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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, β-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. [ABSTRACT FROM AUTHOR]

AbstractPurposeMethodsResultsConclusionMicroglia-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.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.Inducible nitric oxide synthase (iNOS) and IL-1β 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.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. [ABSTRACT FROM AUTHOR]

Abstract Aims To explore the role and mechanism of Notch signaling and ERK1/2 pathway in the inhibitory effect of sacubitril/valsartan on the proliferation of vascular smooth muscle cells (VSMCs). Main methods Human aortic vascular smooth muscle cells (HA-VSMCs) were cultured in vitro. The proliferating VSMCs were divided into three groups as control group, Ang II group and Ang II + sacubitril/valsartan group. Cell proliferation and migration were detected by CCK8 and scratch test respectively. The mRNA and protein expression of PCNA, MMP-9, Notch1 and Jagged-1 were detected by qRT-PCR and Western blot respectively. The p-ERK1/2 expression was detected by Western blot. Key findings Compared with the control group, proliferation and migration of VSMCs and the expression of PCNA, MMP-9, Notch1, Jagged-1 and p-ERK1/2 was increased in Ang II group. Sacubitril/valsartan significantly reduced the proliferation and migration. Additionally, pretreatment with sacubitril/valsartan reduced the PCNA, MMP-9, Notch1, Jagged-1 and p-ERK1/2 expression.

Purpose: To investigate the suppressive influence of mir-186 on multiplication and growth of multiple myeloma (MM), as well as the processes involved. Methods: The U266 cells were divided into control, mir-186 overexpression, and inhibition groups. The latter two were transfected with mir-186 agent and antagonist, respectively. Cell viability, colony formation potential, cell cycle ratio, and Jagged-1 mRNA and protein levels were measured using various assays. Results: Cell growth increased over time in all groups. However, mir-186 overexpression cells showed significantly decreased growth and colony formation capacity, relative to control, while the mir-186 inhibition cells showed significantly higher growth and colony formation capacity. The study revealed a higher proportion of G0/G1 stage cells and lower proportion of S-phase cells in mir-186 overexpression cells than in control cells. The opposite effect was seen in mir-186 inhibition cells. Jagged-1 protein and mRNA levels were significantly lower in mir-186 overexpression cells and higher in mir-186 inhibition cells than in control group. The Jagged-1 knockout group showed significantly higher Jagged-1 mRNA and protein levels than both the control and mir-186 overexpression groups (p < 0.05). Conclusion: When overexpressed, mir-186 inhibits the growth of multiple myeloma cells by inhibiting cell colony-formation capacity and by regulating cell cycle through mir-186-induced regulation of the expression of Jagged-1. there is need for more research to confirm the clinical benefits of therapies based on these findings. [ABSTRACT FROM AUTHOR]

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, β-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.

Objective: Notch signaling, re-activated in β cells from obese mice and causal to β cell dysfunction, is determined in part by transmembrane ligand availability in a neighboring cell. We hypothesized that β cell expression of Jagged1 determines the maladaptive Notch response and resultant insulin secretory defects in obese mice. Methods: We assessed expression of Notch pathway components in high-fat diet-fed (HFD) or leptin receptor-deficient (db/db) mice, and performed single-cell RNA sequencing (scRNA-Seq) in islets from patients with and without type 2 diabetes (T2D). We generated and performed glucose tolerance testing in inducible, β cell-specific Jagged1 gain-of- and loss-of-function mice. We also tested effects of monoclonal neutralizing antibodies to Jagged1 in glucose-stimulated insulin secretion (GSIS) assays in isolated islets. Results: Jag1 was the only Notch ligand that tracked with increased Notch activity in HFD-fed and db/db mice, as well as in metabolically-inflexible β cells enriched in patients with T2D. Neutralizing antibodies to block Jagged1 in islets isolated from HFD-fed and db/db mice potentiated GSIS ex vivo. To demonstrate if β cell Jagged1 is sufficient to cause glucose tolerance in vivo, we generated inducible β cell-specific Jag1 transgenic (β-Jag1TG) and loss-of-function (iβ-Jag1KO) mice. While forced Jagged1 impaired glucose intolerance due to reduced GSIS, loss of β cell Jagged1 did not protect against HFD-induced insulin secretory defects. Conclusions: Jagged1 is increased in islets from obese mice and in patients with T2D, and neutralizing Jagged1 antibodies lead to improved GSIS, suggesting that inhibition of Jagged1-Notch signaling may have therapeutic benefit. However, genetic loss-of-function experiments suggest that β cells are not a likely source of the Jagged1 signal.

During cellular senescence, irreversible cell cycle arrest is accompanied by morphological and genetic alterations. MicroRNAs (miRNAs) play a critical role in regulating senescence by modulating the abundance of crucial senescence regulatory proteins. Therefore, to identify novel senescence-associated miRNAs, we analyzed differentially expressed miRNAs in microvascular endothelial cells (MVEC). Among the 80 differentially expressed miRNAs in replicative senescent MVECs, 16 miRNAs of unknown gene ontology were used in the senescence-associated β-galactosidase assay. Thus, we identified miR-214-5p as having high senescence-inducing activity, inhibiting the proliferation and angiogenesis activity of MVECs. To reveal the senescence-regulating mechanism of miR-214-5p, we searched for target genes through sequence- and literature-based analysis. Molecular manipulation of miR-214-5p demonstrated that miR-214-5p regulated the expression and function of Jagged 1 (JAG1) in senescent MVECs. Silencing JAG1 or downstream genes of JAG1-Notch signaling, accelerated the senescence of MVECs. Additionally, ectopic overexpression of JAG1 reversed the senescence-inducing activity of miR-214-5p. In conclusion, we identified miR-214-5p as a senescence-associated miRNA. Targeting miR-214-5p may be a potential strategy to delay vascular aging and overcome the detrimental effects of senescence and age-related diseases.

Notch signaling plays important roles in the regulation of immune cell functioning during the inflammatory response. Activation of the innate immune signaling receptor NLRP3 promotes inflammation in injured tissue. However, it remains unknown whether Jagged1 (JAG1)-mediated myeloid Notch1 signaling regulates NLRP3 function in acute liver injury. Here, we report that myeloid Notch1 signaling regulates the NLRP3-driven inflammatory response in ischemia/reperfusion (IR)-induced liver injury. In a mouse model of liver IR injury, Notch1-proficient (Notch1FL/FL) mice receiving recombinant JAG1 showed a reduction in IR-induced liver injury and increased Notch intracellular domain (NICD) and heat shock transcription factor 1 (HSF1) expression, whereas myeloid-specific Notch1 knockout (Notch1M-KO) aggravated hepatocellular damage even with concomitant JAG1 treatment. Compared to JAG1-treated Notch1FL/FL controls, Notch1M-KO mice showed diminished HSF1 and Snail activity but augmented NLRP3/caspase-1 activity in ischemic liver. The disruption of HSF1 reduced Snail activation and enhanced NLRP3 activation, while the adoptive transfer of HSF1-expressing macrophages to Notch1M-KO mice augmented Snail activation and mitigated IR-triggered liver inflammation. Moreover, the knockdown of Snail in JAG1-treated Notch1FL/FL livers worsened hepatocellular functioning, reduced TRX1 expression and increased TXNIP/NLRP3 expression. Ablation of myeloid Notch1 or Snail increased ASK1 activation and hepatocellular apoptosis, whereas the activation of Snail increased TRX1 expression and reduced TXNIP, NLRP3/caspase-1, and ROS production. Our findings demonstrated that JAG1-mediated myeloid Notch1 signaling promotes HSF1 and Snail activation, which in turn inhibits NLRP3 function and hepatocellular apoptosis leading to the alleviation of IR-induced liver injury. Hence, the Notch1/HSF1/Snail signaling axis represents a novel regulator of and a potential therapeutic target for liver inflammatory injury.

JAG1 is a ligand that activates the NOTCH signaling pathway which plays a crucial role in determining cell fate behavior through cell-to-cell signaling. JAG1-NOTCH signaling is required for mesenchymal stem cell (MSC) differentiation into cardiomyocytes and cranial neural crest (CNC) cells differentiation into osteoblasts, making it a regenerative candidate for clinical therapy to treat craniofacial bone loss and myocardial infarction. However, delivery of soluble JAG1 has been found to inhibit NOTCH signaling due to the requirement of JAG1 presentation in a bound form. For JAG1-NOTCH signaling to occur, JAG1 must be immobilized within a scaffold and the correct orientation between the NOTCH receptor and JAG1 must be achieved. The lack of clinically translatable JAG1 delivery methods has driven the exploration of alternative immobilization approaches. This review discusses the role of JAG1 in disease, the clinical role of JAG1 as a treatment, and summarizes current approaches for JAG1 delivery. An in-depth review was conducted on literature that used both in vivo and in vitro delivery models and observed the canonical versus non-canonical NOTCH pathway activated by JAG1. Studies were then compared and evaluated based on delivery success, functional outcomes, and translatability. Delivering JAG1 to harness its ability to control cell fate has the potential to serve as a therapeutic for many diseases.

Purpose: To determine the effect of triptolide on rheumatoid arthritis, and the underlying mechanism of action. Methods: Sixty Sprague Dawley (SD) rats were assigned to blank control, model, methotrexate, and triptolide groups (n = 15/group). A rat model of rheumatoid arthritis was established. Each group was given the corresponding treatment. Toe edema and arthritis index of rats were assessed in all the groups. Levels of IL-2, IFN-γ, TNF-α, IL-6, IL-8, and IL-10 were evaluated by enzyme-linked immunosorbent assay (ELISA). Results: Serum levels of IL-2, IFN-γ, TNF-α, IL-6 and IL-8 were significantly higher in model rats than in blank control, while IL-10 level was significantly lower (p < 0.05). In triptolide group, serum IL-2, IFN-γ, TNF-α, IL-6 and IL-8 were significantly lower than the corresponding levels in model group, but IL-10 was significantly higher. The relative mRNA and protein concentrations of Notch1 and Jagged1 were significantly higher in model rats than in blank control rats, but significantly lower, relative to model rats. Conclusion: Tripterygium wilfordii exerts a good therapeutic effect in the treatment of rheumatoid arthritis in rats, most likely by inhibiting the activation of Notch1/Jagged1 signal pathway. Thus, it regulates inflammation by regulating the expressions of various inflammatory cytokines, and may be potential agent for the management of rheumatoid arthritis. [ABSTRACT FROM AUTHOR]

Evolutionary changes in vertebrates are linked to genetic alterations that often affect tooth crown shape, which is a criterion of speciation events. The Notch pathway is highly conserved between species and controls morphogenetic processes in most developing organs, including teeth. Epithelial loss of the Notch-ligand Jagged1 in developing mouse molars affects the location, size and interconnections of their cusps that lead to minor tooth crown shape modifications convergent to those observed along Muridae evolution. RNA sequencing analysis revealed that these alterations are due to the modulation of more than 2000 genes and that Notch signaling is a hub for significant morphogenetic networks, such as Wnts and Fibroblast Growth Factors. The modeling of these tooth crown changes in mutant mice, via a three-dimensional metamorphosis approach, allowed prediction of how Jagged1-associated mutations in humans could affect the morphology of their teeth. These results shed new light on Notch/Jagged1-mediated signaling as one of the crucial components for dental variations in evolution. [ABSTRACT FROM AUTHOR]

Notch 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.

Abstract Background Mesenchymal stem cells (MSCs) have shown therapeutic potential for engraftment to, differentiation into, endothelial cells (ECs). However, low-efficiency yields hinder their use as ECs for therapeutic vascularization. Methods The Notch1 signaling pathway is key to optimal pulmonary development. Recent evidence has shown that this pathway participated in angiogenesis. Herein, we found that in MSCs, Jagged1 was a target for Notch 1, resulting in a positive feedback loop that propagated a wave of ECs differentiation. Results In vitro, Jagged1 was found to be activated by Notch1 in MSCs, resulting in the RBP-Jκ-dependent expression of Jagged1 mRNA, a response that was blocked by Notch1 inhibition. Notch1 promoted the formation of cord-like structures on Matrigel. However, cigarette smoke extract inhibited this process, compared to that in control groups. Moreover, Notch1-overexpressing cells upregulated the expressing of HIF-1α gene. The HIF-1α was an angiogenic factor that clustered with Notch1, underscoring the critical role of Notch1 pathway in vessel assembly. Interestingly, this was abrogated by incubation with Notch1 shRNA. Conclusions Notch signaling pathway promotes differentiation of MSCs in to ECs. It also regulates angiogenesis and transcription of specific markers on ECs. These results provide a mechanism that regulates differentiation of MSCs into ECs phenotypes.

Spontaneous bleeds are a leading cause of death in the pediatric JAG1‐related liver disease Alagille syndrome (ALGS). We asked whether there are sex differences in bleeding events in patients, whether Jag1Ndr/Ndr mice display bleeds or vascular defects, and whether discovered vascular pathology can be confirmed in patients non‐invasively. We performed a systematic review of patients with ALGS and vascular events following PRISMA guidelines, in the context of patient sex, and found significantly more girls than boys reported with spontaneous intracranial hemorrhage. We investigated vascular development, homeostasis, and bleeding in Jag1Ndr/Ndr mice, using retina as a model. Jag1Ndr/Ndr mice displayed sporadic brain bleeds, a thin skull, tortuous blood vessels, sparse arterial smooth muscle cell coverage in multiple organs, which could be aggravated by hypertension, and sex‐specific venous defects. Importantly, we demonstrated that retinographs from patients display similar characteristics with significantly increased vascular tortuosity. In conclusion, there are clinically important sex differences in vascular disease in ALGS, and retinography allows non‐invasive vascular analysis in patients. Finally, Jag1Ndr/Ndr mice represent a new model for vascular compromise in ALGS. Synopsis: Spontaneous bleeds are a significant cause of death in the rare genetic disease Alagille syndrome, but little is known about the risk factors contributing to the bleeding events, or about vascular development and maintenance in the course of the disease. Alagille syndrome is modeled by Jag1Ndr/Ndr mice, including high mortality and sporadic bleeding.More female than male patients with Alagille syndrome are reported with intracranial bleeds, and certain vascular phenotypes are more severe in female Jag1Ndr/Ndr mice.Bleeding risk in Jag1Ndr/Ndr mice may be further modified by thin skull bones, fragile vascular smooth muscle cells, premature vascular aging, and increased venous tortuosity.Developmental and homeostatic vascular defects were detected in both endothelial cells and vascular smooth muscle cells in Jag1Ndr/Ndr mice.The vascular defects observed in Jag1Ndr/Ndr mice could be visualized, quantified, and validated in retinographs from patients, suggesting a non‐invasive method to assess vascular health. [ABSTRACT FROM AUTHOR]

Presenilins (Psen1 and Psen2 in mice) are polytopic transmembrane proteins that act in the γ-secretase complex to make intra-membrane cleavages of their substrates, including the well-studied Notch receptors. Such processing releases the Notch intracellular domain, allowing it to physically relocate from the cell membrane to the nucleus where it acts in a transcriptional activating complex to regulate downstream genes in the signal-receiving cell. Previous studies of Notch pathway mutants for Jagged1, Notch2, and Rbpj demonstrated that canonical signaling is a necessary component of normal mouse lens development. However, the central role of Psens within the γ-secretase complex has never been explored in any developing eye tissue or cell type. By directly comparing Psen single and double mutant phenotypes during mouse lens development, we found a stronger requirement for Psen1, although both genes are needed for progenitor cell growth and to prevent apoptosis. We also uncovered a novel genetic interaction between Psen1 and Jagged1. By quantifying protein and mRNA levels of key Notch pathway genes in Psen1/2 or Jagged1 mutant lenses, we identified multiple points in the overall signaling cascade where feedback regulation can occur. Our data are consistent with the loss of particular genes indirectly influencing the transcription level of another. However, we conclude that regulating Notch2 protein levels is particularly important during normal signaling, supporting the importance of post-translational regulatory mechanisms in this tissue.

Background: Following traumatic bone loss or removal of bone tumors, the failure of bone allograft transplantation for large bone defect repair remains a significant problem in orthopedics. Therefore, new strategies that can efficiently enhance allograft healing and long-term incorporation are critically needed. Method: In this study, we first injected Notch-activating Jagged1 peptide to mice and then isolated bone marrow tissues and cells for proliferation and differentiation assays. Femur bone allograft surgery was also performed in Jagged1 pre-treated mice, and bone defect healing process were monitored by histology, Micro-CT and biomechanical testing. Result: Our results showed that Jagged1 therapeutic injection is sufficient to maximally activate Notch and promote bone marrow stromal cell proliferation in vivo, while no effects on bone structure were observed. More importantly, Jagged1 pre-treatment significantly promoted bone callus formation and increased bone mechanical strength during allograft healing in a femur bone defect mouse model. Conclusion: This study reveals that Notch in vivo activation can be induced by injection of Jagged1 peptide for expansion of local native stromal cells that will significantly enhance bone callus formation. The Translational potential of this Article: The clinical uses of this therapeutic strategy would be immediately applicable for chronic long bone defect repair. More importantly, this devised strategy for expansion of endogenous BMSCs can also be applied to enhance other tissue and organ repair.

Abstract Spontaneous bleeds are a leading cause of death in the pediatric JAG1‐related liver disease Alagille syndrome (ALGS). We asked whether there are sex differences in bleeding events in patients, whether Jag1Ndr/Ndr mice display bleeds or vascular defects, and whether discovered vascular pathology can be confirmed in patients non‐invasively. We performed a systematic review of patients with ALGS and vascular events following PRISMA guidelines, in the context of patient sex, and found significantly more girls than boys reported with spontaneous intracranial hemorrhage. We investigated vascular development, homeostasis, and bleeding in Jag1Ndr/Ndr mice, using retina as a model. Jag1Ndr/Ndr mice displayed sporadic brain bleeds, a thin skull, tortuous blood vessels, sparse arterial smooth muscle cell coverage in multiple organs, which could be aggravated by hypertension, and sex‐specific venous defects. Importantly, we demonstrated that retinographs from patients display similar characteristics with significantly increased vascular tortuosity. In conclusion, there are clinically important sex differences in vascular disease in ALGS, and retinography allows non‐invasive vascular analysis in patients. Finally, Jag1Ndr/Ndr mice represent a new model for vascular compromise in ALGS.

Colorectal cancer (CRC) is characterized by early metastasis, resistance to anti-cancer therapy, and high mortality rate. Despite considerable progress in the development of new treatment options that improved survival benefits in patients with early-stage or advanced CRC, many patients relapse due to the activation of intrinsic or acquired chemoresistance mechanisms. Recently, we reported novel findings about the role of Jagged1 in CRC tumors with Kras signatures. We showed that Jagged1 is a novel proteolytic target of Kras signaling, which induces Jagged1 processing/activation resulting in Jag1-ICD release, which favors tumor development in vivo, through a non-canonical mechanism. Herein, we demonstrate that OXP and 5FU cause a strong accumulation of Jag1-ICD oncogene, through ERK1/2 activation, unveiling a surviving subpopulation with an enforced Jag1-ICD expression, presenting the ability to counteract OXP/5FU-induced apoptosis. Remarkably, we also clarify the clinical ineffectiveness of g-secretase inhibitors (GSIs) in metastatic CRC (mCRC) patients. Indeed, we show that GSI compounds trigger Jag1- ICD release, which promotes cellular growth and EMT processes, functioning as tumorpromoting agents in CRC cells overexpressing Jagged1. We finally demonstrate that Jagged1 silencing in OXP- or 5FU-resistant subpopulations is enough to restore the sensitivity to chemotherapy, confirming that drug sensitivity/resistance is Jag1-ICDdependent, suggesting Jagged1 as a molecular predictive marker for the outcome of chemotherapy. [ABSTRACT FROM AUTHOR]

Objective: Indirect Jagged1 immobilization efficiently activates canonical Notch signaling in human dental pulp stem cells (hDPSCs). This study aimed to investigate the characteristics of the Jagged1-treated hDPSC-derived decellularized extracellular matrix (dECM) and its biological activity on odonto/osteogenic differentiation of stem cells isolated from apical papilla (SCAPs).Methods: Bioinformatic database of Jagged1-treated hDPSCs was analyzed using NetworkAnalyst. hDPSCs seeded on the Jagged1 immobilized surface were maintained with normal or osteogenic induction medium (OM) followed by decellularization procedure, dECM-N, or dECM-OM, respectively. SCAPs were reseeded on each dECM with either the normal medium or the OM. Cell viability was determined by MTT assay. Characteristics of dECMs and SCAPs were evaluated by SEM, EDX, immunofluorescent staining, and alcian blue staining. Mineralization was assessed by alizarin red S, Von Kossa, and alkaline phosphatase staining. Statistical significance was considered at p < 0.05.Results: RNA-seq database revealed upregulation of several genes involved in ECM organization, ECM–receptor interaction, and focal adhesion in Jagged1-treated hDPSCs. Immobilized Jagged1 increased the osteogenesis of the hDPSC culture with OM. dECMs showed fibrillar-like network structure and maintained major ECM proteins, fibronectin, type I-collagen, and glycosaminoglycans. A decrease in calcium and phosphate components was observed in dECMs after the decellularized process. Cell viability on dECMs did not alter by 7 days. Cell attachment and f-actin cytoskeletal organization of SCAPs proliferated on Jagged1-treated dECMs were comparable to those of the control dECMs. SCAPs exhibited significantly higher mineralization on dECM-N in OM and markedly enhanced on dECM-OM with normal medium or OM conditions.Conclusion: Jagged1-treated hDPSC-derived dECMs are biocompatible and increase odonto/osteogenic differentiation of SCAPs. The results suggested the potential of Jagged1 dECMs, which could be further developed into ECM scaffolds for application in regenerative medicine.

Colorectal cancer (CRC) is characterized by early metastasis, resistance to anti-cancer therapy, and high mortality rate. Despite considerable progress in the development of new treatment options that improved survival benefits in patients with early-stage or advanced CRC, many patients relapse due to the activation of intrinsic or acquired chemoresistance mechanisms. Recently, we reported novel findings about the role of Jagged1 in CRC tumors with Kras signatures. We showed that Jagged1 is a novel proteolytic target of Kras signaling, which induces Jagged1 processing/activation resulting in Jag1-ICD release, which favors tumor development in vivo, through a non-canonical mechanism. Herein, we demonstrate that OXP and 5FU cause a strong accumulation of Jag1-ICD oncogene, through ERK1/2 activation, unveiling a surviving subpopulation with an enforced Jag1-ICD expression, presenting the ability to counteract OXP/5FU-induced apoptosis. Remarkably, we also clarify the clinical ineffectiveness of γ-secretase inhibitors (GSIs) in metastatic CRC (mCRC) patients. Indeed, we show that GSI compounds trigger Jag1-ICD release, which promotes cellular growth and EMT processes, functioning as tumor-promoting agents in CRC cells overexpressing Jagged1. We finally demonstrate that Jagged1 silencing in OXP- or 5FU-resistant subpopulations is enough to restore the sensitivity to chemotherapy, confirming that drug sensitivity/resistance is Jag1-ICD-dependent, suggesting Jagged1 as a molecular predictive marker for the outcome of chemotherapy.

Background: Mesenchymal stem cells (MSCs) have shown therapeutic potential for engraftment to, differentiation into, endothelial cells (ECs). However, low-efficiency yields hinder their use as ECs for therapeutic vascularization.Methods: The Notch1 signaling pathway is key to optimal pulmonary development. Recent evidence has shown that this pathway participated in angiogenesis. Herein, we found that in MSCs, Jagged1 was a target for Notch 1, resulting in a positive feedback loop that propagated a wave of ECs differentiation.Results: In vitro, Jagged1 was found to be activated by Notch1 in MSCs, resulting in the RBP-Jκ-dependent expression of Jagged1 mRNA, a response that was blocked by Notch1 inhibition. Notch1 promoted the formation of cord-like structures on Matrigel. However, cigarette smoke extract inhibited this process, compared to that in control groups. Moreover, Notch1-overexpressing cells upregulated the expressing of HIF-1α gene. The HIF-1α was an angiogenic factor that clustered with Notch1, underscoring the critical role of Notch1 pathway in vessel assembly. Interestingly, this was abrogated by incubation with Notch1 shRNA.Conclusions: Notch signaling pathway promotes differentiation of MSCs in to ECs. It also regulates angiogenesis and transcription of specific markers on ECs. These results provide a mechanism that regulates differentiation of MSCs into ECs phenotypes. [ABSTRACT FROM AUTHOR]

Hybrid epithelial/mesenchymal cells (E/M) are key players in aggressive cancer metastasis. It remains a challenge to understand how these cell states, which are mostly non-existent in healthy tissue, become stable phenotypes participating in collective cancer migration. The transcription factor Nrf2, which is associated with tumor progression and resistance to therapy, appears to be central to this process. Here, using a combination of immunocytochemistry, single cell biosensors, and computational modeling, we show that Nrf2 functions as a phenotypic stability factor for hybrid E/M cells by inhibiting a complete epithelial-mesenchymal transition (EMT) during collective cancer migration. We also demonstrate that Nrf2 and EMT signaling are spatially coordinated near the leading edge. In particular, computational analysis of an Nrf2-EMT-Notch network and experimental modulation of Nrf2 by pharmacological treatment or CRISPR/Cas9 gene editing reveal that Nrf2 stabilizes a hybrid E/M phenotype which is maximally observed in the interior region immediately behind the leading edge. We further demonstrate that the Nrf2-EMT-Notch network enhances Dll4 and Jagged1 expression at the leading edge, which correlates with the formation of leader cells and protruding tips. Altogether, our results provide direct evidence that Nrf2 acts as a phenotypic stability factor in restricting complete EMT and plays an important role in coordinating collective cancer migration.

Abstract Background Osteosarcoma is the most common primary bone cancer occurring in young adults and the 5-year survival rate of patients with metastatic osteosarcoma is less than 30% due to high metastatic recurrence and drug resistance. Notch is a highly conserved cell to cell signaling pathway in evolution, and Jagged1 is an important ligand of Notch. Although some studies have found that Notch receptors and ligands including Jagged1 were highly expressed in osteosarcoma tissues and osteosarcoma cells, the role of Jagged1 in osteosarcoma progression and metastasis are still not clear. Methods Tumor tissues were collected from 68 patients and immunohistochemical staining was employed to group these patients by expression of Jagged1. Real-time quantitative PCR and Western blotting were used to detect the expression of Jagged1. We used siRNA to knockdown the expression of Jagged1 in F5M2 cells. Colony formation assay and MTT were employed to detect and analyze the proliferation of F5M2 cells with or without knockdown of Jagged1. Transwell assay were used to detect the migration and invasion of F5M2 cells. Results In this study, we found that the high expression of Jagged1 is closely related to the metastasis and recurrence of osteosarcoma in 68 clinical specimens. The expression of Jagged1 in F5M2 cells with high metastasis was significantly higher than that in F4 cells with low metastasis. Knockdown of Jagged1 led to lower ability of proliferation, migration, and invasion in F5M2 cells. Conclusion The high expression of Jagged1 is closely related to the metastasis and recurrence of osteosarcoma. Knockdown of Jagged1 significantly reduced the proliferation, migration, and invasion of osteosarcoma cells. Our results suggested that knockdown of Jagged1 may be a potentially effective treatment for metastatic osteosarcoma.

Evolutionary changes in vertebrates are linked to genetic alterations that often affect tooth crown shape, which is a criterion of speciation events. The Notch pathway is highly conserved between species and controls morphogenetic processes in most developing organs, including teeth. Epithelial loss of the Notch-ligand Jagged1 in developing mouse molars affects the location, size and interconnections of their cusps that lead to minor tooth crown shape modifications convergent to those observed along Muridae evolution. RNA sequencing analysis revealed that these alterations are due to the modulation of more than 2000 genes and that Notch signaling is a hub for significant morphogenetic networks, such as Wnts and Fibroblast Growth Factors. The modeling of these tooth crown changes in mutant mice, via a three-dimensional metamorphosis approach, allowed prediction of how Jagged1-associated mutations in humans could affect the morphology of their teeth. These results shed new light on Notch/Jagged1-mediated signaling as one of the crucial components for dental variations in evolution.

Summary: Immune checkpoint inhibitor (ICI) therapy is generating remarkable responses in individuals with cancer, but only a small portion of individuals with breast cancer respond well. Here we report that tumor-derived Jagged1 is a key regulator of the tumor immune microenvironment. Jagged1 promotes tumorigenesis in multiple spontaneous mammary tumor models. Through Jagged1-induced Notch activation, tumor cells increase expression and secretion of multiple cytokines to help recruit macrophages into the tumor microenvironment. Educated macrophages crosstalk with tumor-infiltrating T cells to inhibit T cell proliferation and tumoricidal activity. In individuals with triple-negative breast cancer, a high expression level of Jagged1 correlates with increased macrophage infiltration and decreased T cell activity. Co-administration of an ICI PD-1 antibody with a Notch inhibitor significantly inhibits tumor growth in breast cancer models. Our findings establish a distinct signaling cascade by which Jagged1 promotes adaptive immune evasion of tumor cells and provide several possible therapeutic targets.

Aim: To investigate whether TGF‐β/BMP signalling participates in Jagged1‐induced osteogenic differentiation in human dental pulp cells (hDPs). Methodology: Bioinformatic analysis of publicly available RNA sequencing data of Jagged1‐treated hDPs was performed using NetworkAnalyst. The mRNA expression was validated using real‐time polymerase chain reaction. hDPs were seeded on Jagged1 immobilized surfaces in the presence or absence of TGF‐β or BMP inhibitor. Osteogenic differentiation was evaluated using alkaline phosphatase staining, osteogenic marker gene expression and mineralization assay. Statistical analyses were performed using a Kruskal–Wallis test, followed by a pairwise comparison for more than three group comparison. Mann–Whitney U‐test was employed for two group comparison. The statistical significance was considered at p <.05. Results: Jagged1 treatment in growth medium significantly promoted TGFB1, TGFB2 and TGFB3 whilst significantly inhibited BMP2, BMP4 and BMP6 mRNA expression (p <.05). In osteogenic induction medium, Jagged1 significantly up‐regulated TGFB1, TGFB2 and TGFB3 at days 1 and 3 (p <.05). Pre‐treatment with TGF‐β1, TGF‐β2 or TGF‐β3 prior to osteogenic induction resulted in the significant increase of osteogenic marker gene expression, collagen type 1 protein expression, alkaline phosphatase enzymatic activity and mineral deposition (p <.05). However, TGF‐β signalling inhibition with SB431542 (4 μmol L−1) or SB505124 (47 and 129 nmol L−1) failed to attenuate the effect of Jagged1‐induced osteogenic differentiation in hDPs. Dorsomorphin (4 and 8 μmol L−1) treatment significantly abolished the effect of Jagged1 on mineralization by hDPs (p <.05). Conclusion: Notch signalling activation by Jagged1 modulated TGF‐β and BMP ligand expression. Dorsomorphin, but not TGF‐β receptor inhibitor, attenuated Jagged1‐induced osteogenic differentiation in hDPs. [ABSTRACT FROM AUTHOR]

To investigate the mechanism of Pingchuan Granules in the treatment of asthma by observing the changes of Notch2, Jagged1, HES-1 related factors of Notch signaling pathway in chronic asthma rats in each group, as well as the improvement of lung tissue airway remodeling. On the basis of random number table method, 60 rats were divided into normal group, model group, dexamethasone group and Pingchuan granules group on average. Except the normal group, the rest of 3 groups were sensitized by intraperitoneal injection of ovalbumin (OVA) and Al (OH)3, and the asthmatic rat model was duplicated. The normal group was treated with normal saline instead of sensitizing solution. Before modeling, each group was given the corresponding drug gavage treatment. Six weeks later, the rats were killed and their lung tissues were stained with HE. The pathological changes were observed under light microscope. Real-time PCR was used to detect Notch2 and Jagged1 mRNA, and Western blot was used to detect HES-1 protein. Compared with the normal group, significant changes in airway remodeling were observed in the lung tissues of the other groups, and the expressions of Notch2, Jagged1 mRNA and HES-1 protein were significantly increased (P<0.05). Compared with the model group, pingchuan granules and dexamethasone could alleviate airway remodeling, and the expression of each factor was significantly decreased (P<0.05), but there was no significant difference between the two groups (P>0.05). The expression levels of Notch2, Jagged1 and HES-1 are positively correlated with airway remodeling, and the improvement of airway remodeling by reducing the expression levels of Notch2, Jagged1 and HES-1 may be the mechanism for the prevention and treatment of asthma. [ABSTRACT FROM AUTHOR]

In the mammary gland, how alveolar progenitor cells are recruited to fuel tissue growth with each estrus cycle and pregnancy remains poorly understood. Here, we identify a regulatory pathway that controls alveolar progenitor differentiation and lactation by governing Notch activation in mouse. Loss of Robo1 in the mammary gland epithelium activates Notch signaling, which expands the alveolar progenitor cell population at the expense of alveolar differentiation, resulting in compromised lactation. ROBO1 is expressed in both luminal and basal cells, but loss of Robo1 in basal cells results in the luminal differentiation defect. In the basal compartment, ROBO1 inhibits the expression of Notch ligand Jag1 by regulating β-catenin (CTNNB1), which binds the Jag1 promoter. Together, our studies reveal how ROBO1/CTTNB1/JAG1 signaling in the basal compartment exerts paracrine control of Notch signaling in the luminal compartment to regulate alveolar differentiation during pregnancy. [ABSTRACT FROM AUTHOR]

Supplemental Digital Content is available in the text. Background: How the extracellular matrix (ECM) microenvironment modulates the contractile phenotype of vascular smooth muscle cells (VSMCs) and confers vascular homeostasis remains elusive. Methods: To explore the key ECM proteins in the maintenance of the contractile phenotype of VSMCs, we applied protein-protein interaction network analysis to explore novel ECM proteins associated with the VSMC phenotype. By combining in vitro and in vivo genetic mice vascular injury models, we identified nidogen-2, a basement membrane glycoprotein, as a key ECM protein for maintenance of vascular smooth muscle cell identity. Results: We collected a VSMC phenotype–related gene dataset by using Gene Ontology annotation combined with a literature search. A computational analysis of protein-protein interactions between ECM protein genes and the genes from the VSMC phenotype–related gene dataset revealed the candidate gene nidogen-2, a basement membrane glycoprotein involved in regulation of the VSMC phenotype. Indeed, nidogen-2–deficient VSMCs exhibited loss of contractile phenotype in vitro, and compared with wild-type mice, nidogen-2–/– mice showed aggravated post–wire injury neointima formation of carotid arteries. Further bioinformatics analysis, coimmunoprecipitation assays, and luciferase assays revealed that nidogen-2 specifically interacted with Jagged1, a conventional Notch ligand. Nidogen-2 maintained the VSMC contractile phenotype via Jagged1-Notch3 signaling but not Notch1 or Notch2 signaling. Nidogen-2 enhanced Jagged1 and Notch3 interaction and subsequent Notch3 activation. Reciprocally, Jagged1 and Notch3 interaction, signaling activation, and Jagged1-triggered VSMC differentiation were significantly repressed in nidogen-2–deficient VSMCs. In accordance, the suppressive effect of Jagged1 overexpression on neointima formation was attenuated in nidogen-2–/– mice compared with wild-type mice. Conclusions: Nidogen-2 maintains the contractile phenotype of VSMCs through Jagged1-Notch3 signaling in vitro and in vivo. Nidogen-2 is required for Jagged1-Notch3 signaling. [ABSTRACT FROM AUTHOR]

Abstract Background Tumor-associated macrophages (TAMs) and tumor cells are important components of the tumor microenvironment. M2 polarization of TAMs, which is a major actor in breast cancer malignancy and metastasis, can be induced by breast cancer cells. However, the potential mechanisms of the interaction between breast cancer cells and TAMs remain unclear. Methods The candidate breast cancer-associated long non-coding RNAs (lncRNAs) were analyzed using the GEO database. Functional assays, including MTT assay, Transwell assay, and EdU labeling detection, were performed to investigate the oncogenic role of linc00514 in breast cancer progression. The co-culture and ELISA assays were used to assess the role of linc00514 in macrophage recruitment and M2 polarization. RNA immunoprecipitation, RNA pull-down, and luciferase reporter assays were applied to determine the mechanism of linc00514 in breast cancer metastasis. Mouse xenograft models, mouse pulmonary metastatic models, and mouse primary tumor models were used to assess the role of linc00514 in M2 macrophage polarization and breast cancer tumorigenicity. Results Linc00514 was highly expressed in clinical breast cancer tissues and breast cancer cell lines. Overexpression of linc00514 promoted the proliferation and invasion of breast cancer cells and increased xenograft tumor volumes and pulmonary metastatic nodules. Overexpression of linc00514 also increased the percentage of macrophages expressing M2 markers CD206 and CD163. Mechanistically, linc00514 promoted Jagged1 expression in a transcriptional manner by increasing the phosphorylation of a transcription factor STAT3. Subsequently, Jagged1-mediated Notch signaling pathway promoted IL-4 and IL-6 secretions in breast cancer cells and ultimately inducing M2 polarization of macrophages. Conclusion Linc00514 plays an important role in regulating breast cancer tumorigenicity and M2 macrophage polarization via Jagged1-mediated Notch signaling pathway.

Abstract Background The therapeutic effects of adipose-derived mesenchymal stem cell (ADSC) transplantation have been demonstrated in several models of central nervous system (CNS) injury and are thought to involve the modulation of the inflammatory response. However, the exact underlying molecular mechanism is poorly understood. Activation of the Jagged1/Notch signaling pathway is thought to involve inflammatory and gliotic events in the CNS. Here, we elucidated the effect of ADSC transplantation on the inflammatory reaction after spinal cord injury (SCI) and the potential mechanism mediated by Jagged1/Notch signaling pathway suppression. Methods To evaluate the therapeutic effects of ADSC treatment and the potential inhibitory effects of ADSCs on Notch signaling, mice were subjected to contusion SCI, and GFP-labeled ADSCs were injected into the lesion site immediately after the injury. Locomotor function, spinal cord tissue morphology, and the levels of Notch-related proteins and proinflammatory transcripts were compared between groups. To validate the hypothesis that the therapeutic effects of ADSCs are partly due to Notch1 signaling inhibition, a Jagged1 small interfering RNA (siRNA) was injected into the spinal cord to knock down Jagged1/Notch signaling. Neuronal staining and analyses of microglia/macrophage activation and signaling pathways were performed. Results We demonstrated that ADSCs survived in the injured spinal cord for at least 28 days without differentiating into glial or neuronal elements. ADSC treatment resulted in significant downregulation of proinflammatory mediator expression and reduced ionized calcium-binding adapter molecule 1 (IBA1) and ED-1 staining in the injured spinal cord, eventually improving functional recovery. The augmentation of the Jagged1/Notch signaling pathway after SCI was suppressed by ADSC transplantation. The inhibition of the Jagged1/Notch signaling pathway by Jagged1 siRNA resulted in decreases in SCI-induced proinflammatory cytokines and the activation of microglia and an increase in the survival of neurons. Furthermore, Jagged1 knockdown suppressed the phosphorylation of JAK/STAT3 in astrocytes following SCI. Conclusion The results of this study demonstrated that the therapeutic effects of ADSCs in SCI mice were partly due to Jagged1/Notch signaling pathway inhibition and a subsequent reduction in JAK/STAT3 phosphorylation in astrocytes.

Abstract Background Cancer stem cells (CSCs) have been proposed to be responsible for tumor recurrence and chemo-resistance. Previous studies suggested that miR-153 played essential roles in lung cancer. However, the molecular mechanism of miR-153 in regulating the stemness of non-small cell lung cancer (NSCLC) remains poorly understood. In this study, we investigated the role of miR-153 in regulation of the stemness of NSCLC. Methods The stemness property of lung stem cancer cells was detected by sphere formation assay, immunofluorescence, and Western blot. Luciferase reporter assay was performed to investigate the direct binding of miR-153 to the 3′-UTR of JAG1 mRNA. Animal study was conducted to evaluate the effect of miR-153 on tumor growth in vivo. The clinical relevance of miR-153 in NSCLC was evaluated by Rt-PCR and Kaplan-Meier analysis. Results MiR-153 expression was decreased in lung cancer tissues. Reduced miR-153 expression was associated with lung metastasis and poor overall survival of lung cancer patients. Jagged1, one of the ligands of Notch1, is targeted by miR-153 and inversely correlates with miR-153 in human lung samples. More importantly, we found that miR-153 inhibited stem cell-like phenotype and tumor growth of lung adenocarcinoma through inactivating the Jagged1/Notch1 axis. Conclusion MiR-153 suppresses the stem cell-like phenotypes and tumor growth of lung adenocarcinoma by targeting Jagged1 and provides a potential therapeutic target in lung cancer therapy.

Treatments for congenital and acquired craniofacial (CF) bone abnormalities are limited and expensive. Current reconstructive methods include surgical correction of injuries, short-term bone stabilization, and long-term use of bone grafting solutions, including implantation of (i) allografts which are prone to implant failure or infection, (ii) autografts which are limited in supply. Current bone regenerative approaches have consistently relied on BMP2 application with or without addition of stem cells. BMP2 treatment can lead to severe bony overgrowth or uncontrolled inflammation, which can accelerate further bone loss. Bone marrow-derived mesenchymal stem cell-based treatments, which do not have the side effects of BMP2, are not currently FDA approved, and are time and resource intensive. There is a critical need for novel bone regenerative therapies to treat CF bone loss that have minimal side effects, are easily available, and are affordable. In this study we investigated novel bone regenerative therapies downstream of JAGGED1 (JAG1). We previously demonstrated that JAG1 induces murine cranial neural crest (CNC) cells towards osteoblast commitment via a NOTCH non-canonical pathway involving JAK2-STAT5 (1) and that JAG1 delivery with CNC cells elicits bone regeneration in vivo. In this study, we hypothesize that delivery of JAG1 and induction of its downstream NOTCH non-canonical signaling in pediatric human osteoblasts constitute an effective bone regenerative treatment in an in vivo murine bone loss model of a critically-sized cranial defect. Using this CF defect model in vivo, we delivered JAG1 with pediatric human bone-derived osteoblast-like (HBO) cells to demonstrate the osteo-inductive properties of JAG1 in human cells and in vitro we utilized the HBO cells to identify the downstream non-canonical JAG1 signaling intermediates as effective bone regenerative treatments. In vitro, we identified an important mechanism by which JAG1 induces pediatric osteoblast commitment and bone formation involving the phosphorylation of p70 S6K. This discovery enables potential new treatment avenues involving the delivery of tethered JAG1 and the downstream activators of p70 S6K as powerful bone regenerative therapies in pediatric CF bone loss.

Background: Angiogenesis is required for tumor development and metastasis, which is a major part in a pro-tumor microenvironment. Vascular mimicry (VM) is a process in which cancer cells, rather than endothelia, create an alternative perfusion system to support the tumor progression. Objectives: To validate the role of VM and to develop a strategy to inhibit angiogenesis in lung cancer. Methods: In this study, we utilized lung cancer samples to verify the existence of VM and conducted several experimental methods to elucidate the molecular pathways. Results: H1299 and CL1-0 lung cancer cells were unable to form capillary-like structures. VM formation was induced by cancer-associated fibroblast (CAFs) in both in vitro and in vivo experiments. Notch2–Jagged1 cell–cell contact between cancer cells and CAFs contributes to the formation of VM networks, supported by Notch intracellular domain (NICD) 2 nuclear translocation and N2ICD target gene upregulated in lung cancer cells mixed with CAFs. The polarization of tumor-promoting N2-type neutrophil was increased by VM networks consisting of CAF and cancer cells. The intravasation of cancer cells and N2-type neutrophils were increased because of the loose junctions of VM. Disruption of cancer cell–CAF connections by a γ‐secretase inhibitor enforced the anticancer effect of anti‐vascular endothelial growth factor antibodies in a mouse model. Conclusion: This study provides the first evidence that CAFs induce lung cancer to create vascular-like networks. These findings suggest a therapeutic opportunity for improving antiangiogenesis therapy in lung cancer. [ABSTRACT FROM AUTHOR]

Helicobacter pylori (H. pylori) is one of the gram-negative bacteria that mainly colonize the stomach mucosa and cause many gastrointestinal diseases, such as gastritis, peptic ulcer, and gastric cancer. Macrophages play a key role in eradicating H. pylori. Recent data have shown that Notch signaling could modulate the activation and bactericidal activities of macrophages. However, the role of Notch signaling in macrophages against H. pylori remains unclear. In the present study, in the co-culture model of macrophages with H. pylori , the inhibition of Notch signaling using γ-secretase decreased the expression of inducible nitric oxide synthase (iNOS) and its product, nitric oxide (NO), and downregulated the secretion of pro-inflammatory cytokine and attenuated phagocytosis and bactericidal activities of macrophages to H. pylori. Furthermore, we identified that Jagged1, one of Notch signaling ligands, was both upregulated in mRNA and protein level in activated macrophages induced by H. pylori. Clinical specimens showed that the number of Jagged1+ macrophages in the stomach mucosa from H. pylori -infected patients was significantly higher than that in healthy control. The overexpression of Jagged1 promoted bactericidal activities of macrophages against H. pylori and siRNA-Jagged1 presented the opposite effect. Besides, the addition of exogenous rJagged1 facilitated the pro-inflammatory mediators of macrophages against H. pylori , but the treatment of anti-Jagged1 neutralizing antibody attenuated it. Taken together, these results suggest that Jagged1 is a promoting molecule for macrophages against H. pylori , which will provide insight for exploring Jagged1 as a novel therapeutic target for the control of H. pylori infection. [ABSTRACT FROM AUTHOR]

Diabetes exacerbates brain damage in cerebral ischemic stroke. Our previous study has demonstrated that after cerebral ischemia, type 2 diabetes rats displayed worse neurological outcomes, larger cerebral infarction and severer blood-brain barrier disruption. However, our knowledge of the mechanisms of how diabetes impacts the cerebrovascular repair process is limited. This study was aimed to characterize structural alterations and potential mechanisms in brain microvessels before and after ischemic stroke in type 2 diabetic rats treated with high-fat diet and streptozotocin (HFD/STZ). Furtherly, we tested our hypothesis that dysregulated intercellular Jagged1-Notch1 signaling was involved in the dysfunctional cerebral neovascularization both before and after ischemic stroke in HFD/STZ rats. In our study, we found increased yet dysfunctional neovascularization with activated Jagged1-Notch1 signaling in the cerebrovasculature before cerebral ischemia in HFD/STZ rats compared with non-diabetic rats. Furthermore, we observed delayed angiogenesis as well as suppressed Jagged1-Notch1 signaling after ischemic stroke. Our results elucidate the potential mechanisms underlying diabetes-related cerebral microvasculature dysfunction after ischemic stroke. [ABSTRACT FROM AUTHOR]

BackgroundAngiogenesis is required for tumor development and metastasis, which is a major part in a pro-tumor microenvironment. Vascular mimicry (VM) is a process in which cancer cells, rather than endothelia, create an alternative perfusion system to support the tumor progression.ObjectivesTo validate the role of VM and to develop a strategy to inhibit angiogenesis in lung cancer.MethodsIn this study, we utilized lung cancer samples to verify the existence of VM and conducted several experimental methods to elucidate the molecular pathways.ResultsH1299 and CL1-0 lung cancer cells were unable to form capillary-like structures. VM formation was induced by cancer-associated fibroblast (CAFs) in both in vitro and in vivo experiments. Notch2–Jagged1 cell–cell contact between cancer cells and CAFs contributes to the formation of VM networks, supported by Notch intracellular domain (NICD) 2 nuclear translocation and N2ICD target gene upregulated in lung cancer cells mixed with CAFs. The polarization of tumor-promoting N2-type neutrophil was increased by VM networks consisting of CAF and cancer cells. The intravasation of cancer cells and N2-type neutrophils were increased because of the loose junctions of VM. Disruption of cancer cell–CAF connections by a γ‐secretase inhibitor enforced the anticancer effect of anti‐vascular endothelial growth factor antibodies in a mouse model.ConclusionThis study provides the first evidence that CAFs induce lung cancer to create vascular-like networks. These findings suggest a therapeutic opportunity for improving antiangiogenesis therapy in lung cancer.

Diabetes exacerbates brain damage in cerebral ischemic stroke. Our previous study has demonstrated that after cerebral ischemia, type 2 diabetes rats displayed worse neurological outcomes, larger cerebral infarction and severer blood-brain barrier disruption. However, our knowledge of the mechanisms of how diabetes impacts the cerebrovascular repair process is limited. This study was aimed to characterize structural alterations and potential mechanisms in brain microvessels before and after ischemic stroke in type 2 diabetic rats treated with high-fat diet and streptozotocin (HFD/STZ). Furtherly, we tested our hypothesis that dysregulated intercellular Jagged1-Notch1 signaling was involved in the dysfunctional cerebral neovascularization both before and after ischemic stroke in HFD/STZ rats. In our study, we found increased yet dysfunctional neovascularization with activated Jagged1-Notch1 signaling in the cerebrovasculature before cerebral ischemia in HFD/STZ rats compared with non-diabetic rats. Furthermore, we observed delayed angiogenesis as well as suppressed Jagged1-Notch1 signaling after ischemic stroke. Our results elucidate the potential mechanisms underlying diabetes-related cerebral microvasculature dysfunction after ischemic stroke.

Helicobacter pylori (H. pylori) is one of the gram-negative bacteria that mainly colonize the stomach mucosa and cause many gastrointestinal diseases, such as gastritis, peptic ulcer, and gastric cancer. Macrophages play a key role in eradicating H. pylori. Recent data have shown that Notch signaling could modulate the activation and bactericidal activities of macrophages. However, the role of Notch signaling in macrophages against H. pylori remains unclear. In the present study, in the co-culture model of macrophages with H. pylori, the inhibition of Notch signaling using γ-secretase decreased the expression of inducible nitric oxide synthase (iNOS) and its product, nitric oxide (NO), and downregulated the secretion of pro-inflammatory cytokine and attenuated phagocytosis and bactericidal activities of macrophages to H. pylori. Furthermore, we identified that Jagged1, one of Notch signaling ligands, was both upregulated in mRNA and protein level in activated macrophages induced by H. pylori. Clinical specimens showed that the number of Jagged1+ macrophages in the stomach mucosa from H. pylori-infected patients was significantly higher than that in healthy control. The overexpression of Jagged1 promoted bactericidal activities of macrophages against H. pylori and siRNA-Jagged1 presented the opposite effect. Besides, the addition of exogenous rJagged1 facilitated the pro-inflammatory mediators of macrophages against H. pylori, but the treatment of anti-Jagged1 neutralizing antibody attenuated it. Taken together, these results suggest that Jagged1 is a promoting molecule for macrophages against H. pylori, which will provide insight for exploring Jagged1 as a novel therapeutic target for the control of H. pylori infection.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a genetic small vessel disease characterized by NOTCH3 mutation and abnormal aggregation of NOTCH3 mutant proteins around vessel walls. NOTCH3 is a transmembrane receptor that is degraded by JAGGED1 (JAG1) through a process called trans -endocytosis. There are two types of CADASIL-associated NOTCH3 mutations: signal-active (SA) and signal-deficient (SD) mutations. However, the conditions that lead to abnormal aggregation of NOTCH3 mutant proteins remain poorly understood. Performing a coculture assay, we found that the SA NOTCH3 mutants (C49Y, R90C, R141C, and C185R) were degraded and trans -endocytosed by JAG1 similar to wild-type (WT) NOTCH3, but the SD NOTCH3 mutant (C428S) was not degraded or endocytosed by JAG1, suggesting that other environmental factors may be necessary for the aggregation of SA NOTCH3 mutants. Lunatic fringe (LFNG) is a glycosyltransferase of NOTCH3, but whether LFNG affects the aggregation of NOTCH3 mutants remains unknown. Performing a sucrose gradient ultracentrifugation assay, we found that LFNG might decrease the aggregation propensity of WT NOTCH3 but increase that of C185R NOTCH3. In conclusion, the SD NOTCH3 mutant may be more likely to accumulate than the SA NOTCH3 mutants upon interaction with JAG1. Moreover, LFNG may play an important role in promoting the aggregation of SA NOTCH3 mutants. • Signal-active NOTCH3 mutants are degraded and trans -endocytosed by JAGGED1. • The signal-deficient C428S NOTCH3 mutant is not degraded or trans -endocytosed by JAGGED1. • Lunatic fringe can glycosylate signal-active NOTCH3 mutants. • Lunatic fringe may promote the aggregation of signal-active NOTCH3 mutants. [ABSTRACT FROM AUTHOR]

Tumor immunosuppression are prominent characteristics of brain glioma. Current standard modality including surgical resection and chemoradiotherapy do not significantly improve clinical outcomes. Cancer-associated fibroblasts (CAFs) that regard as important stromal cells in tumor microenvironment have been confirmed to play crucial roles in tumor development. However, the effects of CAFs on tumor immunosuppression in glioma are not well expounded. In this study, we report that CAFs contributes to the formation of glioma immunosuppressive microenvironment. Specifically, we found that glioma-derived Jagged1 enhanced the proliferation and PD-L1 expression of CAFs in vitro. Importantly, we discovered that Notch1, c-Myc and PD-L1 expression were significantly increased in high Jagged1-expressing gliomas, moreover, we further confirmed that Notch1 and PD-L1 expression located on the CAFs in glioma tissues. We also found that glioma-derived Jagged1 promotes the increase of tumor-infiltrating macrophages, M2 macrophages and Foxp3 Treg cells, as well as no significance of M1 macrophages and CD8+ T cells, indicating potential immunosuppression. This study opens up novel therapeutic strategies reversing CAF immunosuppression for gliomas. • Identification of fibroblasts in human glioma tissues similar with cancer-associated fibroblasts. • Glioma-derived Jagged1 promotes the PD-L1 expression in CAFs via Notch1 signaling. • Jagged1 facilitates the infiltration of M2 macrophages and Foxp3 Treg cells in glioma tissues. • Strategy reversing CAF-mediated immunosuppression is promising for glioma treatment. [ABSTRACT FROM AUTHOR]

Notch signaling, re-activated in β cells from obese mice and causal to β cell dysfunction, is determined in part by transmembrane ligand availability in a neighboring cell. We hypothesized that β cell expression of Jagged1 determines the maladaptive Notch response and resultant insulin secretory defects in obese mice. We assessed expression of Notch pathway components in high-fat diet-fed (HFD) or leptin receptor-deficient (db/db) mice, and performed single-cell RNA sequencing (scRNA-Seq) in islets from patients with and without type 2 diabetes (T2D). We generated and performed glucose tolerance testing in inducible, β cell-specific Jagged1 gain-of- and loss-of-function mice. We also tested effects of monoclonal neutralizing antibodies to Jagged1 in glucose-stimulated insulin secretion (GSIS) assays in isolated islets. Jag1 was the only Notch ligand that tracked with increased Notch activity in HFD-fed and db/db mice, as well as in metabolically-inflexible β cells enriched in patients with T2D. Neutralizing antibodies to block Jagged1 in islets isolated from HFD-fed and db/db mice potentiated GSIS ex vivo. To demonstrate if β cell Jagged1 is sufficient to cause glucose tolerance in vivo , we generated inducible β cell-specific Jag1 transgenic (β-Jag1TG) and loss-of-function (iβ-Jag1KO) mice. While forced Jagged1 impaired glucose intolerance due to reduced GSIS, loss of β cell Jagged1 did not protect against HFD-induced insulin secretory defects. Jagged1 is increased in islets from obese mice and in patients with T2D, and neutralizing Jagged1 antibodies lead to improved GSIS, suggesting that inhibition of Jagged1-Notch signaling may have therapeutic benefit. However, genetic loss-of-function experiments suggest that β cells are not a likely source of the Jagged1 signal. • Jag1 tracks with increased islet Notch activity in obese mice, and in metabolically inflexible β cells in patients with T2D. • Forced β cell Jagged1 is sufficient to induce glucose intolerance and impair glucose stimulated insulin secretion in mice. • Jagged1 neutralizing antibodies improve glucose stimulatd insulin secretion in islets from obese mice. • Genetic inhibition of β cell Jagged1 does not ameliorate HFD-induced glucose intolerance. [ABSTRACT FROM AUTHOR]

Background/Aims: Jagged1 is the ligands of the Notch signaling and has been shown to promote glioma-initiating cells (GICs) in glioblastoma. The role of Jagged1 in GICs invasion and underlying molecular mechanisms remain unclear. Methods: Survival data from R2 genomics analysis, the Cancer Genome Atlas (TCGA), the Chinese Glioma Genome Atlas (CGGA) and visualization platform database were used to evaluate the effects of Jagged1 on overall patient survival. we investigated Jagged1 induced the GICs cells’ invasion by matrix degradation assays and Transwell cell invasion assays in vitro, then we further explored the underlying molecular mechanisms using Co-immunoprecipitation (co-IP) analysis. Results: High expression of Jagged1 in human glioma was associated with poor survival. Clinical data analysis showed that the Jagged1 was positively correlated with NF-κB(p65). Jagged1-induced invasion of GICs cells through activation of NF-κB(p65) pathway. In vivo, knockdown of Jagged1 could suppress the tumorigenicity of GICs cells through NF-κB(p65) signaling. Conclusion: Insights gained from these findings suggest that Jagged1 plays an important oncogenic role in GICs malignancy by activation of NF-κB(p65) signaling, and Jagged1 could be employed as an effective therapeutic target for GICs.

Background: Osteosarcoma is the most common primary bone cancer occurring in young adults and the 5-year survival rate of patients with metastatic osteosarcoma is less than 30% due to high metastatic recurrence and drug resistance. Notch is a highly conserved cell to cell signaling pathway in evolution, and Jagged1 is an important ligand of Notch. Although some studies have found that Notch receptors and ligands including Jagged1 were highly expressed in osteosarcoma tissues and osteosarcoma cells, the role of Jagged1 in osteosarcoma progression and metastasis are still not clear. Methods: Tumor tissues were collected from 68 patients and immunohistochemical staining was employed to group these patients by expression of Jagged1. Real-time quantitative PCR and Western blotting were used to detect the expression of Jagged1. We used siRNA to knockdown the expression of Jagged1 in F5M2 cells. Colony formation assay and MTT were employed to detect and analyze the proliferation of F5M2 cells with or without knockdown of Jagged1. Transwell assay were used to detect the migration and invasion of F5M2 cells. Results: In this study, we found that the high expression of Jagged1 is closely related to the metastasis and recurrence of osteosarcoma in 68 clinical specimens. The expression of Jagged1 in F5M2 cells with high metastasis was significantly higher than that in F4 cells with low metastasis. Knockdown of Jagged1 led to lower ability of proliferation, migration, and invasion in F5M2 cells. Conclusion: The high expression of Jagged1 is closely related to the metastasis and recurrence of osteosarcoma. Knockdown of Jagged1 significantly reduced the proliferation, migration, and invasion of osteosarcoma cells. Our results suggested that knockdown of Jagged1 may be a potentially effective treatment for metastatic osteosarcoma. [ABSTRACT FROM AUTHOR]

A study was made of the effect that mitomycin C (MitC) treatment of stromal layers of NIH 3T3 cells expressing Jagged1, a ligand of the Notch receptor, exerts on the growth of hematopoietic Lin(–) mouse bone marrow cells in a co-culture system. MitC treatment of stromal cells significantly increased the number of hematopoietic cells and the frequency of colony-forming cells in stromal co-cultures. Transcriptome analysis of control and MitC-treated stromal cell samples was performed by differential RNA sequencing, and genes downregulated by MitC treatment were predominantly associated with the control of cell proliferation, the cell cycle, chromosome segregation, and DNA metabolism. Induction of key hematopoietic cytokines by MitC was not detected by the transcriptome analysis and was therefore not a main factor in the activation of hematopoiesis on the treated stroma. At the same time, the set of the genes most strongly upregulated by MitC treatment is enriched in the genes for cytokines, growth factors, and cell surface proteins, which presumably contribute to enhanced hematopoiesis support on the MitC-treated stroma. Products of some of these genes have been implicated in expansion of hematopoietic stem/progenitor cells in vitro or in vivo. [ABSTRACT FROM AUTHOR]