WO2011111914A1 - Cellules progénitrices endothéliales vasculaires introduites avec le gène d'haptoglobine et composition contenant celui-ci pour favoriser l'angiogenèse - Google Patents

Cellules progénitrices endothéliales vasculaires introduites avec le gène d'haptoglobine et composition contenant celui-ci pour favoriser l'angiogenèse Download PDF

Info

Publication number
WO2011111914A1
WO2011111914A1 PCT/KR2010/006006 KR2010006006W WO2011111914A1 WO 2011111914 A1 WO2011111914 A1 WO 2011111914A1 KR 2010006006 W KR2010006006 W KR 2010006006W WO 2011111914 A1 WO2011111914 A1 WO 2011111914A1
Authority
WO
WIPO (PCT)
Prior art keywords
haptoglobin
vascular endothelial
angiogenesis
endothelial progenitor
progenitor cells
Prior art date
Application number
PCT/KR2010/006006
Other languages
English (en)
Korean (ko)
Inventor
김인숙
박선주
Original Assignee
가톨릭대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 가톨릭대학교 산학협력단 filed Critical 가톨릭대학교 산학협력단
Publication of WO2011111914A1 publication Critical patent/WO2011111914A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • C12N5/0691—Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the present invention relates to a vascular endothelial progenitor cell into which a haptoglobin gene is introduced and an angiogenesis-promoting composition comprising the same, and more particularly, to a vascular endothelial progenitor cell transformed with a recombinant vector comprising a haptoglobin gene and the same. It relates to a composition for promoting angiogenesis and an angiogenesis-promoting method comprising as an active ingredient.
  • EPCs circulating out of the bone marrow migrate to the site of vascular injury and are involved in new blood vessel formation through direct insertion into newly formed blood vessels or through the release of various angiogenesis and nutritional factors (Jujo, K., Ii, M.). and Losordo, DW (2008) Endothelial progenitor cells in neovascularization of infarcted myocardium.J. Mol.Cell.Cardiol .
  • EPCs are attracting attention as potential targets for therapeutic purposes through revascularization (Jujo, K., Ii, M. and Losordo, DW (2008) Endothelial progenitor cells in neovascularization of infarcted myocardium.J. Mol Cell.Cardiol . 45, 530.544.).
  • VEGF vascular endothelial growth factor
  • hypoxia inducer-1 ⁇ hypoxia inducer-1 ⁇
  • -inducible factor-1 ⁇ has been used to increase the pro-angiogenic capacity of EPCs (Iwaguro, H., Yamaguchi, J., Kalka, C., Murasawa, S., Masuda, H., Hayashi, S., Silver, M., Li, T., Isner, JM and Asahara, T.
  • Haptoglobin is an acute-phase glycoprotein in the blood circulation, and the well-known biological function of Hp is the capture of hemoglobin.
  • Hp is known to act to prevent extravascular hemoglobin-stimulatory oxidative tissue damage through the formation of Hp-hemoglobin complexes (Lim, YK, Jenner, A., Ali, AB, Wang, Y., Hsu, SI, Chong, SM, Baumman, H., Halliwell, B. and Lim, SK (2000) Haptoglobin reduces renal oxidative DNA and tissue damage during phenylhydrazine-induced hemolysis.Kidney Int.
  • Haptoglobin preserves the CD163 hemoglobin scavenger pathway by shielding hemoglobin from peroxidative modification. Blood 113, 2578.2586).
  • Acutephase protein haptoglobin is a cell migration factor involved in arterial restructuring.FASEB J. 16, 1123.1125., Lohr , NL, Warltier, DC, chilian, WM and Weihrauch, D. (2005) Haptoglobin expression and activity during coronary collateralization. Am. J. Physiol. Heart Circ. Physiol. 288, H1389. H1395).
  • the present inventors completed the present invention by confirming that the vascular endothelial progenitor cells transformed by introducing the haptoglobin gene have an activity of promoting angiogenesis.
  • Another object of the present invention is to provide a composition for promoting angiogenesis containing the transformed vascular endothelial progenitor cells as an active ingredient.
  • Another object of the present invention is to provide a method for promoting angiogenesis comprising administering the transformed vascular endothelial progenitor cells according to the present invention to a site in need of angiogenesis in mammals other than humans.
  • the present invention provides a vascular endothelial progenitor cell transformed with a recombinant vector comprising a haptoglobin gene encoding the amino acid sequence of SEQ ID NO: 1.
  • the haptoglobin gene may be composed of the nucleotide sequence represented by SEQ ID NO: 2.
  • the recombinant vector may be a pMSCV-Hp vector.
  • the present invention is a composition for promoting angiogenesis containing vascular endothelial progenitor cells transformed with a recombinant vector comprising a haptoglobin gene encoding the amino acid sequence of SEQ ID NO: 1 as an active ingredient To provide.
  • the composition may be a pharmaceutical composition capable of preventing or treating angiogenesis-related diseases.
  • the angiogenesis-related disease may be selected from the group consisting of diabetic ulcer, necrosis, ischemic disease, obstructive vascular disease, cardiovascular disease and ischemia.
  • the present invention also provides a method for promoting angiogenesis comprising administering the transformed vascular endothelial progenitor cells according to the present invention to a site in need of angiogenesis in a mammal other than human.
  • vascular endothelial progenitor cells transformed by introducing the haptoglobin gene according to the present invention promote vascular formation as vascular endothelial progenitor cells are differentiated into mature vascular endothelial cells by haptoglobin expressed in cells. Since the activity is excellent and the tube formation of the cells is also excellent, it can be usefully used for the treatment of diseases caused by the vascular formation.
  • FIG. 1 is a photograph of vascular endothelial progenitor cells isolated in one embodiment of the present invention, (A) by fluorescence microscopy observation that the adherent cells were labeled with DiI-acLDL and FITC-UEA lectin on day 14 of culture It is a photograph taken, (B) is the photograph which observed the form (cobblestone-like) of the cell which grew by cell culture under the microscope.
  • Figure 2a is a photograph showing the analysis of the expression of haptoglobin in a cell transformed with a recombinant vector comprising a haptoglobin prepared in one embodiment of the present invention, Western blot,
  • VEGF vascular endothelial specific markers
  • KDR vascular endothelial specific markers
  • Flt-1 vascular endothelial specific markers
  • vWF vascular endothelial specific markers
  • eNOS vascular endothelial specific markers
  • Figure 2d is a graph showing the results of analyzing the growth of cells transformed with the recombinant vector prepared in one embodiment of the present invention with an MTT assay (assay).
  • Figure 3a shows a photograph of the formation of the tube on Matrigel after transforming the cells using a recombinant vector containing haptoglobin and a vector without haptoglobin in an embodiment of the present invention
  • Figure 3b shows a photograph of fluorescence microscopy of the insertion of the transformed vascular endothelial progenitor cells into the tubular structure.
  • Figure 4a shows that after administering each vector used in one embodiment of the present invention mouse hindlimb ischaemia model, the degree of disease symptoms divided into three groups,
  • Figure 4b is a graph showing the number of mice according to the degree of disease symptoms after administering each vector to the mouse model
  • 4C and 4D show LDPI images of ischemic mouse models.
  • Figure 5a shows a section of muscle tissue obtained from the hind limbs of a mouse model after administration of a recombinant vector expressing haptoglobin, a vector without haptoglobin to ischemia tissue, with alkaline phosphatase. Staining is shown in comparison with the results of normal tissue,
  • 5B shows the staining of sections of muscle tissue with CD31 compared to the results of normal tissue.
  • the present invention is characterized by providing a vascular endothelial progenitor cell transformed with a recombinant vector comprising a haptoglobin gene.
  • Angiogenesis is a highly regulated process that occurs in response to various angiogenic factors such as hypoxia and low pH, as well as growth factors, cytokines and other physiological molecules (Folkman and Shing, J. Biol) Chem ., 267, 10931, 1992).
  • Angiogenesis mechanisms for the development of new blood vessels require the cooperation of various molecules that regulate the degradation and reconstitution, migration, proliferation, differentiation and tube (tube) formation of the extracellular matrix (ECM), and after angiogenesis initiation, Angiogenesis-promoting factors, including VEGF, bFGF, and PDGF, activate endothelial cells through stimulation of cell surface receptors, and activated cells proliferate, increase expression of cell adhesion molecules, increase secretion of proteolytic enzymes, and cell migration. And an increase in intrusion.
  • Angiogenesis-promoting factors including VEGF, bFGF, and PDGF, activate endothelial cells through stimulation of cell surface receptors, and activated cells proliferate, increase expression of cell adhesion molecules, increase secretion of proteolytic enzymes, and cell migration. And an increase in intrusion.
  • angiogenesis-dependent diseases such as cancer, rheumatoid arthritis, psoriasis, ulcers, ischemia, arteriosclerosis, myocardial infarction, angina pectoris and cerebrovascular diseases using factors that induce or inhibit angiogenesis.
  • Actively done (Folkman J., J. Nat. Med. , 1:27, 1995; Jackson JR, et al., FASEB J. , 11: 457, 1997; Risau W., Nature, 386: 671, 1997 Bussolino, F., et al., Trends Biochem. Sci. , 22: 251, 1997; Hanahan D., et al., Cell, 86: 353, 1996).
  • angiogenesis inhibitors such as thrombospondin-1, platelet factor-4, angiostatin, vascular endothelial growth factor, fibroblasts
  • Angiogenesis promoters such as basic fibroblast growth factor usually maintain quantitative equilibrium so that angiogenesis does not occur.However, in the case of wounds or cancers, inhibition of angiogenesis for regeneration of wounded tissues and cancer growth The quantitative equilibrium of factors and promoters is broken and new blood vessels are formed, at which time the overexpression of angiogenic promoters is involved.
  • the role of inducing or promoting new blood vessel formation is very important for reducing tissue damage caused by hypoxic or hypotrophic conditions caused by the formation of blood vessels, and smooth tissue regeneration.
  • angiogenesis must be involved in the necessary wound healing process for the rejuvenation of wounded tissue.
  • inflammatory reactions occur due to necrosis of the cells and destruction of blood vessels, which are accompanied by devascularization of blood components, activation of platelets and coagulation, and kallikrein, thrombin and plasmin. It goes through a series of processes in which biological mediators are formed.
  • angiogenesis treatment angiogenesis promoters such as vascular endothelial growth factor has already been used as a treatment for severe ischemia.
  • angiogenesis factors such as fibroblast growth factor, epidermal growth factor and platelet-derived endothelial growth factor have been studied for clinical treatment.
  • haptoglobin may be usefully used to treat diseases caused by the formation of blood vessels as described above. Especially, in the case of vascular endothelial progenitor cells into which haptoglobin is introduced, Very good first.
  • the present inventors found that in the case of endothelial progenitor cells transformed by haptoglobin introduced, cell differentiation was promoted.
  • endothelial progenitor cells and haptoglobin introduced with haptoglobin are introduced.
  • Microscopic observation of tube formation and tube network formation in untreated endothelial progenitor cells revealed that capillary-like tube formation ability was higher in haptoglobin-induced endothelial progenitor cells than control cells. (See FIG. 3A), the insertion into the network structure also increased (see FIG. 3B).
  • the haptoglobin gene may include a haptoglobin protein expressed by being introduced into the progenitor cells.
  • the “functional equivalent” includes an amino acid sequence variant in which part or all of the amino acid sequence of SEQ ID NO: 1 is substituted or a part of the amino acid is deleted or added, and has substantially the same physiological activity as the haptoglobin protein of SEQ ID NO: 1.
  • the “functional derivative” refers to a protein that has been modified to increase or decrease the physicochemical properties of the haptoglobin protein, and has a physiological activity substantially equivalent to that of the haptoglobin protein of SEQ ID NO: 1.
  • the method for introducing a haptoglobin gene into vascular endothelial progenitor cells in the present invention can be introduced into the cell using a recombinant vector comprising a promoter and a polynucleotide encoding haptoglobin operably linked thereto.
  • Polynucleotide encoding the haptoglobin in the present invention is characterized in that it comprises a nucleotide sequence encoding the haptoglobin protein or a functional equivalent thereof, the base sequence may include all DNA, cDNA and RNA sequences have. Preferably, it is represented by the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, and most preferably represented by the nucleotide sequence of SEQ ID NO: 2.
  • the haptoglobin protein may be prepared using DNA recombination technology, and methods known in the art may be used for this purpose.
  • haptoglobin proteins can be prepared by genetic engineering methods that recombinantly inject into bacterial, yeast and animal cell lines using haptoglobin genes.
  • the recombinant vector refers to a polynucleotide encoding haptoglobin, that is, an expression vector into which a haptoglobin gene is inserted, and the expression vector that can be used in the present invention is not limited thereto, and is known in the art. Plasmids, viruses or other mediators can be used. Preferably retroviral vectors can be used.
  • a recombinant expression vector was prepared using a retroviral vector of the haptoglobin gene of SEQ ID NO: 2, which was named 'pMSCV-Hp', and transfected into endothelial progenitor cells. Introduced.
  • vascular endothelial progenitor cells transformed by introducing haptoglobin according to the present invention are characterized by having an activity of promoting differentiation into mature vascular endothelial cells by expressing haptoglobin.
  • the present invention can provide a composition for promoting angiogenesis containing vascular endothelial progenitor cells transformed with a recombinant vector comprising a haptoglobin gene according to the present invention as an active ingredient.
  • the composition for promoting angiogenesis according to the present invention has the effect of preventing or treating a disease associated with angiogenesis insufficiency
  • the present invention provides a transformed vascular endothelial progenitor cells by introducing the haptoglobin gene according to the present invention It is possible to provide a composition for preventing or treating angiogenesis-related diseases containing as an active ingredient.
  • haptoglobin is not introduced.
  • the group receiving endothelial progenitor cells with haptoclobin showed a significantly lower number of mice with severe leg loss than the control group. Blood vessel density was also shown to increase (see FIGS. 4B and 4C).
  • the present inventors have found that the haptoglobin according to the present invention can be introduced and transformed vascular endothelial progenitor cells can prevent or treat ischemic injury through the activity of promoting angiogenesis. .
  • angiogenesis-related diseases that can be prevented or treated by the composition for promoting angiogenesis according to the present invention, but are not limited thereto, diabetic ulcer, necrosis, ischemic disease, obstructive vascular disease, cardiovascular disease and It may be selected from the group consisting of ischemia.
  • composition for promoting angiogenesis or the composition for preventing or treating angiogenesis-related diseases is a vascular endothelial precursor transformed with a recombinant vector containing a pharmaceutically effective amount of a haptoglobin gene as a bioactive component. It may comprise cells alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents.
  • the term “pharmaceutically effective amount” refers to an amount sufficient to allow the physiologically active ingredient to be administered to an animal or human to exhibit the desired physiological or pharmacological activity. However, the pharmaceutically effective amount may be appropriately changed depending on the age, weight, health condition, sex, route of administration and duration of treatment.
  • pharmaceutically acceptable refers to a physiologically acceptable and, when administered to a human, usually does not cause gastrointestinal disorders, allergic reactions such as dizziness or the like.
  • carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.
  • compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal, and can be formulated for various oral or parenteral administration. It may be formulated in a form.
  • the tablets may include binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally starch, agar, alginic acid or its Disintegrants such as sodium salts, absorbents, colorants, flavors and / or sweeteners may be further included.
  • the formulations may be prepared by conventional mixing, granulating or coating methods.
  • compositions of the present invention may further include auxiliaries such as preservatives, hydrating agents, emulsifiers, salts or buffers for controlling osmotic pressure and other therapeutically useful substances, and may be formulated according to conventional methods.
  • auxiliaries such as preservatives, hydrating agents, emulsifiers, salts or buffers for controlling osmotic pressure and other therapeutically useful substances, and may be formulated according to conventional methods.
  • composition according to the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular, the dosage of the active ingredient being determined by several factors such as the route of administration, the age, sex, weight and severity of the patient. It may be appropriately selected depending on.
  • the composition of the present invention can be administered in parallel with known compounds that can enhance the desired effect.
  • Routes of administration of the compositions according to the invention can be administered to humans and animals orally or parenterally, such as intravenously, subcutaneously, intranasally or intraperitoneally. Oral administration also includes sublingual application. Parenteral administration includes injection and drip methods such as subcutaneous injection, intramuscular injection and intravenous injection.
  • the total effective amount of vascular endothelial progenitor cells transformed with a recombinant vector comprising a haptoglobin gene as a physiologically active component can be administered to a patient in a single dose, and multiple doses Multiple doses may be administered by a fractionated treatment protocol with long term administration.
  • the composition of the present invention may vary the content of the active ingredient according to the degree of disease, but can be repeatedly administered several times a day at an effective dose of 100 ⁇ g to 3,000 mg once administered based on an adult.
  • the effective dose of the bioactive component may be determined in consideration of various factors such as the age, weight, health condition, sex, severity of the disease, diet and excretion rate, as well as the route and frequency of treatment of the drug. have. Therefore, in view of the above, one of ordinary skill in the art can determine an appropriate effective dosage according to the specific use of the bioactive component as a therapeutic agent for the treatment or prevention of angiogenesis-related diseases, and the present invention.
  • the composition according to the present invention is not particularly limited to its formulation, route of administration and method of administration as long as the effect of the present invention is exhibited.
  • Umbilical cord blood and umbilical cord were obtained from donors who received written consent from Gangnam St. Mary's Hospital (Korea). Histopaque-10771 (Sigma, St. Louis, Mo.) was used in accordance with the instructions to separate mononuclear cells from human cord blood by density gradient centrifugation. The cells were dispensed in 6-well plates coated with 0.1 mg / ml of human fibronectin (Sigma) and 5% fetal bovine serum (FBS; Gibco Life Technology, Gaithersburg, MD ) Was added to EGM-2 BulletKit medium (Clonetics, San Diego, Calif.). After 3 days non-adherent cells were removed and the medium changed. To obtain late EPCs, adherent cells were incubated for 25-45 days, changing to fresh medium every 3 days.
  • human umbilical vein endothelial cells were isolated from cord veins and cultured in the same culture as described above (Jaffe, EA, Nachman, RL, Becker, CG and Minick, CR (1973) Culture of human endothelial cells derived from unbilical veins: identification by morphologic and immunologic criteria.J Clin. Invest. 52, 2745.2756).
  • the cells were treated with 2.5 ⁇ g / ml of 1,1′-dioctadecyl-3,3,3 ′, 3′-tetramethylindocarbosi.
  • Non-perchlorate (1,1′-dioctadecyl-3,3,3 ′, 3′-tetramethylindocarbocyanine-perchlorate) -labeled acetylated low-density lipoprotein (DiI-acLDL; Molecular Probes, Eugene, OR) and incubated for 1 hour at 37 °C and fixed for 10 minutes with 1% paraformaldehyde (paraformaldehyde). The cells were again incubated with 10 ⁇ g / ml fluorescein-isothiocyanate-bound UEA lectin (Ulex europaeus agglutinin lectin) (Sigma) for 1 hour. The double labeled cells were then observed by fluorescence microscopy.
  • MSCV-Hp recombinant plasmid
  • cDNA of the human Hp 2 gene Kim, IS, Lee, IH, Lee, JH and Lee, SY (2001) Induction of haptoglobin by alltrans retinoic acid in THP-1 human monocytic cell line. Biochem. Biophys . Res. Commun. 284, 738.742.
  • GFP green fluorescent protein
  • 293 T cells were transfected with MSCVHp plasmid using FuGene 6 sample (Roche Applied Science, Indianapolis, IN).
  • virus containing supernatants were collected and passed through a 0.45 ⁇ m syringe filter (Pall Corporation, East Hills, NY). Viral supernatants were added to EPC medium in the presence of 5 ⁇ g / ml protamine sulphate (Sigma). 72 hours later GFP-positive cells were collected using a flow cytometer (FACS Vantage SE; BD Biosciences, San Diego, Calif.).
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • RT-PCR was performed by RT-PCR (MX- Quantitative real-time RT-PCR using 3000P; Stratagene) was performed using a FullVelocity SYBR Green QPCR master mix (Stratagene, La Jolla, Calif.).
  • EPCs were shown to be positive for DiI-acLDL uptake and UEA-lectin binding, which indicates that the cells isolated in ⁇ 1-1> have the characteristics of EPCs. give.
  • EPCs after 25-40 days after cell division, EPCs showed a gravel-like appearance.
  • EPCs are known as heterogeneous cell populations, and according to Hur et al., The shape of EPCs over time is spindle-shaped early EPCs with low proliferative capacity and gravel type with high expansion capacity.
  • the present inventors have found that the haptoglobin of the present invention has an activity for promoting the differentiation of endothelial progenitor cells, whereas haptoglobin does not affect the proliferation of endothelial progenitor cells.
  • haptoglobin does not affect the proliferation of endothelial progenitor cells.
  • GFP-labeled vector- (control) or Hp-DNA-transformed EPCs (2 ⁇ 10 4 cells / well) were laid on the Matrigel and incubated for 20 hours in EGM-2 medium containing 3% FBS.
  • EPCs were inserted into the vasculature, EPCs (1 ⁇ 10 4 cells) and DiI-acLDL-labeled HUVECs (2 ⁇ 10 4 cells) were laid together on Matrigel and 20 in M199 medium containing 3% FBS. Co-culture for hours. The formation of tubular networks was then observed by fluorescence microscopy.
  • Hp-expressing EPCs showed higher capillary-like tubes than those in the control group (see FIG. 3A).
  • GFP-containing EPCs were co-cultured with DiI-AcLDL-labeled HUVECs in Matrigel, resulting in HUVECs (red) of EPCs (green) in Hp-EPCs.
  • the insertion into the network structure was increased, and the network structure of the tube in HUVECs was found to be better formed in the presence of Hp-introduced EPCs compared to the control (see FIG. 3b).
  • haptoglobin has an activity of promoting angiogenesis of endothelial progenitor cells.
  • femoral blood flow was measured using a laser Doppler blood perfusion imager (LDPI; Perimed PeriScan PIM III, Jarfalla, Sweden). After three scans, the LDPI index was determined as the ratio of ischemia to non-ischemic femoral blood perfusion values.
  • LDPI laser Doppler blood perfusion imager
  • tissue sections cut and cryostat (Leica CM1800; Wetzlar, Germany) were prepared and stained using fast BCIP / NBT solution (Sigma) for alkaline phosphatase measurement.
  • the tissue sections were also prepared using monoclonal rat anti-mouse CD31 antibody (Abcam, Cambridge, UK) and Alexa-Fluor-555-labeled secondary antibody (Invitrogen, Carlsbad, CA). Immunohistostaining was performed.
  • Hp-modified EPCs were locally implanted in the hind limbs after induction of ischemia in nude mice.
  • limb salvage mild loss of limb
  • severe loss of limb as shown in FIG. 4A.
  • the Hp-EPCs transplanted mouse group showed an increase in the number of bridged mice and a decrease in the number of severely lost mice.
  • the measurement of the ratio of blood perfusion values showed that the LDPI index was higher in the Hp-EPC group (0.68 ⁇ 0.22) than the control EPC group (0.44 ⁇ 0.19) or the PBS group (0.35 ⁇ 0.08) (see FIGS. 4B and 4C). ).
  • the present inventors measured capillary density by staining alkaline phosphatase and CD31 in tissue sections to measure neovascularization in ischemia hind limbs.
  • the present inventors have found that in the case of vascular endothelial progenitor cells transformed with haptoglobin, pro-angiogenic factors are increased and the activity of EPCs is increased to form neovascularization. It has been found that it can ultimately be used to prevent or treat angiogenesis-related diseases.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Vascular Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Toxicology (AREA)
  • Epidemiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Developmental Biology & Embryology (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Cardiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des cellules progénitrices endothéliales vasculaires introduites avec un gène d'haptoglobine et une composition contenant celui-ci pour favoriser l'angiogenèse, et plus spécifiquement, des cellules progénitrices endothéliales vasculaires transformées par un vecteur recombinant contenant un gène haptoglobine, une composition pour favoriser l'angiogenèse, contenant celui-ci en tant que principe actif, et un procédé pour favoriser l'angiogenèse. Les cellules progénitrices endothéliales vasculaires transformées par l'introduction d'un gène haptoglobine selon la présente invention sont caractérisées en ce que la différenciation en cellules endothéliales vasculaires matures par l'haptoglobine exprimée dans les cellules est favorisée, une activité pour favoriser l'angiogenèse est excellente et la vasculogenèse est aussi excellente, l'invention pouvant ainsi être utile pour traiter des maladies provoquées par la préformation de vaisseaux sanguins.
PCT/KR2010/006006 2010-03-11 2010-09-03 Cellules progénitrices endothéliales vasculaires introduites avec le gène d'haptoglobine et composition contenant celui-ci pour favoriser l'angiogenèse WO2011111914A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2010-0021998 2010-03-11
KR1020100021998A KR20110102788A (ko) 2010-03-11 2010-03-11 합토글로빈 유전자가 도입된 혈관내피 전구세포 및 이를 포함하는 혈관형성 촉진용 조성물

Publications (1)

Publication Number Publication Date
WO2011111914A1 true WO2011111914A1 (fr) 2011-09-15

Family

ID=44563692

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2010/006006 WO2011111914A1 (fr) 2010-03-11 2010-09-03 Cellules progénitrices endothéliales vasculaires introduites avec le gène d'haptoglobine et composition contenant celui-ci pour favoriser l'angiogenèse

Country Status (2)

Country Link
KR (1) KR20110102788A (fr)
WO (1) WO2011111914A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101438996B1 (ko) * 2013-01-15 2014-09-05 가톨릭대학교 산학협력단 혈관형성 촉진 활성을 갖는 돌연변이형 합토글로빈의 용도
KR102593791B1 (ko) 2021-02-26 2023-10-25 경희대학교 산학협력단 Pdgf-bb를 이용한 혈관 형성능이 증진된 골수 유래 혈관내피전구세포의 제조방법 및 이를 이용한 혈관형성 부전-관련 질환 치료용 세포치료제의 제조방법
KR20220143379A (ko) 2021-04-16 2022-10-25 경희대학교 산학협력단 중간엽 줄기세포 및 혈관내피전구세포를 유효성분으로 포함하는 폐쇄성 혈관질환 또는 이의 합병증 예방 또는 치료용 조성물

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
PARK, S. ET AL., S-5 POSTER OF 19TH FAOBMB SEOUL CONFERENCE, 27 May 2007 (2007-05-27) *
PARK. S. ET AL., FEBS LETTER, vol. 583, 2009, pages 3235 - 3240 *
ROUHL, R. ET AL., CURRENT NEUROVASCULAR RESEARCH, vol. 6, 2009, pages 32 - 41 *
VILLEGAS, A. ET AL., BIOLOGICS: TARGETS & THERAPY, vol. 2, no. 3, 2008, pages 397 - 407 *

Also Published As

Publication number Publication date
KR20110102788A (ko) 2011-09-19

Similar Documents

Publication Publication Date Title
WO2017116205A1 (fr) Conjugué persistant d'un triple activateur activant le récepteur du glucagon, du glp-1 et du gip
WO2016159721A1 (fr) Procédé de production en masse de protéines dans des cellules souches mésenchymateuses
WO2017086627A1 (fr) Procédé de prolongation de la demi-vie d'une protéine
WO2011142514A1 (fr) Composition contenant du pias3 comme ingrédient actif pour la prévention ou le traitement d'un cancer ou d'une maladie immune
WO2020050667A1 (fr) Récepteur antigénique chimérique pour un cancer solide et lymphocytes t exprimant ce récepteur chimérique de l'antigène
WO2020251181A1 (fr) Composition pour favoriser la production d'exosomes dérivés de cellules souches et renforcer le caractère-souche
WO2021145552A1 (fr) Utilisation thérapeutique pour les maladies hépathiques, d'un triple agoniste ayant une activité par rapport à tous les récepteurs du glucagon, du glp-1 et du gip contre les maladies pulmonaires
WO2018208011A2 (fr) PEPTIDE BIOCOMPATIBLE SUPPRIMANT L'AGGRÉGATION DE LA PROTÉINE β-AMYLOÏDE
WO2011111914A1 (fr) Cellules progénitrices endothéliales vasculaires introduites avec le gène d'haptoglobine et composition contenant celui-ci pour favoriser l'angiogenèse
WO2020130749A1 (fr) Composition pharmaceutique comprenant de l'insuline et un triple agoniste ayant une activité par rapport à l'ensemble des récepteurs glp1, glucagon et gip
WO2022025559A1 (fr) Composition comprenant des exosomes dérivés de cellules souches et sa méthode de production
WO2019132547A1 (fr) Composition pharmaceutique permettant de prévenir ou de traiter la métastase du cancer vers le poumon, contenant un inhibiteur de chi3l1 à titre de principe actif
WO2018026212A2 (fr) Procédé de production d'un modèle de fibrose, et utilisation d'un modèle de fibrose
WO2022139493A1 (fr) NOUVEAU PEPTIDE POUVANT INHIBER LA SIGNALISATION DU TGF-β ET SON UTILISATION
WO2016178511A1 (fr) Composition comprenant un polypeptide kal1 ou un gène codant celui-ci pour l'inhibition de l'angiogenèse, et son utilisation
WO2022015115A1 (fr) Utilisation thérapeutique d'une combinaison contenant un conjugué à action prolongée triplement agoniste ou un triple agoniste
WO2017010790A1 (fr) Composition pour l'inhibition de l'angiogenèse contenant un complexe de nanoparticule-protéine à base de corps vitré en tant que substance active, et utilisation de celle-ci
WO2020263063A1 (fr) Utilisation thérapeutique, pour les maladies hépathiques, d'un triple agoniste ayant une activité par rapport à tous les récepteurs du glucagon, de glp-1 et de gip, ou conjugué de ceux-ci
WO2022015082A1 (fr) Utilisation thérapeutique d'un dérivé du glucagon ou d'un conjugué de celui-ci pour une maladie hépatique
WO2018236194A1 (fr) Composition pour la prévention ou le traitement de la fibrose, comprenant la protéine gas6 ou un activateur du récepteur de celle-ci
WO2021006511A1 (fr) Composition de prévention ou de traitement d'un trouble d'hypopigmentation
WO2020214013A1 (fr) Utilisation thérapeutique, pour l'hyperlipidémie, d'un triple agoniste ayant une activité par rapport à tous les récepteurs du glucagon, glp -1 et gip, ou conjugué de ceux-ci
WO2021015584A1 (fr) Procédé pour la préparation d'une lignée de cellules souches immortalisée et son utilisation
WO2009145488A2 (fr) Protéine xbp1(s) agissant en tant que marqueur de différentiation d'adipocyte qui présente un moyen pour réguler une différentiation dans des adipocytes et application correspondante
WO2023059160A1 (fr) Protéine de fusion pour prévenir et traiter la fibrose

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10847548

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10847548

Country of ref document: EP

Kind code of ref document: A1