound healing, the loss of Jag1 in Tregs does not impact the overall abundance or activation profile of immune cell targets in the skin, such as CD4+ and CD8+ T cells, or pro-inflammatory macrophages. This collectively suggests that skin Tregs may utilize Jag1-Notch signalling to co-ordinate innate cell recruitment under conditions of injury but not homeostasis. Overall, our study demonstrates the importance of Jag1 expression in Tregs to facilitate adequate wound repair in the skin.

Notch signaling mediated repressive effects of resveratrol in inducing caspasedependent apoptosis in MCF-7 breast cancer cells.

Resveratrol, a potent anticancer bioactive compound, has been shown to trigger apoptosis in numerous cancer cells. Although Notch signaling promotes breast cancer apoptosis, it is unclear whether resveratrol induces apoptosis in MCF-7 cells via influencing the Notch pathway. This study aimed to evaluate the effect of resveratrol on modulating Notch signaling targets and provide critical information for employing resveratrol in breast cancer therapy. Thus, in this study, we have deciphered the effect of resveratrol against three potent genes (Notch1, Jagged1, and DLL4) of the notch signaling pathway. For mechanistic studies, in silico, and in vitro analysis was executed to investigate the apoptotic-inducing potential of resveratrol against three selected oncogenes involved in the progression of breast cancer. Docking analysis revealed the inhibitory potential of resveratrol against all three selected targets of the Notch pathway (Notch1: -5.0; Jagged-1: -5.9; DLL4: -5.8). In vitro, findings further displayed a significant reduction in cell viability in resveratrol-treated MCF-7 cancer cells, which were concomitantly related to the downregulation of Notch-1, Jagged-1, and DLL4. Moreover, the antiproliferative efficacy of resveratrol was correlated with apoptosis and modulation in the expression of Bax, Bcl-2, cyclin D1, CDK4, p21, and caspase-3 activation. Taken together, these experimental findings suggested that apoptotic inducing potential of resveratrol was mediated through a novel mechanism involving suppression of the Notch signaling pathway.

APOE-NOTCH axis governs elastogenesis during human cardiac valve remodeling.

Valve remodeling is a process involving extracellular matrix organization and elongation of valve leaflets. Here, through single-cell RNA sequencing of human fetal valves, we identified an elastin-producing valve interstitial cell (VIC) subtype (apolipoprotein E (APOE)+, elastin-VICs) spatially located underneath valve endothelial cells (VECs) sensing unidirectional flow. APOE knockdown in fetal VICs resulted in profound elastogenesis defects. In valves with pulmonary stenosis (PS), we observed elastin fragmentation and decreased expression of APOE along with other genes regulating elastogenesis. Cell-cell interaction analysis revealed that jagged 1 (JAG1) from unidirectional VECs activates elastogenesis in elastin-VICs through NOTCH2. Similar observations were made in VICs cocultured with VECs under unidirectional flow. Notably, a drastic reduction of JAG1-NOTCH2 was also observed in PS valves. Lastly, we found that APOE controls JAG1-induced NOTCH activation and elastogenesis in VICs through the extracellular signal-regulated kinase pathway. Our study suggests important roles of both APOE and NOTCH in regulating elastogenesis during human valve remodeling.

Generation of human iPSC-derived 3D bile duct within liver organoid by incorporating human iPSC-derived blood vessel.

In fetal development, tissue interaction such as the interplay between blood vessel (BV) and epithelial tissue is crucial for organogenesis. Here we recapitulate the spatial arrangement between liver epithelial tissue and the portal vein to observe the formation of intrahepatic bile ducts (BDs) from human induced pluripotent stem cells (hiPSC). We co-culture hiPSC-liver progenitors on the artificial BV consisting of immature smooth muscle cells and endothelial cells, both derived from hiPSCs. After 3 weeks, liver progenitors within hiPSC-BV-incorporated liver organoids (BVLO) differentiate to cholangiocytes and acquire epithelial characteristics, including intercellular junctions, microvilli on the apical membrane, and secretory functions. Furthermore, liver surface transplanted-BVLO temporarily attenuates cholestatic injury symptoms. Single cell RNA sequence analysis suggests that BD interact with the BV in BVLO through TGFß and Notch pathways. Knocking out JAG1 in hiPSC-BV significantly attenuates bile duct formation, highlighting BVLO potential as a model for Alagille syndrome, a congenital biliary disease. Overall, we develop a novel 3D co-culture method that successfully establishes functional human BDs by emulating liver epithelial-BV interaction.

Clinical, pathological and genetic characteristics of 17 unrelated children with Alagille Syndrome.

BACKGROUND: Alagille syndrome (ALGS) is a multisystem genetic disorder frequently characterized by hepatic manifestations. This study analyzed the clinical, pathological, and molecular genetic features of ALGS to improve the efficiency of clinical diagnosis. METHODS: We retrospectively analyzed the clinical manifestations, pathological examination findings, and genetic testing results of 17 children diagnosed with ALGS based on the revised criteria and hospitalized at our center from January 2012 to January 2022. RESULTS: The clinical manifestations are as follows: Cholestasis (16/17, 94%), characteristic facies (15/17, 88%), heart disease (12/16, 75%), butterfly vertebrae (12/17, 71%) and posterior embryotoxon (7/12, 58%). Among the 15 patients who underwent liver pathology examination, 13 (87%) were found to have varying degrees of bile duct paucity. Genetic testing was performed on 15 children, and pathogenic variants of the jagged canonical Notch ligand 1 (JAG1) gene were identified in 13 individuals, including 4 novel variants. No pathogenic variant in the notch homolog 2 (NOTCH2) gene were identified, and 2 children exhibited none of the aforementioned gene pathogenic variants. The median follow-up duration was 7 years. Of the remaining 15 patients (excluding 2 lost to follow-up), 11 remained stable, 4 deteriorated, and no patient died during the follow-up period. CONCLUSIONS: Among children diagnosed with ALGS, cholestasis stands as the most common feature. To minimize the risk of misdiagnosis, genetic testing should be performed on children exhibiting cholestasis, followed by the application of the revised diagnostic criteria for ALGS. While pharmacological therapy has shown effectiveness for ALGS patients, liver transplantation may be considered in instances of severe pruritus.

The IGF2BP3/Notch/Jag1 pathway: A key regulator of hepatic stellate cell ferroptosis in liver fibrosis.

INTRODUCTION: Liver fibrosis is primarily driven by the activation of hepatic stellate cells (HSCs), which involves various epigenetic modifications. OBJECTIVES: N6-methyladenosine (m6A), the most prevalent RNA modification in eukaryotic cells, influences numerous physiological and pathological processes. Nevertheless, the role of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), a reader gene mediating m6A modifications, in liver fibrosis remains unclear. METHODS AND RESULTS: This study demonstrated that IGF2BP3 knockout reduces liver fibrosis by promoting HSC ferroptosis (FPT) and inactivating HSCs. Multi-omics analysis revealed that HSC-specific IGF2BP3 knockout decreased m6A content in Jagged1 (Jag1), a key component of the Notch signalling pathway. Furthermore, IGF2BP3 deficiency significantly reduced the expression of hairy and enhancer of split-1 (Hes1), a transcription factor in the Notch/Jag1 signalling pathway, with mRNA levels declining to 35%-62% and protein levels to 28%-35%. Additionally, it suppressed glutathione peroxidase 4 (GPX4) (decreased to approximately 31%-38%), a negative regulator of FPT, thereby facilitating HSC FPT progression and reducing profibrotic gene expression. CONCLUSION: These findings uncover a novel IGF2BP3/Notch/Jag1 signalling pathway involving HSC FPT, suggesting promising targets for ameliorating liver fibrosis. KEY POINTS/HIGHLIGHTS: IGF2BP3 deficiency inactivates Jag1 signalling. IGF2BP3 deficiency-mediated m6A modifications promote HSC ferroptosis. IGF2BP3 inhibition facilitates ferroptosis in HSCs via the Hes1/GPX4 axis. IGF2BP3 deficiency inactivates Jag1/Notch1/3/Hes1 signalling pathway inactivation, leading to the decrease in GPX4, which contributes to HSC ferroptosis.

SOX2, JAGGED1, ß-Catenin, and Vitamin D Receptor Expression Patterns during Early Development and Innervation of the Human Inner Ear.

Sensorineural hearing loss can be caused by lesions to the inner ear during development. Understanding the events and signaling pathways that drive inner ear formation is crucial for determining the possible causes of congenital hearing loss. We have analyzed the innervation and expression of SOX2, JAGGED1, ß-catenin (CTNNB1), and vitamin D receptor (VDR) in the inner ears of human conceptuses aged 5 to 10 weeks after fertilization (W) using immunohistochemistry. The prosensory domains of the human inner ear displayed SOX2 and JAGGED1 expression throughout the analyzed period, with SOX2 expression being more extensive in all the analyzed timepoints. Innervation of vestibular prosensory domains was present at 6 W and extensive at 10 W, while nerve fibers reached the base of the cochlear prosensory domain at 7-8 W. CTNNB1 and VDR expression was mostly membranous and present during all analyzed timepoints in the inner ear, being the strongest in the non-sensory epithelium. Their expression was stronger in the vestibular region compared to the cochlear duct. CTNNB1 and VDR expression displayed opposite expression trends during the analyzed period, but additional studies are needed to elucidate whether they interact during inner ear development.

Overexpression of miR-199b-5p in Colony Forming Unit-Hill's Colonies Positively Mediates the Inflammatory Response in Subclinical Cardiovascular Disease Model: Metformin Therapy Attenuates Its Expression.

Well-controlled type 1 diabetes (T1DM) is characterized by inflammation and endothelial dysfunction, thus constituting a suitable model of subclinical cardiovascular disease (CVD). miR-199b-5p overexpression in murine CVD has shown proatherosclerotic effects. We hypothesized that miR-199b-5p would be overexpressed in subclinical CVD yet downregulated following metformin therapy. Inflammatory and vascular markers were measured in 29 individuals with T1DM and 20 matched healthy controls (HCs). miR-199b-5p expression in CFU-Hill's colonies was analyzed from each study group, and correlations with inflammatory/vascular health indices were evaluated. Significant upregulation of miR-199b-5p was observed in T1DM, which was significantly downregulated by metformin. miR-199b-5p correlated positively with vascular endothelial growth factor-D and c-reactive protein (CRP: nonsignificant). ROC analysis determined miR-199b-5p to define subclinical CVD by discriminating between HCs and T1DM individuals. ROC analyses of HbA1c and CRP showed that the upregulation of miR-199b-5p in T1DM individuals defined subclinical CVD at HbA1c > 44.25 mmol and CRP > 4.35 × 106 pg/mL. Ingenuity pathway analysis predicted miR-199b-5p to inhibit the target genes SIRT1, ETS1, and JAG1. Metformin was predicted to downregulate miR-199b-5p via NFATC2 and STAT3 and reverse its downstream effects. This study validated the antiangiogenic properties of miR-199b-5p and substantiated miR-199b-5p overexpression as a biomarker of subclinical CVD. The downregulation of miR-199b-5p by metformin confirmed its cardio-protective effect.

Association of Very Rare NOTCH2 Variants with Clinical Features of Alagille Syndrome.

BACKGROUND: Alagille syndrome (ALGS) is a rare autosomal dominant genetic disease caused by pathogenic variants in two genes: Jagged Canonical Notch Ligand 1 (JAG1) and Notch Receptor 2 (NOTCH2). It is characterized by phenotypic variability and incomplete penetrance with multiorgan clinical signs. METHODS: Using Next Generation Sequencing (NGS), we analyzed a panel of liver-disease-related genes in a population of 230 patients with cholestasis and hepatopathies. For the rare variants, bioinformatics predictions and pathogenicity classification were performed. RESULTS: We identified eleven rare NOTCH2 variants in 10 patients, two variants being present in the same patient. Ten variants had never been described before in the literature. It was possible to classify only two null variants as pathogenic, whereas the most of variants were missense (8 out of 11) and were classified as uncertain significance variants (USVs). Among patients with ALGS suspicion, two carried null variants, two carried variants predicted to be pathogenic by bioinformatics, one carried a synonymous variant and variants in glycosylation-related genes, and two carried variants predicted as benign in the PEST domain. CONCLUSIONS: Our results increased the knowledge about NOTCH2 variants and the related phenotype, allowing us to improve the genetic diagnosis of ALGS.

Functional characterization of 2,832 JAG1 variants supports reclassification for Alagille syndrome and improves guidance for clinical variant interpretation.

Pathogenic variants in the JAG1 gene are a primary cause of the multi-system disorder Alagille syndrome. Although variant detection rates are high for this disease, there is uncertainty associated with the classification of missense variants that leads to reduced diagnostic yield. Consequently, up to 85% of reported JAG1 missense variants have uncertain or conflicting classifications. We generated a library of 2,832 JAG1 nucleotide variants within exons 1-7, a region with a high number of reported missense variants, and designed a high-throughput assay to measure JAG1 membrane expression, a requirement for normal function. After calibration using a set of 175 known or predicted pathogenic and benign variants included within the variant library, 486 variants were characterized as functionally abnormal (n = 277 abnormal and n = 209 likely abnormal), of which 439 (90.3%) were missense. We identified divergent membrane expression occurring at specific residues, indicating that loss of the wild-type residue itself does not drive pathogenicity, a finding supported by structural modeling data and with broad implications for clinical variant classification both for Alagille syndrome and globally across other disease genes. Of 144 uncertain variants reported in patients undergoing clinical or research testing, 27 had functionally abnormal membrane expression, and inclusion of our data resulted in the reclassification of 26 to likely pathogenic. Functional evidence augments the classification of genomic variants, reducing uncertainty and improving diagnostics. Inclusion of this repository of functional evidence during JAG1 variant reclassification will significantly affect resolution of variant pathogenicity, making a critical impact on the molecular diagnosis of Alagille syndrome.

Cancer-wide in silico analyses using differentially expressed genes demonstrate the functions and clinical relevance of JAG, DLL, and NOTCH.

Notch ligands [jagged (JAG) and, delta-like (DLL) families] and receptors [NOTCH family] are key regulators of Notch signaling. NOTCH signaling contributes to vascular development, tissue homeostasis, angiogenesis, and cancer progression. To elucidate the universal functions of the JAG, DLL, and NOTCH families and their connections with various biological functions, we examined 15 types of cancer using The Cancer Genome Atlas clinical database. We selected the differentially expressed genes (DEGs), which were positively correlated to the JAG, DLL, and NOTCH families in each cancer. We selected positive and negative hallmark signatures across cancer types. These indicated biological features associated with angiogenesis, hypoxia, KRAS signaling, cell cycle, and MYC targets by gene ontology and gene set enrichment analyses using DEGs. Furthermore, we analyzed single-cell RNA sequencing data to examine the expression of JAG, DLL, and NOTCH families and enrichment of hallmark signatures. Positive signatures identified using DEGs, such as KRAS signaling and hypoxia, were enriched in clusters with high expression of JAG, DLL, and NOTCH families. We subsequently validated the correlation between the JAG, DLL, and NOTCH families and clinical stages, including treatment response, metastasis, and recurrence. In addition, we performed survival analysis to identify hallmark signatures that critically affect patient survival when combining the expression of JAG, DLL, and NOTCH families. By combining the DEG enrichment and hallmark signature enrichment in survival analysis, we suggested unexplored regulatory functions and synergistic effects causing synthetic lethality. Taken together, our observations demonstrate the functions of JAG, DLL, and NOTCH families in cancer malignancy and provide insights into their molecular regulatory mechanisms.

Targeting the ZMIZ1-Notch1 signaling axis for the treatment of tongue squamous cell carcinoma.

Zinc finger MIZ-type containing 1 (ZMIZ1) is a transcriptional coactivator related to the protein inhibitors of activated STATs (PIAS) family. Mounting evidence suggests that ZMIZ1 plays a crucial role in the occurrence and development of cancers. The function of ZMIZ1 in tongue squamous cell carcinoma (TSCC) and the mechanisms underpinning its role in this disease have not been fully clarified. We performed qualitative ZMIZ1 protein expression analyses using immunohistochemistry in 20 patient-derived, paraffin-embedded TSCC tissue sections. We used RNAi to knock down ZMIZ1 expression in the CAL-27 TSCC cell line and quantified the impact of ZMIZ1 knock down on proliferation, migration and apoptosis via CCK-8, scratch assay and flow cytometry, respectively. We used qRT-PCR and western blotting to investigate the role of ZMIZ1 in this cell line. Finally, we established a model of lung metastasis in nude mice to replicate the in vitro results. ZMIZ1 protein was significantly more abundant in TSCC case tissue samples. ZMIZ1 knockdown reduced the invasion and metastases of TSCC tumor cells and promoted apoptosis. ZMIZ1 knockdown was associated with the down-regulation of Notch signaling pathway related factors Jagged1 and Notch1, and invasion and metastasis related factors MKP-1, SSBP2 and MMP7 in vitro and in vivo, at the mRNA level. In vitro and in vivo data suggest that knock down of ZMIZ1 may inhibit TSCC invasion and metastasis by modulating Notch signaling. ZMIZ1 inhibition may therefore represent a new therapeutic target for TSCC.

CircVCAN promotes glioma progression through the miR-488-3p/MEF2C-JAGGED1 axis.

Gliomas are the most prevalent primary malignant brain tumors worldwide. Growing evidences indicate that circular RNAs (circRNAs) play an important role in the regulation of biological behavior of tumors. We aimed to investigate the role and mechanism of circVCAN in glioma. RNase R treatment was utilized to assess the cyclic properties of circVCAN. CircVCAN, miR-488-3p, and myocyte enhancer factor 2C (MEF2C) levels in glioma tissues and cells were detected by reverse transcription real-time polymerase chain reaction (RT-qPCR), and the localization of them in glioma cells was determined with fluorescence in situ hybridization. Furthermore, a variety of biologically functional assessments were used to validate the role of circVCAN in glioma. The regulatory mechanisms of circVCAN, miR-488-3p, and MEF2C were further confirmed by double luciferase reporter gene assay, RNA immunoprecipitation and RNA pull-down assay, and the binding of MEF2C to JAGGED1 was revealed by chromatin immunoprecipitation. Additionally, a xenograft tumor model was constructed to demonstrate the effect of circVCAN on tumor growth in vivo. Our results indicated that circVCAN was more stable than its linear RNA and was significantly upregulated in gliomas. CircVCAN overexpression stimulated glioma cells to proliferate and metastasize, but circVCAN silencing exerted the opposite effect. Meanwhile, silencing circVCAN inhibited tumor growth in vivo. Moreover, we found that circVCAN interacted with miR-488-3p to regulate MEF2C expression, and miR-488-3p inhibition or MEF2C overexpression reversed the inhibitory effect on malignant bio-behaviors mediated by circVCAN knockdown in glioma cells. MEF2C promoted the transcription of JAGGED1, and circVCAN knockdown reduced the binding between MEF2C and JAGGED1. Collectively, circVCAN is a carcinogenic circRNA in glioma, and the circVCAN/miR-488-3p/MEF2C-JAGGED1 axis could serve as a potential target for the management of glioma.

C-terminally phosphorylated p27 activates self-renewal driver genes to program cancer stem cell expansion, mammary hyperplasia and cancer.

In many cancers, a stem-like cell subpopulation mediates tumor initiation, dissemination and drug resistance. Here, we report that cancer stem cell (CSC) abundance is transcriptionally regulated by C-terminally phosphorylated p27 (p27pT157pT198). Mechanistically, this arises through p27 co-recruitment with STAT3/CBP to gene regulators of CSC self-renewal including MYC, the Notch ligand JAG1, and ANGPTL4. p27pTpT/STAT3 also recruits a SIN3A/HDAC1 complex to co-repress the Pyk2 inhibitor, PTPN12. Pyk2, in turn, activates STAT3, creating a feed-forward loop increasing stem-like properties in vitro and tumor-initiating stem cells in vivo. The p27-activated gene profile is over-represented in STAT3 activated human breast cancers. Furthermore, mammary transgenic expression of phosphomimetic, cyclin-CDK-binding defective p27 (p27CK-DD) increases mammary duct branching morphogenesis, yielding hyperplasia and microinvasive cancers that can metastasize to liver, further supporting a role for p27pTpT in CSC expansion. Thus, p27pTpT interacts with STAT3, driving transcriptional programs governing stem cell expansion or maintenance in normal and cancer tissues.

Jag1/2 maintain esophageal homeostasis and suppress foregut tumorigenesis by restricting the basal progenitor cell pool.

Basal progenitor cells are crucial for maintaining foregut (the esophagus and forestomach) homeostasis. When their function is dysregulated, it can promote inflammation and tumorigenesis. However, the mechanisms underlying these processes remain largely unclear. Here, we employ genetic mouse models to reveal that Jag1/2 regulate esophageal homeostasis and foregut tumorigenesis by modulating the function of basal progenitor cells. Deletion of Jag1/2 in mice disrupts esophageal and forestomach epithelial homeostasis. Mechanistically, Jag1/2 deficiency impairs activation of Notch signaling, leading to reduced squamous epithelial differentiation and expansion of basal progenitor cells. Moreover, Jag1/2 deficiency exacerbates the deoxycholic acid (DCA)-induced squamous epithelial injury and accelerates the initiation of squamous cell carcinoma (SCC) in the forestomach. Importantly, expression levels of JAG1/2 are lower in the early stages of human esophageal squamous cell carcinoma (ESCC) carcinogenesis. Collectively, our study demonstrates that Jag1/2 are important for maintaining esophageal and forestomach homeostasis and the onset of foregut SCC.

Endothelial to mesenchymal Notch signaling regulates skeletal repair.

We present a transcriptomic analysis that provides a better understanding of regulatory mechanisms within the healthy and injured periosteum. The focus of this work is on characterizing early events controlling bone healing during formation of periosteal callus on day 3 after fracture. Building on our previous findings showing that induced Notch1 signaling in osteoprogenitors leads to better healing, we compared samples in which the Notch 1 intracellular domain is overexpressed by periosteal stem/progenitor cells, with control intact and fractured periosteum. Molecular mechanisms and changes in skeletal stem/progenitor cells (SSPCs) and other cell populations within the callus, including hematopoietic lineages, were determined. Notably, Notch ligands were differentially expressed in endothelial and mesenchymal populations, with Dll4 restricted to endothelial cells, whereas Jag1 was expressed by mesenchymal populations. Targeted deletion of Dll4 in endothelial cells using Cdh5CreER resulted in negative effects on early fracture healing, while deletion in SSPCs using α-smooth muscle actin-CreER did not impact bone healing. Translating these observations into a clinically relevant model of bone healing revealed the beneficial effects of delivering Notch ligands alongside the osteogenic inducer, BMP2. These findings provide insights into the regulatory mechanisms within the healthy and injured periosteum, paving the way for novel translational approaches to bone healing.

Jagged1-Notch1 Signaling Pathway Induces M1 Microglia to Disrupt the Barrier Function of Retinal Microvascular Endothelial Cells.

PURPOSE: Microglia-related inflammation is closely linked to the pathogenesis of retinal diseases. The primary objective of this research was to investigate the impact and mechanism of M1 phenotype microglia on the barrier function of retina microvascular endothelial cells. METHODS: Quantitative polymerase chain reactions and western blot techniques were utilized to analysis the mRNA and protein expressions of M1 and M2 markers of human microglial clone 3 cell line (HMC3), as well as the levels of Notch ligands and receptors under the intervention of lipopolysaccharide (LPS) or interleukin (IL)-4. ELISA was utilized to detect the pro-inflammatory and anti-inflammatory cytokines from HMC3 cells. The cellular tight junction and apoptosis of human retinal microvascular endothelial cells (HRMECs) were assessed by western blot and fluorescein isothiocyanate-dextran permeability assay. The inhibitors of Notch1 and RNA interference (RNAi) targeting Jagged1 were used to assess their contribution to the barrier function of vascular endothelial cells. RESULTS: Inducible nitric oxide synthase (iNOS) and IL-1ß were considerably elevated in LPS-treated HMC3, while CD206 and Arg-1 markedly elevated under IL-4 stimulation. The conditioned medium derived from LPS-treated HMC3 cells promoted permeability, diminished the expression of zonula occludens-1 and Occludin, and elevated the expression of Cleaved caspase-3 in HRMECs. RNAi targeting Jagged1 or Notch1 inhibitor could block M1 HMC3 polarization and maintain barrier function of HRMECs. CONCLUSION: Our findings suggest that Jagged1-Notch1 signaling pathway induces M1 microglial cells to disrupt the barrier function of HRMECs, which may lead to retinal diseases.

[Alagille syndrome associated to JAG1 gene deletion. An unusual etiology]. / Síndrome de Alagille por deleción del gen JAG1. Una causa poco frecuente.

Alagille syndrome (ALGS) is an autosomal dominant, multisystem disorder that typically presents with cholestasis, cardiac, ocular, skeletal, vascular and renal abnormalities, and distinct facial features. Most cases are due to variants in the JAG1 gene, with only a small percentage involving a complete gene deletion. OBJECTIVE: to contribute to the phenotype delineation and interpretation of a microdeletion not previously described in the literature on chromosome 20. CLINICAL CASE: A 4-month-old female patient was diagnosed with a heart murmur. An echocardiogram revealed pulmonary artery stenosis, which, combined with a prominent forehead observed on physical examination, determined her referral to clinical genetics. Because ALGS was suspected, complementary studies were performed, revealing butterfly vertebras and a genetic panel identified a pathogenic heterozygous deletion, encompassing the entire coding sequence of the JAG1 gene. To rule out a more extensive deletion, a chromosome microarray was performed, confirming a pathogenic microdeletion on chromosome 20 of 378 kb (arr[GRCh37] 20p12.2(10414643_10792802)x1). CONCLUSIONS: A targeted sequencing panel followed by confirmation with a chromosome microarray allowed the identification and delineation of a pathogenic microdeletion not previously reported in the literature, including the complete JAG1 gene in a Chilean patient whose phenotype is consistent with ALGS.

Generation of an Alagille Syndrome (ALGS) patient-derived induced pluripotent stem cell line (TRNDi036-A) carrying a heterozygous mutation (p.Cys693*) in the JAG1 gene.

Alagille syndrome (ALGS) is an autosomal dominant, multisystemic disorder due to haploinsufficiency in JAG1 or less frequently, mutations in NOTCH2. The disease has been difficult to diagnose and treat due to variable expression. The generation of this iPSC line (TRNDi036-A) carrying a heterozygous mutation (p.Cys693*) in the JAG1 gene provides a means of studying the disease and developing novel therapeutics towards patient treatment.