Modulation Of Notch Signaling Research Articles

Generation of multilineage liver organoids with luminal vasculature and bile ducts from human pluripotent stem cells via modulation of Notch signaling

BackgroundThe generation of liver organoids recapitulating parenchymal and non-parenchymal cell interplay is essential for the precise in vitro modeling of liver diseases. Although different types of multilineage liver organoids (mLOs) have been generated from human pluripotent stem cells (hPSCs), the assembly and concurrent differentiation of multiple cell types in individual mLOs remain a major challenge. Particularly, most studies focused on the vascularization of mLOs in host tissue after transplantation in vivo. However, relatively little information is available on the in vitro formation of luminal vasculature in mLOs themselves.MethodsThe mLOs with luminal blood vessels and bile ducts were generated by assembling hepatic endoderm, hepatic stellate cell-like cells (HscLCs), and endothelial cells derived entirely from hPSCs using 96-well ultra-low attachment plates. We analyzed the effect of HscLC incorporation and Notch signaling modulation on the formation of both bile ducts and vasculature in mLOs using immunofluorescence staining, qRT-PCR, ELISA, and live-perfusion imaging. The potential use of the mLOs in fibrosis modeling was evaluated by histological and gene expression analyses after treatment with pro-fibrotic cytokines.ResultsWe found that hPSC-derived HscLCs are crucial for generating functional microvasculature in mLOs. HscLC incorporation and subsequent vascularization substantially reduced apoptotic cell death and promoted the survival and growth of mLOs with microvessels. In particular, precise modulation of Notch signaling during a specific time window in organoid differentiation was critical for generating both bile ducts and vasculature. Live-cell imaging, a series of confocal scans, and electron microscopy demonstrated that blood vessels were well distributed inside mLOs and had perfusable lumens in vitro. In addition, exposure of mLOs to pro-fibrotic cytokines induced early fibrosis-associated events, including upregulation of genes associated with fibrotic induction and endothelial cell activation (i.e., collagen I, α-SMA, and ICAM) together with destruction of tissue architecture and organoid shrinkage.ConclusionOur results demonstrate that mLOs can reproduce parenchymal and non-parenchymal cell interactions and suggest that their application can advance the precise modeling of liver diseases in vitro.

Modulation of Notch Signaling at Early Stages of Differentiation of Human Induced Pluripotent Stem Cells to Dopaminergic Neurons.

Elaboration of protocols for differentiation of human pluripotent stem cells to dopamine neurons is an important issue for development of cell replacement therapy for Parkinson's disease. A number of protocols have been already developed; however, their efficiency and specificity still can be improved. Investigating the role of signaling cascades, important for neurogenesis, can help to solve this problem and to provide a deeper understanding of their role in neuronal development. Notch signaling plays an essential role in development and maintenance of the central nervous system after birth. In our study, we analyzed the effect of Notch activation and inhibition at the early stages of differentiation of human induced pluripotent stem cells to dopaminergic neurons. We found that, during the first seven days of differentiation, the cells were not sensitive to the Notch inhibition. On the contrary, activation of Notch signaling during the same time period led to significant changes and was associated with an increase in expression of genes, specific for caudal parts of the brain, a decrease of expression of genes, specific for forebrain, as well as a decrease of expression of genes, important for the formation of axons and dendrites and microtubule stabilizing proteins.

Notch signaling-induced cyclin d1 in diabetes ameliorating effects of the isolated polysaccharide from Rosa canina: In vitro and in vivo studies.

Notch signaling has a role in the expansion of the pancreas and the pathogenesis of diabetes. Modulation of Notch signaling by natural products seems to pave the way for treating diabetes. This research aimed to scrutinize the involvement of the Notch cascade in the diabetes-ameliorating effects of an isolated polysaccharide from Rosa canina. The isolated polysaccharide was characterized using Fourier transform infrared, nuclear magnetic resonance, high-performance gel-permeation chromatography, and liquid chromatography with tandem mass spectrometry techniques. Rat pancreatic β cells and STZ-induced diabetic rats were treated with the isolated polysaccharide. MTT assay, cell cycle analysis, quantative realtime-polymerase chain reaction, immunohistochemistry, and immunoblotting were used to reveal the growth and the expression levels of Notch1, DLL4, Jagged-1, hes1, Ins-1, Pdx-1, and cyclin d1 in treated and untreated pancreatic cells and tissues. The ameliorating effect of the polysaccharide in STZ-treated cells was accomplished by upregulation of cyclin d1 and hes1 as well as cell cycle progression. Notch inhibition by LY-411575 was associated with the downregulation of cyclin d1 which upregulates with polysaccharide treatment. The significant expression of cyclin d1 (90%) and nuclear expression of hes1 in the pancreas of the polysaccharide group were accompanied by improvement of hyperglycemia and associated biochemical factors as well as regeneration of islet cells as compared to untreated diabetic rats. Based on these findings, upregulation of Notch signaling-induced cyclin d1 could be proposed as the underlying diabetes-reducing effects of the isolated polysaccharide derivative implying that cyclin d1 actuation through activation of the Notch-DLL4 circuit may play the causal role in the treatment of diabetes.

Sex-related differential susceptibility to ponatinib-induced cardiotoxicity and its relationship to modulation of Notch signaling in a murine model

Sex-related differential susceptibility to ponatinib-induced cardiotoxicity and its relationship to modulation of Notch signaling in a murine model

Maternal and zygotic ZFP57 regulate coronary vascular formation and myocardium maturation in mouse embryo.

Genomic and epigenomic dynamics both play critical roles during embryogenesis. Zfp57 maintains genomic imprinting with both maternal and zygotic functions. In our previous study, we found that maternal and zygotic Zfp57 modulate NOTCH signaling during cardiac development. In this study, we investigated Zfp57 mutants from E11.5 to E13.5 to delineate its function during cardiac development. Here, we describe novel roles of maternal and zygotic Zfp57 during cardiovascular system development. We found that maternal and zygotic Zfp57 was required for coronary vascular development. Maternal and zygotic loss of Zfp57 perturbed the sprouting of the sinus venosus-derived endothelial cells and led to underdeveloped coronary vasculature, meanwhile, there was an ectopic overproduction of blood islands over the ventricles. Furthermore, loss of Zfp57 and failed vasculature disturbed myocardium maturation. Loss of maternal and zygotic Zfp57 resulted in hyper trabeculation and failed myocardium compaction. Zfp57 zygotic mutant (M+ Z- ) hearts displayed noncompaction cardiomyopathy at E18.5. Our results suggest that maternal and zygotic ZFP57 are essential for coronary vascular formation and myocardium maturation in mice. Our research provides evidence for the role of genomic imprinting during embryogenesis.

Affinity-matured DLL4 ligands as broad-spectrum modulators of Notch signaling.

The Notch pathway regulates cell fate decisions and is an emerging target for regenerative and cancer therapies. Recombinant Notch ligands are attractive candidates for modulating Notch signaling; however, their intrinsically low receptor-binding affinity restricts their utility in biomedical applications. To overcome this limitation, we evolved variants of the ligand Delta-like 4 with enhanced affinity and cross-reactivity. A consensus variant with maximized binding affinity, DeltaMAX, binds human and murine Notch receptors with 500- to 1,000-fold increased affinity compared with wild-type human Delta-like 4. DeltaMAX also potently activates Notch in plate-bound, bead-bound and cellular formats. When administered as a soluble decoy, DeltaMAX inhibits Notch in reporter and neuronal differentiation assays, highlighting its dual utility as an agonist or antagonist. Finally, we demonstrate that DeltaMAX stimulates increased proliferation and expression of effector mediators in T cells. Taken together, our data define DeltaMAX as a versatile tool for broad-spectrum activation or inhibition of Notch signaling.

Dbx1 controls the development of astrocytes of the intermediate spinal cord by modulating Notch signaling.

Significant progress has been made in elucidating the basic principles that govern neuronal specification in the developing central nervous system. In contrast, much less is known about the origin of astrocytic diversity. Here, we demonstrate that a restricted pool of progenitors in the mouse spinal cord, expressing the transcription factor Dbx1, produces a subset of astrocytes, in addition to interneurons. Ventral p0-derived astrocytes (vA0 cells) exclusively populate intermediate regions of spinal cord with extraordinary precision. The postnatal vA0 population comprises gray matter protoplasmic and white matter fibrous astrocytes and a group of cells with strict radial morphology contacting the pia. We identified that vA0 cells in the lateral funiculus are distinguished by the expression of reelin and Kcnmb4. We show that Dbx1 mutants have an increased number of vA0 cells at the expense of p0-derived interneurons. Manipulation of the Notch pathway, together with the alteration in their ligands seen in Dbx1 knockouts, suggest that Dbx1 controls neuron-glial balance by modulating Notch-dependent cell interactions. In summary, this study highlights that restricted progenitors in the dorsal-ventral neural tube produce region-specific astrocytic subgroups and that progenitor transcriptional programs highly influence glial fate and are instrumental in creating astrocyte diversity.

Inhibition of Notch Signaling Stimulates Osteoclastogenesis From the Common Trilineage Progenitor Under Inflammatory Conditions.

Osteoclasts, macrophages and dendritic cells (DCs) can be derived from a common trilineage myeloid progenitor of hematopoietic origin. Progenitor commitment is susceptible to regulation through Notch signaling. Our aim was to determine the effects of Notch modulation on trilineage progenitor commitment and functional properties of differentiated cells under inflammatory conditions. We used the conditional inducible CX3CR1CreERT2 mouse strain to achieve overexpression of the Notch 1 intracellular domain (NICD1) or to inhibit Notch signaling via deletion of the transcription factor RBP-J in a bone marrow population, used as a source of the trilineage progenitor (CD45+Ly6G−CD3−B220−NK1.1−CD11b–/loCD115+). Cre-recombinase, under the control of the CX3CR1 promoter, expressed in the monocyte/macrophage lineage, was induced in vitro by 4-hydroxytamoxifen. Differentiation of osteoclasts was induced by M-CSF/RANKL; macrophages by M-CSF; DCs by IL-4/GM-CSF, and inflammation by LPS. Functionally, DCs were tested for the ability to process and present antigen, macrophages to phagocytose E. coli particles, and osteoclasts to resorb bone and express tartrate-resistant acid phosphatase (TRAP). We found that Notch 1 signal activation suppressed osteoclast formation, whereas disruption of the Notch canonical pathway enhanced osteoclastogenesis, resulting in a higher number and size of osteoclasts. RANK protein and Ctsk gene expression were upregulated in osteoclastogenic cultures from RBP-J+ mice, with the opposing results in NICD1+ mice. Notch modulation did not affect the number of in vitro differentiated macrophages and DCs. However, RBP-J deletion stimulated Il12b and Cd86 expression in macrophages and DCs, respectively. Functional assays under inflammatory conditions confirmed that Notch silencing amplifies TRAP expression by osteoclasts, whereas the enhanced phagocytosis by macrophages was observed in both NICD1+ and RBP-J+ strains. Finally, antigen presentation by LPS-stimulated DCs was significantly downregulated with NICD1 overexpression. This experimental setting allowed us to define a cell-autonomous response to Notch signaling at the trilineage progenitor stage. Although Notch signaling modulation affected the activity of all three lineages, the major effect was observed in osteoclasts, resulting in enhanced differentiation and function with inhibition of canonical Notch signaling. Our results indicate that Notch signaling participates as the negative regulator of osteoclast activity during inflammation, which may be relevant in immune and bone diseases.

Modulation of Notch Signaling Pathway by Bioactive Dietary Agents.

Notch signaling is often aberrantly activated in solid and hematological cancers and regulates cell fate decisions and the maintenance of cancer stem cells. In addition, increased expression of Notch pathway components is clinically associated with poorer prognosis in several types of cancer. Targeting Notch may have chemopreventive and anti-cancer effects, leading to reduced disease incidence and improved survival. While therapeutic agents are currently in development to achieve this goal, several researchers have turned their attention to dietary and natural agents for targeting Notch signaling. Given their natural abundance from food sources, the use of diet-derived agents to target Notch signaling offers the potential advantage of low toxicity to normal tissue. In this review, we discuss several dietary agents including curcumin, EGCG, resveratrol, and isothiocyanates, which modulate Notch pathway components in a context-dependent manner. Dietary agents modulate Notch signaling in several types of cancer and concurrently decrease in vitro cell viability and in vivo tumor growth, suggesting a potential role for their clinical use to target Notch pathway components, either alone or in combination with current therapeutic agents.

Notch signaling enhances bone regeneration in the zebrafish mandible.

Loss or damage to the mandible caused by trauma, treatment of oral malignancies, and other diseases is treated using bone-grafting techniques that suffer from numerous shortcomings and contraindications. Zebrafish naturally heal large injuries to mandibular bone, offering an opportunity to understand how to boost intrinsic healing potential. Using a novel her6:mCherry Notch reporter, we show that canonical Notch signaling is induced during the initial stages of cartilage callus formation in both mesenchymal cells and chondrocytes following surgical mandibulectomy. We also show that modulation of Notch signaling during the initial post-operative period results in lasting changes to regenerate bone quantity one month later. Pharmacological inhibition of Notch signaling reduces the size of the cartilage callus and delays its conversion into bone, resulting in non-union. Conversely, conditional transgenic activation of Notch signaling accelerates conversion of the cartilage callus into bone, improving bone healing. Given the conserved functions of this pathway in bone repair across vertebrates, we propose that targeted activation of Notch signaling during the early phases of bone healing in mammals may both augment the size of the initial callus and boost its ossification into reparative bone.

Research progress of Notch signaling pathway in spinal cord injury

Spinal cord injury is a severe central nervous system disease, which will cause a series of complex pathophysiological changes and activate a variety of signaling pathways including Notch signaling. Studies have evidenced that activation of the Notch signaling pathway is not conducive to nerve repair and symptom improvement after spinal cord injury. Its mechanisms include inhibiting neuronal differentiation and axon regeneration, promoting reactive astrocyte proliferation, promoting M1 macrophage polarization and the release of proinflammatory factors, and inhibiting angiogenesis. Therefore, it has become a promising therapeutic strategy to inhibit Notch signal as a target in the treatment of spinal cord injury. In recent years, some researchers have used drugs, cell transplantation or genetic modification to regulate Notch signaling, which can promote the recovery of nerve function after spinal cord injury, thereby providing new treatment strategies for the treatment of spinal cord injury. This article will summarize the mechanism of Notch signaling pathway in spinal cord injury, and at the same time review the research progress in the treatment of spinal cord injury by modulating Notch signaling pathway in recent years, so as to provide new research ideas for further exploring new strategies for spinal cord injury.

Notch Signaling in HSC Emergence: When, Why and How.

The hematopoietic stem cell (HSC) sustains blood homeostasis throughout life in vertebrates. During embryonic development, HSCs emerge from the aorta-gonads and mesonephros (AGM) region along with hematopoietic progenitors within hematopoietic clusters which are found in the dorsal aorta, the main arterial vessel. Notch signaling, which is essential for arterial specification of the aorta, is also crucial in hematopoietic development and HSC activity. In this review, we will present and discuss the evidence that we have for Notch activity in hematopoietic cell fate specification and the crosstalk with the endothelial and arterial lineage. The core hematopoietic program is conserved across vertebrates and here we review studies conducted using different models of vertebrate hematopoiesis, including zebrafish, mouse and in vitro differentiated Embryonic stem cells. To fulfill the goal of engineering HSCs in vitro, we need to understand the molecular processes that modulate Notch signaling during HSC emergence in a temporal and spatial context. Here, we review relevant contributions from different model systems that are required to specify precursors of HSC and HSC activity through Notch interactions at different stages of development.

3017 – MECHANISMS OF FLOW-DRIVEN TRANSCRIPTIONAL CONTROL OF HEMATOPOIETIC STEM CELL DEVELOPMENT VIA YAP/TAZ REGULATION

Hematopoietic stem cells (HSCs) are born from endothelial cells in the ventral dorsal aorta (VDA) that adopt hematopoietic cell fate during development. Physical forces of wall shear stress (WSS) and cyclic stretch (CS) produced by hemodynamic blood flow in the VDA are required to generate HSCs, but the mechanisms by which these forces are sensed and converted into a “stemness” regulatory program remain unknown. We recently showed that CS from blood flow stimulates activity of the YAP transcription factor (TF) in vitro in human induced pluripotent stem cell-derived CD34+ hemogenic endothelial cells (HE) and in vivo in zebrafish embryos to promote HSC production. YAP, and its paralogous TF TAZ/WWTR1, are members of the Hippo signaling pathway, initiate transcriptional responses downstream of mechanical stimuli, and require DNA binding cofactors to direct activation or repression of target genes. Via gene expression analysis and epistasis experiments modulating Notch signaling in zebrafish, we revealed YAP is responsible for maintenance, not initiation, of the hematopoietic program in newly specified HE. Notably, YAP loss-of-function mimics the phenotype of embryos deficient for the hematopoietic master regulator Runx1, with upregulation of the Notch ligand dll4. YAP/WWTR1 and RUNX TFs can physically interact in certain contexts. Surprisingly, using luciferase-based YAP- and RUNX-responsive reporter assays in HEK293 cells, we saw a potent synergistic effect of TAZ/RUNX1, but not YAP/RUNX1, in transcriptional regulation at RUNX binding elements. These findings position YAP signaling as a key transcriptional regulator of the endothelial-to-hematopoietic transition that produces HSCs from HE, and suggest a previously uninvestigated relationship between alternate utilization of the Hippo TFs and Runx1 that could underly the requirement of hemodynamic forces in this process. Hematopoietic stem cells (HSCs) are born from endothelial cells in the ventral dorsal aorta (VDA) that adopt hematopoietic cell fate during development. Physical forces of wall shear stress (WSS) and cyclic stretch (CS) produced by hemodynamic blood flow in the VDA are required to generate HSCs, but the mechanisms by which these forces are sensed and converted into a “stemness” regulatory program remain unknown. We recently showed that CS from blood flow stimulates activity of the YAP transcription factor (TF) in vitro in human induced pluripotent stem cell-derived CD34+ hemogenic endothelial cells (HE) and in vivo in zebrafish embryos to promote HSC production. YAP, and its paralogous TF TAZ/WWTR1, are members of the Hippo signaling pathway, initiate transcriptional responses downstream of mechanical stimuli, and require DNA binding cofactors to direct activation or repression of target genes. Via gene expression analysis and epistasis experiments modulating Notch signaling in zebrafish, we revealed YAP is responsible for maintenance, not initiation, of the hematopoietic program in newly specified HE. Notably, YAP loss-of-function mimics the phenotype of embryos deficient for the hematopoietic master regulator Runx1, with upregulation of the Notch ligand dll4. YAP/WWTR1 and RUNX TFs can physically interact in certain contexts. Surprisingly, using luciferase-based YAP- and RUNX-responsive reporter assays in HEK293 cells, we saw a potent synergistic effect of TAZ/RUNX1, but not YAP/RUNX1, in transcriptional regulation at RUNX binding elements. These findings position YAP signaling as a key transcriptional regulator of the endothelial-to-hematopoietic transition that produces HSCs from HE, and suggest a previously uninvestigated relationship between alternate utilization of the Hippo TFs and Runx1 that could underly the requirement of hemodynamic forces in this process.

Modulation of Notch signaling and angiogenesis via an isolated polysaccharide from Momordica charantia in diabetic rats.

Given the impact of notch signaling in the modulation of metabolic diseases and normal tissue homeostasis, this study aimed to evaluate whether notch signaling has a role in anti-diabetic and islet regenerative effects of the isolated polysaccharide from Momordica charantia in diabetic rats. The polysaccharide was isolated from M. charantia (MCP) and was characterized by using FTIR and LC-MS/MS. The diabetic model was established by intraperitoneal administration of streptozotocin in male Wistar rats and grouped into control, diabetic, metformin (500mgkg-1 day-1 ), and treatment (10mgkg-1 day-1 ) groups. The levels of Hes1, Notch 1, DLL4, Jagged1, Pdx1, CD34, CD31, and VEGF were analyzed by using immunohistochemistry and real-time PCR. Structural analyses have revealed the polysaccharide structure of the isolated fraction. High blood glucose was normalized by MCP treatment in diabetic rats. MCP scaled up the mRNA levels of Ins1, jagged1, Pdx1, and Hes1 while it scaled down the levels of Notch1, Dll4, and the ratio of Bax/Bcl2 in diabetic rats. Furthermore, the immunohistochemistry staining levels of hes1, cyclin d1, and VEGF proteins were increased in the pancreas of MCP-treated diabetic rats compared to the diabetic group. These findings provide insights into the anti-diabetic potential of MCP through modulation of islets' regeneration and suggest that modulation of notch and angiogenesis pathways may play a pivotal role in the restoration of the islets to relieve diabetes. PRACTICAL APPLICATIONS: Polysaccharides extracted from Momordica charantia could normalize the level of blood glucose in STZ-induced type 2 diabetic rats through modulation of notch and angiogenesis singling pathways. Given that this effect was associated with the increased expression of Pdx-1 and Insulin in the pancreas, the isolated polysaccharide is expected to be introduced as a convenient medicine in the treatment of diabetes through modulation of β-cell regeneration.

12 Sex-related differential susceptibility to ponatinib-induced cardiotoxicity and its relationship to modulation of notch signalling in a muine model

Abstract Aims Ponatinib (PON), a tyrosine kinase inhibitor approved in chronic myeloid leukaemia, has proven cardiovascular toxicity. Although sex is a risk factor for PON-induced cardiotoxicity in humans, little is known about its mechanisms, in general, and sex-related mechanisms in particular. To determine the mechanisms of sex-related PON-induced cardiotoxicity and identify potential rescue strategies in a murine model. Methods Twenty-four-month-old male and female C57B5 mice were treated with 3 mg/kg/day of PON or vehicle via oral gavage for 28 days, with/without siRNA-Notch1 or siRNA-scrambled via tail vein every 3 days. Results PON + scrambled siRNA-treated male mice had a higher number of TUNEL-positive cells, a higher percentage of senescence-associated β-galactosidase positive senescent cardiac areas, as well as a lower reactivity degree for the survival marker Bmi1 than female counterparts. Proteomics analysis of cardiac tissue showed upstream activation of nitric oxide synthase (NOS) type 2, downstream activation of cell death and production of reactive oxygen species in PON + scrambled siRNA- compared to vehicle or PON + Notch1 siRNA-treated male mice. Upstream analysis showed beta-estradiol activation, while downstream analysis showed activation of cell survival and inhibition of cell death in PON + scrambled siRNA compared to vehicle-treated female mice. PON + scrambled siRNA-treated mice also showed a down-regulation of cardiac actin, which was more marked in male; as well as vessel density, which was more marked in female mice. Female hearts showed a greater extent of cardiac fibrosis than male counterparts at baseline, with no significant changes after PON treatment. In contrast, PON + scrambled siRNA-treated mice had less fibrosis than vehicle or PON + Notch1-siRNA-treated mice. Left ventricular systolic dysfunction shown in PON + scrambled siRNA-treated male mice and—to a lesser extent—by female mice was similarly reversed in both PON + Notch1-siRNA-treated male and female mice (Table 1). Conclusions We found a sex-related differential susceptibility and Notch1 modulation in PON-induced cardiotoxicity. This can improve our understanding of sex-related differences and help identify biomarkers in PON cardiotoxicity.

Regulatory Roles of the Notch Signaling Pathway in Liver Repair and Regeneration: A Novel Therapeutic Target.

The liver is one of the significant regenerative organs in the body. Nevertheless, underlying molecular mechanisms regulating liver repair and regeneration following resection or damage remain largely unknown. The Notch signaling pathway is a profoundly evolutionarily well-conserved cell signaling system that mostly involves developing multicellular organisms. Malfunctions in this pathway lead to the progression of several liver disorders, including hepatoblastoma (HB), cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC). The Notch pathway plays a fundamental role in cell fate during the embryonic stage's progression to the adult stage in liver tissue. Modulation of Notch signaling may be used in many patients who succumb to cirrhosis due to chronic hepatitis by virus infection. This review describes the underlying mechanisms of the Notch signaling pathway in liver development and regeneration briefly and discusses how this pathway leads to the betterment of liver disorders in the clinic.

RYBP modulates embryonic neurogenesis involving the Notch signaling pathway in a PRC1-independent pattern.

SummaryRYBP (Ring1 and YY1 binding protein), an essential component of the Polycomb repressive complex 1 (PRC1), plays pivotal roles in development and diseases. However, the roles of Rybp in neuronal development remains completely unknown. In the present study, we have shown that the depletion of Rybp inhibits proliferation and promotes neuronal differentiation of embryonic neural progenitor cells (eNPCs). In addition, Rybp deficiency impairs the morphological development of neurons. Mechanistically, Rybp deficiency does not affect the global level of ubiquitination of H2A, but it inhibits Notch signaling pathway in eNPCs. The direct interaction between RYBP and CIR1 facilitates the binding of RBPJ to Notch intracellular domain (NICD) and consequently activated Notch signaling. Rybp loss promotes CIR1 competing with RBPJ to bind with NICD, and inhibits Notch signaling. Furthermore, ectopic Hes5, Notch signaling downstream target, rescues Rybp-deficiency-induced deficits. Collectively, our findings show that RYBP regulates embryonic neurogenesis and neuronal development through modulating Notch signaling in a PRC1-independent manner.

Sex-related differential susceptibility to ponatinib-induced cardiotoxicity and its relationship to modulation of Notch signaling in a murine model

Abstract Background Ponatinib (PON), a tyrosine kinase inhibitor approved in chronic myeloid leukemia, has proven cardiovascular toxicity. Although sex is a risk factor for PON-induced cardiotoxicity in humans, little is known about its mechanisms in general, and sex-related mechanisms in particular. Objectives To determine the mechanisms of sex-related PON-induced cardiotoxicity and identify potential rescue strategies in a murine model. Methods 24-months-old male and female C57B5 mice were treated with 3 mg/kg/day of PON or vehicle via oral gavage for 28 days, with/without siRNA-Notch1 or siRNA-scrambled via tail vein every 3 days. Results PON + scrambled siRNA-treated male mice had a higher number of TUNEL-positive cells, a higher percentage of senescence-associated β-galactosidase positive senescent cardiac areas, as well as a lower reactivity degree for the survival marker Bmi1 than female counterparts. Proteomics analysis of cardiac tissue showed upstream activation of nitric oxide synthase (NOS) type 2, downstream activation of cell death and production of reactive oxygen species in PON + scrambled siRNA- compared to vehicle or PON + Notch1 siRNA-treated male mice. Upstream analysis showed beta-estradiol activation, while downstream analysis showed activation of cell survival and inhibition of cell death in PON + scrambled siRNA compared to vehicle-treated female mice. PON + scrambled siRNA-treated mice also showed a downregulation of cardiac actin, which was more marked in male; as well as vessel density, which was more marked in female mice. Female hearts showed a greater extent of cardiac fibrosis than male counterparts at baseline, with no significant changes after PON treatment. In contrast, PON + scrambled siRNA-treated mice had less fibrosis than vehicle or PON + Notch1-siRNA-treated mice. Left ventricular systolic dysfunction shown in PON + scrambled siRNA-treated male mice and - to a lesser extent - by female mice was similarly reversed in both PON + Notch1-siRNA-treated male and female mice (Table 1). Conclusions We found a sex-related differential susceptibility and Notch1 modulation in PON-induced cardiotoxicity. This can improve our understanding of sex-related differences and help identify biomarkers in PON cardiotoxicity. Funding Acknowledgement Type of funding sources: None.

Bioengineering Systems for Modulating Notch Signaling in Cardiovascular Development, Disease, and Regeneration.

The Notch intercellular signaling pathways play significant roles in cardiovascular development, disease, and regeneration through modulating cardiovascular cell specification, proliferation, differentiation, and morphogenesis. The dysregulation of Notch signaling leads to malfunction and maldevelopment of the cardiovascular system. Currently, most findings on Notch signaling rely on animal models and a few clinical studies, which significantly bottleneck the understanding of Notch signaling-associated human cardiovascular development and disease. Recent advances in the bioengineering systems and human pluripotent stem cell-derived cardiovascular cells pave the way to decipher the role of Notch signaling in cardiovascular-related cells (endothelial cells, cardiomyocytes, smooth muscle cells, fibroblasts, and immune cells), and intercellular crosstalk in the physiological, pathological, and regenerative context of the complex human cardiovascular system. In this review, we first summarize the significant roles of Notch signaling in individual cardiac cell types. We then cover the bioengineering systems of microfluidics, hydrogel, spheroid, and 3D bioprinting, which are currently being used for modeling and studying Notch signaling in the cardiovascular system. At last, we provide insights into ancillary supports of bioengineering systems, varied types of cardiovascular cells, and advanced characterization approaches in further refining Notch signaling in cardiovascular development, disease, and regeneration.

NOTCH-mediated exvivo expansion of human hematopoietic stem and progenitor cells by culture under hypoxia.

SummaryActivation of NOTCH signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with an engineered Delta-like ligand (DELTA1ext-IgG [DXI]) has enabled ex vivo expansion of short-term HSPCs, but the effect on long-term repopulating hematopoietic stem cells (LTR-HSCs) remains uncertain. Here, we demonstrate that ex vivo culture of human adult HSPCs with DXI under low oxygen tension limits ER stress in LTR-HSCs and lineage-committed progenitors compared with normoxic cultures. A distinct HSC gene signature was upregulated in cells cultured with DXI in hypoxia and, after 21 days of culture, the frequency of LTR-HSCs increased 4.9-fold relative to uncultured cells and 4.2-fold compared with the normoxia + DXI group. NOTCH and hypoxia pathways intersected to maintain undifferentiated phenotypes in cultured HSPCs. Our work underscores the importance of mitigating ER stress perturbations to preserve functional LTR-HSCs in extended cultures and offers a clinically feasible platform for the expansion of human HSPCs.