ABSTRACT
In mice, embryonic dermal lymphatic development is well understood and used to study gene functions in lymphangiogenesis. Notch signaling is an evolutionarily conserved pathway that modulates cell fate decisions, which has been shown to both inhibit and promote dermal lymphangiogenesis. Here, we demonstrate distinct roles for Notch4 signaling versus canonical Notch signaling in embryonic dermal lymphangiogenesis. Actively growing embryonic dermal lymphatics expressed NOTCH1, NOTCH4, and DLL4 which correlated with Notch activity. In lymphatic endothelial cells (LECs), DLL4 activation of Notch induced a subset of Notch effectors and lymphatic genes, which were distinctly regulated by Notch1 and Notch4 activation. Treatment of LECs with VEGF-A or VEGF-C upregulated Dll4 transcripts and differentially and temporally regulated the expression of Notch1 and Hes/Hey genes. Mice nullizygous for Notch4 had an increase in the closure of the lymphangiogenic fronts which correlated with reduced vessel caliber in the maturing lymphatic plexus at E14.5 and reduced branching at E16.5. Activation of Notch4 suppressed LEC migration in a wounding assay significantly more than Notch1, suggesting a dominant role for Notch4 in regulating LEC migration. Unlike Notch4 nulls, inhibition of canonical Notch signaling by expressing a dominant negative form of MAML1 (DNMAML) in Prox1+ LECs led to increased lymphatic density consistent with an increase in LEC proliferation, described for the loss of LEC Notch1. Moreover, loss of Notch4 did not affect LEC canonical Notch signaling. Thus, we propose that Notch4 signaling and canonical Notch signaling have distinct functions in the coordination of embryonic dermal lymphangiogenesis.
Subject(s)
Lymphangiogenesis , Lymphatic Vessels , Animals , Endothelial Cells/metabolism , Lymphangiogenesis/physiology , Lymphatic System/metabolism , Lymphatic Vessels/metabolism , Mice , Receptors, Notch/metabolism , Signal TransductionABSTRACT
Collecting lymphatic ducts contain intraluminal valves that prevent backflow. In mice, lymphatic valve morphogenesis begins at embryonic day 15.5 (E15.5). In the mesentery, Prox1 expression is high in valve-forming lymphatic endothelial cells, whereas cells of the lymphatic ducts express lower levels of Prox1. Integrin α9, fibronectin EIIIA, Foxc2, calcineurin and the gap junction protein Cx37 are required for lymphatic valve formation. We show that Notch1 is expressed throughout the developing mesenteric lymphatic vessels at E16.5, and that, by E18.5, Notch1 expression becomes highly enriched in the lymphatic valve endothelial cells. Using a Notch reporter mouse, Notch activity was detected in lymphatic valves at E17.5 and E18.5. The role of Notch in lymphatic valve morphogenesis was studied using a conditional lymphatic endothelial cell driver either to delete Notch1 or to express a dominant-negative Mastermind-like (DNMAML) transgene. Deletion of Notch1 led to an expansion of Prox1(high) cells, a defect in Prox1(high) cell reorientation and a decrease in integrin α9 expression at sites of valve formation. Expression of DNMAML, which blocks all Notch signaling, resulted in a more severe phenotype characterized by a decrease in valves, failure of Prox1(high) cells to cluster, and rounding of the nuclei and decreased fibronectin-EIIIA expression in the Prox1(high) cells found at valve sites. In human dermal lymphatic endothelial cells, activation of Notch1 or Notch4 induced integrin α9, fibronectin EIIIA and Cx37 expression. We conclude that Notch signaling is required for proper lymphatic valve formation and regulates integrin α9 and fibronectin EIIIA expression during valve morphogenesis.
Subject(s)
Gene Expression Regulation, Developmental , Lymphatic Vessels/embryology , Proto-Oncogene Proteins/physiology , Receptor, Notch1/physiology , Receptors, Notch/physiology , Signal Transduction , Animals , Cell Lineage , Cells, Cultured , Connexins/metabolism , Dermis/metabolism , Fibronectins/metabolism , Gene Deletion , Gene Expression Profiling , Genes, Dominant , Humans , Integrin alpha Chains/metabolism , Lymphatic Vessels/metabolism , Mice , Mice, Transgenic , Receptor, Notch1/metabolism , Receptor, Notch4 , Transgenes , Gap Junction alpha-4 ProteinABSTRACT
In development, lymphatic endothelial cells originate within veins and differentiate via a process requiring Prox1. Notch signaling regulates cell-fate decisions, and expression studies suggested that Jag1/Notch1 signaling functions in veins during lymphatic endothelial specification. Using an inducible lymphatic endothelial Prox1CreER(T2) driver, Notch signaling was suppressed by deleting Notch1 or expressing dominant-negative Mastermind-like in Prox1+ endothelial cells. Either loss of Notch1 or reduced Notch signaling increased Prox1+ lymphatic endothelial progenitor cell numbers in the veins, leading to incomplete separation of venous and lymphatic vessels. Notch loss of function resulted in excessive Prox1+ lymphatic cells emerging from the cardinal vein and significant lymphatic overgrowth. Moreover, loss of one allele of Notch1 in Prox1 heterozygous mice rescued embryonic lethality due to Prox1 haploinsufficiency and significantly increased Prox1+ lymphatic endothelial progenitor cell numbers. Expression of a constitutively active Notch1 protein in Prox1+ cells suppressed endothelial Prox1 from E9.75 to E13.5, resulting in misspecified lymphatic endothelial cells based upon reduced expression of podoplanin, LYVE1 and VEGFR3. Notch activation resulted in the appearance of blood endothelial cells in peripheral lymphatic vessels. Activation of Notch signaling in the venous endothelium at E10.5 did not arterialize the cardinal vein, suggesting that Notch can no longer promote arterialization in the cardinal vein during this developmental stage. We report a novel role for Notch1 in limiting the number of lymphatic endothelial cells that differentiate from the veins to assure proper lymphatic specification.
Subject(s)
Endothelial Cells/metabolism , Gene Expression Regulation, Developmental , Receptor, Notch1/metabolism , Alleles , Animals , Cell Differentiation , Cells, Cultured , Female , Glycoproteins/metabolism , Heterozygote , Homeodomain Proteins/metabolism , Humans , Male , Membrane Glycoproteins/metabolism , Membrane Transport Proteins , Mice , Mice, Transgenic , Microcirculation , Signal Transduction , Time Factors , Tumor Suppressor Proteins/metabolism , Vascular Endothelial Growth Factor Receptor-3/metabolismABSTRACT
Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is a key process in several pathological conditions, including tumour growth and age-related macular degeneration. Vascular endothelial growth factors (VEGFs) stimulate angiogenesis and lymphangiogenesis by activating VEGF receptor (VEGFR) tyrosine kinases in endothelial cells. VEGFR-3 (also known as FLT-4) is present in all endothelia during development, and in the adult it becomes restricted to the lymphatic endothelium. However, VEGFR-3 is upregulated in the microvasculature of tumours and wounds. Here we demonstrate that VEGFR-3 is highly expressed in angiogenic sprouts, and genetic targeting of VEGFR-3 or blocking of VEGFR-3 signalling with monoclonal antibodies results in decreased sprouting, vascular density, vessel branching and endothelial cell proliferation in mouse angiogenesis models. Stimulation of VEGFR-3 augmented VEGF-induced angiogenesis and sustained angiogenesis even in the presence of VEGFR-2 (also known as KDR or FLK-1) inhibitors, whereas antibodies against VEGFR-3 and VEGFR-2 in combination resulted in additive inhibition of angiogenesis and tumour growth. Furthermore, genetic or pharmacological disruption of the Notch signalling pathway led to widespread endothelial VEGFR-3 expression and excessive sprouting, which was inhibited by blocking VEGFR-3 signals. Our results implicate VEGFR-3 as a regulator of vascular network formation. Targeting VEGFR-3 may provide additional efficacy for anti-angiogenic therapies, especially towards vessels that are resistant to VEGF or VEGFR-2 inhibitors.
Subject(s)
Neoplasms/blood supply , Neovascularization, Pathologic/metabolism , Vascular Endothelial Growth Factor Receptor-3/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-3/metabolism , Angiogenesis Inhibitors/pharmacology , Animals , Antibodies, Monoclonal/pharmacology , Cell Line, Tumor , Dipeptides/pharmacology , Down-Regulation , Endothelial Cells/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Ligands , Mice , Mice, Inbred BALB C , Mice, Transgenic , Neoplasms/drug therapy , Neovascularization, Pathologic/genetics , Receptors, Notch/metabolism , Signal TransductionABSTRACT
Primary lymphedema (PL), characterized by tissue swelling, fat accumulation, and fibrosis, results from defects in lymphatic vessels or valves caused by mutations in genes involved in development, maturation, and function of the lymphatic vascular system. Pathogenic variants in various genes have been identified in about 30% of PL cases. By screening of a cohort of 755 individuals with PL, we identified two TIE1 (tyrosine kinase with immunoglobulin- and epidermal growth factor-like domains 1) missense variants and one truncating variant, all predicted to be pathogenic by bioinformatic algorithms. The TIE1 receptor, in complex with TIE2, binds angiopoietins to regulate the formation and remodeling of blood and lymphatic vessels. The premature stop codon mutant encoded an inactive truncated extracellular TIE1 fragment with decreased mRNA stability, and the amino acid substitutions led to decreased TIE1 signaling activity. By reproducing the two missense variants in mouse Tie1 via CRISPR/Cas9, we showed that both cause edema and are lethal in homozygous mice. Thus, our results indicate that TIE1 loss-of-function variants can cause lymphatic dysfunction in patients. Together with our earlier demonstration that ANGPT2 loss-of-function mutations can also cause PL, our results emphasize the important role of the ANGPT2/TIE1 pathway in lymphatic function.
Subject(s)
Loss of Function Mutation , Lymphedema , Receptor, TIE-1 , Lymphedema/genetics , Lymphedema/pathology , Lymphedema/metabolism , Humans , Animals , Mice , Receptor, TIE-1/genetics , Receptor, TIE-1/metabolism , Female , Male , Mutation, Missense , Age of Onset , Middle Aged , Adult , Receptor, TIE-2ABSTRACT
Vascular endothelial growth factor C (VEGF-C) induces lymphangiogenesis via VEGF receptor 3 (VEGFR3), which is encoded by the most frequently mutated gene in human primary lymphedema. Angiopoietins (Angs) and their Tie receptors regulate lymphatic vessel development, and mutations of the ANGPT2 gene were recently found in human primary lymphedema. However, the mechanistic basis of Ang2 activity in lymphangiogenesis is not fully understood. Here, we used gene deletion, blocking Abs, transgene induction, and gene transfer to study how Ang2, its Tie2 receptor, and Tie1 regulate lymphatic vessels. We discovered that VEGF-C-induced Ang2 secretion from lymphatic endothelial cells (LECs) was involved in full Akt activation downstream of phosphoinositide 3 kinase (PI3K). Neonatal deletion of genes encoding the Tie receptors or Ang2 in LECs, or administration of an Ang2-blocking Ab decreased VEGFR3 presentation on LECs and inhibited lymphangiogenesis. A similar effect was observed in LECs upon deletion of the PI3K catalytic p110α subunit or with small-molecule inhibition of a constitutively active PI3K located downstream of Ang2. Deletion of Tie receptors or blockade of Ang2 decreased VEGF-C-induced lymphangiogenesis also in adult mice. Our results reveal an important crosstalk between the VEGF-C and Ang signaling pathways and suggest new avenues for therapeutic manipulation of lymphangiogenesis by targeting Ang2/Tie/PI3K signaling.
Subject(s)
Lymphangiogenesis , Lymphedema , Animals , Endothelial Cells/metabolism , Humans , Lymphangiogenesis/physiology , Lymphedema/metabolism , Mice , Phosphatidylinositol 3-Kinase/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Receptor, TIE-2/genetics , Receptor, TIE-2/metabolism , Receptors, TIE/metabolism , Ribonuclease, Pancreatic/metabolism , Vascular Endothelial Growth Factor C/genetics , Vascular Endothelial Growth Factor C/metabolism , Vascular Endothelial Growth Factor Receptor-3/metabolismABSTRACT
Pericytes regulate vessel stability and pericyte dysfunction contributes to retinopathies, stroke, and cancer. Here we define Notch as a key regulator of pericyte function during angiogenesis. In Notch1(+/-); Notch3(-/-) mice, combined deficiency of Notch1 and Notch3 altered pericyte interaction with the endothelium and reduced pericyte coverage of the retinal vasculature. Notch1 and Notch3 were shown to cooperate to promote proper vascular basement membrane formation and contribute to endothelial cell quiescence. Accordingly, loss of pericyte function due to Notch deficiency exacerbates endothelial cell activation caused by Notch1 haploinsufficiency. Mice mutant for Notch1 and Notch3 develop arteriovenous malformations and display hallmarks of the ischemic stroke disease CADASIL. Thus, Notch deficiency compromises pericyte function and contributes to vascular pathologies.
Subject(s)
Arteriovenous Malformations/genetics , CADASIL/genetics , Pericytes/metabolism , Receptor, Notch1/genetics , Receptors, Notch/genetics , Animals , Arteriovenous Malformations/metabolism , Blotting, Western , CADASIL/metabolism , Cell Differentiation/genetics , Cells, Cultured , Disease Models, Animal , Endothelial Cells/metabolism , Endothelial Cells/pathology , Endothelial Cells/ultrastructure , Gene Expression , HEK293 Cells , Humans , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Confocal , Microscopy, Electron, Transmission , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Pericytes/pathology , Pericytes/ultrastructure , Receptor, Notch1/deficiency , Receptor, Notch3 , Receptor, Platelet-Derived Growth Factor beta/genetics , Receptor, Platelet-Derived Growth Factor beta/metabolism , Receptors, Notch/deficiency , Retinal Vessels/metabolism , Retinal Vessels/pathology , Retinal Vessels/physiopathology , Reverse Transcriptase Polymerase Chain Reaction , Time FactorsABSTRACT
UNLABELLED: A proangiogenic role for Jagged (JAG)-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 Delta-like ligand (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 that 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 antiangiogenic soluble (s) VEGFR1/sFLT1. 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 sVEGFR1-sFLT1 and promoting mural-endothelial cell interactions. Blockade of JAG-class ligands represents a novel, viable therapeutic approach to block tumor angiogenesis and growth. SIGNIFICANCE: This is the first report identifying unique regions of the NOTCH1 extracellular domain that interact with JAG-class and DLL-class ligands. Using this knowledge, we developed therapeutic agents that block JAG-dependent NOTCH signaling and demonstrate for the first time that JAG blockade inhibits experimental tumor growth by targeting tumor angiogenesis.
Subject(s)
Immunoglobulin Fc Fragments/administration & dosage , Intercellular Signaling Peptides and Proteins/metabolism , Neoplasms/blood supply , Neoplasms/therapy , Receptor, Notch1/administration & dosage , Receptors, Notch/antagonists & inhibitors , Recombinant Fusion Proteins/administration & dosage , Angiogenesis Inhibitors/administration & dosage , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/genetics , Animals , Female , Humans , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/genetics , Mice , Mice, Inbred C57BL , Neoplasms/metabolism , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/therapy , Protein Binding , Receptor, Notch1/chemistry , Receptor, Notch1/genetics , Receptors, Notch/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Signal TransductionABSTRACT
BACKGROUND: Notch family members function as both oncogenes and tumor suppressors. NOTCH2 is down-regulated in colon cancer, and reduced expression is associated with a less differentiated, more aggressive phenotype, and reduced overall survival. NOTCH2 has also been shown to have pro-apoptotic and growth suppressive effects in thyroid carcinoma, and carcinoid tumors. The expression pattern of NOTCH2 in ovarian cancer is unknown. METHODS: An immunohistochemical analysis using a polyclonal antibody to the NOTCH2 intracellular domain was performed on a total of 119 ovarian carcinomas, and 7 serous borderline tumors, arranged onto tissue arrays. Normal ovarian and fallopian tube epithelium were used as controls. Specimens were scored as low or high NOTCH2 expression. The score distributions for the subtypes were analyzed with the chi square test. RESULTS: Fifty two of 61 (85.2%) papillary serous, eight of 13 (61.5%) clear cell, and 23 of 30 (76.7%) endometrioid, demonstrated negative or lower NOTCH2 expression than normal fallopian tubal epithelium or ovarian surface epithelium. In contrast, 10 of 15 (66.7%) mucinous carcinomas had a high level of NOTCH2 expression and consistently demonstrated intense polarized staining (P<.001). The apical expression of NOTCH2 protein present in the normal fallopian tube epithelium and many borderline tumors was absent in the high grade carcinomas, most notably in papillary serous. CONCLUSION: Decreased NOTCH2 expression is associated with the poorly differentiated serous epithelial ovarian carcinoma histology. Further studies are needed to assess the functional role of NOTCH2 in ovarian cancer and its effect on prognosis.
ABSTRACT
Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting. Surprisingly, we found that endothelial deletion of Vegfr3, but not VEGFR-3-blocking antibodies, postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch signalling, indicating that VEGFR-3 possesses passive and active signalling modalities. Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and target gene expression, and Foxc2(+/-);Vegfr3(+/-) compound heterozygosity recapitulated homozygous loss of Vegfr3. These results indicate that macrophage-derived VEGF-C activates VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic conversion of endothelial cells at fusion points of vessel sprouts.