WO1989012645A1 - Peptides de potentialisation angiogeniques potentialisant des facteurs angiogeniques - Google Patents

Peptides de potentialisation angiogeniques potentialisant des facteurs angiogeniques Download PDF

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WO1989012645A1
WO1989012645A1 PCT/US1989/002699 US8902699W WO8912645A1 WO 1989012645 A1 WO1989012645 A1 WO 1989012645A1 US 8902699 W US8902699 W US 8902699W WO 8912645 A1 WO8912645 A1 WO 8912645A1
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app
angiogenic
stimulatory effect
growth factor
factor
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PCT/US1989/002699
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English (en)
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Tadahio Kohno
Richard A. Majack
Julie A. Wilson
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Synergen, Inc.
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Publication of WO1989012645A1 publication Critical patent/WO1989012645A1/fr
Priority to KR1019900700363A priority Critical patent/KR900701833A/ko

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • 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
    • A61K38/1816—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This invention relates to angiogenic potentiating peptides which potentiate angiogenic factors, and particular ⁇ ly to platelet-derived endothelial cell growth factors which synergize with fibroblast growth factor to enhance angiogensis, plasminogen activator production, cell migration and DNA synthesis in endothelial cells, and capillary proliferation.
  • angiogenic potentiating peptide refers to a class of relatively small proteins which are capable of potentiating the effects of angiogenic factors. Certain “angiogenic potentiating peptides” may be cell specific growth factors. Two examples of “angiogenic potentiating peptides” are described in greater detail below. These are derived from platelets and cultured vascular smooth muscle cells.
  • growth factor denominates a class of relatively small proteins that by themselves or in combina ⁇ tion with other proteins increase cellular synthesis of nucleic acids and proteins and increase cellular mass. Con ⁇ ventionally, also, growth factors are considerd to be agents which, by themselves or in combination with other proteins, exhibit mitogenic activity. A variety of growth factors have been isolated and at least partially characterized. See Cas ⁇ tor and Cabral, "Growth Factors and Human Disease: The Realities, Pitfalls and Promise," Se . Arthritis Rheum. 15:33-44, 1985.
  • Growth factors are also recognized, respectively, to target a variety of cells.
  • growth factors are known which affect epithelial cells, e.g. , the epidermis and con ⁇ nective tissue.
  • Growth factors that affect endothelial cells which line blood and lymph vessels, are also known.
  • endothelial cells which line blood and lymph vessels.
  • sulfation factor was determined by its growth-promoting activity as measured by the increased sulfated glycosaminoglycan content of carti ⁇ lage extracellular matrix.
  • growth factors are primarily concerned with anabolic activi ⁇ ties, growth factors are more accurately considered not only to augment cell mass but also certain of their extracellular products which have catabolic impact.
  • One growth factor for example, connective tissue activating peptide-III, also stim ⁇ ulates the synthesis and secretion of plasminogen activator, a degradative protease discussed in more detail below.
  • plasminogen activator a degradative protease
  • interleukin-1 also promotes the synthesis and secre ⁇ tion of prostaglandins and collagenase in addition tc its mitogenic effects.
  • a given growth factor may elicit both anabolic and catabolic behavior from a single target cell.
  • Growth factor may be abbreviated "GF”, epidermal cell growth factor as “EGF”, endothelial cell growth factor as “ECGF”, fibroblast growth factor as “FGF” , basic fibroblast growth factor as bFGF, acidic (or anionic) fibroblast growth factor as aFGF, platelet-derived growth factor as “PDGF”, angiogenic factor as “AF”, angiogenic potentiating peptide as “APP”, platelet- derived angiogenic potentiating peptide as "P-APP” , and smooth muscle cell-derived angiogenic potentiating peptide as "SMC-APP.”
  • Growth factors are conventionally named after the types of target cells which they are first identified as affecting, i.e., EGF, ECGF or FGF; after the tissue or cells from which they are derived, i.e.
  • PDGF vascular endothelial growth factor
  • AF angiogenic factors
  • This class of peptide also surprisingly potentiates the angiogenic activity of at least one fibroblast growth factor (FGF) .
  • FGF fibroblast growth factor
  • the basic fibroblast growth factor defined below, exhibits an endothelial cell growth factor (ECGF) activity and stimulates angiogenesis (and is thus also properly termed an angiogenic factor).
  • ECGF endothelial cell growth factor
  • P-APP potentiating platelet-derived factor of the present invention
  • Angiogenic factors have been generally defined as a subset of endothelial cell regulatory factors which may stim ⁇ ulate a group of cellular responses, including (1) an in ⁇ creased rate of endothelial cell proliferation; (2) an in ⁇ creased endothelial cell protease synthesis; (3) chemotactic endothelial cell migration toward a fixed source of the angiogenic factor; and/or (4) capillary proliferation in vivo. It has been observed that substances classified as angiogenic factors may act on DNA synthesis in endothelial cells, thereby increasing the rate of endothelial cell proliferation and thus the rate at which new blood vessels are formed.
  • AF properties Interrelated with the cell proliferation, or itogenic, AF properties is the general ability of endothelial cell growth factors to increase protease synthe ⁇ sis by endothelial cells.
  • Proteases of particular clinical interest include plasminogen activator and collagenase.
  • Angiogenic factors are able to stimulate synthesis of plasminogen activator and latent collagenase by endothelial cells, and it is known that plasminogen activator can con ⁇ vert the zy ogen plasminogen into plasmin, a protease of wide specificity, which in turn can convert latent collagenase into active collagenase.
  • Plasminogen activator particularly tissue plasminogen activator, has great utility in therapy for myocardial infarction by dis ⁇ solving clots which may occlude coronary arteries.
  • Angiogenic factors may also be chemotactic for endothelial cells including capillary endothelial cells.
  • an appropriate angiogenic factor would lead to the development of therapeutc compounds capable of increasing the blood supply to an organ.
  • an angiogenic factor may stimulate healing in decubitus ulcers, surgical incisions and slowly healing wounds, particularly in geriatric and diabetic patients.
  • the application of this material to burns may improve the rate and degree of healing.
  • PAF placental angiogenic factor
  • bFGF basic fibroblast growth- factor
  • the bFGF angiogenic factor of the co-pending appli ⁇ cation has the four above-identified angiogenesis properties, i.e., it is mitogenic, stimulates protease synthesis, is che otactic and causes .in vivo capillary proliferation.
  • This human angiogenic factor was isolated in a substantially purified form from human placental tissues by virtue of its properties of binding strongly to heparin-Sepharose and having a basic pi. The amino acid sequence of this bFGF was also determined. A recombinant-DNA method for the production of this protein was developed.
  • the term bFGF refers to the angiogenic factor of the above-identified copending application and its biological equivalents which stimulate angiogenesis in the same manner although not necessarily to the same degree.
  • bFGF for example, can stimulate the growth of bovine capillary endothelial cells only few-fold. It would be use ⁇ ful, therefore, to develop proteins that synergize with bFGF and will thereby potentiate its angiogenic effects. No one thus far has described a protein that synergizes with bFGF to potentiate angiogenesis.
  • endothelial cell specific growth factors have been purified from platelets. Some of these factors also may be capable of acting as angiogenic potentiating peptides, however, this activity never been previously described. Two endothelial cell growth factors have been described by K. Miyazono e_t al. , in “Purification and Prop ⁇ erties of an Endothelial Cell Growth Factor From Human Platelets," Journal of Biological Chemistry,
  • the present invention relates to APPs capable of potentiating at least one angiogenic factor.
  • this invention relates to a cell- or platelet-derived factor which is an endothelial growth factor capable of po ⁇ tentiating at least one of the angiogenic activities of an angiogenesis factor.
  • This angiogenesis factor is preferably a fibroblast growth factor possessing at least one activity selected from the group consisting of mitogenic activity, chemotactic activity, the ability to stimulate protease syn ⁇ thesis, the ability to stimulate capillary proliferation in vivo, and combinatinos thereof.
  • this fibro ⁇ blast growth factor is the basic fibroblast growth factor identified in ' copending application Serial No. 163,142, and its biological equivalents, or acidic FGF.
  • the APPs of the present invention may be character ⁇ ized in their isolation from blood platelets or smooth muscle cells, but includes other angioge ⁇ esis-potentiating peptides which are biologically equivalent to the factors derived from these sources.
  • the present invention relates to a therapeutically-effective dose of an angiogenic growth factor in admixture with an APP capable of potentiating at least one of the activities of the angiogenic factor, together with suitable pharmaceutical carriers and excipients as may be desirable.
  • the present invention relates to the administration of the medication described above. It is contemplated that the therapeutic and medica ⁇ tion aspects of the present broadly encompass the treatment of pathologic, traumatic and surgical processes in which the regulation of the angiogenesis process, generally, may be beneficial.
  • Fig. 1 illustrates the DEAE-CL6B column chromatography pattern of 0.25m NaCl eluate of Example 3.
  • Fig. 2 illustrates that both the 45,000 dalton and 65,000 dalton fractions contain the P-APP activity. All assays were carried out in the presence of bFGF ( x x line) .
  • Fig. 3 illustrates the synergistic effect of the P-APP of the present invention on the proliferation of bovine capillary endothelial cells (BCE cells).
  • BCE cells bovine capillary endothelial cells
  • the • • line in ⁇ dicates the effect of various concentrations of bFGF on the proliferation of BCE cells.
  • the ° ° line indicates the
  • Fig. 4* presents photomicrographs of bovine endothelial cells cultured on a thick collagen matrix.
  • i ⁇ . 4A shows control cells;
  • Fig. 4B shows the effect of treatment with bFGF.
  • Fig. 4C shows no detectable effect on the morphology of these cells in cultures exposed to a P-APP fraction described herein.
  • Fig.4D shows a marked effect on cultures exposed to P-APP and bFGF simultaneously.
  • Fig. 5 presents photomicrographs of bovine capil ⁇ lary endothelial cells cultured on thick collagen gels.
  • Fig. 5A shows cells maintained in control medium;
  • Fig. 5B shows the effect of treatment with bFGF.
  • Fig. 5 presents photomicrographs of bovine capil ⁇ lary endothelial cells cultured on thick collagen gels.
  • Fig. 5A shows cells maintained in control medium;
  • Fig. 5B shows the effect of treatment with bFGF.
  • FIG. 5C shows no detectable effect on the morphology of these cells in cul ⁇ tures exposed to conditioned medium from rat aortic smooth muscle cells.
  • Fig. 5D shows a marked effect (much greater than that observed with bFGF alone) on cultures exposed to bFGF in the presence of smooth muscle cell conditioned medi ⁇ um.
  • Practice of the present invention requires the iso ⁇ lation and purification of an APP as may be recovered from a platelet lysate, that is capable of potentiating the angiogenic properties of at least one FGF.
  • practice of this invention involves the co- administration of this P-APP, specifically defined herein as P-APP, with another angiogenic factor, such as the basic fibroblast growth factor.
  • platelets are a rich source of cell growth regulatory proteins, including PDGF, transforming growth factor-beta, epidermal growth factor-like protein, endothelial cell growth fator and hepatocyte growth factor, some of which are cationic and others anionic growth factors.
  • the present invention broadly relates to those cell- or platelet-derived APPs which act in a synergistic fashion to potentiate the angiogenic action of other growth factors, particularly the action of fibroblast growth factors.
  • the present invention also relates to a P-APP which is substan ⁇ tially homologous to, immunologically equivalent to, or, most preferably, biologiclaly equivalent to the native poten ⁇ tiating P-APP isolatable from human platelets.
  • biological ⁇ cally equivalent as used in this specification and claims, it is meant that an angiogenesis potentiating peptide pos ⁇ sesses the ability to stimulate the angiogenic properties of FGF in the same manner, although not necessarily to the same degree, as the native P-APP.
  • biological ⁇ cally equivalent it is meant that an angiogenesis potentiating peptide pos ⁇ sesses the ability to stimulate the angiogenic properties of FGF in the same manner, although not necessarily to the same degree, as the native P-APP.
  • the reco binant human bFGF Upon induction with PTG, the reco binant human bFGF in amounts exceeding 2% of the total cell protein or 8 mg/l/ A 600 unit.
  • the synthe ⁇ sized bFGF accumulates in the soluble cell lysate fraction, but when cells were grown at 37°C, a substantial portion of the recombinant protein was found in the insoluble cell lysate fraction.
  • the recombinant bFGF could be purified by a single- step affinity chromatology of the soluble E. coli cell lysate fraction on heparin sepharose.
  • EXAMPLE 3 Isolation of P-APP From Human Platelets
  • a portion of the 0.25 M eluate of the DEAE-A50 col ⁇ umn of Example 3 was dialyzed extensively for 16 hours against 20 mM aP ⁇ , pH 7.4, and applied to a column of DEAE-CL6B (2.5 x 20 cm).
  • the column was washed with 500 ml of 20 mM NaP ⁇ , pH 7.4, an eluted with a linear gradient (500 ml) of NaCl from 0 to 500 mM in 20 mM NaP ⁇ , pH 7.4, at a flow rate of 40 ml/hr.
  • a 280' the ' * line shows growth promoting activity, and the x x line indicates P-APP activity in the presence of
  • EXAMPLE 9 Effect of the P-APP From Human Platelets on the Growth of Bovine Capillary Endothelial Cells Platelet-derived factors of Example 7 were assayed at various concentrations for incorporation of °H-thym ⁇ d ⁇ ne in bovine capillary endothelial cells. Samples were assayed with and without 10 ng/ml bFGF. Without bFGF, the platelet- derived factors alone increased 3H-thymidine incorporation four-fold. The addition of bFGF alone increased 3 H-thymidine incorporation two-fold over the control and the combination of P-APP plus basic FGF surprisingly increased 3 H-thymidine incorporation 16-fold over the control. Similar results for fraction No. 68 of the DEAE-CL6B eluate of Example 5 are sum ⁇ marized in Table I which follows.
  • the acidic FGF was purchased from R&D Sys ⁇ tems, Inc., Minneapolis, Minnesota (Cat. No. 132-FA) .
  • the P-APP synergizes with acidic FGF as well as basic FGF.
  • the addition of either P-APP, acidic FGF or basic FGF alone increased 3 H-thymidine incorporation three- to four-fold over the control, and the combination of DEAE-CL6B fraction No. 67 (P-APP) and acidic FGF increased H-thymidine incorporation 19-fold over the control.
  • EXAMPLE 11 Further Chromatographic Purification of the Eluate of Example 3 An aliquot of the 0.25 M NaCl eluate of the
  • DEAE-A50 column of Example 3 was applied to a column contain ⁇ ing BioRad A1.5m or G75. Proteins were eluted with 20 M NaP0 , pH 7.4, at a flow rate of 50 ml/hr. Fractions of 4-5 ml were collected and analyzed for growth-stimulating activ ⁇ ity on endothelial cells and 3T3 cells with 10 ng/ml bFGF. Growth-promoting activity on 3T3 cells was assayed by the method by Raines and Ross in J. Biol. Chem 277:5154-5160
  • endothelial cell growth factors act synergistically with BFGF on the growth of BCE cells.
  • the two P-APPs do not stimulate the growth of 3T3 fibroblasts, and. do not potentiate the growth promoting effects of bFGF in
  • EXAMPLE 12 Further Chromatographic Purification of the Factor of Example 11 The fractions between Nos. 74 and 86 of the A1.5m column of Example 11 were pooled and dialyzed against 20 mM sodium acetate, pH 4.8, and applied to an SP-C25 column.
  • Bound proteins were eluted with a linear gradient of NaCl from 0 to 1.0 M NaCl. Fractions of 2 ml were collected and assayed for their effect on growth of bovine capillary endothelial cells and 3T3 fibroblast cells. Results indicate that the endothelial cell growth stimulator proteins have no effect on the growth of 3T3 fibroblasts.
  • E XAMPLE 13 Further Purification and Characterization of Platelet-derived Factors of Example 6
  • the proteins 45,000 dalton and 65,000 dalton, will be further purified separately by using SP-C25 column (20mM Na acetate, pH 4.8) and Mono-Q column (25mM Tris-HCl, pH 7.4).
  • the pure proteins will be desalted on HPLC-C8 column and will be subjected to N-terminal amino acid sequencing analysis. Some of the desalted proteins will be treated with proteolytic enzymes such as trypsin to generate small peptide fragments. Amino acid sequence of these peptide fragments will be determined in order to generate oligonucleotide probes for screening tissue or cell sources of these pro ⁇ teins.
  • the proteins will also be used to prepare antibody against the proteins.
  • the anti-platelet-derived APP will be used to screen tissue or cell sources of these protein ' s. Once tissue or cells which contain these proteins are identi ⁇ fied, cDNA libraries will be prepared and screened for the gene or genes which encodes these proteins.
  • EXAMPLE 14 In Vitro Angiogenesis Assay
  • angiogenesis i.e. , activa ⁇ tion of quiescent endothelium, cell migration, and invasion of the pericellular matrix
  • angiogenesis i.e. , activa ⁇ tion of quiescent endothelium, cell migration, and invasion of the pericellular matrix
  • capillary endothelial cells cultured on a thick collagen ma ⁇ trix according to the procedures of Montesano et al. , Cell 42:469-477, 1985; and Motesano et, al. , Proc. Nat'l Acad. Sci. USA 83:7297-7301, 1986, the disclosures of which are expressly incorporated herein by reference in their entireties.
  • Bovine adrenal capillary endothelial cells were grown to confluency on 1.0 ml collagen matrices in 12-well (35 mm diameter) tissue culture plates. Cells were main ⁇ tained in 5% newborn calf serum (NCS) in alpha MEM medium. The collagen matrices were prepared from Types I and III Col ⁇ lagen (obtained from Vitrogen, Collagen Corp.) using the pro ⁇ tocols recommended by the manufacturer. Confluent cells were maintained for 48 hrs in 5% NCS/alpha MEM under the following conditions: (i) without further additions as a control; (ii) with the addition of 10 ng/ml bFGF; (iii) with the addi ⁇ tion of 80 ul/ml of fraction No. 68 of Example 5; and (iv) with the addition of 10 ng/ml bFGF + 80 ul/ml DEAE-CL63 frac ⁇ tion No. 68.
  • Fig. 4 are photomicrographs of the cell cultures.
  • Cells main ⁇ tained on the surface of three-dimensional collagen gels formed and remained as a monolayer of closely opposed cells (Fig. 4A) .
  • bFGf 10 ng/ml
  • numerous endothelial cells could be distinguished by an ir ⁇ regular and dendritic morphology, and a shift in their plane of focus to beneath the monolayer (Fig. 4B) .
  • Addition of the protein from fraction No. 68 alone had no detectable effect on the morphology of confluent endothelial cells (Fig. 4C) .
  • the culture morphology was markedly changed (Fig. 4D). In these cultures, more dentritic cells were observed and, additionally, the cells were organized into short branching chords that formed a discontinuous network under the surface monolayer.
  • P-APP of DEAE-CL6B fraction No. 68 significantly potentiated the ability of bFGF to induce endothelial cell invasion and tube formation, and thus markedly enhances the angiogenic process.
  • EXAMPLE 15 Detection of a factor derived from cultured smooth muscle cells with potentiates the angiogenic effects of bFGF on capillary endothelial cells.
  • Rat aortic smooth muscle cells were grown to overconfluency in 5% NCS/Waymouth's medium in 150-mm diameter tissue culture dishes. The was removed and the cul ⁇ tures were washed, then maintained for 30 hours in Waymouth's medium containing 0.1 mg/ l BSA and no serum. This "condi ⁇ tioned medium" was removed, clarified by centrifugation, and stored at -70°C.
  • In vitro angiogenesis assays were performed exactly as described above in Example 14.
  • Confluent endothelial cells cultured on thick collagen gels were main ⁇ tained for 48 hours in the presence of 5% FBS under the fol ⁇ lowing conditions: (i) in alpha MEM medium without additions as a control; (ii) in alpha MEM medium supplemented with 10 ng/ml bFGF; (iii) in medium conditioned by smooth muscle cells; and (iv) in smooth muscle cell conditioned medium sup ⁇ plemented with 10 ng/ml bFGF.
  • FIG. 5 are photomicrographs of the cell cultures.
  • Control cells Fig. 5A
  • Fig. 5B numerous endothelial cells which had invaded the underlying collagen could be dis ⁇ tinguished by their location (under the monolayered culture) and their irregular and dendritic morphology.
  • Fig. 5C Maintenance of the endothelial cells in medium conditioned by smooth muscle cells had no detectable effect on their morphology.
  • EXAMPLE 16 Isolation of a factor which potentiates the effects of bFGF from smooth muscle cell conditioned medium
  • Ten 150-mm cultures of confluent rat vascular smooth muscle cells will be used to generate 100 ml of serum- free conditioned medium.
  • Cultures will be maintained in 5% NCS/Waymouth's medium, washed and then maintained for 30 hours in serum-free Waymouth's medium containing 0.1 ng/ml BSA.
  • the conditioned medium will be clarified by centrifugation, dialyzed aginst 20 mM NaP ⁇ 4, pH 7.4, then applied to a 2.5 x 9 cm DEAE-A50 column. The column will be washed extensively until the A28O °f e eluant reaches 0.05.
  • Proteins will be recovered from the flow-through fraction and from fractions eluted by step-wise application of 0.25M, 0.5 M, and 1.0 NaCl washes. Each of these fractions will be tested for SMC-APP activity in J H-thym ⁇ d ⁇ ne assays using bovine capillary endothelial cells.
  • the DEA ⁇ -A50 fraction containing SMC-APP activity will be dialyzed exhaustively against 20 M NaP ⁇ 4, pH 7.4 prior to its application to a 2.5 x 20 cm column of DEAE-CL6B. The column will be washed with 500 ml of 20 mM NaP ⁇ 4, pH 7.4.
  • Proteins will be eluted with a linear gradient (500 ml) of NaCl from 0 to 500 mM in the same buffer, at a flow rate of 40 ml/hr. Fractions of 4 ml will be collected and analyzed for growth-potentiating activ ⁇ ity with the bFGF (10 ng/ml) of Example 2, using 3 H-thymidine assays to determine DNA synthesis in BCE cells. The frac ⁇ tions) corresponding to the peak of SMC-APP activity will be concentrated by Centricon-10 to 350 ul and applied to a col ⁇ umn of TSK 4000SW. Proteins will be eluted with 20 mM NaPO , pH 7.4, at a flow rate of 30 ml/hr. Fractions of 0.5 ml will be collected and analyzed for growth-potentiating activity with 10 ng/ml bFGF.
  • SUBSTITUTE SHEET Further purification of the SMC-APP activity will be effected by dialysis against 25mM Tris-HCl, pH 7.4, con ⁇ centration by centricon-10 to a 1.0 ml volume, and separation on a Mono-Q HPLC column. Proteins will be eluted by a linear gradient of 0-0.5M NaCl. Fractions corresponding to the peak of SMC-APP activity, as determining by J ⁇ H-thymidine incorpo ⁇ ration into BCE cells, will be pooled, concentrated, and applied to an HPLC-C8 column. The peak fractions will then be subjected to N-terminal amino acid sequencing either before or after proteolysis and purification of peptides by HPLC.
  • the deduced amino acide sequence will be used as a basis for the generation of oligonucleotide probes which will then be used to isolate clones from a cDNA library prepared from RNA extracted from human vascular SMC.
  • the amino acid sequence of the SMC-APP will be deduced from nucleotide sequencing of appropriate cDNAs.
  • the angiogenic factors potentiated by the APPs used in the process of the present invention will be broadly drawn from the class of angiogenic factors, whether or not also possessing fibroblast growth factor activity or having sub ⁇ stantial homology to the bFGF protein used for illustrative purposes in the foregoing examples.
  • the present inven ⁇ tion is not limited to the foregoing description and exam ⁇ ples, but is broadly encompassing of the following claims and their equivalents.

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Abstract

On a mis au point des peptides de potentialisation angiogéniques stimulant, dans certains cas, la croissance de cellules endothéliales sans affecter la croissance de cellules fibroblastiques, et améliorant considérablement l'activité angiogénique d'un facteur de croissance angiogénique, notamment un facteur de croissance fibroblastique acide ou basique.
PCT/US1989/002699 1988-06-22 1989-06-21 Peptides de potentialisation angiogeniques potentialisant des facteurs angiogeniques WO1989012645A1 (fr)

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KR1019900700363A KR900701833A (ko) 1988-06-22 1990-02-21 맥관형성 인자를 조력하는 맥관형성 조력 펩타이드(angiogenic potentiationg peptide)

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Title
BURGOS, Biol. Abstr. 77(3): 1911,1984 "Angiogenic and Growth Factors in Human Amino-Chorion and Placenta", see Abstract No. 17462. *
JUDITH ABRAHAM, J, of Cell Biochem. Supp. Vol. 6, No. 11, Part A, page 50, 1987 "Cloning and Characterization of the Genes for the Angiogenic Proteins, Basic and Acidic Filbrablast Growth Factor" see the Abstract. *
MOSCATELLI, Proc. Natl. Acad. Sci. USA, Vol. 83, 2091, 1986 "Purification of a Factor from Human Placenta that Stimulates Capillary Endothelial Cell Protease Production, DNA Synthesis and Migration", see the Abstract on page 2091. *
See also references of EP0422104A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0883625A1 (fr) * 1995-10-06 1998-12-16 President And Fellows Of Harvard College Nouvelle proteine activatrice des thrombocytes
EP0883625A4 (fr) * 1995-10-06 2001-04-25 Harvard College Nouvelle proteine activatrice des thrombocytes
US6750323B1 (en) 1995-10-06 2004-06-15 President And Fellows Of Harvard College Platelet activation protein

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Publication number Publication date
EP0422104A4 (en) 1991-11-21
KR900701833A (ko) 1990-12-04
JPH03505581A (ja) 1991-12-05
AU3863689A (en) 1990-01-12
EP0422104A1 (fr) 1991-04-17

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