Notch1 Knockdown Research Articles

Notch3 is involved in adipogenesis of human adipose-derived stromal/stem cells

Human adipose-derived stromal/stem cells (hASCs) have tremendous therapeutic potential and the ability to offer insight into human development and disease. Here we subject human ASCs to siRNA-mediated knockdown of Notch3 cultured under both self-renewing and adipogenic differentiation conditions. Self-renewal was monitored by assessing viability and proliferation rates through staining and alamarBlue assays, respectively. Adipogenesis was measured through Oil-Red O staining, western blot, and quantitative real-time RT-PCR that determined expression levels of multipotency and adipogenic markers over time. Notch3 was expressed in self-renewing hASCs but knockdown, as validated by qRT-PCR and western blot, showed no impact on cell viability, as measured through live-dead staining, or cell proliferation rates, as measured through alamarBlue assays. However, although Notch3 expression was observed to increase during adipogenesis, in the absence of Notch3 there was a significant increase in hASC adipogenesis as demonstrated through an increased number of lipid vesicles, and increased expression of adipogenic markers ppar-γ, adiponectin, fabp4, and plin2. Although Notch3 is only one of four Notch receptors expressed on the surface of hASCs, this receptor appears important for proper regulation of adipogenic differentiation, possibly serving as a negative regulator to prevent inappropriate adipogenesis or promote other lineage commitments of ASCs.

MicroRNA‐944 inhibits migration and invasion in melanoma cells by targeting Notch2

Melanoma is a malignant tumor of melanocytes. The incidence of melanoma is increasing in worldwide. miR‐944 is a primates‐specific microRNA, which plays a role as putative tumor suppressor in cancer cells. However, the function of miR‐944 in melanoma is not reported yet. In this study, we observed that the migration and invasion in melanoma cells transfected with miR‐944 was decreased compared to that in control cells. The expression of cellular invasion‐related genes was decreased in miR‐944 transfected cells. By using bioinformatics program, Notch2 was selected as a target of miR‐944. The overexpression of miR‐944 inhibited the expression of Notch2 in melanoma cells. By reporter assay, we found miR‐944 binding to the 3′UTR of Notch2. Knockdown of Notch2 in melanoma cells also suppressed cellular migration and decreased the expression of cellular invasion‐related genes. Taken together, our results show that miR‐944 suppressed migration of melanoma cells by targeting Notch2. These finding may have important implications concerning the molecular mechanisms of invasion and migration in melanoma.Support or Funding InformationThis research was supported by a grant from Basic Science Research Program through the National Research Foundation of Korea (NRF) (No. 2017R1A2B4009615).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization and Targeting Cancer Stem‐like Cells in Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive diseases. TNBC patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem‐like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Therefore, CSC‐targeted therapies are keenly sought. There is strong evidence for the involvement of canonical‐Notch signaling in TNBC, however the role of non‐canonical Notch signaling in TNBC remains unknown. Notch1 is highly expressed in Basal‐like 1 (BL1) and especially Mesenchymal (M) TNBCs. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Moreover, strong evidence supports key roles of different Notch paralogs in breast CSCs. Here, we demonstrate that Notch activation by Jagged1‐expressing stromal cells enhances transcription of the anti‐apoptotic gene cIAP‐2 (BIRC3), a known NF‐κB target. This event is dependent on recruitment to the cIAP‐2 promoter of NF‐κB subunits, IKKα and Notch1. Short term exposure of MDA‐MB‐231 cells (M, PTEN wild‐type), but not MDA‐MB‐468 cells (BL1, PTEN‐null) to recombinant Jagged1 leads to AKT phosphorylation. This is suppressed by AKT inhibitors, IKK inhibitors, and dual mTORC1/2 inhibitors but not an mTORC1‐selective inhibitor. These observations support a model where canonical and non‐canonical mechanisms downstream of Notch1 trigger AKT phosphorylation and NF‐κB activation in PTEN wild type TNBC cells. Importantly, we find co‐localization of Notch1 with Mitochondria in MDA‐MB‐231 cells by confocal microscopy and Western blot of isolated mitochondrial fractions. We demonstrate that recombinant Jagged1 increases the metabolism of TNBC cells. Knockdown of Notch1 or IKKα by siRNA decreases mitochondrial respiration and glycolysis. CSCs derived from MDA‐MB‐231 cells have increased Notch1, p‐AKT, and oxidative metabolism compared to non‐stem cells. AKT inhibition or IKK inhibition decreases both mitochondrial respiration and glycolysis of TNBC derived CSCs. Pharmacological inhibition of Notch cleavage by gamma secretase inhibitor (PF‐03084014) in combination with AKT inhibitor (MK‐2206) or IKK inhibitor (Bay11‐7082) blocks CD90hi or CD44+CD24low sorted secondary mammospheres formation. Notably, we find similar results in TNBC patient derived xenograft (PDX) models. However, Notch inhibition may have adverse effects on tumor immunity, due to the role of Notch in CD4 and CD8 T‐cell activation. Thus, we explored an immunocompetent tumor mouse model to study the effects of these pharmacological interventions in that setting. We developed an immunocompetent TNBC mouse model by injecting TNBC mouse cells (C0321) into mammary fat pads of isogenic FVB female mice. Combination treatments with GSI (PF‐03084014) and AKT inhibitor (MK‐2206) significantly delayed tumor growth as well as decreased CSCs number in that model. These data suggest that combination treatments affecting the intersection of Notch and AKT pathways have potential therapeutic applications in targeting CSCs in TNBC cases with high Notch1 expression. The possible immunological effects of such combination treatments require additional investigation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of notch-1 knockdown on the proliferation and the differentiation of human osteoarthritis chondrocytes

Effects of notch-1 knockdown on the proliferation and the differentiation of human osteoarthritis chondrocytes

Serum microRNA-139-5p is downregulated in lung cancer patients with lytic bone metastasis.

Bone remodeling can be interrupted by tumor cells which leads to an inappropriate balance of osteoblasts and osteoclasts. As the progenitors of osteoblasts, mesenchymal stem cells (MSCs) have been reported to exhibit an abnormal osteogenic differentiation potential in some cancer‑related bone lesions. However, the evidence is very limited in terms of the biological alterations of MSCs in the bone metastasis of non‑small cell lung cancers (NSCLC). We investigated the expression and function of miR‑139‑5p in MSC osteogenic differentiation invitro in normal and NSCLC-exposed condition. Then, we compared the serum miR‑139‑5p in stageIV lung adenocarcinoma cancer patients with and without lytic bone metastasis. We found that MSCs exhibited a significant increase in miR‑139‑5p expression after exposure to osteogenic differentiation induction medium. However, Notch1, which was confirmed as a target of miR‑139‑5p by luciferase and western blot assays, showed a marked downregulated expression together with its pathway downstream factors during MSC osteogenesis. miR‑139‑5p positively regulated MSC osteogenic differentiation but this effect was abrogated significantly by Notch1 knockdown. After exposure to conditions of lung cancer cells A549 and L9981, MSCs exhibited significant downregulation of miR‑139‑5p expression and early osteogenic marker ALP activity. Furthermore, we demonstrated that the expression of serum miR‑139‑5p from lung adenocarcinoma patients with lytic bone metastasis was significantly lower compared to that in patients with metastases in other organs. The potential roles of miR‑139‑5p as a biomarker and treatment target in monitoring and controlling bone metastasis in lung cancer patients are worthy of being further explored.

Knockdown of MALAT1 enhances chemosensitivity of ovarian cancer cells to cisplatin through inhibiting the Notch1 signaling pathway

Long non-coding RNAs (lncRNAs) are critical regulators in chemoresistance of various tumors including ovarian cancer. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been reported to be upregulated and contributed to ovarian cancer tumorigenesis. The aim of this study was to explore the roles of MALAT1 and the underlying molecular regulatory mechanism in the chemoresistance of ovarian cancer cells. Our data demonstrated that MALAT1 and Notch1 mRNA were upregulated in ovarian cancer tissues, as well as cisplatin (CDDP)-resistant ovarian cancer cells. A positive correlation between MALAT1 and Notch1 mRNA expression was observed. MALAT1 knockdown significantly attenuated CDDP resistance, and enhanced CDDP-induced apoptosis in CDDP-resistant ovarian cancer cells. MALAT1 knockdown enhanced CDDP-induced apoptosis in vivo, as indicated by upregulation of Bax protein expression and downregulation of Bcl-2 protein expression. Additionally, MALAT1 knockdown inhibited the Notch1 pathway and ABCC1 expression in CDDP-resistant ovarian cancer cells. MALAT1 was demonstrated to interact with Notch1. Notch1 knockdown attenuated CDDP resistance, and downregulated the protein expression of ABCC1 in ovarian cancer cells. Taken together, our findings suggested that knockdown of MALAT-1 enhanced chemosensitivity of ovarian cancer cells to CDDP through inhibiting Notch1 signaling pathway.

Cerebral ischemia induces angiogenesis in the peri-infarct regions via Notch1 signaling activation

The Notch1 signaling pathway is considered as one of important regulators of angiogenesis during development, but its role in cerebral ischemia-induced angiogenesis is less well understood. Here, we used human and rodent brains to explore whether Notch1 signaling was involved in the angiogenesis after focal cerebral ischemia. Using immunohistochemistry on surgically resected ischemic stroke brain tissue, we found that the area, volume, and length of the blood vessels in the peri-infarct regions were significantly increased after ischemic stroke in humans, compared with non-ischemic stroke specimens. In addition, the expression of the activated form of Notch1 (Notch intracellular domain; NICD) was increased in endothelial cells of the peri-infarct region. The Notch1 ligand, Jagged1, also increased in abundance in the peri-infarct regions in human. We further confirmed that Notch1 signaling was activated in the peri-infarct regions in a mouse distal middle cerebral artery occlusion (dMCAO) model. Lentivirus-mediated Notch1 knockdown reduced ischemia-induced angiogenesis in the peri-infarct regions of the brain. Our findings suggest that ischemic stroke in human can also induce angiogenesis in the peri-infarct regions as does in animal models of focal ischemia and that Notch1 signaling plays a critical role in mediating this process, which may provide fundamental knowledge regarding the potential mechanisms underlying angiogenesis after ischemic stroke.

Maintenance of hematopoietic stem and progenitor cells in fetal intra-aortic hematopoietic clusters by the Sox17-Notch1-Hes1 axis

The aorta-gonad-mesonephros region, from which definitive hematopoiesis first arises in midgestation mouse embryos, has intra-aortic hematopoietic clusters (IAHCs) containing hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). We previously reported expression of the transcription factor Sox17 in IAHCs, and overexpression of Sox17 in CD45lowc-KIThigh cells comprising IAHCs maintains the formation of cell clusters and their multipotency in vitro over multiple passages. Here, we demonstrate the importance of NOTCH1 in IAHC formation and maintenance of the HSC/HPC phenotype. We further show that Notch1 expression is positively regulated by SOX17 via direct binding to its gene promoter. SOX17 and NOTCH1 were both found to be expressed in vivo in cells of IAHCs by whole mount immunostaining. We found that cells transduced with the active form of NOTCH1 or its downstream target, Hes1, maintained their multipotent colony-forming capacity in semisolid medium. Moreover, cells stimulated by NOTCH1 ligand, Jagged1, or Delta-like protein 1, had the capacity to form multilineage colonies. Conversely, knockdown of Notch1 and Hes1 led to a reduction of their multipotent colony-forming capacity. These results suggest that the Sox17-Notch1-Hes1 pathway is critical for maintaining the undifferentiated state of IAHCs.

Inhibition of Endothelial NOTCH1 Signaling Attenuates Inflammation by Reducing Cytokine-Mediated Histone Acetylation at Inflammatory Enhancers.

Endothelial upregulation of adhesion molecules serves to recruit leukocytes to inflammatory sites and appears to be promoted by NOTCH1; however, current models based on interactions between active NOTCH1 and NF-κB components cannot explain the transcriptional selectivity exerted by NOTCH1 in this context. Observing that Cre/Lox-induced conditional mutations of endothelial Notch modulated inflammation in murine contact hypersensitivity, we found that IL (interleukin)-1β stimulation induced rapid recruitment of RELA (v-rel avian reticuloendotheliosis viral oncogene homolog A) to genomic sites occupied by NOTCH1-RBPJ (recombination signal-binding protein for immunoglobulin kappa J region) and that NOTCH1 knockdown reduced histone H3K27 acetylation at a subset of NF-κB-directed inflammatory enhancers. Our findings reveal that NOTCH1 signaling supports the expression of a subset of inflammatory genes at the enhancer level and demonstrate how key signaling pathways converge on chromatin to coordinate the transition to an infla mmatory endothelial phenotype.

Abstract P6-07-06: Targeting cancer stem-like cells metabolism via non-canonical notch signaling pathways in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive diseases. TNBC patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem-like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Therefore CSC-targeted therapies are keenly sought. There is strong evidence for the involvement of Notch signaling in TNBC. Notch1 is highly expressed in Basal-like 1 (BL1) and especially Mesenchymal-Stem-Like (MSL) TNBCs. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Moreover, strong evidence supports key roles of different Notch paralogs in breast CSCs. Here, we demonstrate that Notch activation by Jagged1-expressing stromal cells enhances transcription of the anti-apoptotic gene cIAP-2 (BIRC3), a known NF-κB target. This event is dependent on recruitment to the cIAP-2 promoter of NF-κB subunits, IKKα and Notch1. Short term exposure of MDA-MB-231 cells (MSL, PTEN wild-type), but not MDA-MB-468 cells (BL1, PTEN-null) to recombinant Jagged1 leads to AKT phosphorylation. This is suppressed by AKT inhibitors, IKK inhibitors, and dual mTORC1/2 inhibitors but not an mTORC1-selective inhibitor. These observations support a model where canonical and non-canonical mechanisms downstream of Notch1 trigger AKT phosphorylation and NF-κB activation in PTEN wild type TNBC cells. Rapid AKT phosphorylation downstream of Notch1 requires mTORC2, PI3K and IKKα, and contributes to NF-κB activation. This suggests a bidirectional crosstalk between the IKKα and AKT arms of this Jagged1-activated pathway. Importantly, we find co-localization of Notch1 with Mitochondria in MDA-MB-231 cells by confocal microscopy and Western blot of isolated mitochondrial fractions. We demonstrate that recombinant Jagged1 increases metabolism of TNBC cells. Knockdown of Notch1 or IKKα by siRNA decreases mitochondrial respiration and glycolysis. CSCs derived from MDA-MB-231 cells have increased Notch1, p-AKT, and oxidative metabolism compared to non-stem cells. AKT inhibition or IKK inhibition decreases both mitochondrial respiration and glycolysis of TNBC derived CSCs. Pharmacological inhibition of Notch cleavage by gamma secretase inhibitor (PF-03084014) in combination with AKT inhibitor (MK-2206) or IKK inhibitor (Bay11-7082) blocks CD90hi or CD44+CD24low sorted secondary mammospheres formation. Notably, we find similar results in TNBC patient derived xenograft (PDX) models. These data suggest that combination treatments affecting the intersection of Notch, NF-kB and AKT pathways have potential therapeutic importance in targeting CSCs in TNBC cases with high Notch1 expression. Citation Format: Hossain F, Sorrentino C, Ucar Bilyeu AD, Matossian M, Crabtree J, Pannuti A, Burow M, Golde T, Osborne B, Miele L. Targeting cancer stem-like cells metabolism via non-canonical notch signaling pathways in triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-07-06.

Notch1 knockdown disturbed neural oscillations in the hippocampus of C57BL mice

Neural oscillations and their interactions are associated with the coordination of neural groups, which provide a mechanism underlying information processing of brain functions. Notch1 receptor is involved in the neurological and psychiatric disorders, such as neurodevelopmental deficits, cerebral ischemia, Alzheimer's disease and depression. Here, we investigated the dynamics of neural oscillations in hippocampus of Notch1+/− mice in urethane-anesthetized state. Notch1 knockdown altered the distribution of power in the hippocampal DG areas, reduced theta (3–8 Hz) power and enhanced low gamma (LG, 30–50 Hz) and high gamma (HG, 50–100 Hz) power. Moreover, theta-gamma phase-amplitude coupling in the hippocampal DG area was markedly impaired in the Notch1+/− mice. The data further showed that the expression of NR2B was decreased, and the expressions of GABAARα1, GAD67 and parvalbumin were considerably increased after Notch1 knockdown. Taken together, our results suggest that Notch1 genetic deficiency significantly impaired the corss-frequency coupling of neural oscillations, and their interactions in the hippocampal DG region by means of disrupting the balance of excitatory and inhibitory receptors, which could be an underlying mechanism of cognitive impairment in neuropsychiatric disorders.

γ-Secretase Inhibition Induces Muscle Hypertrophy in a Notch-Independent Mechanism.

A wide variety of cellular processes and signaling events are regulated by the proteolytic enzyme γ-secretase. Notch-1 is one of the substrates of γ-secretase and its role in the regulation of muscle differentiation has been well described. Importantly, besides Notch-1, a number of proteins have been identified to undergo proteolysis by γ-secretase. To date, the specific role of γ-secretase during embryonic skeletal muscle differentiation has not been studied. Therefore, we address this question through the analysis of in vitro grown chick myogenic cells during the formation of multinucleated myotubes. The γ-secretase inhibitor DAPT (N-N[-(3,5-Difluorophenacetyl-l-alanyl)]-S-328 phenylglycine-t-butyl-ester) induces muscle hypertrophy. Knockdown of Notch-1 using siRNA specific to chick shows no significant effect in myotube size, suggesting that γ-secretase-dependent effects on muscle hypertrophy in chick myogenic cells are Notch-1-independent. We also investigate the effects of γ-secretase inhibition in the whole proteomic profile of chick myogenic cells. We identified 276 differentially expressed proteins from Label-free proteomic approach. Data overview of interaction network obtained from STRING show that after γ-secretase inhibition cells exhibited imbalance in protein metabolism, cytoskeleton/adhesion, and Sonic Hedgehog signaling. The collection of these results provides new insights into the role of γ-secretase in skeletal muscle hypertrophy.

Regulation of pancreatic stellate cell activation by Notch3

BackgroundActivated pancreatic stellate cells (PaSCs) are the key cellular source of cancer-associated fibroblasts in the pancreatic stroma of patients with pancreatic ductal adenocarcinoma (PDAC), however, the activation mechanism of PaSCs is not yet known. The Notch signaling pathway, components of which are expressed in stromal cells, is involved in the fibrosis of several organs, including the lung and liver. In the current study, we investigated whether Notch signal transduction is involved in PaSC activation in PDAC.MethodsThe expression of Notch signaling pathway components in human PDAC was examined via immunohistochemical staining and assessed in mouse PaSCs using RT-qPCR and western blotting. Notch3 expression in both PDAC stromal cells and activated mouse PaSCs was evaluated using immunofluorescence, RT-qPCR and western blotting. The impact of siRNA-mediated Notch3 knockdown on PaSC activation was detected with RT-qPCR and western blotting, and the impact on PaSC proliferation and migration was detected using CCK-8 assays and scratch experiments. The effect of conditioned medium from PaSCs activated with Notch3 siRNA on pancreatic cancer (LTPA) cells was also detected with CCK-8 assays and scratch experiments. The data were analyzed for statistical significance using Student’s t-test.ResultsNotch3 was overexpressed in both human PDAC stromal cells and activated mouse PaSCs, and Notch3 knockdown with Notch3 siRNA decreased the proliferation and migration of mouse PaSCs. The levels of markers related to PaSC activation, such as α-smooth muscle actin (α-SMA), collagen I and fibronectin, decreased in response to Notch3 knockdown, indicating that Notch3 plays an important role in PaSC activation. Furthermore, we confirmed that inhibition of PaSC activation via Notch3 siRNA reduced the proliferation and migration of PaSC-induced mouse pancreatic cancer (LTPA) cells.ConclusionsNotch3 inhibition in PaSCs can inhibit the activation, proliferation and migration of PaSCs and reduce the PaSC-induced pro-tumorigenic effect. Therefore, Notch3 silencing in PaSCs is a potential novel therapeutic option for patients with PDAC.

Inhibition of Notch1 induces population and suppressive activity of regulatory T cell in inflammatory arthritis.

Inhibition of Notch signalling has shown anti-inflammatory properties in vivo and in vitro models of rheumatoid arthritis (RA). The objective of this study was to determine whether Notch1 might play a role in regulating T-regulatory cells (Tregs) in animal models of RA.Methods: Collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA) were induced in C57BL/6, Notch1 antisense transgenic (NAS) or DBA1/J mice. We examined whether pharmacological inhibitors of γ-secretase (an enzyme required for Notch1 activation) and antisense-mediated knockdown of Notch1 could attenuate the severity of inflammatory arthritis in CIA and CAIA mice. Proportions of CD4+CD25+Foxp3+ Treg cells were measured by flow cytometry. To assess the suppressive capacity of Treg toward responder cells, CFSE-based suppression assay of Treg was performed.Results: γ-secretase inhibitors and antisense-mediated knockdown of Notch1 reduced the severity of inflammatory arthritis in both CIA and CAIA mice. Pharmacological and genetic inhibition of Notch1 signalling induced significant elevation of Treg cell population in CIA and CAIA mice. We also demonstrated that inhibition of Notch signalling suppressed the progression of inflammatory arthritis through modulating the expansion and suppressive function of regulatory T (Treg) cells.Conclusion: Pharmacological and genetic inhibition of Notch1 signalling suppresses the progression of inflammatory arthritis through modulating the population and suppressive function of Treg cells in animal models of RA.

Adropin preserves the blood-brain barrier through a Notch1/Hes1 pathway after intracerebral hemorrhage in mice.

Adropin is expressed in the CNS and plays a crucial role in the development of stroke. However, little is currently known about the effects of adropin on the blood-brain barrier (BBB) function after intracerebral hemorrhage (ICH). In this study, the role of adropin in collagenase-induced ICH was investigated in mice. At 1-h post-ICH, mice were administered with recombinant human adropin by intranasal. Brain water +content, BBB permeability, and neurological function were measured at different time intervals. Proteins were quantified using western blot analysis, and the localizations of adropin and Notch1 were visualized via immunofluorescence staining. It is shown that adropin reduced brain water content and improved neurological functions. Adropin preserved the functionality of BBB by increasing N-cadherin expression and reducing extravasation of albumin. Moreover, invivo knockdown of Notch1 and Hes1 both abolished the protective effects of adropin. Taken together, our data demonstrate that adropin constitutes a potential treatment value for ICH by preserving BBB and improving functional outcomes through the Notch1 signaling pathway.

The Pericytic Phenotype of Adipose Tissue-Derived Stromal Cells Is Promoted by NOTCH2.

Long-term diabetes leads to macrovascular and microvascular complication. In diabetic retinopathy (DR), persistent hyperglycemia causes permanent loss of retinal pericytes and aberrant proliferation of microvascular endothelial cells (ECs). Adipose tissue-derived stromal cells (ASCs) may serve to functionally replace retinal pericytes and normalize retinal microvasculature during disease progression. We hypothesized that Notch signaling in ASC underlies regulation and stabilization of dysfunctional retinal microvascular networks such as in DR. ASC prominently and constitutively expressed NOTCH2. Genetic knockdown of NOTCH2 in ASC (SH-NOTCH2) disturbed the formation of vascular networks of human umbilical cord vein endothelial cells both on monolayers of ASC and in organotypical three-dimensional cocultures with ASC. On ASC SH-NOTCH2, cell surface platelet-derived growth factor receptor beta was downregulated which disrupted their migration toward the chemoattractant platelet-derived growth factor beta subunits (PDGF-BB) as well as to conditioned media from EC and bovine retinal EC. This chemoattractant is secreted by pro-angiogenic EC in newly formed microvascular networks to attract pericytes. Intravitreal injected ASC SH-NOTCH2 in oxygen-induced retinopathy mouse eyes did not engraft in the preexisting retinal microvasculature. However, the in vivo pro-angiogenic capacity of ASC SH-NOTCH2 did not differ from controls. In this respect, multifocal electroretinography displayed similar b-wave amplitudes in the avascular zones when either wild type ASC or SH-NOTCH2 ASC were injected. In conclusion, our results indicate that NOTCH2 is essential to support in vitro vasculogenesis via juxtacrine interactions. In contrast, ongoing in vivo angiogenesis is influenced by paracrine signaling of ASC, irrespective of Notch signaling. Stem Cells 2018;36:240-251.

Area-Specific Regulation of Quiescent Neural Stem Cells by Notch3 in the Adult Mouse Subependymal Zone.

In the adult mammalian brain, neural stem cells (NSCs) generate new neurons throughout the mammal's lifetime. The balance between quiescence and active cell division among NSCs is crucial in producing appropriate numbers of neurons while maintaining the stem cell pool for a long period. The Notch signaling pathway plays a central role in both maintaining quiescent NSCs (qNSCs) and promoting cell division of active NSCs (aNSCs), although no one knows how this pathway regulates these apparently opposite functions. Notch1 has been shown to promote proliferation of aNSCs without affecting qNSCs in the adult mouse subependymal zone (SEZ). In this study, we found that Notch3 is expressed to a higher extent in qNSCs than in aNSCs while Notch1 is preferentially expressed in aNSCs and transit-amplifying progenitors in the adult mouse SEZ. Furthermore, Notch3 is selectively expressed in the lateral and ventral walls of the SEZ. Knockdown of Notch3 in the lateral wall of the adult SEZ increased the division of NSCs. Moreover, deletion of the Notch3 gene resulted in significant reduction of qNSCs specifically in the lateral and ventral walls, compared with the medial and dorsal walls, of the lateral ventricles. Notch3 deletion also reduced the number of qNSCs activated after antimitotic cytosine β-D-arabinofuranoside (Ara-C) treatment. Importantly, Notch3 deletion preferentially reduced specific subtypes of newborn neurons in the olfactory bulb derived from the lateral walls of the SEZ. These results indicate that Notch isoforms differentially control the quiescent and proliferative steps of adult SEZ NSCs in a domain-specific manner.SIGNIFICANCE STATEMENT In the adult mammalian brain, the subependymal zone (SEZ) of the lateral ventricles is the largest neurogenic niche, where neural stem cells (NSCs) generate neurons. In this study, we found that Notch3 plays an important role in the maintenance of quiescent NSCs (qNSCs), while Notch1 has been reported to act as a regulator of actively cycling NSCs. Furthermore, we found that Notch3 is specifically expressed in qNSCs located in the lateral and ventral walls of the lateral ventricles and regulates neuronal production of NSCs in a region-specific manner. Our results indicate that Notch3, by maintaining the quiescence of a subpopulation of NSCs, confers a region-specific heterogeneity among NSCs in the adult SEZ.

Trichostatin A ameliorates renal tubulointerstitial fibrosis through modulation of the JNK-dependent Notch-2 signaling pathway

Renal fibrosis is the final common pathological feature in a variety of chronic kidney disease. Trichostatin A (TSA), a histone deacetylase inhibitor, reportedly attenuates renal fibrosis in various kidney disease models. However, the detailed molecular action of TSA in ameliorating renal fibrotic injury is not yet fully understood. In a cultured renal fibroblastic cell model, we showed that TGF-β1 triggers upregulation of α-SMA and fibronectin, two hallmarks of myofibroblastic activation. During the course of TGF-β1 treatment, activation of Smad2/3, p38, ERK, JNK and Notch-2 was also detected. Under the conditions, administration of TSA significantly decreased TGF-β1-stimulated expression of α-SMA, fibronectin, phospho-JNK, and cleaved Notch-2; however, the levels of phospho-Smad2/3, phospho-p38 and phospho-ERK remained unchanged. Pharmacological inhibition of different signaling pathways and genetic knockdown of Notch-2 further revealed JNK as an upstream effector of Notch-2 in TGF-β1-mediated renal fibrosis. Consistently, we also demonstrated that administration of TSA or a γ-secretase inhibitor RO4929097 in the mouse model of unilateral ureteral obstruction significantly ameliorated renal fibrosis through suppression of the JNK/Notch-2 signaling activation. Taken together, our findings provide further insights into the crosstalk among different signaling pathways in renal fibrosis, and elucidate the molecular action of TSA in attenuating fibrogenesis.

NOTCH3 regulates stem-to-mural cell differentiation in infantile hemangioma.

Infantile hemangioma (IH) is a vascular tumor that begins with rapid vascular proliferation shortly after birth, followed by vascular involution in early childhood. We have found that NOTCH3, a critical regulator of mural cell differentiation and maturation, is expressed in hemangioma stem cells (HemSCs), suggesting that NOTCH3 may function in HemSC-to-mural cell differentiation and pathological vessel stabilization. Here, we demonstrate that NOTCH3 is expressed in NG2+PDGFRβ+ perivascular HemSCs and CD31+GLUT1+ hemangioma endothelial cells (HemECs) in proliferating IHs and becomes mostly restricted to the αSMA+NG2loPDGFRβlo mural cells in involuting IHs. NOTCH3 knockdown in HemSCs inhibited in vitro mural cell differentiation and perturbed αSMA expression. In a mouse model of IH, NOTCH3 knockdown or systemic expression of the NOTCH3 inhibitor, NOTCH3 Decoy, significantly decreased IH blood flow, vessel caliber, and αSMA+ perivascular cell coverage. Thus, NOTCH3 is necessary for HemSC-to-mural cell differentiation, and adequate perivascular cell coverage of IH vessels is required for IH vessel stability.

Voluntary running-enhanced synaptic plasticity, learning and memory are mediated by Notch1 signal pathway in C57BL mice

It is well known that voluntary running can enhance synaptic plasticity and improve learning and memory abilities. The Notch1 receptor is also reported to be associated with these processes, but its role in running-induced alterations is unclear. In this study, we aimed to investigate whether the Notch1 signalling pathway was involved in voluntary running-induced enhancement of synaptic plasticity, learning and memory. Notch1 heterozygous deficient (Notch1+/-) mice and wildtype (WT) C57BL littermates were randomly divided into runner group and non-runner group. Mice were given free access to running wheels for 14days in both the Notch1+/- runner group and the WT runner group. Our results demonstrate that Notch1 knockdown impairs the performance in the novel object recognition (NOR) test and Morris water maze test and decreases the synaptic plasticity. Voluntary running improves spatial learning and memory abilities, promotes synaptic plasticity and increases expressions of postsynaptic proteins in WT mice but not in Notch1+/- mice. Our results suggest that Notch1 plays a vital role in spatial learning and memory, synaptic plasticity under normal physiological conditions and voluntary running conditions. These findings will set the groundwork and fill in some gaps for understanding the role of Notch1 signalling in voluntary running-induced phenomena.