WO1998057621A1 - Modulation angiogenique par transduction du signal de notch - Google Patents

Modulation angiogenique par transduction du signal de notch Download PDF

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Publication number
WO1998057621A1
WO1998057621A1 PCT/US1998/013050 US9813050W WO9857621A1 WO 1998057621 A1 WO1998057621 A1 WO 1998057621A1 US 9813050 W US9813050 W US 9813050W WO 9857621 A1 WO9857621 A1 WO 9857621A1
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notch4
protein
cells
ligand
notch4 protein
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PCT/US1998/013050
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English (en)
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Jan Kitajewski
Hendrik Uyttendaele
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The Trustees Of Columbia University In The City Ofnew York
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Priority to AU81628/98A priority Critical patent/AU8162898A/en
Publication of WO1998057621A1 publication Critical patent/WO1998057621A1/fr
Priority to US09/467,997 priority patent/US6379925B1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • int-3 was originally identified based on its oncogenic effects in the mouse mammary gland. int-3 is a frequent target for insertional activation by MMTV proviral DNA in MMTV- induced mammary gland tumors
  • Tumor specific transcripts derived from the int-3 gene encode a protein homologous to the intracellular part of the Notch family of cell surface receptors. .
  • Exogenous expression of the int-3 oncoprotein has been shown to affect the growth and development of mammary epithelial cells. Over expression of the int-3 oncoprotein in a mouse mammary epithelial cells (HC11) promotes anchorage independent growth (Robbins et al . , 1992) .
  • int-3 as an MMTV-LTR driven transgene in the mouse mammary gland results in abnormal development of the mammary gland and rapid development of undifferentiated mammary carcinomas (Jhappan et al . , 1992) .
  • endogenous int-3 protein has been detected in mammary stroma and epithelium (Smith et al . , 1995).
  • Drosophila Notch regulates multiple cell fate decisions that involve cell-cell interactions during fly development, for instance, control of cell fate decisions involving neural/epidermal specification in proneural clusters (Artavanis-Tsakonas and Simpson, 1991) .
  • elegans lin-12 and glp-1 proteins are structurally related to Notch, and are also involved in cell fate specifications during development in the nematode (Greenwald, 1985; Yochem and Greenwald, 1989). Genetic analysis of Notch/lin-12 genes suggest that this family of genes controls binary cell fate decisions and inductive signaling that depend on cell-cell interactions
  • Notch/lin-12 genes have been proposed to block cell differentiation, thus maintaining the competence of cells for subsequent cell-fate determination (Coffman et al . , 1993; Fortini et al . , 1993).
  • Notch/lin-12 genes encode transmembrane receptor proteins characterized by highly repeated, conserved domains.
  • the amino terminus of Notch proteins encodes the extracellular domain and contains as many as 36 repeats of an EGF-like motif involved in ligand binding (Rebay et al . , 1993), and three tandem copies of a Notch/lin-12 sequence motif of unknown function.
  • the intracellular portion of Notch proteins is characterized by six tandem copies of a cdclO/ankyrin motif, thought to be a protein- protein interaction domain (Michaely and Bennett, 1992), and a PEST sequence motif which may represent a protein degradation signal (Rogers et al . , 1986).
  • Notch/lin-12 proteins that contain an intact intracellular domain without most of the extracellular domain behave as constitutively activated receptors (reviewed in Artavanis-Tsakonas et al . , 1995; Greenwald, 1994).
  • the human Notch 1 orthologue, TAN-1 was first identified in independently isolated translocation breakpoints in acute T lymphoblastic leukemia and is predicted to encode a truncated product that has an intact intracellular domain but lacks most of the extracellular domain (Ellisen et al . , 1991) .
  • the int-3 oncoprotein encodes the intracellular domain of a Notch-like protein and thus has been proposed to act as an activated Notch receptor (Robbins et al . , 1992).
  • Notch-related genes have been isolated from mammals; including mouse (Franco Del Amo et al . , 1993; Lardelli et al . , 1994; Lardelli and Lendahl, 1993; Reaume et al., 1992), rat ( einmaster et al . , 1992; einmaster et al., 1991), and human (Ellisen et al . , 1991; Stifani et al., 1992; Sugaya et al . , 1994).
  • Notchl has been identified in the mouse and their embryonic expression patterns display partially overlapping but distinct patterns of expression that are consistent with a potential role in the formation of the mesoderm, somites, and nervous system (Williams et al . , 1995) .
  • Abundant expression of Notchl, Notch2 , and Notch3 is found in proliferating neuroepithelium during central nervous system development.
  • Targeted disruption of the Notchl gene in mice results in embryonic death during the second half of gestation (Conlon et al . , 1995; Swiatek et al .
  • homozygous mutant embryos display delayed somitogenesis as well as widespread cell death, preferentially in neuroepithelium and neurogenic neural crest (Conlon et al . , 1995; Swiatek et al . , 1994).
  • the identification and expression analysis of a fourth murine Notch homologue is reported here.
  • the fourth murine Notch homologue has been named Notch4 and the int- 3_ nomenclature has been reserved for the truncated oncogene.
  • the intracellular domain of the int-3 oncoprotein shares homology with the Notch/Lin- 12 protein family, a comparison of the full length Notch4 protein to that of the int-3 oncoprotein is now provided.
  • the activated int-3 protein encodes only the transmembrane and intracellular domain of the Notch4 protein.
  • Notch4 contains the conserved features of all Notch proteins, however Notch4 has 7 fewer EGF-like repeats compared to Notchl and Notch2 and contains a significantly shorter intracellular domain. Notch4 is expressed primarily in embryonic endothelium and in adult endothelium and male germ cells .
  • This invention uses an established cell line, Rat Brain Microvessel Endothelial cells (RBE4 cells) , to test RBE4 cells.
  • RBE4 cells grown on collagen coated plates in the absence of bFGF or with low concentrations of bFGF (lng/ml) display a cobblestone morphology.
  • bFGF low concentrations of bFGF
  • RBE4 cells reach confluency they growth arrest and cells retain their cobblestone morphology.
  • Post-confluent RBE4 cell cultures grown in the presence of 5ng/ml bFGF develop ulticellular aggregates from the cobblestone monolayer.
  • the RBE4 cell sprouts and three-dimensional structures contain high activity of several enzymatic markers that are specific for differentiated microvessels .
  • bFGF a known angiogenic agent, induces angiogenesis of RBE4 cells which upon treatment with high concentrations of bFGF develop structures resembling capillaries (based on their morphological appearance and based on their expression of differentiated endothelial cell markers) .
  • RBE4 cells express an endogenous Notch, since RBE4 cells that are programmed to express Jagged (a mammalian Notch ligand) have a similar phenotype when compared to RBE4 cells that express a constitutive activated Notch (int-3) . This is consistent with previously published data showing that Notch4 is expressed in endothelial cells in vivo .
  • RBE4 cells do not express sufficient levels of Notch4 ligand to activate the Notch4 receptor, since RBE4 cells programmed to express Notch4 do not exhibit a spindle form morphology when grown in the absence of bFGF.
  • This invention also demonstrates that over expression of Notch4 protein in RBE4 cells does not result in activation of the Notch4 receptor, because otherwise one would expect that these cells would have a similar phenotype to those cells that express a constitutive activated form of the Notch4 receptor.
  • This invention also demonstrates that bFGF mediated angiogenesis is unrelated to Notch4 mediated angiogenesis. This invention further suggests that Jagged or activated Notch4 induced activity can synergize or cooperate with bFGF.
  • Notch signaling is typically associated with cell fate decision. The finding that Notch activation stimulates capillary outgrowth suggests that Notch receptors must be activated to allow this process to occur. Therefore, Notch modulation provides a novel paradigm for regulating angiogenesis. Specifically, modulation of Notch signaling can be used to modulate angiogenesis either positively, by activating Notch signaling to stimulate angiogenesis or negatively, by blocking Notch signaling to block angiogenesis . This modulation would be distinct from previous proteins known to modulate angiogenesis. The induction or inhibition of angiogenesis in vivo can be used as a therapeutic means to treat a variety of diseases, including but not limited to cancer, diabetes, wound repair and arteriosclerosis.
  • Notch signaling is distinct from FGF signaling, this offers a different strategy to affect angiogenesis that may or may not be more effective then FGFs .
  • Notch signaling establishes cell fate decision, by blocking Notch, angiogenesis may be blocked regardless of what other angiogenic factors are present.
  • Notch and FGF may be used synergistically to modulate angiogenesis.
  • This invention provides an isolated nucleic acid molecule, encoding a Notch4 protein.
  • This invention provides a vector comprising the above-described nucleic acid.
  • This invention also provides an isolated Notch4 protein.
  • This invention also provides a plasmid which comprises the regulatory elements necessary for expression of DNA in a mammalian cell operatively linked to the DNA encoding Notch4 protein so as to permit expression thereof.
  • This invention also provides a mammalian cell comprising the above-described plasmid or vector.
  • This invention also provides a nucleic acid probe comprising a nucleic acid of at least 12 nucleotides capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid encoding Notch4 protein.
  • This invention also provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to mRNA encoding Notch4 protein so as to prevent translation of the mRNA.
  • This invention also provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to mRNA encoding activated Notch4 protein so as to prevent translation of the mRNA.
  • This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to the genomic DNA encoding a Notch4 protein or an activated Notch4 protein.
  • This invention provides an antibody directed to a Notch4 protein.
  • This invention provides a pharmaceutical composition comprising an amount of the oligonucleotide effective to reduce activity of Notch4 protein by passing through a cell membrane and binding specifically with mRNA encoding Notch4 protein in the cell so as to prevent its translation and a pharmaceutically acceptable carrier capable of passing through a cell membrane.
  • This invention provides a pharmaceutical composition comprising an amount of an antagonist effective to reduce the activity of Notch4 protein and a pharmaceutically acceptable carrier.
  • This invention provides a pharmaceutical composition comprising an amount of an agonist effective to increase activity of Notch4 protein and a pharmaceutically acceptable carrier.
  • This invention provides the above-described pharmaceutical composition which comprises an amount of the antibody effective to block binding of a ligand to the Notch4 protein and a pharmaceutically acceptable carrier.
  • This invention also provides a method for determining whether a ligand can specifically bind to Notch4 protein comprising the steps of: a) contacting Notch4 protein with the ligand under conditions permitting formation of specific complexes between Notch4 protein and known Notch4 protein-binding ligands; b) determining whether complexes result from step (a) , the presence of such complexes indicating that the ligand specifically binds to Notch4 protein .
  • This invention provides a method for determining whether a ligand can specifically bind to a Notch4 protein which comprises preparing a cell extract from cells transfected with and expressing DNA encoding the Notch4 protein, isolating a membrane fraction from the cell extract, contacting the membrane fraction with the ligand under conditions permitting binding of ligands to such Notch4 protein, detecting the presence of the ligand specifically bound to Notch4 protein, and thereby determining whether the ligand specifically binds to Notch4 protein.
  • This invention provides a method for determining whether a ligand is a Notch4 protein agonist which comprises contacting a cell transfected with and expressing nucleic acid encoding Notch4 protein with the ligand under conditions permitting activation of a functional Notch4 protein response by ligands known to be agonists of Notch4 protein, and detecting whether a functional increase in Notch4 protein activity occurs so as to determine whether the ligand is a Notch4 agonist.
  • This invention provides a method for determining whether a ligand is a Notch4 protein antagonist which comprises contacting a cell transfected with and expressing DNA encoding Notch4 protein with the ligand under conditions permitting the activation of a functional Notch4 protein, and detecting whether a functional decrease in Notch4 activity occurs so as to determine whether the ligand is a Notch4 protein antagonist.
  • This invention provides a method of treating an abnormality in a subject, wherein the abnormality is alleviated by the inhibition of Notch4 protein activity which comprises administering to a subject an amount of Notch4 antagonist effective to inhibit Notch4 protein activity.
  • This invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by the activation of Notch4 activity which comprises administering to a subject an amount of a Notch4 agonist effective to promote Notch4 protein activity.
  • This invention provides a method of preparing isolated Notch4 protein which comprises: a) inserting nucleic acid encoding Notch4 protein in a suitable vector which comprises the regulatory elements necessary for expression of the nucleic acid operatively linked to the nucleic acid encoding Notch4 protein; b) inserting the resulting vector in a suitable host cell so as to obtain a cell which produces Notch4 protein; c) recovering the Notch4 protein produced by the resulting cell; and d) purifying the protein so recovered.
  • This invention also provides a method of modulating angiogenesis in a subject comprising administering to the subject an effective amount of agonist or antagonist of the NotchA protein so as to promote or inhibit angiogenesis in the subject .
  • This invention also provides a method of promoting angiogenesis in a subject comprising administering to the subject an amount of Notch4 protein agonist effective to promote angiogenesis in the subject.
  • This invention also provides method of inhibiting angiogenesis in a subject comprising administering to the subject an amount of Notch4 protein antagonist effective to inhibit angiogenesis in the subject.
  • This invention also provides method of promoting angiogenesis comprising transducing selected cells, wherein the cells express activated Notch4 protein in an amount sufficient promote angiogenesis in the cells.
  • This invention provides a method of promoting angiogenesis comprising transducing selected cells which express Notch4 protein, wherein the cells express a Notch4 ligand in an amount sufficient to promote angiogenesis in the cells.
  • This invention provides a method of promoting angiogenesis comprising transducing selected cells, wherein the cells express a Notch4 protein and a Notch4 ligand in an amount sufficient to promote angiogenesis in the cells.
  • This invention provides a method of inhibiting angiogenesis comprising administering to cells expressing Notch4 protein an amount of a specific antibody effective to block binding of a ligand to Notch4 protein.
  • This invention provides a method of inhibiting angiogenesis comprising administering to cells expressing Notch4 protein an amount of a fragment of a specific antibody effective to block binding of a ligand to Notch4 protein.
  • This invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by inhibiting angiogenesis comprising blocking Notch4 protein signaling in the subject.
  • This invention also provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by promoting angiogenesis comprising stimulating the Notch4 signaling pathway.
  • Figure 1 Deduced amino acid sequence of Notch4 (GenBank accession number U43691) .
  • the boxed regions indicate the major structural elements of the Notch family of proteins, as follows: 29 epidermal growth factor (EGF)-like repeats; 3 Notch/linl2 repeats; a transmembrane domain; and 6 cdclO/ankyrin repeats.
  • EGF epidermal growth factor
  • Putative glycosylation sites are underlined.
  • a putative PEST domain is doubly underlined.
  • the two cysteines thought to promote dimerization are marked with asterisks.
  • the initiating methionine of the int-3 oncoprotein is in bold and marked by an arrow.
  • FIG. 2 Schematic structural comparison of the four murine Notch proteins.
  • the EGF-like repeats are numbered according to their position in each different protein. Where equivalent EGF-like repeats can be identified, connecting lines are placed to compare the relationship between these repeats in different Notch proteins (see EGF- repeat equivalents) .
  • Notch4 contains seven EGF-like repeats, fewer than Notchl and Notch2. One of the missing EGF-like repeats (#25) in Notch4 is derived from equivalent repeats #31 and #32 of Notchl/Notch2 , creating a novel and hybrid EGF-like repeat. Eight of the EGF-like repeats of Notch4 (#14 to #21) have no identifiable equivalent repeats in Notchl/Notch2. The region of Notch4 from the end of the ceclO/ankyrin repeats to the carboxy terminus is shorter when compared to Notchl, 2 and 3.
  • FIG. 3 Amino acid sequence comparison of EGF-like repeat #11 of mouse Notchl, 2, 3 and 4. Residues conserved between the mouse Notch proteins are shaded and the putative calciu-binding sites are marked with arrows. A region with in EGF-like repeat #11 of the Notch proteins containing non-conserved and variable numbers of residues is boxed. The leucine to proline mutation in Xenopus Notch that obliterates binding to Delta is marked with an asterisk ( * ) .
  • Figure 4 Expression analysis of Notch4 in adult mouse tissues.
  • A Northern blot using a riboprobe transcribed from the 3' UTR of Notch4 (probe D in Fig. 5).
  • B The same blot reprobed with a GAPDH probe.
  • the transcript sizes of 6.7 kb, 1.5 kb and 1.1 kb are indicated and were estimated with reference to 28S and 18S rRNA migration.
  • Notch4 testis transcripts are expressed in post-meiotic germ cells. Northern blot analysis from staged and germ cell-deficient testes with probe C and a GAPDH probe. Note that GAPDH transcripts appear as two isoforms in the adult testis. RNA was isolated from testes of day 7 p.n., day 17 p.n., adult, W v /W and W/+ mice, as indicated.
  • B Northern blot analysis of several adult tissues with probe A, derived from the 5' UTR of Notch4 and a GAPDH probe.
  • C Schematic representation of truncated Notch4 transcripts as compared to the full-length coding potential.
  • Notch4 is expressed in embryonic endothelial cells.
  • A,B Phase contrast and dark-field photomicrograph of a horizontal section of a 9 d.p.c. embryo hybridized with a cRNA probe corresponding to Notch4. Strong labeling is detectable over the anterior cardinal vein (white/black arrows) . Diffuse labeling is also present throughout the developing nervous system and at higher levels over the tip of the neural folds (red arrows) .
  • C-F Phase and darkfields images of a horizontal section of a 13.5 d.p.c embryo hybridized for Notch4 , showing the venous and arterial system anterior to the lung, including dorsal aorta arch, aortic and pulmonary tract.
  • E and F are higher magnifications of the area framed in C. Embryonic vessels are labeled and, as shown in E and F, labeling is restricted to the endothelial cells lining the vessels. Arrows denote the gut, which does not have a detectable signal in the epithelium.
  • Notch4 is expressed in adult lung endothelial cells.
  • A, B Phase contrast and darkfield photomicrographs of an adult mouse lung hybridized with a cRNA probe corresponding to Notch4. Punctate staining is observed over the alveolar walls, which are predominantly composed of capillaries. No labeling is observed over the pseudostratified squamous epithelium (black and white arrows) nor over the smooth muscle cells (red arrows) .
  • Figure 8 RBE4 cell lines grown on collagen coated plates in the absence of bFGF.
  • Fig 10 Immunohistochemical analysis on adult mouse kidney sections. Endothelial cells within the cortical kidney glomeruli can be detected by using either an anti-PECAM antibody (B) or an antibody was used in panel B, and both panel A and B were processed for horse radish peroxidase detection which results in a brown staining. Pre- immune serum at identical dilution was used in panel C, and both panel C and D were processed for alkaline phosphatase detection which results in a blue-purple staining. Panels C and D were counter stained with eosin.
  • B anti-PECAM antibody
  • Pre- immune serum at identical dilution was used in panel C, and both panel C and D were processed for alkaline phosphatase detection which results in a blue-purple staining.
  • Panels C and D were counter stained with eosin.
  • FIG 11 Schematic representation of the notch4, Notch4/int- 3 and Jagged-1 proteins (A) . conserveed domains within each proteins are indicated. Immunoblot analysis on lysates of 293 cells transiently transfected with either epitope tagged or non-epitope tagged cDNA' s of Notch4/int or Notch4
  • Notch4 using the anti-HA antibody demonstrates expression of each respective protein (D) .
  • Fig 12 RBE4 cells that are programmed to express either LacZ (A) or Notch.4 (B) display a cobble stone morphology when grown on collagen coated plates.
  • FIG. 13 Histochemical analysis of alkaline phosphatase (B) and gamma-glutamyl transpeptidase ® activities in microvessel structures induced by RBE4 cells expressing Notch4/int-3 (B) or Jagged-1 (C) . RBE4 cells surrounding the microvessel structures do not express either enzyme activity (B and C) . Panel A is control.
  • RNA RNA from RBE4 cells programmed to express either LacZ, Notch4/int-3 , Notch4 or Jagged-1
  • riboprobes for either Jagged-1, Notchl, Notch4 or ⁇ -actin.
  • LTR-driven transcripts are denoted (LTR) .
  • the activity of a protein such as a Notch4 protein may be measured using any of a variety of appropriate functional assays in which activation of the protein in question results in an observable change in the level of some second messenger system.
  • This invention provides an isolated nucleic acid encoding Notch4 protein.
  • the Notch4 nucleic acid provides the Notch4 nucleic acid, wherein the nucleic acid is a DNA.
  • the Notch4 nucleic acid has the nucleic acid sequence as described in figure 9.
  • the DNA is a cDNA.
  • the DNA is a genomic DNA.
  • the DNA is synthetic DNA.
  • the nucleic acid is RNA.
  • the nucleic acid encodes Notch4 protein.
  • Notch4 protein has the amino acid sequence as described in Figure 1.
  • Notch4 protein includes any polypeptide having Notch4 protein activity and having an amino acid sequence homologous to the amino acid sequence of Notch4.
  • this term includes any such polypeptides whether naturally occurring and obtained by purification from natural sources or non-naturally occurring and obtained synthetically, e.g. by recombinant DNA procedures.
  • the term includes any such polypeptide where its sequence is substantially the same as, or identical to the sequence of any mammalian homolog of the human polypeptide, e.g. murine, bovine, porcine, etc. homologs .
  • the term includes mutants or other variants of any of the foregoing which retain at least some of the biological activity of nonmutants or nonvariants .
  • This invention also encompasses DNAs and cDNAs which encode amino acid sequences which differ from those of Notch4 protein, but which should not produce phenotypic changes.
  • this invention also encompasses DNAs and cDNAs which hybridize to the DNA and cDNA of the subject invention. Hybridization methods are well known to those of skill in the art.
  • This invention provides an isolated nucleic acid encoding an activated Notch4 protein.
  • the nucleic acid of the subject invention also include DNA coding for polypeptide analogs, fragments or derivatives of polypeptides which differ from naturally-occurring forms in terms of the identity or location of one or more amino acid residues (deletion analogs containing less than all of the residues specified for the protein, substitution analogs wherein one or more residues specified are replaced by other residues and addition analogs where in one or more amino acid residues is added to a terminal or medial portion of the polypeptides) and which share some or all properties of naturally-occurring forms.
  • nucleic acids include: the incorporation of codons "preferred" for expression by selected non-mammalian hosts; the provision of sites for cleavage by restriction endonuclease enzymes; and the provision of additional initial, terminal or intermediate DNA sequences that facilitate construction of readily expressed vectors.
  • nucleic acids described and claimed herein are useful for the information which they provide concerning the amino acid sequence of the polypeptide and as products for the large scale synthesis of the polypeptide by a variety of recombinant techniques.
  • the nucleic acid is useful for generating new cloning and expression vectors, transformed and transfected prokaryotic and eukaryotic host cells, and new and useful methods for cultured growth of such host cells capable of expression of the polypeptide and related products .
  • This invention also provides an isolated Notch4 protein.
  • the Notch4 protein has the amino acid sequence as shown in Figure 1.
  • This invention provides a vector comprising the above- described nucleic acid.
  • Vectors which comprise the isolated nucleic acid described hereinabove also are provided.
  • Suitable vectors comprise, but are not limited to, a plasmid or a virus. These vectors may be transformed into a suitable host cell to form a host cell vector system for the production of a polypeptide having the biological activity of Notch4 protein.
  • This invention provides the above-described vector adapted for expression in a cell which further comprises the regulatory elements necessary for expression of the nucleic acid in the cell operatively linked to the nucleic acid encoding Notch4 protein as to permit expression thereof .
  • This invention provides the above-described vector adapted for expression in a bacterial cell which further comprises the regulatory elements necessary for expression of the nucleic acid in the bacterial cell operatively linked to the nucleic acid encoding Notch4 protein so as to permit expression thereof.
  • This invention provides the above-described vector adapted for expression in a yeast cell which comprises the regulatory elements necessary for expression of the nucleic acid in the yeast cell operatively linked to the nucleic acid encoding Notch4 protein so as to permit expression thereof .
  • This invention provides the above-described vector adapted for expression in a plant cell which comprises the regulatory elements necessary for expression of the nucleic acid in the insect cell operatively linked to the nucleic acid encoding Notch4 protein so as to permit expression thereof.
  • the vector is adapted for expression in a animal cell which comprises the regulatory elements necessary for expression of the DNA in the animal cell operatively linked to the DNA encoding animal Notch4 protein so as to permit expression thereof .
  • This invention provides a plasmid which comprises the regulatory elements necessary for expression of DNA in a cell operatively linked to the DNA encoding Notch4 protein as to permit expression thereof designated pBS-Notch4 (ATCC Accession No . ) .
  • This plasmid (pBS-Notch4) was deposited on June 12, 1997, with the American Type Culture Collection (ATCC) , 12301
  • This invention provides a mammalian cell comprising the above-described plasmid or vector.
  • the mammalian cell is a Rat Brain Microvessel endothelial (RBE4) cell.
  • This invention provides a method of transforming host cells produced by transfecting host cells with the above- described vector.
  • This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 12 nucleotides capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding Notch4 protein.
  • the nucleic acid is DNA.
  • the nucleic acid probe can either be DNA or RNA.
  • specifically hybridizing means the ability of a nucleic acid to recognize a nucleic acid sequence complementary to its own and to form double - helical segments through hydrogen bonding between complementary base pairs.
  • nucleic acid of at least 12 nucleotides capable of specifically hybridizing with a sequence of a nucleic acid encoding Notch4 can be used as a probe.
  • Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary greatly in length and may be labeled with a detectable label, such as a radioisotope or fluorescent dye, to facilitate detection of the probe.
  • DNA probe molecules may be produced by insertion of a DNA molecule which encodes Notch4 into suitable vectors, such as plasmids or bacteriophages, followed by transforming into suitable bacterial host cells, replication in the transformed bacterial host cells and harvesting of the DNA probes, using methods well known in the art. Alternatively, probes may be generated chemically from DNA synthesizers.
  • RNA probes may be generated by inserting the DNA which encodes Notch4 protein downstream of a bacteriophage promoter such as T3 , T7 or SP6. Large amounts of RNA probe may be produced by incubating the labeled nucleotides with the linearized fragment where it contains an upstream promoter in the presence of the appropriate RNA polymerase .
  • This invention also provides a nucleic acid of at least 12 nucleotides capable of specifically hybridizing with a sequence of a nucleic acid which is complementary to the mammalian nucleic acid encoding Notch4 protein.
  • This nucleic acid may either be a DNA or RNA molecule.
  • This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to mRNA encoding Notch4 protein so as to prevent translation of the mRNA.
  • This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to the genomic DNA of Notch4 protein or activated Notch4 protein.
  • This invention provides an antisense oligonucleotide of Notch4 comprising chemical analogues of nucleotides.
  • This invention provides an antibody directed to Notch4 protein. This invention also provides an antibody directed to activated Notch4 protein.
  • antibody encompasses fragments of the antibody such that fragments are capable of binding to the specific antigen.
  • Antibody also includes polypeptides which have the antigen binding domains. Such polypeptide may contain a single chain known as single-chain antibody.
  • peptide encompasses both polypeptide and oligopeptide .
  • Polyclonal antibodies against these peptides may be produced by immunizing animals using the selected peptides.
  • Monoclonal antibodies are prepared using hybridoma technology by fusing antibody producing B cells from immunized animals with myeloma cells and selecting the resulting hybridoma cell line producing the desired antibody.
  • monoclonal antibodies may be produced by in vitro techniques known to a person of ordinary skill in the art. These antibodies are useful to detect the expression of mammalian PSM antigen in living animals, in humans, or in biological tissues or fluids isolated from animals or humans.
  • This invention provides a monoclonal antibody directed to an epitope of Notch4 present on the surface of a Notch4 receptor expressing cell.
  • This invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an amount of the oligonucleotide effective to reduce activity of Notch4 protein by passing through a cell membrane and binding specifically with mRNA encoding Notch4 in the cell so as to prevent its translation and a pharmaceutically acceptable carrier capable of passing through a cell membrane.
  • the oligonucleotide is coupled to a substance which inactivates mRNA.
  • the pharmaceutically acceptable carrier capable of passing through a cell membrane comprises a structure which binds to a receptor specific for a selected cell type and is thereby taken up by cells of the selected cell type.
  • This invention provides a pharmaceutical composition comprising an amount of an antagonist effective to reduce the activity of Notch4 protein and a pharmaceutically acceptable carrier.
  • This invention provides a pharmaceutical composition comprising an amount of an agonist effective to increase activity of Notch4 protein and a pharmaceutically acceptable carrier.
  • This invention provides the above-described pharmaceutical composition which comprises an amount of the antibody effective to block binding of a ligand to the Notch4 protein and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers means any of the standard pharmaceutically acceptable carriers. Examples include, but are not limited to, phosphate buffered saline, physiological saline, water and emulsions, such as oil/water emulsions.
  • This invention also provides a method for determining whether a ligand can specifically bind to Notch4 which comprises contacting a cell transfected with and expressing DNA encoding Notch4 with the ligand under conditions permitting binding of ligands to Notch4, detecting the presence of any such ligand specifically bound to Notch4, and thereby determining whether the ligand specifically binds to Notch4.
  • This invention provides a method for determining whether a ligand can specifically bind to Notch4 protein comprising the steps of: a) contacting Notch4 protein with the ligand under conditions permitting formation of specific complexes between Notch4 protein and known Notch4 protein- binding ligands; b) determining whether complexes result from step (a) , the presence of such complexes indicating that the ligand specifically binds to Notch4 protein.
  • the Notch4 protein in step (a) described above is reconstituted in liposomes.
  • the ligand is not previously known to be a ligand which can specifically bind to Notch4 protein.
  • This invention provides a method for determining whether a ligand can specifically bind to Notch4 which comprises contacting a cell transfected with and expressing DNA encoding Notch4 with the ligand under conditions permitting binding of ligands to Notch4, detecting the presence of any such ligand specifically bound to Notch4 , and thereby determining whether the ligand specifically binds to Notch4, such Notch4 having substantially the same amino acid sequence shown in Figure 1.
  • This invention provides a method for determining whether a ligand can specifically bind to Notch4 which comprises contacting a cell transfected with and expressing DNA encoding Notch4 with the ligand under conditions permitting binding of ligands to Notch4, detecting the presence of any such ligand specifically bound to Notch4 , and thereby determining whether the ligand specifically binds to Notch4, such Notch4 being characterized by an amino acid sequence in the transmembrane region having 60% homology or higher to the amino acid sequence in the transmembrane region of Notch4 shown in Figure 1.
  • This invention provides a method for determining whether a ligand can specifically bind to Notch4 which comprises preparing a cell extract from cells transfected with and expressing DNA encoding Notch4 , isolating a membrane fraction from the cell extract, contacting the membrane fraction with the ligand under conditions permitting binding of ligands to such protein, detecting the presence of the ligand specifically bound to Notch4, and thereby determining whether the ligand specifically binds to Notch4.
  • This invention provides a method for determining whether a ligand can specifically bind to Notch4 which comprises preparing a cell extract from cells transfected with and expressing DNA encoding Notch4 , isolating a membrane fraction from the cell extract, contacting the membrane fraction with the ligand under conditions permitting binding of ligands to Notch4, detecting the presence of the ligand specifically bound to Notch4, and thereby determining whether the ligand can specifically bind to Notch4, such Notch4 having substantially the same amino acid sequence shown in Figure 1.
  • This invention provides a method for determining whether a ligand is a Notch4 protein agonist which comprises contacting a cell transfected with and expressing Notch4 protein with the ligand under conditions permitting activation of a functional Notch4 protein response by ligands known to be agonists of Notch4 protein, and detecting whether a functional increase in Notch4 protein activity occurs so as to determine whether the ligand is a Notch4 protein agonist .
  • agonist is used throughout this application to indicate any peptide or non-peptidyl compound which increases the activity of any of the receptors/proteins of the subject invention.
  • This invention provides a method for determining whether a ligand is a Notch4 protein antagonist which comprises contacting a cell transfected with and expressing Notch4 protein with the ligand under conditions permitting activation of a functional Notch4 protein response by ligands known to be agonists of Notch4 protein, and detecting whether a functional decrease in Notch4 protein activity occurs so as to determine whether the ligand is a Notch4 antagonist.
  • antagonist is used throughout this application to indicate any peptide or non-peptidyl compound which decreases the activity of any of the receptors/proteins of the subject invention.
  • This invention provides a pharmaceutical composition which comprises an amount of a Notch4 protein agonist effective to increase the activity of a Notch4 protein within a cell and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises a peptide fragment of Jagged protein capable of increasing the activity of Notch4 protein.
  • This invention also provides a pharmaceutical composition which comprises an amount of a Notch4 protein antagonist effective to decrease activity of Notch4 protein within a cell and a pharmaceutically acceptable carrier.
  • This invention provides a method of modulating angiogenesis in a subject comprising administering to the subject an amount of agonist or antagonist of Notch4 protein effective to promote or inhibit angiogenesis in the subject.
  • This invention provides a method of promoting angiogenesis in a subject comprising administering to the subject an amount of Notch4 protein agonist effective to promote angiogenesis in the subject.
  • This invention provides a method of inhibiting angiogenesis in a subject comprising administering to the subject an amount of Notch4 protein antagonist effective to inhibit angiogenesis in the subject.
  • This invention provides a method of promoting angiogenesis comprising transducing selected cells, wherein the cells express activated Notch4 protein in an amount sufficient promote angiogenesis in the cells.
  • This invention provides a method of promoting angiogenesis comprising transducing selected cells which express Notch4 protein, wherein the cells express a Notch4 ligand in an amount sufficient to promote angiogenesis in the cells.
  • This invention provides a method of promoting angiogenesis comprising transducing selected cells, wherein the cells express a Notch4 protein and a Notch4 protein ligand in an amount sufficient to promote angiogenesis in the cells.
  • the Notch4 protein ligand is Jagged protein or a peptide fragment thereof capable of increasing the activity of Notch4 protein.
  • This invention provides a method of inhibiting angiogenesis comprising administering to cells expressing Notch4 protein an amount of an antibody of effective to block binding of a ligand to Notch4 protein.
  • This invention also provides a method of inhibiting angiogenesis comprising administering to cells expressing Notch4 protein an amount of a fragment of antibody effective to block binding of a ligand to Notch4 protein.
  • This invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by inhibiting angiogenesis comprising blocking Notch4 protein signaling in the subject.
  • the above- described method comprises administering to the subject an amount of an antibody effective to block binding of a ligand to Notch4 protein.
  • the above -described method of treating an abnormality in a subject comprises administering to the subject an amount of a fragment of an antibody effective to block binding of a ligand to Notch4 protein.
  • the abnormality is a solid tumor, hemangioma, hemangiosarcoma or Kaposi's Sarcoma.
  • This invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by promoting angiogenesis comprising stimulating the Notch4 signaling pathway.
  • the above-described method of treatment comprises transducing selected cells within the subject, wherein the cells express activated Notch4 protein.
  • the above-described method of treatment comprises transducing selected cells within the subject expressing a Notch4 protein such that the cells express a Notch4 protein ligand.
  • the above-described method of treatment comprises transducing selected cells within the subject such that the cells express a Notch4 protein and a Notch4 protein ligand.
  • the ligand is Jagged protein or a peptide fragment thereof capable of increasing the activity of Notch4 protein.
  • the abnormality is an ischemic disorder, gangrene, diabetes ulceratis, chronic ulceration, Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) , Vascular Dementia or a wound .
  • CADASIL Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy
  • This invention provides a method of detecting expression of Notch4 by detecting the presence of mRNA coding for Notch4 which comprises obtaining total mRNA from the cell and contacting the mRNA so obtained with the above-described nucleic acid probe under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of Notch4 by the cell.
  • This invention provides a method of detecting the presence of Notch4 on the surface of a cell which comprises contacting the cell with the antibody capable of binding to Notch4 under conditions permitting binding of the antibody to the receptor, detecting the presence of the antibody bound to the cell, and thereby detecting the presence of Notch4 on the surface of the cell.
  • a 1680 bp fragment was amplified by PCR from adult mouse testis cDNA (RT-PCR) using specific primers (5' primer: CGTCCTGCTGCGCTTCCTTGCA (Seq. I.D. No. ) and 3' primer CCGGTGCCTAGTTCAGATTTCTTA) (Seq. I.D. No. ) designed from the int-3 cDNA sequence (Robbins et al . , 1992). This cDNA fragment corresponds to the previously cloned int-3 oncogene.
  • Hybridization solution contained 60% Formamide, 5x SSC, 5x Denhardt's solution, 1% SDS, 20 mM NaH 2 P04 (pH 6.8), 0.1 mg/ml salmon sperm DNA, 100 ug/ml yeast tRNA, 10 ug/ml poly-A mRNA and 7% dextran sulfate and was done for 14 hours at 65°C.
  • Washing solution contained 2x SSC and 1% SDS and was done at room temperature and 50°C for 15 minutes each, followed by a 2 hour wash at 80°C with a solution containing 0.2x SSC and 1% SDS.
  • Membranes were exposed on X-ray film (X-OMAT AR, Kodak ® ) . The integrity of the RNA, as well as comparable amounts of RNA were tested by rehybridization with a GAPDH probe.
  • Staged embryos ranging from 9 days to birth were obtained from timed breedings of CD-I mice. Morning of the vaginal plugs was counted as 0.5 days post coitum (p.c.) . Preparation of tissue and subsequent procedures for in situ hybridization were done as previously described (Marazzi and Buckley, 1993; Sassoon and Rosenthal, 1993). After hybridization and procedures, sections were dehydrated rapidly and processed for standard autoradiography using NTB-2 Kodak ® emulsion and exposed for two weeks at 4°C. Analysis was carried out using both light and dark field optics on a Leica ® DA microscope.
  • Notch4 cDNA clones
  • the int-3 mammary oncogene encodes a truncated protein that is highly homologous to the intracellular part of the Notch receptor proteins.
  • the full length int-3 gene referred to as Notch4
  • Notch4 had been proposed to encode a novel member of the Notch protein family (Robbins et al . , 1992) .
  • cDNAs containing the complete coding potential of the Notch4 gene have been cloned.
  • RT-PCR was used to isolate a 2.4 kb int-3 cDNA encoding the putative intracellular portion of the receptor.
  • cDNAs were isolated by 5' RACE and by screening a mouse lung cDNA library. A total of 37 overlapping cDNA clones were analyzed and sequenced to obtain a 6677 bp cDNA sequence. This sequence encodes one long open reading frame of 1964 amino acids, starting with an initiator methionine at nucleotide 347 and terminating with a stop codon at nucleotide 6239.
  • the 6677 bp cDNA corresponds in size to that of Notch4 transcripts detected by Northern blot analysis; thus, this suggests that the cloned cDNA represents the full length Notch4 gene .
  • Notch4 contains EGF-like repeats, Notch/lin-12 repeats, a transmembrane domain, cdclO/ankyrin repeats and a putative PEST domain.
  • GCG Bestfit, gap weight 3.0, length weight 0.1
  • the Notch4 protein is approximately 60% similar and 43% identical to other vertebrate Notch proteins, and 58% similar and 40% identical to Drosophila Notch. Lower homologies were found when compared to the C . elegans lin-12 and glp-l proteins (49% similar and 29% identical) .
  • a hydrophobic region in the Notch4 protein sequence was identified by hydropathy analysis (Kyte Doolittle algorithm, data not shown) .
  • An N-terminal region contains 19 hydrophobic residues that could function as a signal peptide sequence (Fig. 1) and a putative signal peptidase cleavage site was identified at residue 20.
  • a second hydrophobic region from amino acid residues 1441 to 1465 is of sufficient length (25 amino acids) to behave as a membrane spanning domain and is immediately followed by five consecutive arginine residues that are consistent with a stop transfer signal (Fig. 1) .
  • EGF-like repeats are defined by a cysteine-rich consensus sequence and generally occur in analogous locations in two different Notch proteins. Since analogous repeats are more homologous to each other than to their neighboring EGF-like repeats, they have been referred to in Notch proteins as equivalent EGF-like repeats.
  • FIG. 2 schematizes the relationship of EGF- equivalents between Notch4 and Notchl/Notch2.
  • EGF-like repeats 1-13 of Notch4 are equivalent to EGF-like repeats 1-13 of Notchl/Notch2
  • EGF-like repeats 22-24 of Notch4 correspond to EGF-like repeats 28-30 of Notchl/Notch2
  • EGF-like repeats 26-29 of Notch4 are equivalent to EGF-like repeats 33-36 of Notchl/Notch2.
  • Comparison of Notch4 to other Notch proteins revealed no clear-cut identification of the seven particular equivalent EGF-like repeats that are absent in Notch4.
  • EGF-like repeats The amino acid sequence of equivalent EGF-like repeats has diverged between different Notch homologues and orthologues (Maine et al . , 1995), sometimes resulting in loss of a clear-cut equivalent repeat consensus.
  • Six of the unassigned EGF-like repeats of Notch4 appear to be derived from EGF-like repeats 14-27 of Notchl and Notch2 (Fig. 2) .
  • EGF-like repeat 25 of Notch4 may be a hybrid EGF-like repeats derived from parts of EGF-like repeats 31 and 32 of Notchl/Notch2 (Fig. 2) .
  • a discussion of the relationship between Notch3 and Notchl/Notch2 (shown in Fig. 2) is described in Lardelli et al., 1994.
  • EGF-like repeats 11 and 12 of Drosophila Notch have been shown to be necessary and sufficient for Notch to bind Delta and Serrate proteins in vitro (Rebay et al . , 1991) . These two equivalent EGF-like repeats are present in Notch4 (Fig 2).
  • the putative calcium-binding residues (Handford et al . , 1991) in EGF-like repeat 11 are also conserved in Notch4 (Fig. 3) .
  • the residues between the first and second cysteine of EGF-like repeat 11 have been shown in Xenopus Notch to be important in ligand binding, and are divergent between Notch proteins (Fig. 3) .
  • Notch4 has additional residues and is unique when compared to other murine Notch proteins .
  • Notch4 also contains three Notch/lin-12 repeats which are approximately 53% identical to the Notch/lin-12 repeats found in other murine Notch proteins. Between the Notch/lin-12 repeats and the transmembrane domain of Notch4 are 2 cysteines at positions 1388 and 1397 that are conserved among all Notch proteins and may promote receptor dimerization upon ligand binding (Greenwald and Seydoux, 1990) .
  • the intracellular domain of Notch4 contains the 6 ankyrin/cdclO repeats found in other Notch proteins.
  • the ankyrin repeat domain of Notch4 is 48%, 52%, and 55% identical to the ankyrin repeat domains of Notchl, Notch2, and Notch3 respectively.
  • the number of amino acids between the transmembrane domain and the ankyrin/cdclO repeats is 110 residues, as it is in Notch4 (Fig. 1) .
  • Notch4 contains a C- terminal PEST domain, albeit of shorter length.
  • Notch4 lacks a recognizable opa repeat (Fig. 1), such as that found in Drosophila Notch.
  • Notch4 displays little homology to other Notch proteins and no significant homology to other known proteins. This C-terminal is also much shorter in Notch4 than in other Notch proteins, containing 177 residues, compared to 457 in Notchl, 437 in Notch2, and 329 in Notch3.
  • Notch4 transcripts were examined for the presence of Notch4 transcripts by Northern blot analysis. To minimize cross-hybridization with other mouse Notch transcripts, a riboprobe derived from the 3' UTR of Notch4 was used. In most tissues analyzed, a single hybridizing species of 6.7 kb was detected (Fig. 4), which roughly corresponds in size to the cloned Notch4 cDNA. The 6.7 kb transcript is most highly expressed in lung, at lower levels in heart and kidney, and at detectable levels in ovary and skeletal muscle. Very low levels of the 6.7 kb transcript were observed in several other adult tissues; including brain, intestine, liver, testis (Fig. 4) and spleen (data not shown) . In adult testis, two abundant transcripts of 1.5 kb and 1.1 kb were observed. Thus, Notch4 expression varies widely in adult tissues. Other than in testis, transcript size variation in different tissues was not detected.
  • Mice that carry two mutations at the white-spotting locus (W/W y ) are devoid of germ cells, but have the normal complement of somatic cell types, including Leydig, Sertoli, and peritubular myoid cells (Mintz and Russell, 1957) .
  • Heterozygous litter mates (W/+) have normal somatic and germ cell complements.
  • RNA from germ cell- deficient testes W/W y
  • testes with normal germ cells W/+ and adult (+/+)
  • a riboprobe derived from the 3' UTR probe D in Fig. 5C
  • Transcripts of 1.5 kb and 1.1 kb were detected in RNA from the testes of adult wild- type mice and W/+ mice (Fig. 5A) .
  • neither transcript was detected in RNA from homozygous mutant testes, suggesting that these transcripts were likely to be specific to the germinal compartment.
  • Notch4 transcripts of 1.5 kb and 1.1 kb are absent m day 7 p.n. and day 17 p.n. testis, but are present adult testis (Fig. 5A) . These results indicate that the expression of the 1.5 kb and 1.1 kb Notch4 transcripts are restricted to post-meiotic germ cells.
  • Notch4 transcripts are either derived from an alternate mtronic promoter that is active in post-meiotic germ cells or they may be driven by the same promoter as the 6.7 kb transcript and consist of spliced products derived from a 5' untranslated region upstream of what has currently been identified.
  • testis Notch4 transcripts with novel 5' sequence does not contain a methionine that could function as a translation initiator; therefore, these transcripts are unlikely to encode for protein products.
  • the testis transcripts may thus represent aberrant transcriptional events in post- meiotic germ cells, as has been described previously (Davies and Willison, 1993) .
  • Notch4 is highly expressed in endothelial cells . Intense labeling for Notch4 is observed in embryonic blood vessels at 9.0 days p.c. (Fig. 6A, B) . As shown in Fig.
  • Notch4 contains all the conserved domains characteristic of Notch proteins (Figs 1 and 2) . However, Notch4 contains only 29 EGF-like repeats within its extracellular domain as compared to the 36 repeats found in Notchl and Notch2. In addition, the C-terminal tail of Notch4 , beyond the ankyrin/cdclO repeats, is shorter and unique when compared to all other Notch proteins, however, little is known of the function of this region in Notch proteins .
  • Notch4 also contains a distinct EGF-like repeat 11 which has been proposed to be crucial for ligand binding. Structural variation in this repeat, and differences in the number of EGF-like repeats between murine Notch proteins, may be important for ligand specificity among the different possible Notch ligands. It must be noted that Notch/lin-12 proteins of varying structure have been demonstrated to be functionally interchangeable; C. elegans glp-1 can fully substitute for lin-12 (Fitzgerald et al . , 1993) for instance. Therefore, Notch4 may be functionally interchangeable with other murine Notch proteins despite structural differences between these Notch proteins.
  • Notch4 is distinct from other Notch family proteins based on its expression pattern during embryonic development and in the adult mouse. In situ hybridization demonstrates endothelial-specific embryonic expression of Notch4. This endothelial-specific expression of Notch4 remains in the adult mouse. A weak and transient labeling is seen in the neural tube between day 9 p.c. and 11.5 p.c., with a more intense labeling at the tips of neural folds. This region of the neural tube is a highly plastic area where cells will probably participate in the fusion process of the neural tube and/or migrate as neural crest . The Notch4 expression pattern is in sharp contrast to the expression patterns of Notchl , 2 . , and 3_.
  • Notch genes are expressed in a variety of different embryonic tissues such as the developing brain and spinal cord, presomitic and somitic mesoderm, and a variety of epithelial cells and mesenchymal derived tissues (Weinmaster et al . , 1991; Williams et al . , 1995).
  • Notchl is the only other Notch gene reported to be expressed in endothelial cells (Reaume et al . , 1992). Endothelial expression has not been reported for Jagged and Dll-l, two putative mammalian Notch ligands (Bettenhausen et al . , 1995; Lindsell et al . , 1995) . Expression of Notchl and 4 in endothelial cells might reflect either redundancy of function or distinct biological functions in endothelial development .
  • Notch4 Since Notch proteins have been implicated in binary cell fate specification regulating how equivalent cells can give rise to cells with different fates, a putative biological function of Notch4 might be to govern the cell fate decisions during endothelial growth and development. In amniotes, endothelial and hematopoietic cells appear synchronously in the blood islands. In zebra fish, lineage data have shown that individual cells of the early blastula can give rise to both endothelial and blood cells, suggesting a common embryonic precursor which has been referred to as the "hemangioblast.” The occurrence of binary cell fate decision events in the hemangioblast is supported by analysis of the endothelial and/or hematopoietic cell lineages.
  • Cloche , bloodless , and spadetail are mutants isolated in zebra fish that display phenotypes defective in either the hematopoietic development or both hematopoietic and endothelial development (Stainier et al . , 1995) .
  • the Flk-1 and the Flt-1 genes encode receptor tyrosine kinases that are expressed in embryonic endothelium (Shalaby et al., 1995, Fong et al . , 1995).
  • Null mutants for the Flk-1 gene are defective in endothelial and blood cell development (Shalaby et al .
  • null mutants for the Flt-1 gene display only hematopoietic cell development defects (Fong et al . , 1995). Mutational analysis of the Notch4 gene in whole animals would help to define the role of Notch4 in endothelial cell growth and development .
  • Notch4 contains the canonical ankyrin/cdclO repeats
  • RBP-Jk or RBP-Jk homologues and mammalian Deltex homologues may interact with the cdclO/ankyrin repeats of Notch4.
  • Su(H) or RBP-Jk are activated and translocate to the nucleus where they may regulate transcription of target genes (Goodbourn, 1995) .
  • activated Notch proteins encoding the intracellular domain have been reported to localize to the nucleus (Kopan et al . , 1994; Struhl et al . , 1993) suggesting a nuclear function for this domain.
  • int-3 oncoprotein is also localized to the nucleus when expressed in cultured 293T cells, as determined by immunofluorescence (unpublished data) . This finding may indicate that int-3 can bind to cytoplasmic proteins that are then translocated to the nucleus.
  • Notch4 protein with the extracellular domain deleted (EGF-like repeats and Notch/lin-12 repeats) , providing the first comparison of a naturally-activated murine Notch protein and its normal counterpart.
  • the oncogenic affects are likely the result of both over expression or ectopic expression of Notch.4 mRNA as well as functional activation of the Notch4 protein.
  • a structural comparison of the mutant int-3 protein to the normal Notch4 protein is reminiscent of the structural alterations reported to activate the effector function of Drosophila Notch and C.
  • glp-1 can substitute for lin-12 in specifying cell fate decisions in Caenorhabditis elegans. Development 119, 1019-1027.
  • the gene Serrate encodes a putative EGF-like transmembrane protein essential for proper ectodermal development in Drosophila melanogaster . Genes & Development 4, 2188-2201.
  • Notch receptor blocks cell- fate commitment in the developing Drosophila eye. Nature 365, 555-557.
  • Greenwald, I. (1985) . lin-12 a nematode homeotic gene, is homologues to a set of mammalian proteins that includes epidermal growth factor. Cell 43, 583-590.
  • lag-2 may encode a signaling ligand for the GLP-1 and LIN-12 receptors of C. elegans. Development 120, 2913- 2924.
  • the novel Notch homologue mouse Notch3 lacks specific epidermal growth factor-repeats and is expressed in proliferating neuroepithelium. Mechanism of Development 46, 123-136.
  • Deltex acts as a positive regulator of Notch signaling through interactions with the Notch ankyrin repeats. Development 121, 2633-2644. Mello, C. C, Draper, B. W. and Priess, J. R. (1994). The maternal genes apx-1 and glp-1 and establishment of dorsal - ventral polarity in the early C. elegans embryo. Cell 77, 95-106.
  • the ANK repeat a ubiquitous motif involved in macromolecular recognition. Trends Cell Biol. 2, 127-129.
  • Notch 1 is essential for postimplantation development in mice. Genes & Development 8, 707-719.
  • Tax F. E., Yeargers, J. J. and Thomas, J. H. (1994) . Sequence of C. elegans lag-2 reveals a cell-signaling domain shared with Delta and Serrate of Drosophila. Nature 368, 150-154.
  • the neurogenic gene Delta of Drosophila melanogaster is expressed in neurogenic territories and encodes for a putative transmembrane protein with EGF-like repeats. EMBO J. 6, 3431-3440.
  • Notch 2 a second mammalian Notch gene. Development 116, 931-941.
  • bFGF basic fibroblast growth factor
  • Rat tail collagen solution was obtained from Upstate Biotechnology Inc. (Lake Placid, NY) .
  • Anti-HA monoclonal antibody (12CA5) was obtained from Berkeley
  • the murine int-3 cDNA corresponds to a truncated Notch4 cDNA as described previously.
  • the int-3 nucleotide sequence corresponds to nucleotide 4551 to nucleotide 6244 of Notch4.
  • An oligonucleotide sequence encoding the haemagglutinin (HA) antigenic determinant was appended to the 3' end of the int-3 and Notch4 cDNA' s .
  • These eighteen codons specified the amino acid sequence SMAYPYDVPDYASLGPGPGP (Sequence I.D. No. ), including the nine amino acid-long HA epitope, as underlined.
  • HA-tagged int-3 and Notch4 cDNAs were created by subcloning each cDNA into Bluescript KS
  • each oligonucleotide is complimentary to the C-terminus of int-3 or Notch4 cDNA and their 3' ends anneal to the beginning of the HA epitope-encoding sequence (underlined in oligonucleotide) .
  • Mutagenesis was accomplished using the Muta-Gene phagemid in vitro mutagenesis kit (Bio Rad, Richmond, CA) . The presence of each fusion was confirmed by DNA sequencing.
  • Rat Brain Microvessel endothelial cells were obtained from Dr. Francoise Roux ( INSERM U26, Hopital F. Widal, Paris) and were routinely plated on collagen coated dishes and maintained in Alpha MEM/Ham's F10 (1:1) supplemented with 2mM glutamine, 10% fetal calf serum, lng/ml basic fibroblast growth factor (bFGF) , and penicilline/streptomycin in humidified 5% C02/95% air at 37C. Medium was changed twice a week, and cells were passaged at a split ratio of 1:4.
  • HA-tagged cDNAs (int-3HA, JaggedHA, and Notch4HA) were inserted into the retroviral vector pLHTCX wherein hygromycin-resistance/thymidine kinase fusion gene phosphotransf rase gene expression is controlled by the murine leukemia virus (MLV) long terminal repeat (LTR) , and cDNA transcription is controlled by an internal cytomegalovirus (CMV) enhancer/promoter.
  • MMV murine leukemia virus
  • LTR long terminal repeat
  • CMV internal cytomegalovirus
  • Retroviral infection of RBE4 cells was accomplished by culturing these cells the presence of viral supernatants obtained from the transfected packaging cells two day post- transfection. Infections were carried out in the presence of 4 ⁇ g/ml polybrene for 12 hours after which the medium was replaced to regular medium. One day post -infection the culture medium was replaced to regular medium containing 100 ⁇ g/ml hygromycin B (Sigma Chemical Co.) . Colonies appeared 5 days later and were pooled into medium containing 100 ⁇ g/ml hygromycin B.
  • HA epitope tagged int-3, Jagged and Notch4 proteins from lysates of RBE4 cell populations were analyzed by immunoblotting.
  • RBE4 cells were treated with 2mM sodium butyrate for 16 hours prior to lysis. Cells were washed twice with cold PBS and, subsequently, removed from the dish in 1.5 ml PBS using a rubber policeman. Cells were pelleted by centrifugation at
  • Proteins were transferred from gels onto nitrocellulose filters by electroblotting, and subsequently, blocked overnight at 4°C in TBST (10 M Tris, pH 8.0, 150 mM NaCl, 0.2% Tween-20) containing 1% bovine serum albumin (fraction V) . Blots were then incubated in anti-HA monoclonal antibody (12CA5) diluted 1:200 in TBST at room temperature. After four hours, the primary antibody was removed by washing three times for 5 min. each in TBST at temperatures identical to the primary antibody incubation. Blots were exposed to a 1:16,000 dilution of horseradish peroxidase-conjugated sheep anti-mouse immunoglobulin G. Excess secondary antibody was removed in the same manner as the primary antibody.
  • TBST 10 M Tris, pH 8.0, 150 mM NaCl, 0.2% Tween-20
  • bovine serum albumin fraction V
  • Blots were then incubated 1-2 min. in enhanced chemiluminescence detection reagents (Amersham Inc, Arlington Heights, IL) and exposed to X-ray film (Fujifilm, Fuji Photo Film Co., LTD., Tokyo) .
  • RBE4 cell lines were harvested using trypsin-EDTA, centrifuged, and plated on collagen coated dishes at equal cell densities (50% confluence at plating) with or without bFGF (5ng/ml) RBE4 cell culture was done in the absence of 2mM Sodium Butyrate . After 3 to 5 days, cell cultures were photographed with a Nikon ELWD 0.3 phase contrast microscope on Kodak T-Max film (100 X magnification) .
  • RBE4 cell lines programmed to express the int-3, Jagged, and Notch4 proteins were generated using either the retroviral vector pLHTCX which drives expression from the CMV promoter, or by direct transfection of the cDNA in target RBE4 cells.
  • the int-3, Jagged and Notch4 cDNA were fused at the carboxy termini to the haemagglutinin-epitope (HA), allowing us to detect int-3, Jagged and Notch4 proteins in immunoblot analysis using the anti-HA monoclonal antibody.
  • RBE4 cells programmed to express LacZ or Notch4HA have an identical cell morphology when compared to wild type RBE4 cells (Fig 8) .
  • RBE4 cells programmed to express either LacZ or Notch4 are grown in high concentrations of bFGF, they exhibit a spindle shape morphology and form modest sprouting and capillaries, a phenotype that is indistinguishable with wild type RBE4 cells grown under identical conditions (data not shown) .
  • RBE4 cells programmed to express JaggedHA display a modest spindle shape morphology when grown in the absence of bFGF
  • RBE4 cells programmed to express int-3HA display a striking spindle shape morphology when grown in the absence of bFGF (Fig 8).
  • RBE4 cells programmed to express either JaggedHA or int-3 HA exhibit a morphology in the absence of bFGF that is similar (although more extensive) to the bFGF induced morphological change of wild type RBE4 cells, or RBE4 cells programmed to express either LacZ or Notch4.
  • the spindle shape morphology of the cells becomes even more accentuated (data not shown) .
  • RBE4 cells programmed to express JaggedHA develop sprouts and three-dimensional capillary- like structures when grown in the absence of bFGF (see Fig 8) .
  • the capillary structures formed by RBE4 cells programmed to express Jagged either float (although originating from a mount formed on the tissue culture plate) in the cell culture medium or are attached to the tissue culture plate.
  • RBE4 cells programmed to express int-3HA develop similar three-dimensional structures when grown in the absence of bFGF (Fig 8) , however the extent is even greater then when compared to RBE4 cells programmed to express Jagged.
  • RBE4 cells programmed to express either JaggedHA or int-3 HA exhibit a morphology in the absence of bFGF that is similar (although more extensive) to the bFGF induced morphological change of wild type RBE4 cells, or RBE4 cells programmed to express either LacZ or Notch4.
  • RBE4 cells programmed to express Jagged or int-3 are grown in the presence of high concentrations of bFGF, the development of the three dimensional capillary structures becomes even more accentuated (data not shown) .
  • RBE4 cells express an endogenous Notch, since RBE4 cells that are programmed to express Jagged (a mammalian Notch ligand) have a similar phenotype when compared to RBE4 cells that express a constitutive activated Notch (int-3) . This is consistent with our previously published data that Notch4 is expressed in endothelial cells in vivo.
  • RBE4 cells do not express the Notch4 ligand since RBE4 cells programmed to express Notch4 do not exhibit a spindle form morphology when grown in the absence of bFGF.
  • Notch4/int-3 a truncated form of Notch4 , acts as a constitutive activated Notch receptor.
  • Rat brain miscrovessel endothelial cells RBE4
  • Both Notch4/int-3 and Jagged-1 were able to induce cellular structures with morphological and biochemical properties of endothelial microvessels .
  • Ectopic expression of full length Notch4 did not have any discernible effect in RBE4 cells.
  • Activation of the notch signal transduction pathway was measured by the induction of endogenous Notch4 and Jagged-1 genes.
  • the observed morphological changes to RBE4 cells correlated with endogenous Notch4 and Jagged-1 gene activation, demonstrating a link between Notch signaling and biological activity. Our observations demonstrate that Notch signaling can promote endothelial cell differentiation.
  • Notch4 is a member of the Notch/lin-12 family of transmembrane receptors that are involved in cell fate determination (1,2). Like other Notch proteins, Notch4 extracellular domain is characterized by both Epidermal Growth Factor (EGF)- like repeats and lin-12/Notch repeats (LNR) , and the intracellular domain contains ankyrin/cdclO repeats (3) . Analysis of invertebrate Notch/lin-12 mutants support a function for Notch/lin-12 receptors in intercellular signaling events that control cell fate. Mutants that delete the extracellular domain of C. elegans lin-12 or Drosophila Notch result in dominant gain-of- function phenotypes .
  • EGF Epidermal Growth Factor
  • LNR lin-12/Notch repeats
  • Notch4/int- 3 The int-3 form of Notch4, Notch4/int- 3, was identified based on its oncogenic effects in the mouse mammary gland and encodes only the transmembrane and intracellular domain of Notch4 (3-5) .
  • Notch4/int-3 behaves as a constitutively activated receptor (6-8) .
  • Similar activating mutations in the Notchl gene have been identified in T lymphoblastic leukemia and can lead to neoplastic transformation in vitro (9,10).
  • Notch ligands Based on homology to the Drosophila ligands, Notch ligands have also been identified in vertebrates. Jagged- ______ a rat homologue of Drosophila Serrate, contains the hallmarks of a Notch ligand (15) .
  • Other putative mouse Notch ligands Delta-like 1 (16) and Delta-like 3 (17) have been identified and are closely related to Drosophila Delta. More recently, a human Jagged- 2 gene has been identified (18) .
  • Notch ligands and Notch receptors have been identified in mammals, it is not clear if ligands display distinct specificities towards different receptors
  • Notch4 is expressed primarily in endothelial cells of the vasculature of mouse embryos and adult tissues (3, 19) .
  • Notchl and Jagged-1 are also expressed in the endothelium, as well as a variety of other tissues (20-23) .
  • Jagged-1 was identified as gene induced during angiogenesis in vitro (24) .
  • a role of Notch in vascular development is suggested by analysis of mice with targeted disruption of genes encoding Notch ligands or Notch regulatory components. Mice deficient in either Delta like-1 (Dill) (25,26) or Jagged-1 develop severe hemorrhages (T. Gridley and G.
  • CADASIL Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephelopathy
  • CADASIL Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephelopathy
  • Notch receptors and their ligands expressed in endothelium may similarly control cell fate decisions during vascular development .
  • FBE4 Rat brain endothelial cell line
  • Ectopic expression of an activated Notch4/int-3 or of Jagged-1 in RBE4 cells induced cellular structures with morphological and biochemical properties of endothelial microvessels .
  • activation of the Notch signaling pathway can promote endothelial differentiation.
  • Frozen mouse kidney sections were fixed in 100% acetone and rinsed in 0.1% Triton X-100 in PBS (PBST) . Tissue sections were incubated in blocking solution (5% goat serum, 3% BSA in PBS) for 1 hour. Sections were covered with 1° antibody in dilution solution (1% goat serum, 3% BSA in PBS) for 12 hours at 4° C.
  • the anti-PECAM) antibody (PharMingen) was used at a concentration of 3 ⁇ g/ml, the anti-mouse Notch4 immune- serum and pre-immune serum were diluted 50 OX. Tissue sections were rinsed in PBST and 2° antibodies
  • alkaline phosphatase labeled goat anti-rabbit IgG was detected as previously described (30) . Sections for alkaline phosphatase staining were counter stained with eosine .
  • the Notch4/int-3 cDNA corresponds to a truncated Notch4 cDNA as described previously (3).
  • An oligonucleotide sequence encoding the haemagglutinin (HA) eptope was appended to the 3' end of the Notch4/int-3 and Notch4 cDNA' s as previously described (6). The presence of each fusion was confirmed by DNA sequencing. Similarly, a Jagged-1 cDNA was modified to encode an HA-tag at the carboxy terminus .
  • RBE4 cells were maintained in Alpha MEM/Ham's F10 (1:1) supplemented with 2 mM glutamine, 10% fetal calf serum and penicillin/streptomycin.
  • RBE4 cell lines were plated at equal ceil densities (50% confluence) . After 3 to 5 days, cell cultures were photographed (100X or 40X magnification) .
  • HA-tagged cDNAs were inserted into the retroviral vector pLHTCX wherein hygromycin-resistance gene is controlled by the murine leukemia virus (MLV) long terminal repeat (LTR) , and cDNA transcription is controlled by an internal cytomegalovirus (CMV) promoter.
  • MLV murine leukemia virus
  • CMV internal cytomegalovirus
  • Distinct populations of RBE4 cells either expressing Notch4/int-3 or Jagged-1 cDNA, were prepared by retroviral infection as described previously (6) . The resultant populations, each comprised of at least 50 clones, were used in cellular and biochemical assays described below. Notch4 expressing cell lines were established by transfecting RBE4 cells directly by calcium phosphate transfection.
  • HA epitope-tagged or non-tagged Notch4/int-3 Jagged-1 and Notch4 proteins from lysates of RBE4 cells were analyzed by immunoblotting as described previously (6) . Lysates containing 20 ⁇ g protein were electophoresed and transferred to nitrocellulose filters, and subsequently, blocked in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, .02% Tween-20) containing 1% BSA (TBST-BSA) .
  • Blots were then incubated with 1° antibody diluted (1:100 for 12CA5 (Berkeley Antibody Co.); 1:2000 for anti-Notch4) in TBST- BSA for 4 hours, washed in TBST, and incubated with 2° antibody in TBST-BSA for 1 hour. After 3 washes, the signal was visualized by chemiluminescence (Amersham, ECL) .
  • GTP Gamma-glutamyl transpeptidase activity was demonstrated histochemically in cell cultures fixed in acetone, as described previously (31) : L-gamma-glutamyl- 4methoxy-2-naphthylamide was used as substrate and glycyl- glycine as acceptor. In a simultaneous azo coupling reaction with Fastblue BB, a red azo dye is produced. Alkaline phosphatase (ALP) activity was determined in cell cultures fixed in citrate-buffered acetone, using the reaction mixture Sigma Kit N85, with naphtol ASMX phosphate as substrate and fast blue RR salt as diazonium salt, as previously described (31) .
  • ALP alkaline phosphatase
  • Notch4 mRNA was expressed in mouse embryos and adult mouse lung, as analyzed in si tu hybridization (3) .
  • an anti- Notch4 rabbit antiserum directed against a GST fusion protein containing the carboxy-terminus of Notch4 (32) .
  • the presence of endogenous Notch4 protein was analyzed in mouse kidney sections, as the kidney contains characteristic glomeruli with endothelial cell clusters in the renal cortex.
  • PECAM Platelet Endothelial Cell Adhesion Molecule-1
  • Antibodies against PECAM were used as a positive control in these experiments as they stained glomeruli (Fig. IB) .
  • anti-Notch4 antibody specifically detects glomeruli in the kidney cortex in a similar pattern as the anti-PECAM antibody (Fig. IB) .
  • data presented here demonstrates endothelial expression of Notch4 protein in the kidney.
  • the anti-Notch4 antibody was used to detect ectopically expressed Notch4 and Notch4/int-3 proteins by immunoblot analysis (Fig. 2C) .
  • Transiently transfected 293 cells expressed either HA epitope-tagged or non-epitope-tagged versions of Notch4 and Notch4/int-3.
  • Immunoblot analysis of extracts from these cells displayed the full length Notch4 and Notch4/int-3 proteins which migrate with an approximate molecular weight of 220 and 70 kD respectively
  • RBE4 microvessel outgrowth is induced by activated Notch4 and Jagged-1
  • bFGF Growth Factor
  • ALP alkaline phosphatase
  • GTP gamma glutamyl transpeptidase
  • RBE4 cell lines were programmed to express the HA-tagged Notch4/int-3 , Jagged-1 and LacZ proteins using the retroviral vector pLHTCX.
  • RBE4 cell lines programmed to express Notch4 were generated by direct transfection of the HA-tagged Notch4 cDNA. All three recombinant proteins were detected by immunoblot analysis of the respective RBE4 cell lines and migrated with their predicted molecular weight (220kD for Notch4, 70kD for Notch4/int-3 , 150 kD for Jagged-1, see Fig. 2D) .
  • RBE4 cells The consequences of activating the Notch signaling pathway by ectopic expression of ligand (Jagged-1) or an activated receptor (Notch4/int-3 ) was analyzed in RBE4 cells.
  • bFGF 5 ng/ml
  • RBE-LacZ were indistinguishable from wild type RBE4 cells grown under identical conditions.
  • RBE-Notch4/int-3 or RBE-Jagged-1 cells displayed a more spindle shape morphology and do not form any microvessel structures (Fig. 3B) .
  • RBE-Notch4/int- 3 or RBE-Jagged-1 cells displayed a more spindle shape morphology and no not form the cobblestone monolayer, as observed in RBE-LacZ or RBE-Notch4 (compare Fig. 3C and D with A or B) .
  • This morphological change is similar to when wild type RBE4 cells are treated with bFGF (31) .
  • Microvessel structures induced by Jagged-1 and Notch4/int-3 in RBE4 cells were analyzed histochemically for the activities of two blood-brain barrier-associated enzymes,
  • GTP and ALP are specifically expressed in the brain vasculature, and their expression is induced by bFGF treatment of RBE4 cells (31) .
  • ALP activity was detected in microvessel structures induced by either Notch4/int-3 (Fig. 4B) or by Jagged-1 (data not shown) .
  • GTP activity was also detected in both RBE-Jagged-1 (Fig. 4C) and RBE-Notch4/int-3 (data not shown) .
  • the detection of both ALP and GTP activities was specific for microvessel structures and not found in surrounding monolayer cells.
  • Notch signaling has been reported to induce endogenous genes encoding Notch receptors and ligands (18) . This induction can serve as a measure of signal activation and may lead to positive feedback control of Notch genes by their ligands (33) .
  • Notch signaling output in RBE4 cells we assessed the level of endogenous Notch receptor and Notch ligand gene transcripts by northern blot analysis (Fig. 5B) .
  • RBE-Notch4/int-3 have increased levels of endogenous Notch4 (6.7 kb) and Jagged-1 transcripts (5.9 kb) (Fig. 5B, lane 2) .
  • the Notch4 -specific riboprobe corresponds to the carboxy-terminal domain of Notch4 , and can detect both ectopic Notch4/int-3 transcripts (1.8 kb) and endogenous Notch4 transcripts (6.7 kb) .
  • Notch4/int-3 expression in RBE cells specifically led to increased levels of endogenous Notch4 transcripts (Fig. 5B, lane 2) and not endogenous Notchl (Fig. 5B, lane 2 ) or Notch3 transcripts (data not shown) .
  • RBE-Jagged-1 cells also have increased levels of endogenous Notch4 transcripts (Fig. 5B, lane 4) .
  • RBE4 cell lines were generated with retroviral vectors designed to express genes via an internal CMV promoter; however, gene expression may also be driven by the retroviral LTR leading to expression of two different sized transcripts.
  • CMV-driven transcripts correspond to 1.8 kb for Notch4/int-3 6.7 kb for Notch 4 and 5.9 kb for Jagged-1 (Fig. 5B) .
  • Larger transcripts encompassing the complete retroviral genome originate from the LTF resulting in transcripts that are 3.5 kb larger than the CMV-driven transcripts.
  • the LTR-driven transcripts are denoted in fig.
  • Notch4/mt-3 and Jagged-1 induce a biological activity m RBE4 cells that is similar to that induced by a known angiogenic agent bFGF.
  • Notch activation results m an angiogenic response characterized by microvessel formation
  • Notch activation may regulate the angiogenic process endothelium where Notch4 is found to be expressed, such as the vasculature of the adult kidney and lung.
  • Notch4/mt-3 is a constitutive activated Notch allele as it induces a phenotype that is identical to when endogenous Notch receptors are activated by Jagged-1
  • ectopic expression of full length Notch.4 RBE4 cells does not result m Notch signal activation, thus receptor over expression does not lead to constitutive receptor activation. It may also suggest that RBE4 cells do not express a Notch4 ligand Alternately RBE4 cells may not express Notch4 ligands at levels sufficient to activate the ectopically expressed Notch receptor.
  • RBE4 cells must express endogenous Notch receptors which can be activated by Jagged-1.
  • Northern blot analysis demonstrated that the Notchl, Notch3 and Notch4 genes are expressed in RBE4 cells.
  • Notchl and Notch4 are known to be expressed in endothelium in vivo (3, 19-23) and Notch3 may function in the vasculature (29) .
  • RBE4 cells also express Jagged-1 when analyzed by Northern blot.
  • RBE4 cells express endogenous transcripts that encode for both Notch proteins and one of their ligands, they do not spontaneously form microvessel structures. This observation may suggest that the levels of Notch proteins or ligands in RBE4 cells are insufficient to activate the Notch pathway.
  • Notch signal transduction pathway is activated in RBE4 cells expressing either Notch4/int-3 or Jagged-1. Activation of Notch signaling results in up- regulation of the endogenous Notch4 and Jagged- 1 transcripts. Thus, the observed microvessel induction of RBE4 cells correlated with Notch signal activation. Both Jagged-1 and Notch4/int-3 expression resulted in a similar up-regulation of the endogenous Notch4 gene but neither altered the levels of Notchl and Notch3 transcripts. Activation of a particular Notch signaling pathway, in this case Notch4, led to transcriptional activation events that are specific for distinct endogenous Notch genes.
  • Jagged-1 results in an identical transcriptional activation pattern as activated Notch4/int- 3 may suggest that Jagged-1 is a Notch4 ligand.
  • activation of the Notch signal transduction pathway in mammalian cells inhibits differentiative or morphogenetic events. For instance, activated Notchl and Notch2 are able to inhibit myogenic differentiation (15,34), and myeloid differentiation (35), and activated Notch4 (Notch4/int-3 ) is able to inhibit branching morphogenesis of mammary epithelial cells (6) .
  • activated Notch4/int-3 promoted the differentiation of RBE4 cells.
  • Cell fate determination may involve either inhibition or promotion of differentiative steps. Our data is thus a clear demonstration that Notch signal activation can regulate cell fate decisions that involve the promotion of differentiation in mammalian cells.

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Abstract

Cette invention porte sur des procédés de modulation de l'angiogenèse consistant à activer ou inhiber l'angiogenèse, par exemple, en association avec un traitement des anomalies telles que des hémangiomes, des hémangiosarcomes, le sarcome de Kaposi, les troubles ischémiques et les blessures. Ces procédés consistent à administrer des composés qui sont des agonistes ou des antagonistes sélectifs de la protéine Notch4. Cette invention porte également sur une molécule d'acide nucléique isolée codant une Notch4, une protéine Notch4 isolée, des vecteurs comprenant une molécule d'acide nucléique isolée codant une protéine Notch4, des cellules comprenant ces vecteurs, des anticorps dirigés contre la protéine Notch4, des sondes d'acide nucléique utilisées dans la détection des acides nucléiques codant la protéine Notch4 et des oligonucléotides antisens complémentaires à des séquences uniques quelconques d'une molécule d'acide nucléique qui code la protéine Notch4.
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