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1.  Notch4 is Activated in Endothelial and Smooth Muscle Cells in Human Brain Arteriovenous Malformations 
Journal of cellular and molecular medicine  2013;17(11):10.1111/jcmm.12115.
Upregulation of Notch4 was observed in the endothelial cells in the arteriovenous malformations (AVMs) in mice. However, whether Notch4 is also involved in brain AVMs in humans remains unclear. Here, we performed immunohistochemistry on normal brain vascular tissue and surgically-resection brain AVMs and found that Notch4 was upregulated in the subset of abnormal vessels of the brain AVM nidus, compared with control brain vascular tissue. Two-photon confocal images show that Notch4 was expressed not only in the endothelial but also in the smooth muscle cells of the vascular wall in brain AVMs. Western blotting shows that Notch 4 was activated in brain AVMs, but not in middle cerebral artery of normal human brain, which was confirmed by immunostaining. Our findings suggest a possible contribution of Notch4 signaling to the development of brain AVMs in human.
PMCID: PMC3877925  PMID: 24373503
Notch4; AVM; human; brain; signaling
2.  Notch4 is activated in endothelial and smooth muscle cells in human brain arteriovenous malformations 
Up-regulation of Notch4 was observed in the endothelial cells in the arteriovenous malformations (AVMs) in mice. However, whether Notch4 is also involved in brain AVMs in humans remains unclear. Here, we performed immunohistochemistry on normal brain vascular tissue and surgically resected brain AVMs and found that Notch4 was up-regulated in the subset of abnormal vessels of the brain AVM nidus, compared with control brain vascular tissue. Two-photon confocal images show that Notch4 was expressed not only in the endothelial but also in the smooth muscle cells of the vascular wall in brain AVMs. Western blotting shows that Notch4 was activated in brain AVMs, but not in middle cerebral artery of normal human brain, which was confirmed by immunostaining. Our findings suggest a possible contribution of Notch4 signalling to the development of brain AVMs in human.
PMCID: PMC3877925  PMID: 24373503
Notch4; AVM; human; brain; signalling
3.  Intraovarian regulation of gonadotropin-dependent folliculogenesis depends on notch receptor signaling pathways not involving Delta-like ligand 4 (Dll4) 
In-situ hybridisation studies demonstrate that Notch receptors and ligands are expressed in granulosa cells (GCs) and in the theca layer vasculature of growing follicles. Notch signaling involves cell-to-cell interaction mediated by transmembrane receptors and ligands. This signaling pathway may represent a novel intraovarian regulator of gonadotropin-dependent follicular development to the preovulatory stage. We hypothesized that blocking Notch pathways would disrupt follicular maturation in the mouse ovary.
Hypophysectomized CD21 female mice were administered pregnant mare serum gonadotropin (PMSG) for 3 days to stimulate follicular development. In one experiment, a pan-notch inhibitor, compound E, was initiated 2 days prior to and throughout stimulation (n = 10), while in a second experiment, a humanized phage Dll4 blocking antibody, YW152F, was used (n = 5). After sacrifice, ovarian histology, serum estradiol levels and uterine weights were compared to controls. The ovarian morphology was evaluated with hematoxylin/eosin staining and immunohistochemistry was performed for Notch1, Notch2, Notch3, Notch4, Jagged1, Dll4, platelet endothelial cell adhesion molecule (PECAM) and alpha-smooth muscle actin (α-SMA) detection.
We localized specific Notch ligands and receptors in the following structures: Dll4 is specific to theca layer endothelial cells (ECs); Notch1/Notch4 and Jagged1 are expressed in theca layer ECs and vascular smooth muscle cells (VSMCs), whereas Notch3 is restricted to VSMCs; Notch2 is expressed mostly on GCs of small follicles. Administration of a pan-Notch inhibitor, compound E, inhibits follicular development to the preovulatory stage (8.5 preovulatory follicles in treatment vs. 3.4 preovulatory follicles in control, p < 0.01; average number per ovary) with significant secondary effects on ovarian and uterine weight and estradiol secretion in a setting of uninhibited vascular proliferation, but disorganized appearance of ECs and VSMCs. Inhibition of endothelial Notch1 function through the inactivation of its ligand Dll4 with the blocking antibody YW152F induces mild disorganisation of follicular vasculature, but has no significant effect on gonadotropin-dependent folliculogenesis.
Our experiments suggest that the complete blockage of the Notch signaling pathway with compound E impairs folliculogenesis and induces disruption of gonadotropin stimulated angiogenesis. It seems the mechanism involves Notch1 and Notch3, specifically, causing the improper assembly of ECs and VSMCs in the theca layer, although the potential role of non-angiogenic Notch signaling, such as Jagged2 to Notch2 in GCs, remains to be elucidated.
PMCID: PMC3662615  PMID: 23675950
Notch; Dll4; Jagged; Folliculogenesis; Ovary; Gamma-secretase inhibitor; YW152F
4.  Notch1 and 4 Signaling Responds to an Increasing Vascular Wall Shear Stress in a Rat Model of Arteriovenous Malformations 
BioMed Research International  2014;2014:368082.
Notch signaling is suggested to promote the development and maintenance of cerebral arteriovenous malformations (AVMs), and an increasing wall shear stress (WSS) contributes to AVM rupture. Little is known about whether WSS impacts Notch signaling, which is important for understanding the angiogenesis of AVMs. WSS was measured in arteriovenous fistulas (AVF) surgically created in 96 rats at different time points over a period of 84 days. The expression of Notch receptors 1 and 4 and their ligands, Delta1 and 4, Jagged1, and Notch downstream gene target Hes1 was quantified in “nidus” vessels. The interaction events between Notch receptors and their ligands were quantified using proximity ligation assay. There was a positive correlation between WSS and time (r = 0.97; P < 0.001). The expression of Notch receptors and their ligands was upregulated following AVF formation. There was a positive correlation between time and the number of interactions between Notch receptors and their ligands aftre AVF formation (r = 0.62, P < 0.05) and a positive correlation between WSS and the number of interactions between Notch receptors and their ligands (r = 0.87, P < 0.005). In conclusion, an increasing WSS may contribute to the angiogenesis of AVMs by activation of Notch signaling.
PMCID: PMC3915856  PMID: 24563863
5.  Implications of Dll4-Notch signaling activation in primary glioblastoma multiforme 
Neuro-Oncology  2013;15(10):1366-1378.
Glioblastoma multiforme (GBM) is a highly aggressive brain tumor characterized by massive neovascularization, necrosis, and intense resistance to therapy. Deregulated Notch signaling has been implicated in the formation and progression of different malignancies. The present study attempted to investigate the activation status of Dll4-Notch signaling in primary human GBM and its association with vascular and clinical parameters in patients.
Major components of Dll4-Notch signaling were examined by real-time reverse-transcription polymerase chain reaction (PCR), Western blotting, and immunohistochemistry in GBM (n = 26) and control (n = 11) brain tissue. The vascular pattern (VP) and microvascular density (MVD) were analyzed after laminin immunostaining. O6-Methylguanine-methyltransferase (MGMT) promoter methylation in GBM samples was detected by methylation-specific PCR.
The mRNA levels of Dll4, Jagged1, Notch1, Notch4, Hey1, Hey2, Hes1, and VEGF were 3.12-, 3.58-, 3.37-, 5.77-, 4.89-, 3.13-, 6.62-, and 32.57-fold elevated, respectively, in GBM samples, compared with the controls. Western blotting revealed a 4-, 3.7-, and 45.6-fold upregulation of Dll4, Notch1, and Hey1, respectively, accompanied by a downregulation of PTEN expression and an increase in the expression of p-Akt and VEGF. Immunostaining located the immunoreactivity of Dll4 and Notch1 in endothelial cells, microglia/macrophages, tumor cells, and astrocytes. Furthermore, the upregulation of Dll4-Notch signaling components was correlated to a low MVD and was potentially related to a classic VP, tumor edema, and MGMT promoter methylation.
The upregulation of Dll4-Notch signaling components was found in a subset of GBM samples and was associated with some angiogenic and clinical parameters. These findings highlight this signaling pathway as a potential therapeutic target for patients with GBM who show an activation of Dll4-Notch signaling.
PMCID: PMC3779034  PMID: 23787764
Dll4-Notch signaling; macrophage/microglia; microvascular density; primary glioblastoma multiforme; vascular pattern
6.  A Receptor-Specific Function for Notch2 in Mediating Vascular Smooth Muscle Cell Growth Arrest Through p27kip1 
Circulation research  2013;113(8):10.1161/CIRCRESAHA.113.301272.
Deregulated vascular smooth muscle cell (VSMC) proliferation contributes to multiple vascular pathologies, and Notch signaling regulates VSMC phenotype.
Previous work focused on Notch1 and Notch3 in VSMC during vascular disease; however, the role of Notch2 is unknown. Because injured murine carotid arteries display increased Notch2 in VSMC as compared to uninjured arteries, we sought to understand the impact of Notch2 signaling in VSMC.
Methods and Results
In human primary VSMC, Jagged-1 (Jag-1) significantly reduced proliferation through specific activation of Notch2. Increased levels of p27kip1 were observed downstream of Jag-1/Notch2 signaling, and required for cell cycle exit. Jag-1 activation of Notch resulted in increased phosphorylation on serine 10, decreased ubiquitination and prolonged half-life of p27kip1. Jag-1/Notch2 signaling robustly decreased S-phase kinase associated protein (Skp2), an F-box protein that degrades p27kip1 during G1. Over expression of Skp2 prior to Notch activation by Jag-1 suppressed the induction of p27kip1. Additionally, increased Notch2 and p27kip1 expression was co-localized to the non-proliferative zone of injured arteries as indicated by co-staining with proliferating cell nuclear antigen (PCNA), whereas Notch3 was expressed throughout normal and injured arteries, suggesting Notch2 may negatively regulate lesion formation.
We propose a receptor specific function for Notch2 in regulating Jag-1-induced p27kip1 expression and growth arrest in VSMC. During vascular remodeling, co-localization of Notch2 and p27kip1 to the non-proliferating region supports a model where Notch2 activation may negatively regulate VSMC proliferation to lessen the severity of the lesion. Thus Notch2 is a potential target for control of VSMC hyperplasia.
PMCID: PMC3882755  PMID: 23965337
Smooth muscle cell; Notch receptor; proliferation; neointima; neointimal hyerplasia
7.  Endothelial Notch Signaling is Upregulated in Human Brain Arteriovenous Malformations and a Mouse Model of the Disease 
Brain arteriovenous malformations (BAVMs) can cause lethal hemorrhagic stroke and have no effective treatment. The cellular and molecular basis for this disease is largely unknown. We have previously shown that expression of constitutively-active Notch4 receptor in the endothelium elicits and maintains the hallmarks of BAVM in mice, thus establishing a mouse model of the disease. Our work suggested that Notch pathway could be a critical molecular mediator of BAVM pathogenesis. Here, we investigated the hypothesis that upregulated Notch activation contributes to the pathogenesis of human BAVM. We examined expression of the canonical Notch downstream target Hes1 in the endothelium of human BAVMs by immunofluorescence, and showed increased levels relative to either autopsy or surgical biopsy controls. We then analyzed receptor activity using an antibody to the activated form of the Notch1 receptor, and found increased levels of activity. These findings suggest that Notch activation may promote the development and even maintenance of BAVM. We also detected increases in Hes1 and activated Notch1 expression in our mouse model of BAVM induced by constitutively-active Notch4, demonstrating molecular similarity between the mouse model and the human disease. Our work suggests that activation of Notch signaling is an important molecular candidate in BAVM pathogenesis and further validates that our animal model provides a platform to study the progression as well as the regression of the disease.
PMCID: PMC3095492  PMID: 19546852
angiogenesis; arteriovenous malformation; arterial-venous differentiation; cell signaling; endothelial cell; Notch signaling; stroke
8.  Isolation and functional analysis of a cDNA for human Jagged2, a gene encoding a ligand for the Notch1 receptor. 
Molecular and Cellular Biology  1997;17(10):6057-6067.
Signaling through Notch receptors has been implicated in the control of cellular differentiation in animals ranging from nematodes to humans. Starting from a human expressed sequence tag-containing sequence resembling that of Serrate, the gene for a ligand of Drosophila melanogaster Notch, we assembled a full-length cDNA, now called human Jagged2, from overlapping cDNA clones. The full-length cDNA encodes a polypeptide having extensive sequence homology to Serrate (40.6% identity and 58.7% similarity) and even greater homology to several putative mammalian Notch ligands that have subsequently been described. When in situ hybridization was performed, expression of the murine Jagged2 homolog was found to be highest in fetal thymus, epidermis, foregut, dorsal root ganglia, and inner ear. In Northern blot analysis of RNA from tissues of 2-week-old mice, the 5.0-kb Jagged2 transcript was most abundant in heart, lung, thymus, skeletal muscle, brain, and testis. Immunohistochemistry revealed coexpression of Jagged2 and Notch1 within thymus and other fetal murine tissues, consistent with interaction of the two proteins in vivo. Coculture of fibroblasts expressing human Jagged2 with murine C2C12 myoblasts inhibited myogenic differentiation, accompanied by increased Notch1 and the appearance of a novel 115-kDa Notch1 fragment. Exposure of C2C12 cells to Jagged2 led to increased amounts of Notch mRNA as well as mRNAs for a second Notch receptor, Notch3, and a second Notch ligand, Jagged1. Constitutively active forms of Notchl in C2C12 cells also induced increased levels of the same set of mRNAs, suggesting positive feedback control of these genes initiated by binding of Jagged2 to Notch1. This feedback control may function in vivo to coordinate differentiation across certain groups of progenitor cells adopting identical cell fates.
PMCID: PMC232455  PMID: 9315665
9.  Regulation of Ocular Angiogenesis by Notch Signaling: Implications in Neovascular Age-Related Macular Degeneration 
This study identifies the Notch pathway as a putative molecular target for therapeutic intervention in wet age-related macular degeneration.
Wet age-related macular degeneration (AMD), which accounts for most AMD-related vision loss, is characterized by choroidal neovascularization (CNV). The underlying mechanism of CNV is poorly understood, but evidence indicates pathologic recruitment of normal angiogenic signaling pathways such as the VEGF pathway. Recent evidence suggests that the VEGF pathway regulates angiogenesis in concert with Notch signaling. Here, the authors examined the role of Notch signaling in CNV in the backdrop of Notch signaling–mediated regulation of retinal angiogenesis.
Choroid sclera complexes, after laser-induced CNV, were examined for changes in CNV lesion volume and in proangiogenic and antiangiogenic gene expression after perturbation in Notch signaling. Retinal vessels and angiogenic gene expression in retinal endothelial cells were analyzed in postnatal rats after perturbations in Notch signaling. Notch signaling was activated and inhibited by intravitreal or systemic injection of Jagged1 peptide and gamma secretase inhibitor DAPT, respectively.
The authors demonstrated that activation of the canonical Notch pathway reduced the volume of CNV lesions as it attenuated the development of postnatal retinal vasculature. In contrast, inhibition of the Notch pathway exacerbated CNV lesions as it led to the development of hyperdense retinal vasculature. The authors also identified genes associated with proangiogenesis (Vegfr2, Ccr3, and Pdgfb) and antiangiogenesis (Vegfr1 and Unc5b) as targets of Notch signaling–mediated vascular homeostasis, the disruption of which might underlie CNV.
This study suggests that Notch signaling is a key regulator of CNV and thus a molecular target for therapeutic intervention in wet AMD.
PMCID: PMC3109005  PMID: 21228388
10.  Loss of CCM3 impairs DLL4-Notch signalling: implication in endothelial angiogenesis and in inherited cerebral cavernous malformations 
CCM3, a product of the cerebral cavernous malformation 3 or programmed cell death 10 gene (CCM3/PDCD10), is broadly expressed throughout development in both vertebrates and invertebrates. Increasing evidence indicates a crucial role of CCM3 in vascular development and in regulation of angiogenesis and apoptosis. Furthermore, loss of CCM3 causes inherited (familial) cerebral cavernous malformation (CCM), a common brain vascular anomaly involving aberrant angiogenesis. This study focused on signalling pathways underlying the angiogenic functions of CCM3. Silencing CCM3 by siRNA stimulated endothelial proliferation, migration and sprouting accompanied by significant downregulation of the core components of Notch signalling including DLL4, Notch4, HEY2 and HES1 and by activation of VEGF and Erk pathways. Treatment with recombinant DLL4 (rhDLL4) restored DLL4 expression and reversed CCM3-silence-mediated impairment of Notch signalling and reduced the ratio of VEGF-R2 to VEGF-R1 expression. Importantly, restoration of DLL4-Notch signalling entirely rescued the hyper-angiogenic phenotype induced by CCM3 silence. A concomitant loss of CCM3 and the core components of DLL4-Notch signalling were also demonstrated in CCM3-deficient endothelial cells derived from human CCM lesions (CCMEC) and in a CCM3 germline mutation carrier. This study defined DLL4 as a key downstream target of CCM3 in endothelial cells. CCM3/DLL4-Notch pathway serves as an important signalling for endothelial angiogenesis and is potentially implicated in the pathomechanism of human CCMs.
PMCID: PMC3823022  PMID: 23388056
CCM3/PDCD10; DLL4-Notch signalling; angiogenesis; endothelium; cerebral cavernous malformation
11.  Cis-interactions between Notch and its ligands block ligand-independent Notch activity 
eLife  null;3:e04415.
The Notch pathway is integrated into numerous developmental processes and therefore is fine-tuned on many levels, including receptor production, endocytosis, and degradation. Notch is further characterized by a twofold relationship with its Delta-Serrate (DSL) ligands, as ligands from opposing cells (trans-ligands) activate Notch, whereas ligands expressed in the same cell (cis-ligands) inhibit signaling. We show that cells without both cis- and trans-ligands can mediate Notch-dependent developmental events during Drosophila oogenesis, indicating ligand-independent Notch activity occurs when the receptor is free of cis- and trans-ligands. Furthermore, cis-ligands can reduce Notch activity in endogenous and genetically induced situations of elevated trans-ligand-independent Notch signaling. We conclude that cis-expressed ligands exert their repressive effect on Notch signaling in cases of trans-ligand-independent activation, and propose a new function of cis-inhibition which buffers cells against accidental Notch activity.
eLife digest
Many biological processes require cells to send messages to one another. Typically, this is achieved when molecules are released from one cell and make contact with companion molecules on another cell. This triggers a chemical or biological reaction in the receiving cell.
One of the most common examples of this is the Notch pathway, which is used throughout the animal kingdom and plays an important role in helping cells and embryos to develop. The Notch protein itself is a ‘receptor’ protein that is embedded in the surface of a cell, and relays signals from outside the cell to activate certain genes inside the cell. In fruit flies, two proteins called Serrate and Delta act as ‘ligands’ for Notch—by binding to Notch, they can change how this receptor works.
If Serrate or Delta are present on the outside of one cell, they can activate Notch (and hence the Notch signaling pathway) in an adjacent cell. However, if the Serrate or Delta ligands are present on the surface of the same cell as Notch they turn the receptor off, rather than activate it. Notch can also work without being activated by Serrate or Delta, but whether the ligands can inhibit this ‘ligand-independent’ Notch activation if they are on the surface of the same cell as the Notch receptor was unknown.
Palmer et al. study Notch signaling in the fruit fly equivalent of the ovary, in cells that are naturally deficient in Serrate and from which Delta was artificially removed. The Notch protein was activated when these ligands were not present. Furthermore, the developmental processes that are activated by Notch were able to proceed as normal when triggered by ligand-independent Notch signaling. In total, Palmer et al. investigated three different types of fruit fly cell, and found that ligand-independent Notch signaling can occur in all of them.
Reintroducing Delta to the same cell as Notch turns the receptor off, suggesting that ligands on the surface of the same cell as the receptor can inhibit ligand-independent Notch activity. Many genetic diseases and cancers have been linked to Notch being activated when it should not be; therefore, understanding how Notch is controlled could help guide the development of new treatments for these conditions.
PMCID: PMC4286723  PMID: 25486593
Notch pathway; signal transduction; oogenesis; D. melanogaster
12.  A switch in Notch Gene Expression Parallels Stem Cell to Endothelial Transition in Infantile Hemangioma 
Angiogenesis  2010;13(1):15-23.
Infantile hemangioma (IH) is the most common benign tumor of infancy, yet its pathogenesis is poorly understood. Notch family members are known to play a role in vascular development during embryogenesis and postnatal tumor angiogenesis, yet the role of Notch signaling in the pathogenesis of IH has not been investigated. This study aims to survey Notch expression in IH.
Materials & Methods
RNA from resected hemangioma tissue and hemangioma-derived stem cells (HemSCs) and endothelial cells (HemECs) were used for gene expression analyses by real-time PCR. Results were confirmed with immunofluorescence (IF) for protein expression in tissue.
Real-time PCR showed that Notch family gene expression in IH is distinct from placenta and skin. Notch3 is expressed in HemSCs, but not in HemECs, indicating Notch3 is downregulated as HemSCs differentiate into HemECs. Moreover, expression of endothelial-associated Notch proteins, Notch1, -4, and Jagged-1 are increased in involuting hemangiomas and HemECs, suggesting that as hemangioma progresses towards involution, it acquires more differentiated endothelium. A subset of cells stained double positive for Notch3 and CD31, pointing to a potential intermediate between the HemSC cellular differentiation into HemEC.
HemSCs have distinct Notch expression patterns from differentiated HemECs and from normal human endothelial cells. Notch3 is expressed in HemSCs while Notch1, Notch4, and Jagged-1 have higher expression levels in HemECs. Notch3 was localized to the interstitial cells outside of the nascent vascular channels in proliferating IH tissue sections, but became more apparent in the perivascular cells in involuting IH. In summary, the pattern of Notch gene expression mirrors the progression from immature cells to endothelial-lined vascular channels (i.e., endothelial differentiation) that characterizes the growth and involution of IH.
PMCID: PMC2846229  PMID: 20069356
endothelial cell; infantile hemangioma; Notch gene expression; stem cell
13.  Endovascular treatment of cerebral arteriovenous malformations with emphasis on the curative role of embolisation 
Interventional Neuroradiology  2005;11(Suppl 1):37-43.
Valavanis A, Pangalu A, Tanaka M. Endovascular treatment of cerebral arteriovenous malformations with emphasis on the curative role of embolisation. Schweiz Arch Neurol Psychiatr 2004;155:341-7.
Cerebral arteriovenous malformations are complex and only partially understood vascular lesions of the central nervous system with a natural history characterised by significant morbidity and mortality mainly due to an increased haemorrhagic risk. Microneurosurgical removal, radiosurgical obliteration and neuroendovascular embolisation are the principal therapeutic modalities applied individually or in various combinations according to varying selection criteria for the treatment of cerebral arteriovenous malformations. In this context embolisation plays a central role either as a complementary or as the sole treatment technique. This report summarises the evolutive 18 years of continuous experience of the senior author with the neuroradiological evaluation and endovascular treatment of 644 patients with a cerebral arteriovenous malformation. Special emphasis is given to the underlying concepts and specific endovascular techniques developed for the complete, i.e. curative embolisation of cerebral arteriovenous malformations.
Precise angiographic analysis of the vascular composition and intrinsic angioarchitecture of the nidus of the arteriovenous malformation by superselective microcatheterisation is required to identify the types of feeding arteries and patterns of their supply, the number and vascular connections of nidal compartments, the types of arteriovenous shunts, the morphology of the vascular spaces composing the nidus and the number and exit patterns of draining veins. Complete angiographic investigation for recognition of secondarily induced phenomena of the cerebral vasculature, such as arterial and venous high-flow angiopathy and so-called perinidal angiogenesis is essential for a comprehensive evaluation and assessment of the associated haemorrhagic risk.
Based on a precise topographic classification, detailed angioarchitectural analysis, application of superselective multimicrocatheterisation techniques along with a controlled intranidal injection of non-absorbable liquid embolic materials, nearly 40% of cerebral arteriovenous malformations can be completely and stably obliterated and therefore curatively treated by single session or multistaged embolisation with a morbidity of 1.3% and a mortality of 1.3%, which are lower than the known natural history of this disease.
PMCID: PMC3404764  PMID: 20584458
cerebral arteriovenous malformations; embolisation; angioarchitecture; endovascular treatment; treatment outcome
14.  Regulation of Notch1 and Dll4 by Vascular Endothelial Growth Factor in Arterial Endothelial Cells: Implications for Modulating Arteriogenesis and Angiogenesis 
Molecular and Cellular Biology  2003;23(1):14-25.
Notch and its ligands play critical roles in cell fate determination. Expression of Notch and ligand in vascular endothelium and defects in vascular phenotypes of targeted mutants in the Notch pathway have suggested a critical role for Notch signaling in vasculogenesis and angiogenesis. However, the angiogenic signaling that controls Notch and ligand gene expression is unknown. We show here that vascular endothelial growth factor (VEGF) but not basic fibroblast growth factor can induce gene expression of Notch1 and its ligand, Delta-like 4 (Dll4), in human arterial endothelial cells. The VEGF-induced specific signaling is mediated through VEGF receptors 1 and 2 and is transmitted via the phosphatidylinositol 3-kinase/Akt pathway but is independent of mitogen-activated protein kinase and Src tyrosine kinase. Constitutive activation of Notch signaling stabilizes network formation of endothelial cells on Matrigel and enhances formation of vessel-like structures in a three-dimensional angiogenesis model, whereas blocking Notch signaling can partially inhibit network formation. This study provides the first evidence for regulation of Notch/Delta gene expression by an angiogenic growth factor and insight into the critical role of Notch signaling in arteriogenesis and angiogenesis.
PMCID: PMC140667  PMID: 12482957
15.  17β-Estradiol Enhances Signalling Mediated by VEGF-A-Delta-Like Ligand 4-Notch1 Axis in Human Endothelial Cells 
PLoS ONE  2013;8(8):e71440.
Estrogens play a protective role in coronary artery disease. The mechanisms of action are still poorly understood, although a role for estrogens in stimulation of angiogenesis has been suggested. In several cell types, estrogens modulate the Notch pathway, which is involved in controlling angiogenesis downstream of vascular endothelial growth factor A (VEGF-A). The goal of our study was to establish whether estrogens modulate Notch activity in endothelial cells and the possible consequences on angiogenesis. Human umbilical vein endothelial cells (HUVECs) were treated with 17β-estradiol (E2) and the effects on Notch signalling were evaluated. E2 increased Notch1 processing as indicated by i) decreased levels of Notch1 transmembrane subunit ii) increased amount of Notch1 in nuclei iii) unaffected level of mRNA. Similarly, E2 increased the levels of the active form of Notch4 without altering Notch4 mRNA. Conversely, protein and mRNA levels of Notch2 were both reduced suggesting transcriptional repression of Notch2 by E2. Under conditions where Notch was activated by upregulation of Delta-like ligand 4 (Dll4) following VEGF-A treatment, E2 caused a further increase of the active form of Notch1, of the number of cells with nuclear Notch1 and of Hey2 mRNA. Estrogen receptor antagonist ICI 182.780 antagonized these effects suggesting that E2 modulation of Notch1 is mediated by estrogen receptors. E2 treatment abolished the increase in endothelial cells sprouting caused by Notch inhibition in a tube formation assay on 3D Matrigel and in mouse aortic ring explants. In conclusion, E2 affects several Notch pathway components in HUVECs, leading to an activation of the VEGF-A-Dll4-Notch1 axis and to a modulation of vascular branching when Notch signalling is inhibited. These results contribute to our understanding of the molecular mechanisms of cardiovascular protection exerted by estrogens by uncovering a novel role of E2 in the Notch signalling-mediated modulation of angiogenesis.
PMCID: PMC3742772  PMID: 23967210
16.  A Role for Notch Signaling in Salivary Acinar Cell Growth and Differentiation 
The Notch pathway is crucial for stem/progenitor cell maintenance, growth and differentiation in a variety of tissues. The Notch signaling is essential for Drosophila salivary gland development but its role in mammalian salivary gland remains unclear. The human salivary epithelial cell line, HSG, was studied to determine the role of Notch signaling in salivary epithelial cell differentiation. HSG expressed Notch 1 to 4, and the Notch ligands Jagged 1 and 2 and Delta 1. Treatment of HSG cells with inhibitors of γ-secretase, which is required for Notch cleavage and activation, blocked vimentin expression, an indicator of HSG differentiation. HSG differentiation was also associated with Notch downsteam signal Hes-1 expression, and Hes-1 expression was inhibited by γ-secretase inhibitors. siRNA corresponding to Notch 1 to 4 was used to show that silencing of all four Notch receptors was required to inhibit HSG differentiation. Normal human submandibular gland expressed Notch 1 to 4, Jagged 1 and 2, and Delta 1, with nuclear localization indicating Notch signaling in vivo. Hes-1 was also expressed in the human tissue, with staining predominantly in the ductal cells. In salivary tissue from rats undergoing and recovering from ductal obstruction, we found that Notch receptors and ligands were expressed in the nucleus of the regenerating epithelial cells. Taken together, these data suggest that Notch signaling is critical for normal salivary gland cell growth and differentiation.
PMCID: PMC2671016  PMID: 19235730
Salivary Gland; Epithelial Cell Differentiation; Notch; Hes1
17.  A Notch1 Ectodomain Construct Inhibits Endothelial Notch Signaling, Tumor Growth, and Angiogenesis 
Cancer research  2008;68(12):4727-4735.
Notch signaling is required for vascular development and tumor angiogenesis. Although inhibition of the Notch ligand Delta-like 4 can restrict tumor growth and disrupt neo-vasculature, the effect of inhibiting Notch receptor function on angiogenesis has yet to be defined. In this study, we generated a soluble form of the Notch1 receptor (Notch1 decoy) and assessed its effect on angiogenesis in vitro and in vivo. Notch1 decoy expression reduced signaling stimulated by the binding of three distinct Notch ligands to Notch1 and inhibited morphogenesis of endothelial cells overexpressing Notch4. Thus, Notch1 decoy functioned as an antagonist of ligand-dependent Notch signaling. In mice, Notch1 decoy also inhibited vascular endothelial growth factor–induced angiogenesis in skin, establishing a role for Notch receptor function in this process. We tested the effects of Notch1 decoy on tumor angiogenesis using two models: mouse mammary Mm5MT cells overexpressing fibroblast growth factor 4 (Mm5MT-FGF4) and NGP human neuroblastoma cells. Exogenously expressed FGF4 induced Notch ligand expression in Mm5MT cells and xenografts. Notch1 decoy expression did not affect tumorigenicity of Mm5MT-FGF4 cells in vitro but restricted Mm5MT-FGF4 xenograft growth in mice while markedly impairing neoangiogenesis. Similarly, Notch1 decoy expression did not affect NGP cells in vitro but disrupted vessels and decreased tumor viability in vivo. These results strongly suggest that Notch receptor signaling is required for tumor neoangiogenesis and provides a new target for tumor therapy.
PMCID: PMC3690602  PMID: 18559519
18.  Characterization of Notch1 Antibodies That Inhibit Signaling of Both Normal and Mutated Notch1 Receptors 
PLoS ONE  2010;5(2):e9094.
Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target.
Principal Findings
Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC50 values as low as 5±3 nM and 0.13±0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR “class I” point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare “class II” or “class III” mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors.
Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation.
PMCID: PMC2817004  PMID: 20161710
19.  Notch Signaling in Descending Thoracic Aortic Aneurysm and Dissection 
PLoS ONE  2012;7(12):e52833.
Descending thoracic aortic aneurysm and dissection (DTAAD) is characterized by progressive medial degeneration, which may result from excessive tissue destruction and insufficient repair. Resistance to tissue destruction and aortic self-repair are critical in preventing medial degeneration. The signaling pathways that control these processes in DTAAD are poorly understood. Because Notch signaling is a critical pathway for cell survival, proliferation, and tissue repair, we examined its activation in DTAAD.
We studied descending thoracic aortic tissue from patients with sporadic thoracic aortic aneurysm (TAA; n = 14) or chronic thoracic aortic dissection (TAD; n = 16) and from age-matched organ donors (n = 12). Using western blot, real-time RT-PCR, and immunofluorescence staining, we examined aortic tissue samples for the Notch ligands Delta-like 1, Delta-like 4 (DLL1/4), and Jagged1; the Notch receptor 1 (Notch1); the Notch1 intracellular domain (NICD); and Hes1, a downstream target of Notch signaling.
Western blots and RT-PCR showed higher levels of the Notch1 protein and mRNA and the NICD and Hes1 proteins in both TAA and TAD tissues than in control tissue. However, immunofluorescence staining showed a complex pattern of Notch signaling in the diseased tissue. The ligand DLL1/4 and Notch1 were significantly decreased and NICD and Hes1 were rarely detected in medial vascular smooth muscle cells (VSMCs) in both TAA and TAD tissues, indicating downregulation of Notch signaling in aortic VSMCs. Interestingly Jagged1, NICD, and Hes1 were highly present in CD34+ stem cells and Stro-1+ stem cells in aortas from TAA and TAD patients. NICD and Hes1 were also detected in most fibroblasts and macrophages that accumulated in the aortic wall of DTAAD patients.
Notch signaling exhibits a complex pattern in DTAAD. The Notch pathway is impaired in medial VSMCs but activated in stem cells, fibroblasts, and macrophages.
PMCID: PMC3530510  PMID: 23300792
20.  Abnormal recruitment of extracellular matrix proteins by excess Notch3ECD: a new pathomechanism in CADASIL 
Brain  2013;136(6):1830-1845.
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, one of the most common inherited small vessel diseases of the brain, is characterized by a progressive loss of vascular smooth muscle cells and extracellular matrix accumulation. The disease is caused by highly stereotyped mutations within the extracellular domain of the NOTCH3 receptor (Notch3ECD) that result in an odd number of cysteine residues. While CADASIL-associated NOTCH3 mutations differentially affect NOTCH3 receptor function and activity, they all are associated with early accumulation of Notch3ECD-containing aggregates in small vessels. We still lack mechanistic explanation to link NOTCH3 mutations with small vessel pathology. Herein, we hypothesized that excess Notch3ECD could recruit and sequester functionally important proteins within small vessels of the brain. We performed biochemical, nano-liquid chromatography-tandem mass spectrometry and immunohistochemical analyses, using cerebral and arterial tissue derived from patients with CADASIL and mouse models of CADASIL that exhibit vascular lesions in the end- and early-stage of the disease, respectively. Biochemical fractionation of brain and artery samples demonstrated that mutant Notch3ECD accumulates in disulphide cross-linked detergent-insoluble aggregates in mice and patients with CADASIL. Further proteomic and immunohistochemical analyses identified two functionally important extracellular matrix proteins, tissue inhibitor of metalloproteinases 3 (TIMP3) and vitronectin (VTN) that are sequestered into Notch3ECD-containing aggregates. Using cultured cells, we show that increased levels or aggregation of Notch3 enhances the formation of Notch3ECD–TIMP3 complex, promoting TIMP3 recruitment and accumulation. In turn, TIMP3 promotes complex formation including NOTCH3 and VTN. In vivo, brain vessels from mice and patients with CADASIL exhibit elevated levels of both insoluble cross-linked and soluble TIMP3 species. Moreover, reverse zymography assays show a significant elevation of TIMP3 activity in the brain vessels from mice and patients with CADASIL. Collectively, our findings lend support to a Notch3ECD cascade hypothesis in CADASIL disease pathology, which posits that aggregation/accumulation of Notch3ECD in the brain vessels is a central event, promoting the abnormal recruitment of functionally important extracellular matrix proteins that may ultimately cause multifactorial toxicity. Specifically, our results suggest a dysregulation of TIMP3 activity, which could contribute to mutant Notch3ECD toxicity by impairing extracellular matrix homeostasis in small vessels.
PMCID: PMC3673461  PMID: 23649698
CADASIL; Notch3; protein aggregation; extracellular matrix proteins; cerebrovasculature
21.  Notch1 Signaling Modulates Neuronal Progenitor Activity in the Subventricular Zone in Response to Aging and Focal Ischemia 
Aging cell  2013;12(6):10.1111/acel.12134.
Neurogenesis diminishes with aging and ischemia-induced neurogenesis also occurs, but reduced in aged brain. Currently, the cellular and molecular pathways mediating these effects remain largely unknown. Our previous study has shown that Notch1 signaling regulates neurogenesis in subventricular zone (SVZ) of young-adult brain after focal ischemia, but whether a similar effect occurs in aged normal and ischemic animals is unknown. Here, we used normal and ischemic aged rat brains to investigate whether Notch1 signaling was involved in the reduction of neurogenesis in response to aging and modulates neurogenesis in aged brains after focal ischemia. By Western blot, we found that Notch1 and Jagged1 expression in the SVZ of aged brain was significantly reduced compared with young-adult brain. Consistently, the activated form of Notch1(Notch intracellular domain;NICD) expression was also declined. Immunohistochemistry confirmed that expression and activation of Notch1 signaling in the SVZ of aged brain were reduced. Double or triple immunostaining showed that that Notch1 was mainly expressed in DCX-positive cells, whereas Jagged1 was predominantly expressed in astroglial cells in the SVZ of normal aged rat brain. In addition, disruption or activation of Notch1 signaling altered the number of proliferating cells labeled by bromodeoxyuridine (BrdU) and doublecortin (DCX) in the SVZ of aged brain. Moreover, ischemia-induced cell proliferation in the SVZ of aged brain was enhanced by activating the Notch1 pathway, and was suppressed by inhibiting the Notch1 signaling. Reduced infarct volume and improved motor deficits were also observed in Notch1 activator-treated aged ischemic rats. Our data suggest that Notch1 signaling modulates the SVZ neurogenesis in aged brain in normal and ischemic conditions.
PMCID: PMC3838489  PMID: 23834718
Notch1 signaling pathway; aged rat brain; neurogenesis; focal cerebral ischemia
22.  Notch1 activation in mice causes arteriovenous malformations phenocopied by ephrinB2 and EphB4 mutants 
Genesis (New York, N.Y. : 2000)  2010;48(3):146-150.
Notch signaling is essential for embryonic vascular development in mammals and other vertebrates. Here we show that mouse embryos with conditional activation of the Notch1 gene in endothelial cells (Notch1 gain of function embryos) exhibit defects in vascular remodeling, increased diameter of the dorsal aortae, and form arteriovenous malformations. Conversely, embryos with either constitutive or endothelial cell-specific Notch1 gene deletion also have vascular defects, but exhibit decreased diameter of the dorsal aortae and form arteriovenous malformations distinctly different from the Notch1 gain of function mutants. Surprisingly, embryos homozygous for mutations of the ephrinB/EphB pathway genes Efnb2 and Ephb4 exhibit vascular defects and arteriovenous malformations that phenocopy the Notch1 gain of function mutants. These results suggest that formation of arteriovenous malformations in Notch1 gain of function mutants and ephrinB/EphB pathway loss of function mutant embryos occurs by different mechanisms.
PMCID: PMC2849749  PMID: 20101599
angiogenesis; arteriovenous malformation; EphrinB2; EphB4; Notch signaling pathway; vascular morphogenesis
23.  Evidence of endothelial progenitor cells in the human brain and spinal cord arteriovenous malformations 
Neurosurgery  2010;67(4):1029-1035.
Brain and spinal cord arteriovenous malformations (AVMs) are characterized by aberrant angiogenesis and vascular remodeling. Endothelial progenitor cells (EPCs) can be recruited by stromal cell-derived factor-1 (SDF-1), and participate in vascular remodeling in both physiological and pathological settings. We hypothesized that there was increased EPC levels in the brain and spinal cord AVM nidus.
Microsurgical specimens without endovascular embolization and radiosurgery from the brain (n=12) and spinal cord (n=5) AVMs were examined. Hemangioblastoma, meningioma, cerebral cortex obtained from epilepsy surgery, and the basilar artery (BA) from the autopsy were chosen for control comparisons. EPCs were identified as cells that were double-positive for the stem cell marker CD133 and the endothelial cell marker VEGFR-2 (vascular endothelial growth factor receptor-2 or KDR). In addition, SDF-1 was characterized by immunohistochemistry.
Both brain and spinal AVM tissues displayed more CD133, SDF-1, and CD68-positive signals than epilepsy and basilar artery control tissues. The level of EPCs was increased in the brain and spinal cord AVM nidus, mainly at the edge of the vessel wall. The expression of SDF-1 was co-localized with CD31-positive and α-smooth muscle cells, and was predominantly found within the vessel wall.
Our data demonstrate that EPCs are present in the nidus of the brain and spinal cord AVMs, which may mediate pathological vascular remodeling and impact the clinical course of AVMs.
PMCID: PMC2949301  PMID: 20881566
Angiogenesis; Endothelial progenitor cell; Stromal cell-derived factor-1; Vascular malformation
24.  Notch signaling regulates tumor angiogenesis by diverse mechanisms 
Oncogene  2008;27(38):5132-5137.
The Notch signaling pathway is fundamental to proper cardiovascular development and is now recognized as an important player in tumor angiogenesis. Two key Notch ligands have been implicated in tumor angiogenesis, Delta-like 4 and Jagged1. We introduce the proteins and how they work in normal developing vasculature and then discuss differing models describing the action of these Notch ligands in tumor angiogenesis. Endothelial Dll4 expression activates Notch resulting in restriction of new sprout development; for instance, in growing retinal vessels. In agreement with this activity, inhibition of Dll4-mediated Notch signaling in tumors results in hypersprouting of nonfunctional vasculature. This Dll4 inhibition may paradoxically lead to increased angiogenesis but poor tumor growth because the newly growing vessels are not functional. In contrast, Jagged1 has been described as a Notch ligand expressed in tumor cells that can have a positive influence on tumor angiogenesis, possibly by activating Notch on tumor endothelium. A novel Notch inhibitor, the Notch1 decoy, which blocks both Dll4 and Jagged1 has been recently shown to restrict tumor vessel growth. We discuss these models and speculate on therapeutic approaches.
PMCID: PMC3893692  PMID: 18758482
angiogenesis; Notch; Jagged1; Dll4; sprout; VEGFR-2
25.  Notch3 regulates the activation of hepatic stellate cells 
AIM: To investigate whether Notch signaling is involved in liver fibrosis by regulating the activation of hepatic stellate cells (HSCs).
METHODS: Immunohistochemistry was used to detect the expression of Notch3 in fibrotic liver tissues of patients with chronic active hepatitis. The expression of Notch3 in HSC-T6 cells treated or not with transforming growth factor (TGF)-β1 was analyzed by immunofluorescence staining. The expression of Notch3 and myofibroblastic marker α-smooth muscle actin (α-SMA) and collagen I in HSC-T6 cells transfected with pcDNA3.1-N3ICD or control vector were detected by Western blotting and immunofluorescence staining. Moreover, effects of Notch3 knockdown in HSC-T6 by Notch3 siRNA were investigated by Western blotting and immunofluorescence staining.
RESULTS: The expression of Notch3 was significantly up-regulated in fibrotic liver tissues of patients with chronic active hepatitis, but not detected in normal liver tissues. Active Notch signaling was found in HSC-T6 cells. TGF-β1 treatment led to up-regulation of Notch3 expression in HSC-T6 cells, and over-expression of Notch3 increased the expression of α-SMA and collagen I in HSC-T6 without TGF-β1 treatment. Interestingly, transient knockdown of Notch3 decreased the expression of myofibroblastic marker and antagonized TGF-β1-induced expression of α-SMA and collagen I in HSC-T6.
CONCLUSION: Notch3 may regulate the activation of HSCs, and the selective interruption of Notch3 may provide an anti-fibrotic strategy in hepatic fibrosis.
PMCID: PMC3319968  PMID: 22493555
Notch signaling; Myofibroblast; Liver fibrosis; Hepatic stellate cells; siRNA

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