The roles of the Notch pathway proteins in normal adult vascular physiology and the pathogenesis of brain arteriovenous malformations are not well-understood. Notch 1 and 4 have been detected in human and mutant mice vascular malformations respectively. Although mutations in the human Notch 3 gene caused a genetic form of vascular stroke and dementia, its role in arteriovenous malformations development has been unknown. In this study, we performed immunohistochemistry screening on tissue microarrays containing eight surgically resected human brain arteriovenous malformations and 10 control surgical epilepsy samples. The tissue microarrays were evaluated for Notch 1–4 expression. We have found that compared to normal brain vascular tissue Notch-3 was dramatically increased in brain arteriovenous malformations. Similarly, Notch 4 labelling was also increased in vascular malformations and was confirmed by western blot analysis. Notch 2 was not detectable in any of the human vessels analysed. Using both immunohistochemistry on microarrays and western blot analysis, we have found that Notch-1 expression was detectable in control vessels, and discovered a significant decrease of Notch 1 expression in vascular malformations. We have demonstrated that Notch 3 and 4, and not Notch 1, were highly increased in human arteriovenous malformations. Our findings suggested that Notch 4, and more importantly, Notch 3, may play a role in the development and pathobiology of human arteriovenous malformations.
cell signalling; BAVMs; endothelial cells; vascular malformations
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.
Notch4; AVM; human; brain; signaling
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.
Notch4; AVM; human; brain; signalling
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.
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.
Notch; Dll4; Jagged; Folliculogenesis; Ovary; Gamma-secretase inhibitor; YW152F
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.
Smooth muscle cell; Notch receptor; proliferation; neointima; neointimal hyerplasia
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.
CCM3/PDCD10; DLL4-Notch signalling; angiogenesis; endothelium; cerebral cavernous malformation
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.
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.
endothelial cell; infantile hemangioma; Notch gene expression; stem cell
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.
Dll4-Notch signaling; macrophage/microglia; microvascular density; primary glioblastoma multiforme; vascular pattern
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.
angiogenesis; arteriovenous malformation; arterial-venous differentiation; cell signaling; endothelial cell; Notch signaling; stroke
Cyclosporin A (CSA) suppresses immune function by blocking the cyclophilin A and calcineurin/NFAT signaling pathways. In addition to immunosuppression, CSA has also been shown to have a wide range of effects in the cardiovascular system including disruption of heart valve development, smooth muscle cell proliferation, and angiogenesis inhibition. Circumstantial evidence has suggested that CSA might control Notch signaling which is also a potent regulator of cardiovascular function. Therefore, the goal of this project was to determine if CSA controls Notch and to dissect the molecular mechanism(s) by which CSA impacts cardiovascular homeostasis. We found that CSA blocked JAG1, but not Dll4 mediated Notch1 NICD cleavage in transfected 293T cells and decreased Notch signaling in zebrafish embryos. CSA suppression of Notch was linked to cyclophilin A but not calcineurin/NFAT inhibition since N-MeVal-4-CsA but not FK506 decreased Notch1 NICD cleavage. To examine the effect of CSA on vascular development and function, double transgenic Fli1-GFP/Gata1-RFP zebrafish embryos were treated with CSA and monitored for vasculogenesis, angiogenesis, and overall cardiovascular function. Vascular patterning was not obviously impacted by CSA treatment and contrary to the anti-angiogenic activity ascribed to CSA, angiogenic sprouting of ISV vessels was normal in CSA treated embryos. Most strikingly, CSA treated embryos exhibited a progressive decline in blood flow that was associated with eventual collapse of vascular luminal structures. Vascular collapse in zebrafish embryos was partially rescued by global Notch inhibition with DAPT suggesting that disruption of normal Notch signaling by CSA may be linked to vascular collapse. However, multiple signaling pathways likely cause the vascular collapse phenotype since both cyclophilin A and calcineurin/NFAT were required for normal vascular function. Collectively, these results show that CSA is a novel inhibitor of Notch signaling and vascular function in zebrafish embryos.
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.
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.
Recent studies have implicated aberrant Notch signaling in breast cancers. Yet, relatively little is known about the pattern of expression of various components of the Notch pathway, or its mechanism of action. To better understand the role of the Notch pathway in breast cancer, we have undertaken a detailed expression analysis of various Notch receptors, their ligands, and downstream targets at different stages of breast cancer progression.
We report here that there is a general increase in the expression levels of Notch 1, 2, 4, Jagged1, Jagged2, and Delta-like 4 proteins in breast cancers, with simultaneous upregulation of multiple Notch receptors and ligands in a given cancer tissue. While Notch3 and Delta-like1 were undetectable in normal tissues, moderate to high expression was detected in several cancers. We detected the presence of active, cleaved Notch1, along with downstream targets of the Notch pathway, Hes1/Hes5, in ~75% of breast cancers, clearly indicating that in a large proportion of breast cancers Notch signaling is aberrantly activated. Furthermore, we detected cleaved Notch1 and Hes1/5 in early precursors of breast cancers - hyperplasia and ductal carcinoma in situ - suggesting that aberrant Notch activation may be an early event in breast cancer progression. Mechanistically, while constitutively active Notch1 alone failed to transform immortalized breast cells, it synergized with the Ras/MAPK pathway to mediate transformation. This cooperation is reflected in vivo, as a subset of cleaved Notch positive tumors additionally expressed phopsho-Erk1/2 in the nuclei. Such cases exhibited high node positivity, suggesting that Notch-Ras cooperation may lead to poor prognosis.
High level expression of Notch receptors and ligands, and its increased activation in several breast cancers and early precursors, places Notch signaling as a key player in breast cancer pathogenesis. Its cooperation with the Ras/MAPK pathway in transformation offers combined inhibition of the two pathways as a new modality for breast cancer treatment.
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.
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.
CADASIL; Notch3; protein aggregation; extracellular matrix proteins; cerebrovasculature
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.
angiogenesis; arteriovenous malformation; EphrinB2; EphB4; Notch signaling pathway; vascular morphogenesis
In the vasculature, Notch signaling functions as a downstream effecter of Vascular Endothelial Growth Factor (VEGF) signaling. VEGF regulates sprouting angiogenesis in part by inducing and activating matrix metalloproteases (MMPs). This study sought to determine if VEGF regulation of MMPs was mediated via Notch signaling and to determine how Notch regulation of MMPs influenced endothelial cell morphogenesis.
Methods and Results
We assessed the relationship between VEGF and Notch signaling in cultured human umbilical vein endothelial cells. Overexpression of VEGF-induced Notch4 and the Notch ligand, Dll4, activated Notch signaling, and altered endothelial cell morphology in a fashion similar to that induced by Notch activation. Expression of a secreted Notch antagonist (Notch1 decoy) suppressed VEGF-mediated activation of endothelial Notch signaling and endothelial morphogenesis. We demonstrate that Notch mediates VEGF-induced matrix metalloprotease activity via induction of MMP9 and MT1-MMP expression and activation of MMP2. Introduction of a MMP inhibitor blocked Notch-mediated endothelial morphogenesis. In mice, analysis of VEGF-induced dermal angiogenesis demonstrated that the Notch1 decoy reduced perivascular MMP9 expression.
Taken together, our data demonstrate that Notch signaling can act downstream of VEGF signaling to regulate endothelial cell morphogenesis via induction and activation of specific MMPs. In a murine model of VEGF-induced dermal angiogenesis, Notch inhibition led to reduced MMP9 expression.
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.
angiogenesis; Notch; Jagged1; Dll4; sprout; VEGFR-2
A pro-angiogenic role for Jagged-dependent activation of Notch signaling in the endothelium has yet to be described. Using proteins that encoded different NOTCH1 EGF-like repeats, we identified unique regions of DLL-class and JAG-class ligand/receptor interactions, and developed Notch decoys that function as ligand-specific Notch inhibitors. N110-24 decoy blocked JAG1/JAG2-mediated NOTCH1 signaling, angiogenic sprouting in vitro and retinal angiogenesis, demonstrating JAG-dependent Notch signal activation promotes angiogenesis. In tumors, N110-24 decoy reduced angiogenic sprouting, vessel perfusion, pericyte coverage, and tumor growth. JAG/NOTCH signaling uniquely inhibited expression of anti-angiogenic sVEFGFR-1/sFlt-1. N11-13 decoy interfered with DLL1/DLL4-mediated NOTCH1 signaling and caused endothelial hypersprouting in vitro, in retinal angiogenesis and in tumors. Thus, blockade of JAG- or DLL-mediated Notch signaling inhibits angiogenesis by distinct mechanisms. JAG/Notch signaling positively regulates angiogenesis by suppressing sVEGFR-1/sFlt-1 and promoting mural/endothelial cell interactions. Blockade of JAG-class ligands represents a novel, viable therapeutic approach to block tumor angiogenesis and growth.
Soluble Notch; Jagged; Delta-like; tumor; angiogenesis
Arteriovenous differentiation is a key event during vascular development and hemodynamic forces play an important role. Arteriovenous gene expression is present before the onset of flow, however it remains plastic and flow can alter arteriovenous identity. Notch signaling is especially important in the genetic determination of arteriovenous identity. Nevertheless, the effect of the onset of circulation on Notch expression and signaling has not been studied. The aim of this study is therefore to investigate the interaction of Notch1 signaling and hemodynamic forces during early vascular development. We find that the onset of Notch1 expression coincides with the onset of flow, and that expression is pan-endothelial at the onset of circulation in mouse embryos and only becomes arterial-specific after remodeling has occurred. When we ablate flow in the early embryo, endothelial cells fail to express Notch1. We show that low and disturbed flow patterns upregulate Notch1 expression in endothelial cells in vitro, but that higher shear stress levels do not (≥10 dynes/cm2). Using siRNA, we knocked down Notch1 to investigate the role of Notch1 in mechanotransduction. When we applied shear stress levels similar to those found in embryonic arteries, we found an upregulation of Klf2, Dll1, Dll4, Jag1, Hey1, Nrp1 and CoupTFII but that only Dll4, Hey1, Nrp1 and EphB4 required Notch1 for flow-induced expression. Our results therefore indicate that Notch1 can modulate mechanotransduction but is not a critical mediator of the process since many genes mechanotransduce normally in the absence of Notch1, including genes involved in arteriovenous differentiation.
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.
cerebral arteriovenous malformations; embolisation; angioarchitecture; endovascular treatment; treatment outcome
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.
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.
Salivary Gland; Epithelial Cell Differentiation; Notch; Hes1