Inhibition Of Notch Signaling Research Articles

Immune Cell Plasticity Allows for Resetting of Phenotype From Effector to Regulator With Combined Inhibition of Notch/eIF5A Pathways.

Type 1 diabetes (T1D) results from the destruction of pancreatic β-cells caused by an altered immune balance in the pancreatic microenvironment. In humans as well as in mouse models, T cells are well recognized as key orchestrators of T1D, which is characterized by T helper (Th) 1 and Th17 cell bias and/or low/defective T-regulatory cells (Treg), and culminates in cytotoxic T-cell (CTL)-mediated destruction of β-cells. Refitting of immune cells toward the non-inflammatory phenotype in the pancreas may represent a way to prevent/treat T1D. Recently we developed a unique spontaneous humanized mouse model of type 1 diabetes, wherein mouse MHC-II molecules were replaced by human DQ8, and β-cells were made to express human glutamic acid decarboxylase (GAD) 65 auto-antigen. The mice spontaneously developed T1D resembling the human disease. Humanized T1D mice showed hyperglycemic (250–300 mg/dl) symptoms by the 4th week of life. The diabetogenic T cells (CD4, CD8) present in our model are GAD65 antigen-specific in nature. Intermolecular antigen spreading recorded during 3rd–6th week of age is like that observed in the human preclinical period of T1D. In this paper, we tested our hypothesis in our spontaneous humanized T1D mouse model. We targeted two cell-signaling pathways and their inhibitions: eIF5A pathway inhibition influences T helper cell dynamics toward the non-inflammatory phenotype and Notch signaling inhibition enrich Tregs and targets auto-reactive CTLs, rescues the pancreatic islet structure, and increases the functionality of β-cells in terms of insulin production. We report that inhibition of (eIF5A + Notch) signaling mediates suppression of diabetogenic T cells by inducing plasticity in CD4 + T cells co-expressing IL-17 and IFNγ (IL-17 + IFNγ +) toward the Treg cells phenotype.

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.

Dexamethasone-mediated inhibition of Notch signalling blocks the interaction of leukaemia and mesenchymal stromal cells.

Acute myeloid leukaemia (AML) is a haematological malignancy characterized by a poor prognosis. Bone marrow mesenchymal stromal cells (BM MSCs) support leukaemic cells in preventing chemotherapy-induced apoptosis. This encouraged us to investigate leukaemia-BM niche-associated signalling and to identify signalling cascades supporting the interaction of leukaemic cells and BM MSC. Our study demonstrated functional differences between MSCs originating from leukaemic (AML MSCs) and healthy donors (HD MSCs). The direct interaction of leukaemic and AML MSCs was indispensable in influencing AML cell proliferation. We further identified an important role for Notch expression and its activation in AML MSCs contributing to the enhanced proliferation of AML cells. Supporting this observation, overexpression of the intracellular Notch domain (Notch ICN) in AML MSCs enhanced AML cells' proliferation. From a therapeutic point of view, dexamethasone treatment impeded Notch signalling in AML MSCs resulting in reduced AML cell proliferation. Concurrent with our data, Notch inhibitors had only a marginal effect on leukaemic cells alone but strongly influenced Notch signalling in AML MSCs and abrogated their cytoprotective function on AML cells. In vivo, dexamethasone treatment impeded Notch signalling in AML MSCs leading to a reduced number of AML MSCs and improved survival of leukaemic mice. In summary, targeting the interaction of leukaemic cells and AML MSCs using dexamethasone or Notch inhibitors might further improve treatment outcomes in AML patients.

Abstract 10425: Epigenetic Regulation of Notch Signaling by Prenatal Alcohol Exposure Impacts Cardiac Outflow Tract Defect Development

Introduction: Prenatal alcohol exposure (PAE) leads to congenital heart defects (CHD) with incomplete penetrance and phenotypic heterogeneity. We have demonstrated a gene-environment interaction in this context wherein acute PAE synergizes with second heart field (SHF) haploinsufficiency of Notch signaling, resulting in highly penetrant outflow tract (OFT) defects. Hypothesis: We hypothesize that acute PAE-induced hyperacetylation at histone marker 3 lysine residue 9 (aH3K9) epigenetically inhibits Notch signaling. This potentiates the impact of a pre-existing genetic defect that results in partial loss of Notch signaling leading to loss of viability of SHF progenitors necessary for OFT elongation and alignment. Methods: We studied the effects of acute PAE in vivo in our murine model (two injections of 3g/kg 30% ethanol on E6.5) on acetylation, SHF viability, OFT length, and Notch intracellular domain (NICD) localization. Complimentarily, we evaluated alcohol exposure in vitro using our established thoracic organ culture model. Acetylation was reversed using pan-histone acetyltransferase inhibitor anacardic acid. Results: At E9.5, acute PAE resulted in a significant increase in H3K9 acetylation in SHF associated with restriction of NICD to the cell membrane. This resulted in a 0.28-fold reduction in Notch signaling, as evidenced by reduced Notch reporter expression, and 0.52-fold reduction in SHF cell proliferation (both p<0.001). Loss of SHF progenitors needed for incorporation into the developing OFT reduced OFT length 26% (p<0.001). In vitro thoracic organ culture analyses recapitulated increased acetylation, NICD restriction to cell membrane and reduced SHF proliferation by 60% (p<0.001). Anacardic acid reversed the increase of aH3K9 and allowed NICD nuclear translocation. Notch signaling and SHF proliferation returned to baseline. Conclusions: Acute PAE induces hyperacetylation in the SHF and inhibits Notch signaling by restricting NICD translocation. Lack of Notch signaling reduces SHF viability and prevents elongation of the developing OFT. This hereto unexplored impact of alcohol on the Notch pathway provides a novel area of investigation regarding gene-environment interaction in alcohol teratogenesis.

Abstract 12266: Endothelial Cells Senescence Exacerbates Pulmonary Hypertension Through Notch-Induced Pulmonary Arterial Smooth Muscle Cells Proliferation and Migration

Background: Endothelial cell (EC) dysfunction is critically involved in the pathogenesis of pulmonary arterial hypertension (PAH) by mediating structural changes in vasculatures. Various stress that promotes EC dysfunction also induces premature senescence in EC; however, the role of EC senescence in the development of PAH remains unknown. Hypothesis: We hypothesized that senescent ECs play a significant role in the development of PAH. Methods: We generated EC-specific progeroid mice that overexpress the dominant-negative form of telomere repeat binding factor 2 under the control of the VE-cadherin promoter. Following chronic hypoxia exposure, PH phenotypes were assessed by RVSP and lung histology. Gene expression was assessed by RT-qPCR. The interaction of pulmonary artery ECs (PAECs) and pulmonary artery smooth muscle cells (PASMCs) was analyzed using indirect and direct co-culture systems. In addition, gamma-secretase inhibitor (DAPT) was used to inhibit Notch signaling both in the in-vitro and in-vivo studies. Results: The transgene expression was specifically expressed and fairly distributed in various pulmonary blood vessels in EC-specific progeroid mice. After three weeks of hypoxia exposure, EC-specific progeroid mice exhibited exacerbated pulmonary hypertension (PH) phenotype, accompanied by the prominent medial thickening and enhanced proliferation capacity of SMCs in the distal pulmonary arteries. Interestingly, contact-mediated interaction with senescent ECs increased proliferation and migration capacities in SMCs, while the absence of EC-SMC contact abolished those effects. Mechanistically, senescent ECs expressed higher Notch ligands than in young ECs, leading to the enhanced Notch signaling in PASMCs. Consistently, pharmacological inhibition of Notch signaling attenuated senescent ECs’ effects on SMCs in vitro and the PH phenotype in EC-specific progeroid mice in vivo. Conclusions: These findings established the critical roles of senescent ECs in the PAH pathogenesis, suggesting a novel pathological-targeted therapeutic approach for PAH.

Identification of 1β,2α-epoxytagitinin C as a Notch inhibitor, oxidative stress mechanism and its anti-leukemia activity.

Notch signaling plays crucial roles in cell differentiation and proliferation, but aberrant activation of this signaling results in tumorigenesis and cancer progression. Notch signaling is thus a promising drug target for oncotherapy, and the development of Notch signaling inhibitors is eagerly awaited. Notch inhibitory activity-guided fractionation of a Spilanthes acmella extract led to the identification of five sesquiterpene lactones: tagitinin A (1), 1β,2α-epoxytagitinin C (2), tagitinin C (3), orizabin (4), and 2α-hydroxytirotundin (5). 1β,2α-Epoxytagitinin C (2) exhibited Notch signaling inhibition, with an IC50 of 25.6μM, and was further evaluated for its activity against HPB-ALL, a Notch-activated leukemia cell line. Compound 2 showed potent cytotoxicity against HPB-ALL (IC50 1.7μM) and arrested the cell cycle at the G2/M phase, but did not induce apoptotic cell death. Notch inhibitory mechanism analysis suggested that compound 2 transcriptionally suppresses Notch1 mRNA. In addition, we found that oxidative stress induction is critical for Notch signaling inhibition and the cytotoxicity of compound 2. This is the first mechanism of small molecule Notch inhibition. Our results demonstrate that 1β,2α-epoxytagitinin C (2) is a potential anti-leukemia agent and further investigation of this compound is warranted.

Fetal growth restriction impairs hippocampal neurogenesis and cognition via Tet1 in offspring.

Fetal growth restriction (FGR) increases the risk for impaired cognitive function later in life. However, the precise mechanisms remain elusive. Using dexamethasone-induced FGR and protein restriction-influenced FGR mouse models, we observe learning and memory deficits in adult FGR offspring. FGR induces decreased hippocampal neurogenesis from the early post-natal period to adulthood by reducing the proliferation of neural stem cells (NSCs). We further find a persistent decrease of Tet1 expression in hippocampal NSCs of FGR mice. Mechanistically, Tet1 downregulation results in hypermethylation of the Dll3 and Notch1 promoters and inhibition of Notch signaling, leading to reduced NSC proliferation. Overexpression of Tet1 activates Notch signaling, offsets the decline in neurogenesis, and enhances learning and memory abilities in FGR offspring. Our data indicate that a long-term decrease in Tet1/Notch signaling in hippocampal NSCs contributes to impaired neurogenesis following FGR and could serve as potential targets for the intervention of FGR-related cognitive disorders.

Management of intrauterine adhesions using human amniotic mesenchymal stromal cells to promote endometrial regeneration and repair through Notch signalling.

Intrauterine adhesions (IUAs) severely hamper women's reproductive functions. Human amniotic mesenchymal stromal cell (hAMSC) transplantation is effective in treating IUAs. Here, we examined the function of Notch signalling in IUA treatment with hAMSC transplantation. Forty‐five Sprague‐Dawley female rats were randomly divided into the sham operation, IUA, IUA + E2, IUA + hAMSCs and IUA + hAMSCs + E2 groups. After IUA induction in the rats, hAMSCs promoted endometrial regeneration and repair via differentiation into endometrial epithelial cells. In all groups, the expression of key proteins in Notch signalling was detected in the uterus by immunohistochemistry. The results indicated Notch signalling activation in the hAMSCs and hAMSCs + E2 groups. We could also induce hAMSC differentiation to generate endometrial epithelial cells in vitro. Furthermore, the inhibition of Notch signalling using the AdR‐dnNotch1 vector suppressed hAMSC differentiation (assessed by epithelial and mesenchymal marker levels), whereas its activation using the AdR‐Jagged1 vector increased differentiation. The above findings indicate Notch signalling mediates the differentiation of hAMSCs into endometrial epithelial cells, thus promoting endometrial regeneration and repair; Notch signalling could have an important function in IUA treatment.

Propranolol inhibits stemness of hemangioma through Jagged1.

BackgroundPropranolol is used clinically to treat infantile hemangioma (IH), although the exact mechanism that underlies its effectiveness is not fully understood. The Jagged1/Notch signaling pathway is downstream of the β2-adrenergic receptor (β2-AR). Propranolol is a non-selective β2-AR blocker that was shown to inhibit demethylation adrenaline-induced Jagged1 expression. A previous study has shown that propranolol dose-dependently inhibits the growth of IH. However, the effects of propranolol on stemness of IH are not known and are thus addressed in the current study.MethodsWe analyzed the expression of Jagged1 and Notch3 in IH specimens, using genetic tools to alter Notch signaling. The transduced IH cells were treated with different doses of propranolol, and the effects on IH cell proliferation, migration, and potential for tumor sphere formation were investigated. The effects of altered Notch signaling on tumor formation in vivo were also assessed.ResultsNotch3 and Jagged1 were significantly upregulated in IH. Augmented Notch signaling in IH cells increased cell proliferation, migration, the potential for tumor sphere formation and in vivo tumor formation. On the other hand, reduced Notch signaling in IH cells decreased cell proliferation, migration, the potential for tumor sphere formation and in vivo tumor formation.ConclusionsJagged1/Notch signaling regulated the stemness of IH, and propranolol inhibited it through suppression of Notch signaling.

Schwann Cells Are Required for Efficient Corneal Wound Healing

BACKGROUND: Corneal nerves play a crucial role in maintaining corneal health, which includes regulation of activity of limbal stem cell (LSC). Their loss leads to neurotrophic keratopathy (NK), with corneal ulceration, scarring, and ultimately, blindness.1 Having identified nerve-ensheathing Schwann cells (SC) in the corneal limbus, we hypothesize that SCs, via paracrine interaction with LSC, play a key role in corneal epithelial maintenance and healing. In this study we wanted to (1) Define the role of SCs in corneal healing; (2) Determine the paracrine interaction between the limbal SCs and LSC. METHODS: (1) Local corneal ablation of SCs was induced in a genetically modified mouse where the topical application of tamoxifen induced SCs apoptosis.2 The corneal epithelium was then removed with an Amoils brush under anesthesia and fluorescein was used to assess healing over 4 days. (2) We performed single-cell RNA expression analysis of 10,000 cells derived from dissociated rat limbus with droplet-based high throughput 10× Genomics to identify ~3000 genes.3 We used the data to predict possible ligand-receptor interactions between the limbal SCs and LSC. RESULTS: (1) Ablation of SCs impaired corneal wound healing in mouse cornea, suggesting the involvement of SC in innervation-dependent corneal epithelial recovery. (2) Genomic analysis suggested the presence of paracrine crosstalk between SCs and LSCs, and relevant downstream intracellular signaling events in LSCs. The latter included activation of Notch signaling and VEGF-mediated cell migration and inhibition of apoptosis.4 Further expression analysis comparing the limbal region of healthy and wounded corneas indicated significant changes in the expression of jag1, Pdgfa, Tgfb1, and Ptn genes by SCs. All of these genes could potentially play a role in corneal recovery. CONCLUSIONS: Our findings (i) describe the presence of a high volume of SCs at the limbus, located in close spatial vicinity to LSCs, (ii) demonstrate the importance of the limbal SCs for corneal wound healing, and (iii) suggest the presence of paracrine SC-LSC interaction that may be responsible for the limbal nerve-mediated activation of LSCs during homeostasis or the epithelial wound healing after injury. These findings suggest new therapeutic targets for treating NK. REFERENCES: 1. Dua HS, Said DG, Messmer EM, et al. Neurotrophic keratopathy. Prog Retin Eye Res. 2018 Sep;66:107-131. 2. Johnston APW, Naska S, Jones K, et al. Sox2-mediated regulation of adult neural crest precursors and skin repair. Stem Cell Rep. 2013;1(1):38–45. doi:10.1016/j.stemcr.2013.04.004 3. Yuzwa SA, Borrett MJ, Innes BT, et al. Developmental emergence of adult neural stem cells as revealed by single-cell transcriptional profiling. Cell Rep. 2017;21(13):3970–3986. doi:10.1016/j.celrep.2017.12.017 4. Lindsell CE, Shawber CJ, Boulter J, et al. Jagged: A mammalian ligand that activates notch1. Cell. 1995;80(6):909–917. doi:10.1016/0092-8674(95)90294-5

Neuron-Radial Glial Cell Communication via BMP/Id1 Signaling Is Key to Long-Term Maintenance of the Regenerative Capacity of the Adult Zebrafish Telencephalon.

The central nervous system of adult zebrafish displays an extraordinary neurogenic and regenerative capacity. In the zebrafish adult brain, this regenerative capacity relies on neural stem cells (NSCs) and the careful management of the NSC pool. However, the mechanisms controlling NSC pool maintenance are not yet fully understood. Recently, Bone Morphogenetic Proteins (BMPs) and their downstream effector Id1 (Inhibitor of differentiation 1) were suggested to act as key players in NSC maintenance under constitutive and regenerative conditions. Here, we further investigated the role of BMP/Id1 signaling in these processes, using different genetic and pharmacological approaches. Our data show that BMPs are mainly expressed by neurons in the adult telencephalon, while id1 is expressed in NSCs, suggesting a neuron-NSC communication via the BMP/Id1 signaling axis. Furthermore, manipulation of BMP signaling by conditionally inducing or repressing BMP signaling via heat-shock, lead to an increase or a decrease of id1 expression in the NSCs, respectively. Induction of id1 was followed by an increase in the number of quiescent NSCs, while knocking down id1 expression caused an increase in NSC proliferation. In agreement, genetic ablation of id1 function lead to increased proliferation of NSCs, followed by depletion of the stem cell pool with concomitant failure to heal injuries in repeatedly injured mutant telencephala. Moreover, pharmacological inhibition of BMP and Notch signaling suggests that the two signaling systems cooperate and converge onto the transcriptional regulator her4.1. Interestingly, brain injury lead to a depletion of NSCs in animals lacking BMP/Id1 signaling despite an intact Notch pathway. Taken together, our data demonstrate how neurons feedback on NSC proliferation and that BMP1/Id1 signaling acts as a safeguard of the NSC pool under regenerative conditions.

Unique functions for Notch4 in murine embryonic lymphangiogenesis

In mice, embryonic dermal lymphatic development is well understood and used to study gene functions in lymphangiogenesis. Notch signaling is an evolutionarily conserved pathway that modulates cell fate decisions, which has been shown to both inhibit and promote dermal lymphangiogenesis. Here, we demonstrate distinct roles for Notch4 signaling versus canonical Notch signaling in embryonic dermal lymphangiogenesis. Actively growing embryonic dermal lymphatics expressed NOTCH1, NOTCH4, and DLL4 which correlated with Notch activity. In lymphatic endothelial cells (LECs), DLL4 activation of Notch induced a subset of Notch effectors and lymphatic genes, which were distinctly regulated by Notch1 and Notch4 activation. Treatment of LECs with VEGF-A or VEGF-C upregulated Dll4 transcripts and differentially and temporally regulated the expression of Notch1 and Hes/Hey genes. Mice nullizygous for Notch4 had an increase in the closure of the lymphangiogenic fronts which correlated with reduced vessel caliber in the maturing lymphatic plexus at E14.5 and reduced branching at E16.5. Activation of Notch4 suppressed LEC migration in a wounding assay significantly more than Notch1, suggesting a dominant role for Notch4 in regulating LEC migration. Unlike Notch4 nulls, inhibition of canonical Notch signaling by expressing a dominant negative form of MAML1 (DNMAML) in Prox1+ LECs led to increased lymphatic density consistent with an increase in LEC proliferation, described for the loss of LEC Notch1. Moreover, loss of Notch4 did not affect LEC canonical Notch signaling. Thus, we propose that Notch4 signaling and canonical Notch signaling have distinct functions in the coordination of embryonic dermal lymphangiogenesis.

M6A associated TSUC7 inhibition contributed to Erlotinib resistance in lung adenocarcinoma through a notch signaling activation dependent way

BackgroundThe small tyrosine kinase inhibitors (TKIs) subversively altered the lung cancer treatments, but patients will inevitably face the therapy resistance and disease recurrence. We aim to explore the potential roles of non-coding RNAs in sensitizing the TKIs effects. Methods: Multiple cellular and molecular detections were applied to confirm the mechanistic regulations and intracellular connections.ResultsWe explored the specific gene features of candidates in association with resistance, and found that m6A controlled the stemness of EMT features through METTL3 and YTHDF2. The miR-146a/Notch signaling was sustained highly activated in a m6A dependent manner, and the m6A regulator of YTHDF2 suppressed TUSC7, both of which contributed to the resistant features. Functionally, the sponge type of TUSC7 regulation of miR-146a inhibited Notch signaling functions, and affected the cancer progression and stem cells’ renewal in Erlotinib resistant PC9 cells (PC9ER) and Erlotinib resistant HCC827 cells (HCC827ER) cells. The Notch signaling functions manipulated the cMYC and DICER inner cytoplasm, and the absence of either cMYC or DICER1 lead to TUSC7 and miR-146a decreasing respectively, formed the closed circle to maintain the balance.ConclusionPC9ER and HCC827ER cells harbored much more stem-like cells, and the resistance could be reversed by Notch signaling inactivation. The intrinsic miR-146 and TUSC7 levels are monitored by m6A effectors, the alternation of either miR-146 or TUSC7 expression could lead to the circling loop to sustain the new homeostasis. Further in clinics, the combined delivery of TKIs and Notch specific inhibitory non-coding RNAs will pave the way for yielding the susceptibility to targeted therapy in lung cancer.

Targeting Notch to Maximize Chemotherapeutic Benefits: Rationale, Advanced Strategies, and Future Perspectives.

Simple SummaryThe Notch signaling pathway regulates cell proliferation, apoptosis, stem cell self-renewal, and differentiation in a context-dependent fashion both during embryonic development and in adult tissue homeostasis. Consistent with its pleiotropic physiological role, unproper activation of the signaling promotes or counteracts tumor pathogenesis and therapy response in distinct tissues. In the last twenty years, a wide number of studies have highlighted the anti-cancer potential of Notch-modulating agents as single treatment and in combination with the existent therapies. However, most of these strategies have failed in the clinical exploration due to dose-limiting toxicity and low efficacy, encouraging the development of novel agents and the design of more appropriate combinations between Notch signaling inhibitors and chemotherapeutic drugs with improved safety and effectiveness for distinct types of cancer.Notch signaling guides cell fate decisions by affecting proliferation, apoptosis, stem cell self-renewal, and differentiation depending on cell and tissue context. Given its multifaceted function during tissue development, both overactivation and loss of Notch signaling have been linked to tumorigenesis in ways that are either oncogenic or oncosuppressive, but always context-dependent. Notch signaling is critical for several mechanisms of chemoresistance including cancer stem cell maintenance, epithelial-mesenchymal transition, tumor-stroma interaction, and malignant neovascularization that makes its targeting an appealing strategy against tumor growth and recurrence. During the last decades, numerous Notch-interfering agents have been developed, and the abundant preclinical evidence has been transformed in orphan drug approval for few rare diseases. However, the majority of Notch-dependent malignancies remain untargeted, even if the application of Notch inhibitors alone or in combination with common chemotherapeutic drugs is being evaluated in clinical trials. The modest clinical success of current Notch-targeting strategies is mostly due to their limited efficacy and severe on-target toxicity in Notch-controlled healthy tissues. Here, we review the available preclinical and clinical evidence on combinatorial treatment between different Notch signaling inhibitors and existent chemotherapeutic drugs, providing a comprehensive picture of molecular mechanisms explaining the potential or lacking success of these combinations.

Senescent endothelial cells exacerbate pulmonary hypertension through notch-mediated juxtacrine interaction with pulmonary artery smooth muscle cells

Abstract Background Despite recently developed clinical therapies, vascular remodelling in pulmonary arterial hypertension (PAH) progressively worsen. Hemodynamic unloading has been proposed to normalize the remodelled pulmonary vascular structures in the lungs. Recently, it has been reported that cellular senescence was associated with the irreversibility of pulmonary vascular structures after hemodynamic unloading. Purpose This study aims to elucidate the role of senescent endothelial cells (ECs) in the pathogenesis of PAH. Methods We generated EC-specific progeroid mice in which ECs undergo premature senescence by overexpressing the dominant-negative form of telomere repeat-binding factor 2 under the control of the VE-cadherin promoter. Following three weeks of hypoxia exposure, the PH phenotypes were assessed by RVSP, lung histology, and RT-qPCR. The interaction of human pulmonary artery ECs (hPAECs) and human pulmonary artery smooth muscle cells (hPASMCs) was indirectly and directly explored through the co-culture system. Gamma-secretase inhibitor (DAPT) was administrated to inhibit Notch signalling both in the in-vitro and in-vivo study. Results EC-specific progeroid mice showed exacerbated pulmonary hypertension after chronic hypoxia exposure, accompanied by the enhanced medial SMCs proliferation in the distal pulmonary arteries. Contact-mediated interaction with senescent hPAECs increased proliferation and migration capacities in hPASMCs, while no such effects were detected in the absence of ECs-SMCs contact. Consistently, senescent ECs highly expressed Notch ligands, thus activated Notch signalling in hPASMCs, leading to increased Notch target genes in hPASMCs. Pharmacological inhibition of Notch signalling attenuated the enhanced SMCs proliferation and migration induced by senescent hPAECs, as well as the worsened PH phenotypes in EC-specific progeroid mice. Conclusions Our data established a crucial role of senescent ECs in the PAH pathogenesis through the dysregulated SMC functions via juxtacrine signaling. Senescent ECs are attracting targets for further pathological-targeted therapy to cure PAH completely. Funding Acknowledgement Type of funding sources: None.

Notch1 Signaling Contributes to Mechanical Allodynia Associated with Cyclophosphamide-Induced Cystitis by Promoting Microglia Activation and Neuroinflammation.

Aims Notch1 signaling regulates microglia activation, which promotes neuroinflammation. Neuroinflammation plays an essential role in various kinds of pain sensation, including bladder-related pain in bladder pain syndrome/interstitial cystitis (BPS/IC). However, the impact of Notch1 signaling on mechanical allodynia in cyclophosphamide- (CYP-) induced cystitis is unclear. This study is aimed at determining whether and how Notch1 signaling modulates mechanical allodynia of CYP-induced cystitis. Methods CYP was peritoneally injected to establish a bladder pain syndrome/interstitial cystitis (BPS/IC) rat model. A γ-secretase inhibitor, DAPT, was intrathecally injected to modulate Notch1 signaling indirectly. Mechanical withdrawal threshold in the lower abdomen was measured with von Frey filaments using the up-down method. The expression of Notch1 signaling, Iba-1, OX-42, TNF-α, and IL-1β in the L6-S1 spinal dorsal horn (SDH) was measured with Western blotting analysis and immunofluorescence staining. Results Notch1 and Notch intracellular domain (NICD) were both upregulated in the SDH of the cystitis group. Moreover, the expression of Notch1 and NICD was negatively correlated with the mechanical withdrawal threshold of the cystitis rats. Furthermore, treatment with DAPT attenuated mechanical allodynia in CYP-induced cystitis and inhibited microglia activation, leading to decreased production of TNF-α and IL-1β. Conclusion Notch1 signaling contributes to mechanical allodynia associated with CYP-induced cystitis by promoting microglia activation and neuroinflammation. Our study showed that inhibition of Notch1 signaling might have therapeutic value for treating pain symptoms in BPS/IC.

Nr0b1 (DAX1) loss of function in zebrafish causes hypothalamic defects via abnormal progenitor proliferation and differentiation

The nuclear receptor DAX-1, encoded by the NR0B1 gene, is presented in the hypothalamic tissues in humans and other vertebrates. Human patients with NR0B1 mutations often have hypothalamic-pituitary defects, but the involvement of NR0B1 in hypothalamic development and function is not well understood. Here, we report the disruption of the nr0b1 gene in zebrafish causes abnormal expression of gonadotropins, a reduction in fertilization rate, and an increase in postfasting food intake, which are indicators of abnormal hypothalamic functions. We find that loss of nr0b1 increases the number of prodynorphin (pdyn)-expressing neurons but decreases the number of pro-opiomelanocortin (pomcb)-expressing neurons in the zebrafish hypothalamic arcuate region (ARC). Further examination reveals that the proliferation of progenitor cells is reduced in the hypothalamus of nr0b1 mutant embryos accompanying the decreased expression of genes in the Notch signaling pathway. Additionally, the inhibition of Notch signaling in wild-type embryos increases the number of pdyn neurons, mimicking the nr0b1 mutant phenotype. In contrast, ectopic activation of Notch signaling in nr0b1 mutant embryos decreases the number of pdyn neurons. Taken together, our results suggest that nr0b1 regulates neural progenitor proliferation and maintenance to ensure normal hypothalamic neuron development.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Low-density-lipoprotein-receptor-related protein 1 mediates Notch pathway activation.

The Notch signaling pathway controls cell growth, differentiation, and fate decisions, and its dysregulation has been linked to various human genetic disorders and cancers. To comprehensively understand the global organization of the Notch pathway and identify potential drug targets for Notch-related diseases, we established a protein interaction landscape for the human Notch pathway. By combining and analyzing genetic and phenotypic data with bioinformatics analysis, we greatly expanded this pathway and identified many key regulators, including low-density-lipoprotein-receptor-related protein 1 (LRP1). We demonstrated that LRP1 mediates the ubiquitination chain linkage switching of Delta ligands, which further affects ligand recycling, membrane localization, and stability. LRP1 inhibition led to Notch signaling inhibition and decreased tumorigenesis in leukemia models. Our study provides a glimpse into the Notch pathway interaction network and uncovers LRP1 as one critical regulator of the Notch pathway, as well as a possible therapeutic target for Notch-related cancers.

Inhibition of Notch activity promotes pancreatic cytokeratin 5-positive cell differentiation to beta cells and improves glucose homeostasis following acute pancreatitis

Some individuals develop prediabetes and/or diabetes following acute pancreatitis (AP). AP-induced beta-cell injury and the limited regenerative capacity of beta cells might account for pancreatic endocrine insufficiency. Previously, we found that only a few pancreatic cytokeratin 5 positive (Krt5+) cells differentiated into beta cells in the murine AP model, which was insufficient to maintain glucose homeostasis. Notch signaling determines pancreatic progenitor differentiation in pancreas development. This study aimed to examine whether Notch signaling inhibition could promote pancreatic Krt5+ cell differentiation into beta cells and improve glucose homeostasis following AP. Pancreatic tissues from patients with acute necrotizing pancreatitis (ANP) were used to evaluate beta-cell injury, Krt5+ cell activation and differentiation, and Notch activity. The murine AP model was induced by cerulein, and the effect of Notch inhibition on Krt5+ cell differentiation was evaluated both in vivo and in vitro. The results demonstrated beta-cell loss in ANP patients and AP mice. Krt5+ cells were activated in ANP pancreases along with persistently elevated Notch activity, which resulted in the formation of massive duct-like structures. AP mice that received Notch inhibitor showed that impaired glucose tolerance was reversed 7 and 15 days following AP, and increased numbers of newborn small islets due to increased differentiation of Krt5+ cells to beta cells to some extent. In addition, Krt5+ cells isolated from AP mice showed increased differentiation to beta cells by Notch inhibition. Collectively, these findings suggest that beta-cell loss contributes to pancreatic endocrine insufficiency following AP, and inhibition of Notch activity promotes pancreatic Krt5+ cell differentiation to beta cells and improves glucose homeostasis. The findings from this study may shed light on the potential treatment of prediabetes/diabetes following AP.