EP1011328A4 - Stimulation modulation et/ou inhibition de l'activite protheolithique endotheliale et/ou de l'activite angioginique - Google Patents

Stimulation modulation et/ou inhibition de l'activite protheolithique endotheliale et/ou de l'activite angioginique

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Publication number
EP1011328A4
EP1011328A4 EP98940875A EP98940875A EP1011328A4 EP 1011328 A4 EP1011328 A4 EP 1011328A4 EP 98940875 A EP98940875 A EP 98940875A EP 98940875 A EP98940875 A EP 98940875A EP 1011328 A4 EP1011328 A4 EP 1011328A4
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European Patent Office
Prior art keywords
vegf
cells
endothelial cells
cytokines
endothelial
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EP98940875A
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German (de)
English (en)
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EP1011328A1 (fr
Inventor
Michael S Pepper
Kari Alitalo
Ulf Eriksson
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Universite de Geneve
Ludwig Institute for Cancer Research Ltd
Helsinki University Licensing Ltd
Ludwig Institute for Cancer Research New York
Original Assignee
Universite de Geneve
Ludwig Institute for Cancer Research Ltd
Helsinki University Licensing Ltd
Ludwig Institute for Cancer Research New York
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Publication of EP1011328A1 publication Critical patent/EP1011328A1/fr
Publication of EP1011328A4 publication Critical patent/EP1011328A4/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • C07K14/503—Fibroblast growth factor [FGF] basic FGF [bFGF]
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons

Definitions

  • Angiogenesis is the formation of new capillary blood vessels by a process of sprouting from pre-existing vessels, and occurs during development as well as in a number of physiological and pathological settings. Angiogenesis is thus necessary for tissue growth, wound healing and female reproductive function, and is also a component of pathological processes such as tumor growth, hemangioma formation and ocular neovascularization [see Folkman, New Engl . J. Med . , 333:1757-1763 (1995); Pepper, Arterioscler. Thromb . Vase . Biol . , 17:605-619 (1997)]. A similar although far less well studied process also occurs in the lymphatic system, and is sometimes referred to as lymphangiogenesis .
  • Angiogenesis begins with localized breakdown of the basement membrane of the parent vessel, which is followed by the migration and outgrowth of endothelial cells into the surrounding extracellular matrix, resulting in the formation of a capillary sprout. A lumen is subsequently formed, and constitutes an essential element in functional sprout formation. Sprout maturation is completed after reconstitution of the basement membrane.
  • Alterations in at least three endothelial cell functions occur during this series of events: 1) modulation of interactions with the extracellular matrix, which requires alterations in cell -matrix contacts and the production of matrix-degrading proteolytic enzymes (plasminogen activators (PAs) and matrix metalloproteinases) ; 2) an initial increase and subsequent decrease in locomotion (migration) , which allows the cells to translocate towards the angiogenic stimulus and to stop once they reach their destination; 3) an increase in proliferation, which provides new cells for the growing and elongating vessel, and a subsequent return to the quiescent state once the vessel is formed. Together, these cellular functions contribute to the process of capillary morphogenesis, i.e.
  • VEGF vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • VEGF and bFGF have been demonstrated to synergize in the induction of angiogenesis in vi tro [Pepper et al . , Biochem . Biophys . Res . Commun . , 189:824-831 (1992); Goto et al . , Lab . Invest . 69:508-517 (1993)], and this observation been confirmed in vivo in the rabbit in a model of hind-limb ischemia [Asahara et al . , Circulation, 92 (Supp.II) : II .365-71 (1995)] and in the rat in a sponge implant model [Hu et al . , A . Br . J. Pharmacol . , 114:262-268 (1995)].
  • Second, the in vitro angiogenic effect of VEGF as well as its capacity to induce PA activity are both dependent on endogenous bFGF produced by endothelial cells.
  • VEGFR-1 Flt-1
  • VEGFR-2 KDR/Flk-1
  • VEGFR-3 Flt-4
  • VEGFRs are expressed in many adult tissues, despite the apparent lack of constitutive angiogenesis. VEGFRs are however clearly upregulated in endothelial cells during development and in certain angiogenesis-associated/dependent pathological situations including tumor growth [see Dvorak et al . , Amer. J. Pathol .
  • VEGFR-1-deficient mice die in utero at mid-gestation due to inefficient assembly of endothelial cells into blood vessels, resulting in the formation of abnormal vascular channels [Fong et al . , Nature, 376:66-70 (1995)].
  • VEGFR-2-deficient mice die in utero between 8.5 and 9.5 days post-coitum, and in contrast to VEGFR-1, this appears to be due to abortive development of endothelial cell precursors [Shalaby et al . , Nature 376:62-66 (1995)].
  • the importance of VEGFR-2 in tumor angiogenesis has also been clearly demonstrated by using a dominant-negative approach [Millauer et al . , Nature, 367:576-579 (1994); Millauer et al . , Cancer Res . 56:1615-1620 (1996)].
  • the phenotype of VEGFR-3 -deficient mice has not yet been reported.
  • the ligands for VEGFR-1 include VEGF and placenta growth factor (PlGF) ; ligands for VEGFR-2 include VEGF and VEGF-C; while the only ligand reported so far for VEGFR-3 is VEGF-C [see Mustonen et al . , J. Cell Biol . , 129:895-898 (1995); Thomas, J. Biol . Chem . , 271:603-606 (1996)] .
  • VEGF-C is a recently described member of the VEGF family of angiogenic cytokines .
  • VRP VEGF-related protein
  • EST expressed sequence tag
  • VEGF-C and VRP are the same protein, and will be referred to as VEGF-C from here on.
  • VEGF-C displays a high degree of similarity with VEGF, including conservation of the eight cysteine residues involved in intra- and intermolecular disulfide bonding.
  • the cysteine-rich C-terminal half which increases the length of the VEGF-C/VRP polypeptide relative to other ligands of this family, shows a pattern of spacing of cysteine residues reminiscent of the Balbiani ring 3 protein repeat.
  • the C-terminal propeptide also contains short motifs of VEGF-like domains, which may promote the interaction of secreted VEGF-C with the extracellular matrix.
  • VEGF-C binds to the extracellular domain of VEGFR-3 and induces VEGFR-3 tyrosine phosphorylation. In addition to VEGFR-3, VEGF-C binds to and induces phosphorylation of VEGFR-2.
  • VEGF-C promotes the growth of human and bovine endothelial cells, although it is less active than VEGF in this assay.
  • VEGF-C has been reported to induce endothelial cell migration in three-dimensional collagen gels [Joukov et al . , EMBO J. , 16:3898-911 (1997)].
  • VEGF-C transcripts are detectable in many adult and fetal human tissues and in a number of cell lines. Human VEGF-C has been mapped to chromosome 4q34 [Paavonen et al . , Circulation 93:1079-82 (1996)].
  • VEGF-C One of the striking features of VEGF-C is that its mRNA is first translated into a precursor from which the mature ligand is derived by cell -associated proteolytic processing. Following biosynthesis, VEGF-C rapidly associates into a 58 kDa antiparallel homodimer linked both by disulfide and non-covalent bonds. This is followed by proteolytic processing of both N- and C-terminal propeptides in the terminal portion of the secretory pathway and at the cell membrane, giving rise to a number of incompletely processed intermediates. Mature VEGF-C is then released from cells as a 21 kDa homodimer containing two VEGF-homology domains linked by non-covalent interactions.
  • VEGF-C acquires the ability to bind to and activate VEGFR-2, and also increases its affinity for and activating properties towards VEGFR-3. Intracellular proteolytic cleavage is not a prerequisite for VEGF-C secretion. Based on these observations, it has been suggested that the synthesis of VEGF-C as a precursor allows it to signal preferentially through VEGFR-3
  • VEGF-C may acquire the additional capacity to signal through VEGFR-2, thereby providing an additional level of regulation of VEGF-C activity.
  • Signalling through VEGFRs requires receptor dimerization.
  • Proteolytic processing might provide a further regulatory mechanism by indirectly promoting the formation of VEGFR-2/VEGFR-3 heterodimers .
  • TGF- ⁇ l has a biphasic effect [Pepper et al . , Exp . Cell Res . , 204:356-363 (1993) ] .
  • Another object of the invention is to develop a method of synergistically inducing the angiogenic response of endothelial cells .
  • a further object of the invention is to provide a method of influencing the proteolytic properties of endothelial cells.
  • Yet another object of the invention is to provide a method of inhibiting the activity of proteolytic enzymes. It is a specific object of the invention to provide a method of inhibiting the activity of plasminogen activators.
  • a still further object of the invention is to provide a method of inhibiting tumor growth.
  • vascular endothelial growth factor-B vascular endothelial growth factor-B
  • vascular endothelial growth factor-C vascular endothelial growth factor- C
  • bFGF basic fibroblast growth factor
  • VEGF-C induces angiogenesis in endothelial cells in vi tro and that both VEGF-B and VEGF-C synergistically promote angiogenesis in endothelial cells when co-administered with bFGF or VEGF.
  • VEGF-C with bFGF may be applied in regulation of physiological and pathological angiogenesis, as well as in the planning of therapeutic strategies against tumors and other pathological conditions .
  • bovine cell lines used in the following tests show a clear response to the human VEGF, VEGF-B and VEGF-C tested indicates that the bovine receptors are sufficiently homologous to the human receptors to give rise to a high degree of inter-species cross reactivity. In view of this, a similar or greater response certainly can be expected in human tissues which express the human receptors.
  • VEGF-B and VEGF-C induce gene expression of various proteolytic enzymes such as plasminogen activators (PAs) , which may damage surrounding tissues and thereby facilitate invasion of those tissues by tumor cells.
  • PAs plasminogen activators
  • VEGF-C can act as a chemoattractant for endothelial cells, this could lead to invasion and permeation by lymphatic vessels (which are composed of endothelial cells) and ultimately support the onset of tumor metastases. Consequently, VEGF-B and VEGF-C antagonists could be used therapeutically to inhibit the action of VEGF-B and/or VEGF-C and thereby retard or prevent permeation, endothelial cell invasion and/or metastasis.
  • 2-antiplasmin can counteract the angiogenic action of VEGF-B and/or VEGF-C and could therefore be used in accordance with one aspect of the invention as an anti-metastatic agent.
  • Another aspect of the invention involves providing an anti- sense nucleotide sequence which is complementary to at least a part of the DNA sequence for VEGF-B and/or VEGF-C, which promote proliferation of endothelial cells.
  • the invention thus embraces antisense oligonucleotides that selectively bind to a nucleic acid molecule encoding VEGF-B and/or VEGF-C protein, and their use to decrease transcription and/or translation of VEGF-B or VEGF-C genes, respectively.
  • VEGF-B and/or VEGF-C gene product expression is desirable, including to reduce any aspect of a tumor cell phenotype attributable to VEGF-B and/or VEGF-C gene expression.
  • Antisense molecules can be used in this manner to retard or arrest such aspects of a tumor cell phenotype.
  • antisense oligonucleotide or “antisense” describes an oligonucleotide that is an oligoribonucleotide, or a modified oligodeoxyribonucleotide, which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene and which thereby inhibits transcription of that gene and/or translation of that RNA.
  • Antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with that gene.
  • the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence. It is preferred that the antisense oligonucleotide be constructed and arranged so as to bind selectively with the target under physiological conditions, i.e. to hybridize substantially more to the target sequence than to another sequence in the target cell under physiological conditions. Based upon the published DNA sequences of VEGF-B and VEGF-C, one of skill in the art can readily select and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention.
  • antisense oligonucleotides should comprise at least 7, and more preferably at least 15, consecutive bases which are complementary to the target [see Wagner et al . , Nature Biotechnology, 14:840-844 (1996)]. Most preferably, the antisense oligonucleotides comprise a complementary sequence of from 20 to 30 bases. Although oligonucleotides may be chosen which are antisense to any region of the gene or mR ⁇ A transcripts, in preferred embodiments the antisense oligonucleotides correspond to ⁇ -terminal or 5' upstream sites such as translation initiation, transcription initiation or promoter sites. In addition, 3'- untranslated regions may be targeted.
  • the antisense sequence preferably is targeted to sites in which mRNA secondary structure is not expected [see Sainio et al . , Cell Mol . Neurobiol . , 14 (5) : 439-457 (1994)] and at which proteins are not expected to bind.
  • antisense nucleotides which are complementary to a cDNA of the desired gene or antisense nucleotides which hybridize with genomic DNA of the desired gene, since one of ordinary skill in the art may readily derive the genomic DNA corresponding to a cDNA or vice versa .
  • antisense to allelic or homologous DNAs may be obtained without undue experimentation.
  • Antisense oligonucleotides used in the invention may be composed of "natural" deoxyribonucleotides, ribonucleotides, or any combination thereof. That is the 5' end of one native nucleotide and the 3 ' end of another native nucleotide may be covalently linked, as in natural systems, via a phosphyodiester internucleoside linkage. These oligonucleotides may be prepared by art recognized methods which may be carried out manually or by an automated synthesizer. They also may be produced recombinantly by vectors. In preferred embodiments, however, antisense oligonucleotides used in the invention also may include "modified" oligonucleotides. That is, the oligonucleotides may be modified in a number of ways which do not prevent them from hybridizing to their target, but which enhance their stability or targeting or which otherwise enhance their therapeutic effectiveness.
  • modified oligonucleotide as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic internucleoside linkage (i.e. a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide” and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide.
  • a synthetic internucleoside linkage i.e. a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide
  • Preferred synthetic internucleoside linkages are phosphorothioates , alklphosphonates , phosphorodithioates, phosphate esters, alkylphosphonothioates , phosphoramidates , carbamates, carbonates, phosphate triesters, acetamidates, peptides, and carboxymethyl esters.
  • modified oligonucleotide also encompasses oligonucleotides with a covalently modified base and/or sugar.
  • modified oligonucleotides include oligonucleotides having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3 ' position and other than a phosphate group at the 5' position.
  • modified oligonucleotides may include a 2 ' -O-alkylated ribose group.
  • modified oligonucleotides may include sugars such as arabinose instead of ribose.
  • Modified oligonucleotides also can include base analogs such as C-5 propyne modified bases [see Wagner et al . , Nature Biotechnology, 14:844 (1996)] .
  • the present invention contemplates pharmaceutical preparations containing modified antisense molecules that are complementary to and hybridize under physiological conditions with nucleic acids encoding VEGF-B and/or VEGF-C proteins, together with pharmaceutically acceptable carriers.
  • Antisense oligonucleotides may be administered as part of a pharmaceutical composition.
  • a pharmaceutical composition may include the antisense oligonucleotides in combination with any standard physiologically and/or pharmaceutically acceptable carriers known in the art .
  • the compositions should be sterile and contain a therapeutically effective amount of the antisense oligonucleotides in a unit of weight or volume suitable for administration to a patient.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.
  • physiologically acceptable refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue or organism. The characteristics of the carrier will depend on the route of administration.
  • Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.
  • a vector comprising such an anti-sense sequence may be used to inhibit, or at least mitigate expression of the relevant cytokine.
  • the use of a vector of this type to inhibit expression of one or more angiogenic cytokines may be advantageous in instances where expression of the cytokine is associated with a disease such as in instances where tumors produce VEGF-B and/or VEGF-C in order to provide for angiogenesis.
  • Transformation of such tumor cells with a vector containing an anti-sense nucleotide sequence would suppress or retard the endothelial cell proliferating activity and could thereby inhibit permeation, endothelial invasion and/or metastasis and consequently inhibit or retard tumor growth.
  • Figures 2a and 2b are graphs showing in vi tro angiogenesis in bovine microvascular endothelial (BME) cells induced by VEGF-C alone or in combination with bFGF.
  • Figures 3a and 3b are graphs showing in vi tro angiogenesis in BME cells induced by VEGF-B alone or in combination with bFGF.
  • Figures 4a through 4c are graphs showing in vi tro angiogenesis in bovine aortic endothelial (BAE) cells induced by VEGF-C alone or in combination with various concentrations of bFGF.
  • BAE bovine aortic endothelial
  • Figures 5a and 5b are graphs showing in vi tro angiogenesis in BAE cells induced by VEGF-B alone or in combination with bFGF.
  • Figure 6 is a graph showing in vi tro angiogenesis induced by VEGF-C in bovine lymphatic endothelial (BLE) cells.
  • Figure 7 is a graph showing the effects of VEGF and VEGF-C individually and in conjunction with each other on BAE cell collagen invasion.
  • Figures 8a and 8b are graphs which also show the effects of VEGF, VEGF-B and VEGF-C individually and in various combinations on BAE cell collagen invasion.
  • Figures 9a and 9b are zymographic analyses of the induction of tissue-type plasminogen activator (tPA) activity and urokinase- type plasminogen activator (uPA) activity
  • Figure 9c is a reverse zymographic analysis of the induction of plasminogen activator inhibitor-1 (PAI-1) by VEGF-C, VEGF and bFGF.
  • Figure 10a is a zymographic analysis of the induction of tPA activity and uPA activity
  • Figure 10b is a reverse zymographic analysis of the induction of PAI-1 activity, by VEGF-B, VEGF and bFGF.
  • Figure 11 shows the increase in steady state levels of tPA, uPA and PAI-1 mRNA induced by VEGF-C in BAE cells.
  • Figure 12 shows the increase in steady state levels of uPA and PAI-1 mRNA induced by VEGF-B in BAE cells.
  • Figure 13 is a graph showing the inhibiting effect of 2 - antiplasmin on endothelial cell invasion of collagen gels induced by VEGF-C and bFGF in BME cells.
  • Recombinant human VEGF-B 167 and VEGF-B 186 can be produced as described in Eriksson et al . , WO 96/26736, the disclosure of which is incorporated herein by reference.
  • VEGF-C Recombinant human VEGF-C was produced in the baculovirus system using the Sf9 insect cell line (National Public Health Institute, Helsinki) and baculovirus shuttle vectors derived from the transfer plasmid pFASTBACl as described in Jeltsch M. , Functional Analysis of VEGF-B and VEGF-C, Helsinki University (1997) .
  • Recombinant human VEGF-C ⁇ N ⁇ C was produced in the yeast Picha pastoris (strain GS115) using the pIC9 expression vector (Invitrogen) as described by Joukov et al . , EMBO J. , 16:3898-911 (1997) .
  • Recombinant human VEGF (165-amino acid homodimeric species - VEGF 165 ) was purchased from Peprotech.
  • Recombinant human bFGF (155 amino acid form) was provided by Dr. P. Sarmientos (Farmitalia Carlo Erba, Milan, Italy).
  • Bovine uPA, bovine uPAR and human tPA cDNA clones were provided by Dr. W.-D. Schleuning.
  • Example 1 Cell Culture of Microvascular Endothelial Cells.
  • Bovine adrenal cortex-derived microvascular endothelial (BME) cells (Furie et al . , 1984) were obtained from Drs . M.B. Furie and S.C Silverstein and were grown in minimal essential medium, alpha modification (Gibco AG, Basel, Switzerland), supplemented with 15% heat-inactivated donor calf serum (DCS) (Flow Laboratories, Baar, Switzerland) , penicillin (500 U/ml) and streptomycin (lOO ⁇ g/ml) . Cells were used between passages 16 and 19.
  • DCS heat-inactivated donor calf serum
  • penicillin 500 U/ml
  • streptomycin lOO ⁇ g/ml
  • Example 2 Cell Culture of Aortic Endothelial Cells.
  • Bovine aortic endothelial (BAE) cells isolated from scrapings of adult bovine thoracic aortas and cloned by limiting dilution as previously described in Pepper et al . , Am. J. Physiol . 262:C1246-57 (1992), were cultured in low glucose Dulbecco ' s modified minimal essential medium (DMEM, Gibco) supplemented with 10% DCS and antibiotics. Cells were used between passages 10 and 15.
  • DMEM low glucose Dulbecco ' s modified minimal essential medium
  • Example 3 Cell Culture of Lymphatic Endothelial Cells.
  • Bovine lymphatic endothelial (BLE) cells were isolated from mesenteric lymphatic vessels and provided by Dr. S. Wasi. Passage 3 cells were cloned by limiting dilution as described in Pepper et al . , Exp . Cell Res . , 210:298-305 (1994). BLE cells were cultured in DMEM supplemented with 1 mM sodium pyruvate, 10% donor calf serum and antibiotics. Cells were used between passages 21 and 26.
  • Example 4 Cell Culture of Pulmonary Artery Endothelial Cells.
  • Calf pulmonary artery endothelial (CPAE) cells were purchased from American Type Culture Collection (Rockville, MD) , and were cultured in medium 199 supplemented with 20% DCS. Cells were used at passage 23 .
  • CPAE Calf pulmonary artery endothelial
  • All endothelial cell lines were maintained in 1.5% gelatin-coated tissue culture flasks (Falcon Labware, Becton-Dickinson Company, Lincoln Park, NJ) and subcultured at a split ratio of 1/3 or 1/4.
  • the endothelial nature of all four cell lines has previously been confirmed by Dil-Ac-LDL (Paesel and Lorei, Frankfurt, Germany) uptake and immunostaining with a rabbit polyclonal antiserum against human von Willebrand factor (Nordic Immunology, Tilburg, The Netherlands) .
  • Example 5 Stimulation of Angiogenesis in BME Cells by VEGF or VEGF-C Alone or in Combination with bFGF.
  • VEGF or VEGF-C Alone or in Combination with bFGF were prepared as described by Montesano et al . , Cell 42:469-477 (1985) .
  • VEGF vascular endothelial growth factor
  • VEGF-C vascular endothelial growth factor
  • bFGF bovine fibroblast growth factor
  • Figure la is a phase contrast view of an untreated control .
  • Figure lb shows a sample treated with 30 ng/ml of VEGF. The resultant formation of cell cords within the collagen gel is clearly apparent, the plane of focus being beneath the surface of the gel.
  • Figure lc shows a collagen gel treated with 30 ng/ml VEGF-C.
  • Figure Id shows a sample treated 10 ng/ml bFGF alone. Again a definite invasive response is visible.
  • Figure le shows a gel treated with a combination of 30 ng/ml VEGF and 10 ng/ml bFGF, and Figure If shows a gel treated with the combination of 30 ng/ml VEGF-C and 10 ng/ml bFGF.
  • the synergistic effects achieved by co- administration of VEGF and bFGF or of VEGF-C and bFGF are readily seen.
  • Example 6 Stimulation of Angiogenesis in BME Cells by VEGF-B in Combination with bFGF.
  • the procedure of Example 5 was repeated, except that BME cell monolayers were treated with VEGF-B 167 at concentrations of 1, 10 and 100 ng/ml and VEGF-B 186 at concentrations of 1, 10 and 100 ng/ml alone and in combination with 10 ng/ml bFGF.
  • the shorter 167 amino acid form of VEGF-B lacks the hydrophobic region and the O-glycosylation site seen in the longer 186 amino acid form. The results are illustrated graphically in Figure 3.
  • VEGF-B alone did not produce measurable angiogenesis in this test
  • VEGF- B 167 at a concentration of 100 ng/ml and VEGF-B 186 at all concentrations tested produced a more than additive angiogenesis effect in combination with 10 ng/ml bFGF.
  • the effects of administration of VEGF and VEGF-C alone and in combination with 10 ng/ml bFGF are shown by the bars labelled V-A and V-C at the right of each graph, respectively.
  • Example 7 Stimulation of Angiogenesis in BAE Cells by VEGF-C Alone and in Combination with bFGF.
  • Example 5 The procedure of Example 5 was again repeated except that the surface of the gel in each well was seeded with a culture of bovine aortic endothelial (BAE) cells, and VEGF-C was tested both alone and in combination with bFGF at concentrations of 1 ng/ml and 10 ng/ml.
  • BAE bovine aortic endothelial
  • VEGF-C was tested both alone and in combination with bFGF at concentrations of 1 ng/ml and 10 ng/ml.
  • Example 8 Stimulation of Angiogenesis in BAE Cells by VEGF-B in Combination with bFGF.
  • the procedure of Example 6 was repeated except that the surface of the gel in each well was seeded with a culture of bovine aortic endothelial (BAE) cells and bFGF was used at a concentration of 3 ng/ml.
  • BAE bovine aortic endothelial
  • bFGF was used at a concentration of 3 ng/ml.
  • the results are depicted graphically in Figures 5 (a) and 5 (b) . Again VEGF-B alone did not produce an apparent angiogenic effect in this test, but both VEGF-B 167 and
  • VEGF-B 186 produced a more than additive (i.e. synergistic) angiogenic effect when co-administered with bFGF.
  • results achieved with VEGF (V-A) and VEGF-C (V-C) alone and in combination with bFGF are shown at the right side of each graph.
  • Example 9 Stimulation of Angiogenesis in BLE Cells by VEGF-C.
  • Bovine lymphatic endothelial (BLE) cell monolayers formed by seeding three-dimensional gels as described in Example 5 with BLE cell cultures were treated with VEGF-C and/or VEGF. After 4 days treatment, randomly selected fields of the gels were photographed at a single level beneath the surface monolayer as described above. Endothelial cell invasion was quantified as described in conjunction with the preceding examples. The results are shown graphically in Figure 6. It can be seen that VEGF-C produced a definite stimulation of angiogenic activity.
  • Examples 5 through 9 demonstrate that the angiogenesis-inducing properties of VEGF-B and VEGF-C can be mediated via a direct effect on endothelial cells and that there is a potent synergistic effect between VEGF-B or VEGF-C and bFGF in the induction of angiogenesis.
  • FIGS. lc and 2a when added to confluent monolayers of BME cells on three-dimensional collagen gels, the invasive response induced by VEGF-C was barely measurable.
  • Figures 4a and 6 show that VEGF-C induced a definite dose dependent invasive response in BAE and BLE cells, respectively, which was accompanied by the formation of branching tube-like structures as seen by phase-contrast microscopy by focussing beneath the surface monolayer.
  • VEGF-C Assuming a M r of 42,000 for VEGF-C, when compared at equimolar (0.6-0.7 nM) concentrations, VEGF-C (30 ng/ml) was slightly less potent than VEGF (30 ng/ml) , which in turn was about half as potent as bFGF (10 ng/ml) (Cf. Figures 4(a) & (b) , 6 and 7).
  • VEGF-C When co-administered with bFGF, VEGF-C induced a synergistic in vi tro angiogenic response.
  • VEGF-C when added to BME cells, in which VEGF-C alone has little or no effect, VEGF-C potentiated the effect of bFGF, with a maximal 2.5-fold increase at 30 ng/ml VEGF-C as shown in Figures If and 2.
  • co-administration of VEGF-C and bFGF induced a greater-than- additive response as shown, for example, in Figure 4.
  • Example 10 Co-Administration of VEGF and VEGF-C.
  • FIG. 8a shows the individual effects of the three cytokines . It can be seen that 30 ng/ml of VEGF-B alone produced a negligible effect. VEGF (30 ng/ml) alone produced a marked effect, but the greatest individual effect was produced by 30 ng/ml of VEGF-C. As can be seen from Figure 8b, when VEGF and VEGF-B were co-administered, the result was clearly greater than the sum of their individual effects.
  • VEGF-B co-administration of VEGF-B with VEGF-C clearly potentiated the collagen gel invasion inducing activity of VEGF-C.
  • Example 11 Stimulation of PA and PAI-1 Activity by VEGF-C in
  • BME and BAE cells Cell extracts and culture supernatants prepared from BME and BAE cells exposed to VEGF-C at concentrations 0, 1 3 10 30 and 100 ng/ml for 15 hours, were subjected to zymography and reverse zymography.
  • Confluent monolayers of endothelial cells in 35 mm gelatin-coated tissue culture dishes were washed twice with serum- free medium, and the respective cytokines were added in serum-free medium containing Trasylol (200 KlU/ml) .
  • Figure 9a shows a zymographic analysis of cell extracts from the BME cells.
  • Figure 9b shows the same zymography incubated for a longer time period at 37°C.
  • Figure 9c shows a reverse zymographic analysis of culture supernatant from BAE cells.
  • Example 12 Stimulation of PA and PAI-1 Activity by VEGF-B in
  • Example 11 BME and BAE Cells.
  • the procedure of Example 11 was repeated except that the culture supernatants were taken from cells exposed to VEGF-B at concentrations of 0, 1, 3, 10, 30 and 100 ng/ml.
  • the results are shown in Figures 10a and 10b.
  • the results of exposures to 30 ng/ml VEGF and 10 ng/ml bFGF are also shown.
  • PAI-1 activity in BME and BAE cells PAI-1 activity in BME and BAE cells.
  • the zymography and reverse zymography results shown in Figures 10a and 10b demonstrate that VEGF-B induces uPA and PAI-1 activity in endothelial cells. The induction of uPA and PAI-1 activity was less than that seen with approximately equimolar concentrations of VEGF.
  • the zymography test results also confirm that bFGF has little or no effect on the expression of tPA.
  • the effect of VEGF-C on PAI-1 activity in BME cells, on uPA activity in BAE cells and on tPA and PAI-1 activity in BLE cells was also tested and was found to be less marked than what is shown in Figures 9a through 9c .
  • Example 13 RNA preparat ion , in vi tro transcript ion , and
  • RNA integrity and uniformity of loading were determined by staining the filters with methylene blue after transfer and cross-linking as shown by the 28S and 18S ribosomal RNAs at the bottom of the Figures 11 and 12.
  • Northern blots, UV cross-linking and methylene blue staining of filters, in vitro transcription, hybridization and post-hybridization washes were as previously described by Pepper et al . , J " . Cell Biol . ,
  • [ 32 P] -labelled cRNA probes were prepared from bovine urokinase-type plasminogen activator (u-PA) [Kraetzschmar et al . , Gene, 125:177-83 (1993)], bovine u-PA receptor (u-PAR)
  • the Northern blot analysis shown in Figure 11 indicates that VEGF-C increases steady state levels of uPA, uPAR, tPA and PAI-1 mRNAs in BAE cells.
  • the kinetics of PA, uPAR and PAI-1 induction in were rapid (within 1 hour) and transient (baseline levels were attained between 3 and 9 hours for uPA and uPAR, and between 9 and 24 hours for PAI-1) .
  • uPA and uPAR this is in contrast to the more sustained increase reported with VEGF and bFGF [see Pepper et al . , J. Cell Biol . , 111:743-755 (1990); Mandriota et al . , J. Biol . Chem . , 270:9709-16 (1995)].
  • PAI-1 the results are similar to VEGF and bFGF.
  • the Northern blot test results for the tests with VEGF-B are shown in Figure 12.
  • protease inhibition which is manifest by increased synthesis of PAI-1
  • proteolytic balance which recognizes that while proteases are necessary for cell migration and morphogenesis, protease inhibitors play an equally important permissive role by protecting the extracellular matrix from inappropriate destruction.
  • Example 16 Inhibition of Endothelial Cell Angiogenesis in the
  • VEGF-C and bFGF Presence of VEGF-C and bFGF by 2 -antiplasmin.
  • BME cell monolayers were co-treated for 4 days with bFGF and VEGF-C as well as 2 -antiplasmin at concentrations of 0 , 1, 3, 10 and 30 ⁇ g/ml . Randomly selected fields of the treated gels were then photographed at a single level beneath the surface monolayer. Endothelial cell invasion was quantified as described above in Example 5.
  • Figure 13 shows the results in graph form. A clear decrease in angiogenic activity can be seen as the concentration of 2 -antiplasmin increases. It is thus apparent that 2 - antiplasmin inhibits the induction of angiogenesis by co-treatment with VEGF-C and bFGF in a dose-dependent manner.
  • Circulation 92 (suppl.II) : II-365-II371.
  • the FLT4 gene encodes a transmembrane tyrosine kinase related to the vascular endothelial growth factor receptor.
  • VEGF-C vascular endothelial growth factor
  • Neovascularization is associated with a switch to the export of bFGF in the multistep development of fibrosarcoma. Cell 66: 1095-1104.
  • Vascular endothelial growth factor-related protein a ligand and specific activator of the tyrosine kinase receptor FLT4. Proc. Natl. Acad. Sci. USA 93: 1988-1992.
  • Transforming growth factor- ⁇ l downregulates vascular endothelial growth factor receptor-2/flk-1 expression in vascular endothelial cells. J. Biol. Chem. 271: 11500-11505.
  • VEGF vascular endothelial growth factor
  • Vascular endothelial growth factor increases urokinase receptor expression in vascular endothelial cells. J. Biol. Chem. 270: 9709-9716. Millauer, B., Longhi , M.P., Plate, K.H., Shawver, L.K., Risau, W. , Ullrich, A. and Strawn, L.M. (1996) Dominant negative inhibition of Flk-1 suppresses the growth of many tumor types in vivo. Cancer Res. 56: 1615-1620.
  • VEGF Vascular endothelial growth factor
  • Leukemia inhibitory factor is a potent inhibitor of in vitro angiogenesis. J Cell Sci 108: 73-83.
  • Angiogenesis a paradigm for balanced extracellular proteolysis during cell migration and morphogenesis.
  • Fetal liver kinase-1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium. Proc. Natl. Acad. Sci. USA 90: 7533-7537. Risau, W. (1997) Mechanisms of angiogenesis. Nature 386: 671-674. Roberts, W.G. and Palade, G.E. (1995) Increased microvascular permeability and endothelial fenestration induced by vascular endothelial growth factor. J. Cell Sci. 108: 2369-2379. Roberts, W.G. and Palade, G.E. (1997) Neovasculature induced by vascular endothelial growth factor is fenestrated. Cancer Res. 57: 765 - 772 .
  • Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 219: 983-985.
  • Vascular permeability factor/vascular endothelial growth factor inhibits anchorage-disruption- induced apoptosis in microvascular endothelial cells by inducing scaffold formation.

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Abstract

Le facteur B de croissance endothéliale vasculaire (VEGF-B) et le facteur C de croissance endothéliale vasculaire (VEGF-C) sont des polypeptides angiogéniques. On a découvert que VEGF-B et VEGF-C sont angiogéniques in vitro plus particulièrement en combinaison avec b(FGF). VEGE-C augmente aussi l'activité des activateurs du plasminogène (PA) et ceci s'accompagne d'une augmentation correspondante de l'inhibiteur-1 PA. L'apport d'alpha-2-antiplasmine aux cellules endothéliales bovines traitées conjointement avec bFGF et VEGF-C permet d'inhiber partiellement l'invasion de gel collagène.
EP98940875A 1997-08-15 1998-08-14 Stimulation modulation et/ou inhibition de l'activite protheolithique endotheliale et/ou de l'activite angioginique Withdrawn EP1011328A4 (fr)

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US7109308B1 (en) 1994-03-08 2006-09-19 Human Genome Sciences, Inc. Antibodies to human vascular endothelial growth factor 2
US6040157A (en) 1994-03-08 2000-03-21 Human Genome Sciences, Inc. Vascular endothelial growth factor 2
US5932540A (en) 1994-03-08 1999-08-03 Human Genome Sciences, Inc. Vascular endothelial growth factor 2
US7153827B1 (en) 1994-03-08 2006-12-26 Human Genome Sciences, Inc. Vascular endothelial growth factor 2 and methods of use
ATE309360T1 (de) 1994-03-08 2005-11-15 Human Genome Sciences Inc Vaskularer endothelialer wachstumsfaktor 2
US7186688B1 (en) 1994-03-08 2007-03-06 Human Genome Sciences, Inc. Methods of stimulating angiogenesis in a patient by administering vascular endothelial growth factor 2
US6608182B1 (en) 1994-03-08 2003-08-19 Human Genome Sciences, Inc. Human vascular endothelial growth factor 2
US6734285B2 (en) 1994-03-08 2004-05-11 Human Genome Sciences, Inc. Vascular endothelial growth factor 2 proteins and compositions
ATE250427T1 (de) * 1998-10-08 2003-10-15 Childrens Hosp Medical Center Zusammensetzungen und deren verwendung zur hemmung von angiogenese
US7223724B1 (en) 1999-02-08 2007-05-29 Human Genome Sciences, Inc. Use of vascular endothelial growth factor to treat photoreceptor cells
EP1374889A1 (fr) * 1999-06-25 2004-01-02 Yissum Research Development Company Of The Hebrew University Of Jerusalem Procédé permettant d'induire une angiogénèse au moyen des micro-organes
NZ518077A (en) 2000-08-04 2003-11-28 Human Genome Sciences Inc Biologically active fragments, analogues and derivatives of VEGF-2 for the treatment of peripheral artery diseases such as critical limb ischemia and coronary disease
AU2002224471A1 (en) * 2000-11-01 2002-05-15 Aventis Pharma S.A. In vivo stimulation of angiogenic activity
WO2002083704A1 (fr) 2001-04-13 2002-10-24 Human Genome Sciences, Inc. Facteur de croissance 2, endothelial, vasculaire
US7402312B2 (en) 2001-04-13 2008-07-22 Human Genome Sciences, Inc. Antibodies to vascular endothelial growth factor 2 (VEGF-2)
US20080075750A1 (en) * 2001-05-11 2008-03-27 The Nemours Foundation Methods for producing three-dimensional tissue-engineered cardiac constructs and uses regarding same
CA2493572A1 (fr) 2002-07-23 2004-01-29 Ludwig Institute For Cancer Research Procedes et compositions pour l'activation ou l'inhibition du facteur de croissance endothelial vasculaire d et du facteur de croissance endothelial vasculaire c
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