KR20110102788A - Haptoglobin gene introduced endothelial progenitor cells and compositions for promoting angiogenesis comprising the same - Google Patents

Abstract

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. The vascular endothelial progenitor cells transformed by introducing the haptoglobin gene according to the present invention have the effect of promoting differentiation into mature vascular endothelial cells by haptoglobin expressed in the cell, and promoting vascular formation. It is excellent and can be usefully used for the treatment of diseases caused by the formation of blood vessels because of the excellent characteristics of the tube formation of the cells.

Description

Haptoglobin gene introduced endothelial progenitor cells and compositions for promoting angiogenesis comprising the same

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.

In the case of ischemic tissue injury, new blood vessels must be formed at the damaged site in order for the tissue to recover. After birth, new blood vessels are produced by angiogenesis, arteriogenesis, and vasculogenesis. Angiogenesis processes are associated with the proliferation and migration of vascular endothelial cells that grow from already existing mature endothelial cells, and angiogenesis correlates the already existing arterial connections to collateral vessels. (Carmeliet, P. (2000) Mechanisms of angiogenesis and arteriogenesis. Nat. Med . 6, 389.395). Angiogenesis, on the other hand, proceeds through the differentiation of endothelial progenitor cells (EPCs) into mature vascular endothelial cells (Asahara, T. and Kawamoto, A. (2004) Endothelial progenitor cells for postnatal vasculogenesis. Am. J. Physiol. Cell Physiol . 287, 572.579.). 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. 45, 530.544, Urbich, C., Aicher, A., Heeschen, C., Dernbach, E., Hofmann , WK, Zeiher, AM and Dimmeler , S. (2005) Soluble factors released by endothelial progenitor cells promote migration of endothelial cells and cardiac resident progenitor cells.. J. Mol. Cell. Cardiol. 39, 733.742).

Therefore, EPCs are attracting attention as a potential target for therapeutic angiogenesis through the material (revascularization) (Jujo, K., Ii, M. and Losordo, DW (2008) Endothelial progenitor cells in neovascularization of infarcted myocardium. J. Mol .. Cell. Cardiol. 45, 530.544).

In addition, recent studies have focused on ex vivo gene-modified EPCs to increase cell function, vascular endothelial growth factor (VEGF) and hypoxia inducer-1α (hypoxia). -inducible factor-1α) 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. (2002) Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration.Circulation 105, 732.738., Jiang, M., Wang, B., Wang, C., He, B., Fan, H., Guo, TB, Shao, Q., Gao, L. and Liu, Y. (2008) Angiogenesis by transplantation of HIF-1a modified EPCs into ischemic limbs. J. Cell . Biochem . 103, 321.334.).

Haptoglobin (Hp), on the other hand, 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 . 58, 1033.1044., Buehler, PW, Abraham, B., Vallelian, F., Linnemayr, C., Pereira, CP, Cipollo, JF, Jia, Y., Mikolajczyk, M., Boretti, FS, Schoedon, G. , Alayash, AI and Schaer, DJ (2009) Haptoglobin preserves the CD163 hemoglobin scavenger pathway by shielding hemoglobin from peroxidative modification. Blood 113, 2578.2586).

Hp can also be expressed in arteries (Smeets, MB, Pasterkamp, G., Lim, SK, Velema, E., van Middelaar, B. and de Kleijn, DPV (2002) Nitric oxide synthesis is involved in arterial haptoglobin expression after sustained flow changes.FEBS Lett. 529, 221.224.), known to act as cell migration factors associated with arterial restructuring (de Kleijn, DPV, Smeets, MB, Kemmeren, PPCW, Lim, SK) , van Middelaar, BJ, Velema, E., Schoneveld, A., Pasterkamp, G. and Borst, C. (2002) 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).

However, there have been no reports on the induction of neovascularization using vascular endothelial progenitor cells introduced with haptoglobin.

Accordingly, 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.

It is therefore an object of the present invention to provide a vascular endothelial progenitor cell transformed with a recombinant vector comprising a haptoglobin gene encoding the amino acid sequence of SEQ ID NO: 1.

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.

In order to achieve the above object of the present invention, 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.

In one embodiment of the present invention, the haptoglobin gene may be composed of the nucleotide sequence represented by SEQ ID NO: 2.

In one embodiment of the present invention, the recombinant vector may be a pMSCV-Hp vector.

In one embodiment of the present invention, the transformed vascular endothelial progenitor cells may have activity that promotes angiogenesis by promoting differentiation into mature vascular endothelial cells by expressed haptoglobin.

In addition, 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.

In one embodiment of the invention, the composition may be a pharmaceutical composition capable of preventing or treating angiogenesis-related diseases.

In one embodiment of the present invention, 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.

The 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 vascular dysplasia.

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,
2b and 2c shows haptoglobin and vascular endothelial specific markers (VEGF, KDR, Flt-1, vWF, VE-cadherin, eNOS, in cells transformed with the recombinant vector prepared in one embodiment of the present invention). GAPDH) expression is shown respectively by RT-PCR analysis results,
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. In addition, many molecules, including members of the integrin, selectin and immunoglobulin gene superfamily for cell attachment, as well as proteolytic enzymes such as matrix metalloproteases and serine proteases to degrade ECM, promote proliferation and invasion ( Brooks, Eur. J. Cancer, 32A, 2423, 1996), and furthermore, lumen formation and differentiation into mature blood vessels by signal transduction mechanisms derived from receptors on the cell surface that interact with ECM components and soluble factors. Induction, angiogenesis is formed.

Recently, there have been attempts to treat 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).

In normal adults, the replacement time of endothelial cells that make up blood vessels generally varies from 47 days to 20,000 days and is under very strict control. In general, 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.

Therefore, excessive blood vessel formation may be a major cause of disease exacerbation, but the formation of blood vessels is also a cause of serious disease. Angiogenesis is an essential phenomenon for wound healing or tissue regeneration. For example, placenta with poorly formed blood vessels is an important cause of miscarriage, and necrosis, ulceration and ischemia due to the formation of blood vessels can lead to tissue or organ damage. It may cause dysfunction or death. In addition, diseases such as arteriosclerosis, myocardial infarction and angina are caused by poor blood supply.

Therefore, 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.

In particular, angiogenesis must be involved in the necessary wound healing process for the rejuvenation of wounded tissue. In the early stages of the wound, 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.

In addition, the treatment of biological diseases using angiogenesis is also called angiogenesis treatment, angiogenesis promoters such as vascular endothelial growth factor has already been used as a treatment for severe ischemia. In addition, angiogenesis factors such as fibroblast growth factor, epidermal growth factor and platelet-derived endothelial growth factor have been studied for clinical treatment.

Meanwhile, in the present invention, 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.

According to one embodiment of the present invention, in order to investigate whether haptoglobin affects the differentiation of endothelial progenitor cells, after separating endothelial progenitor cells from umbilical cord blood, recombination is made so that haptoglobin can be expressed in cells. After transforming the endothelial progenitor cells using a vector and comparing the cell differentiation with endothelial progenitor cells without haptoglobin, the endothelial progenitor cells transformed with haptoglobin were compared with control cells. MRNA expression of endothelial markers VEGF, KDR, Flt-1, vWF, VE-cadherin, and eNOS was increased (see FIGS. 2B and 2C), whereas haptoglobin was induced by the introduction of haptoglobin. The growth of overexpressed endothelial progenitor cells was not significantly different from the control group (see FIG. 2D).

Therefore, the present inventors found that in the case of endothelial progenitor cells transformed by haptoglobin introduced, cell differentiation was promoted.

In addition, according to another embodiment of the present invention, in order to investigate whether haptoglobin has activity to promote angiogenesis by endothelial progenitor cells, 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).

Based on the above results, the present inventors found that the transformed vascular endothelial progenitor cells into which the haptoglobin of the present invention was introduced can promote the formation of neovascularization due to the excellent tube forming and insertion effect into the vascular structure. .

Therefore, the present invention can provide a vascular endothelial progenitor cell transformed with a recombinant vector comprising a haptoglobin gene, and the haptoglobin may be a gene encoding the amino acid sequence of SEQ ID NO: 1.

Therefore, in the case of vascular endothelial progenitor cells introduced with the haptoglobin gene according to the present invention, the haptoglobin gene may include a haptoglobin protein expressed by being introduced into the progenitor cells.

The haptoglobin protein may include a haptoglobin protein having an amino acid sequence represented by SEQ ID NO: 1 or a protein having a substantially equivalent physiological activity, and a protein having a substantially equivalent physiological activity may include a sum of SEQ ID NO: 1 Toglobin proteins and their functional equivalents and functional derivatives.

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. Say.

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.

In addition, 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.

In addition, in the present invention, the haptoglobin protein may be prepared using DNA recombination technology, and methods known in the art may be used for this purpose. Specifically, haptoglobin proteins can be prepared by genetic engineering methods that recombinantly inject into bacterial, yeast and animal cell lines using haptoglobin genes.

In the present invention, 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.

The recombinant vector according to the present invention can be introduced into cells using methods known in the art. For example, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran- DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene guns and other known methods for introducing nucleic acids into cells By the method (Wu et al., J. Bio. Chem., 267: 963-967, 1992; Wu and Wu, J. Bio. Chem., 263: 14621-14624, 1988).

In one embodiment of the present invention, 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.

The 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.

In addition, the present invention provides a method for promoting angiogenesis comprising administering vascular endothelial progenitor cells introduced with the haptoglobin gene according to the present invention to a site in need of angiogenesis in mammals other than humans. Can be.

Furthermore, 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.

As described above, vascular endothelial progenitor cells transformed by introducing haptoglobin according to the present invention not only have an excellent activity of promoting differentiation into vascular endothelial cells, but also form tube and endothelial cells into vascular structures. It is characterized by the formation of neovascularization through the action of increasing insertion.

Therefore, 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.

According to one embodiment of the present invention, after administering endothelial progenitor cells into which haptoglobin is introduced in an animal model in which ischemia occurs due to angiogenesis, haptoglobin is not introduced. In comparison with the control group treated with untreated endothelial progenitor cells and the degree of ischemia symptoms, 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).

Based on the above results, 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. .

In addition, 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.

The composition for promoting angiogenesis or the composition for preventing or treating angiogenesis-related diseases according to the present invention 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.

As used herein, 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.

In addition, as used herein, "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. Examples of such 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. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

In addition, the 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.

Representative of parenteral formulations are injectable formulations, preferably aqueous isotonic solutions or suspensions. Injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving in saline or buffer. In addition, oral dosage forms include, for example, intake tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers, and these formulations may contain, in addition to the active ingredients, diluents (e.g., lactose, dextrose). , Sucrose, mannitol, sorbitol, cellulose and / or glycine) and a suspending agent such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols. The tablets may comprise 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.

In addition, the 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.

The 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. In addition, 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.

In the composition of the present invention, 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 μg to 3,000 mg once administered based on an adult. However, 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.

Hereinafter, the present invention will be described in detail by way of examples. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.

< Example  1>

Haptoglobin (Hp) EPC  Impact on Differentiation

<1-1> Cell Culture

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.

In addition, human umbilical vein endothelial cells (HUVECs) 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).

<1-2> Acetylation  Low density lipoprotein ( acetylated low - density lipoprotein ) Absorption and Ulex  europaeus lectin  Combination

After culturing the cells prepared by the method of <1-1> for 14 days, the cells were treated with 2.5 μg / 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 ℃ and fixed for 10 minutes with 1% paraformaldehyde (paraformaldehyde). The cells were again incubated with 10 μg / ml fluorescein-isothiocyanate-bound UEA lectin (Ulex europaeus agglutinin lectin) (Sigma) for 1 hour. The double labeled cells were then observed by fluorescence microscopy.

<1-3> person Hp  Gene Of EPCs  transform( modification )

Humans show allelic polymorphism for Hp. According to the two major alleles Hp ¹ and Hp ² , Hp is Hp 1-1 ( Hp ¹ / Hp ¹ ), Hp 2-1 ( Hp ¹ / Hp ² ) and Hp 2-2 ( Hp ² / Hp ² ) it is expressed by the three major traits (Maeda, N., Yang, F., Barnett, DR, Bowman, BH and Smithies, O. (1984) Duplication within the haptoglobin gene Hp2. Nature 309, 131.135.). Hp 2-2 has a larger angiogenesis activity compared to Hp 1-1, and (Cid, et al. (1993 ) Identification of haptoglobin as an angiogenic factor in sera from patients with systemic vasculitis. J. Clin. Invest. 91, 977.985.) Therefore, in the present invention, the human Hp ² gene was transduced and the effect of the expressed Hp 2-2 on the EPC function was investigated.

To this end, the preparation of the recombinant plasmid (MSCV-Hp) was carried out by 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.) were sub-cloned to the green fluorescent protein (GFP) of MSCVneoEB retroviral vector containing the gene EcoRI and XhoI site. 293 T cells were transfected with MSCVHp plasmid using FuGene 6 sample (Roche Applied Science, Indianapolis, IN). 48, 60 and 72 hours after transfection, virus containing supernatants were collected and passed through a 0.45 μm syringe filter (Pall Corporation, East Hills, NY). Viral supernatants were added to EPC medium in the presence of 5 μg / ml protamine sulphate (Sigma). 72 hours later GFP-positive cells were collected using a flow cytometer (FACS Vantage SE; BD Biosciences, San Diego, Calif.).

<1-4> RT -PCR ( reverse transcriptase - 중합체 chain reaction )

Using the primers of Table 1, conventional methods (Lee, MY, Kim, SY, Choi, JS, Lee, IH, Choi, YS, Jin, JY, Park, SJ, Sung, KW, Chun, MH and Kim) , IS (2002) Upregulation of haptoglobin in reactive astrocytes after transient forebrain ischemia in rats. J. Cereb. Blood Flow Metab . 22, 1176.1180. RT-PCR was performed according to RT-PCR, and RT-PCR was run on a real-time PCR machine (MX-3000P; Stratagene) to perform quantitative real-time RT-PCR FullVelocity SYBR Green QPCR master mix (Stratagene, La Jolla). , CA).

 Primers used for RT-PCR Name of the primer Primer sequence SEQ ID NO: Hp F  5'-ATGAGTGCCCTGGGAGCTGTCATT-3 ' 3 Hp R  5'-GCATTAGTTCTCAGCTATGGTCTT-3 ' 4 VEGF F  5'-AGGAGGGCAGAATCATCACG-3 ' 5 VEGF R  5'-CAAGGCCCACAGGGATTTTCT-3 ' 6 KDR F  5'-CTGGCATGGTCTTCTGTG-3 ' 7 KDR R  5'-AATGGGATTGGTAAGGATG-3 ' 8 Flt-1 F  5'-AGCAAGTGGGAGTTTGC-3 ' 9 Flt-1 R  5'-AGGTCCCGATGAATGC-3 ' 10 vWF F  5'-GAGGCTGAGTTTGAAGTGC-3 ' 11 vWF R  5'-CTGCTCCAGCTCATCCAC-3 ' 12 VE-cadherin F  5'-AAGACATCAATGACAACTTCC-3 ' 13 VE-cadherin R  5'-CCTCCACAGTCAGGTTATACC-3 ' 14 eNOS F  5'-AAGACATTTTCGGGCTCAC-3 ' 15 eNOS R  5'-GGCACTTTAGTAGTTCTCC-3 ' 16 GAPDH F  5'-ACCACAGTCCATGCCATCAC-3 ' 17 GAPDH R  5'-TCCACCACCCTGTTGCTGTA -3 ' 18

<1-5> Statistics Processing

Student's t-test and one-way analysis of variance were used to analyze the differences between the values obtained in experimental and control conditions. P <0.05 was considered significant.

<1-6> EPC  On eruption Haptoglobin  Impact Analysis

In order to identify the isolated cells as typical characteristics of EPCs, the cells cultured under the conditions of <1-1> were cultured on day 14 of the cultivation, according to the method of <1-2>, according to the method of <I-2>, -Lectin binding was detected.

As a result, as shown in FIG. 1A, the cells 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. In addition, as shown in Figure 1b, after 25-40 days after cell division, EPCs showed a gravel-like appearance. On the other hand, in general, 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. There are two types of cobblestone-shaped late EPCs (Hur, J., Yoon, CH, Kim, HS, Choi, JH, Kang, HJ, Hwang, KK, Oh, BH, Lee, MM). and Park, YB (2004) Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis.Arterioscler.Thromb. Vasc. Biol. 24, 288.293.), according to the present invention, the results of the cells as shown in Figure 1b The shape shows a gravel shape, which can be expected to be rapidly differentiated into mature endothelial cells in the form of cells with high expansion capacity.

In addition, looking at the results of FIG. 2, in the case of EPC cells into which the human haptoglobin gene was introduced, it was confirmed that haptoglobin (Hp) protein was expressed in the cells and secreted into the culture medium (see FIG. 2A). ), Hp-expressing EPC cells showed increased mRNA expression of various endothelial markers, ie, VEGF, KDR, Flt-1, vWF, VE-cadherin, and eNOS compared to controls (FIGS. 2B and 2C). Reference). On the other hand, when Hp was introduced into EPC cells, overexpression of Hp did not appear to significantly affect the growth of EPC (see FIG. 2D).

Therefore, 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. Could know.

< Example  2>

Haptoglobin EPC Of effects on neovascularization of rats

Experiments not described in the following examples, such as cell culture, EPC cell modification, and statistical treatment, were performed in the same manner as in Example 1, and the effect of haptoglobin on EPC neovascularization on haptoglobin on Matrigel was examined. Was observed by performing a method of observing the degree of in vitro tube formation in vitro. For this method, a cold Matrigel (BD Biosciences, San Jose, Calif.) Was first used in a 48-well plate well. In each, and polymerized at 37 degreeC for 30 minutes. 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. To determine if 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.

As a result, when EPCs were cultured in Matrigel with reduced growth factors, Hp-expressing EPCs showed higher capillary-like tubes than those in the control group (see FIG. 3A). In addition, to measure the insertion of EPCs into endothelial vasculature, 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).

Therefore, the present inventors found out that haptoglobin has an activity of promoting angiogenesis of endothelial progenitor cells.

< Example  3>

Haptoglobin EPC Induced angiogenesis ( EPC - induced neovascularization ) Impact analysis

Experiments not described in the examples below, such as cell culture, EPC cell modification, statistical treatment, were performed by the methods described in Examples 1 and 2 above.

<3-1> Mouse hindlimb ischaemia

All animal experiments were conducted with the approval of the Catholic University Ethics Committee. 18-22 g male athymic nude mice (6 weeks old) were used. Zoletile 50  (10 mg / kg; Vibac, Carros, France) and the right femoral artery (right proximal femoral artery) and distal saphenous artery were joined with 6.0 silk suture (Ethicon, Somerville, NJ), and The femoral artery and attached lateral branches were excised. The left hindlimb was left untouched and used as a non-ischaemic area. 24 hours after induction of hind limb ischemia, the mice were optionally divided into three groups (1, 2, and 3) and 5 × 10 ⁵ carrier vector-EPCs and Hp-EPCs cells per mouse were group 1 (n = 8). ) And 2 (n = 8) mice were injected locally at two different points of the femoral muscle with ischemia. Group 3 (n = 4) mice were injected with the same volume of PBS. Four weeks after EPC implantation, 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.

<3-2> Blood vessel density measurement

After 4 weeks of ischemia, ischemia and non-ischemic leg muscles were removed from mice and placed in Tissue-Tek (Sakura Finetek Europe, Zoeterwoude, The Netherlands). Frozen 5 μm-thick 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.

<3-3> mouse hindlimb ischaemia  In the model Hp of EPC -Induced angiogenesis increased activity

To investigate the effect of haptoglobin of the present invention on neovascularization in vivo, Hp-modified EPCs were locally implanted in the hind limbs after induction of ischemia in nude mice. As a result, we classified the experimental mice into three groups, limb salvage, mild loss of limb, and severe loss of limb, as shown in FIG. 4A. Compared to the control EPC mice and the PBS mouse group, 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.

In addition, 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). ).

In addition, the present inventors measured capillary density by staining alkaline phosphatase and CD31 in tissue sections to measure neovascularization in ischemia hind limbs.

As a result, the blood vessel density in the tissue sections obtained from the legs treated with Hp-EPCs was significantly increased compared to other controls (see FIG. 5).

Based on the above experimental results, 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.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

<110> Industry Academic Cooperation Foundation of Catholic University <120> Haptoglobin gene introduced endothelial progenitor cells and          compositions for promoting angiogenesis comprising the same <160> 18 <170> KopatentIn 1.71 <210> 1 <211> 406 <212> PRT <213> Artificial Sequence <220> 223 haptoglobin amino acid sequence <400> 1 Met Ser Ala Leu Gly Ala Val Ile Ala Leu Leu Leu Trp Gly Gln Leu   1 5 10 15 Phe Ala Val Asp Ser Gly Asn Asp Val Thr Asp Ile Ala Asp Asp Gly              20 25 30 Cys Pro Lys Pro Pro Glu Ile Ala His Gly Tyr Val Glu His Ser Val          35 40 45 Arg Tyr Gln Cys Lys Asn Tyr Tyr Lys Leu Arg Thr Glu Gly Asp Gly      50 55 60 Val Tyr Thr Leu Asn Asp Lys Lys Gln Trp Ile Asn Lys Ala Val Gly  65 70 75 80 Asp Lys Leu Pro Glu Cys Glu Ala Asp Asp Gly Cys Pro Lys Pro Pro                  85 90 95 Glu Ile Ala His Gly Tyr Val Glu His Ser Val Arg Tyr Gln Cys Lys             100 105 110 Asn Tyr Tyr Lys Leu Arg Thr Glu Gly Asp Gly Val Tyr Thr Leu Asn         115 120 125 Asn Glu Lys Gln Trp Ile Asn Lys Ala Val Gly Asp Lys Leu Pro Glu     130 135 140 Cys Glu Ala Val Cys Gly Lys Pro Lys Asn Pro Ala Asn Pro Val Gln 145 150 155 160 Arg Ile Leu Gly Gly His Leu Asp Ala Lys Gly Ser Phe Pro Trp Gln                 165 170 175 Ala Lys Met Val Ser His His Asn Leu Thr Thr Gly Ala Thr Leu Ile             180 185 190 Asn Glu Gln Trp Leu Leu Thr Thr Ala Lys Asn Leu Phe Leu Asn His         195 200 205 Ser Glu Asn Ala Thr Ala Lys Asp Ile Ala Pro Thr Leu Thr Leu Tyr     210 215 220 Val Gly Lys Lys Gln Leu Val Glu Ile Glu Lys Val Val Leu His Pro 225 230 235 240 Asn Tyr Ser Gln Val Asp Ile Gly Leu Ile Lys Leu Lys Gln Lys Val                 245 250 255 Ser Val Asn Glu Arg Val Met Pro Ile Cys Leu Pro Ser Lys Asp Tyr             260 265 270 Ala Glu Val Gly Arg Val Gly Tyr Val Ser Gly Trp Gly Arg Asn Ala         275 280 285 Asn Phe Lys Phe Thr Asp His Leu Lys Tyr Val Met Leu Pro Val Ala     290 295 300 Asp Gln Asp Gln Cys Ile Arg His Tyr Glu Gly Ser Thr Val Pro Glu 305 310 315 320 Lys Lys Thr Pro Lys Ser Pro Val Gly Val Gln Pro Ile Leu Asn Glu                 325 330 335 His Thr Phe Cys Ala Gly Met Ser Lys Tyr Gln Glu Asp Thr Cys Tyr             340 345 350 Gly Asp Ala Gly Ser Ala Phe Ala Val His Asp Leu Glu Glu Asp Thr         355 360 365 Trp Tyr Ala Thr Gly Ile Leu Ser Phe Asp Lys Ser Cys Ala Val Ala     370 375 380 Glu Tyr Gly Val Tyr Val Lys Val Thr Ser Ile Gln Asp Trp Val Gln 385 390 395 400 Lys Thr Ile Ala Glu Asn                 405 <210> 2 <211> 1221 <212> DNA <213> Artificial Sequence <220> <223> haptoglobin gene sequence <400> 2 atgagtgccc tgggagctgt cattgccctc ctgctctggg gacagctttt tgcagtggac 60 tcaggcaatg atgtcacgga tatcgcagat gacggctgcc cgaagccccc cgagattgca 120 catggctatg tggagcactc ggttcgctac cagtgtaaga actactacaa actgcgcaca 180 gaaggagatg gagtatacac cttaaatgat aagaagcagt ggataaataa ggctgttgga 240 gataaacttc ctgaatgtga agcagatgac ggctgcccga agccccccga gattgcacat 300 ggctatgtgg agcactcggt tcgctaccag tgtaagaact actacaaact gcgcacagaa 360 ggagatggag tgtacacctt aaacaatgag aagcagtgga taaataaggc tgttggagat 420 aaacttcctg aatgtgaagc agtatgtggg aagcccaaga atccggcaaa cccagtgcag 480 cggatcctgg gtggacacct ggatgccaaa ggcagctttc cctggcaggc taagatggtt 540 tcccaccata atctcaccac aggtgccacg ctgatcaatg aacaatggct gctgaccacg 600 gctaaaaatc tcttcctgaa ccattcagaa aatgcaacag cgaaagacat tgcccctact 660 ttaacactct atgtggggaa aaagcagctt gtagagattg agaaggttgt tctacaccct 720 aactactccc aggtagatat tgggctcatc aaactcaaac agaaggtgtc tgttaatgag 780 agagtgatgc ccatctgcct accttcaaag gattatgcag aagtagggcg tgtgggttat 840 gtttctggct gggggcgaaa tgccaatttt aaatttactg accatctgaa gtatgtcatg 900 ctgcctgtgg ctgaccaaga ccaatgcata aggcattatg aaggcagcac agtccccgaa 960 aagaagacac cgaagagccc tgtaggggtg cagcccatac tgaatgaaca caccttctgt 1020 gctggcatgt ctaagtacca agaagacacc tgctatggcg atgcgggcag tgcctttgcc 1080 gttcacgacc tggaggagga cacctggtat gcgactggga tcttaagctt tgataagagc 1140 tgtgctgtgg ctgagtatgg tgtgtatgtg aaggtgactt ccatccagga ctgggttcag 1200 aagaccatag ctgagaacta a 1221 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Hp F-primer <400> 3 atgagtgccc tgggagctgt catt 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Hp R-primer <400> 4 gcattagttc tcagctatgg tctt 24 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> 223 VEGF F-primer <400> 5 aggagggcag aatcatcacg 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> 223 VEGF R-primer <400> 6 caaggcccac agggattttc t 21 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> KDR F-primer <400> 7 ctggcatggt cttctgtg 18 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> KDR R-primer <400> 8 aatgggattg gtaaggatg 19 <210> 9 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Flt-1 F-primer <400> 9 agcaagtggg agtttgc 17 <210> 10 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Flt-1 R-primer <400> 10 aggtcccgat gaatgc 16 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> vWF F-primer <400> 11 gaggctgagt ttgaagtgc 19 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> vWF R-primer <400> 12 ctgctccagc tcatccac 18 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> 223 VE-cadherin F-primer <400> 13 aagacatcaa tgacaacttc c 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> 223 VE-cadherin R-primer <400> 14 cctccacagt caggttatac c 21 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> eNOS F-primer <400> 15 aagacatttt cgggctcac 19 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> eNOS R-primer <400> 16 ggcactttag tagttctcc 19 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH F-primer <400> 17 accacagtcc atgccatcac 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH R-primer <400> 18 tccaccaccc tgttgctgta 20

Claims (8)

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 method of claim 1,
The haptoglobin gene is vascular endothelial progenitors, characterized in that consisting of the nucleotide sequence represented by SEQ ID NO: 2. The method of claim 1,
The recombinant vector is a vascular endothelial progenitor cell, characterized in that the pMSCV-Hp vector. The method of claim 1,
The transformed vascular endothelial progenitor cells are characterized in that the expression of haptoglobin is promoted differentiation into mature vascular endothelial cells and has an activity of promoting angiogenesis. The composition for promoting angiogenesis comprising the vascular endothelial progenitor cell of any one of claims 1 to 4 as an active ingredient. The method of claim 5,
The composition is a composition for promoting angiogenesis, characterized in that the pharmaceutical composition capable of preventing or treating angiogenesis-related diseases. The method of claim 6,
The angiogenesis-related disease is an angiogenesis-promoting composition, characterized in that selected from the group consisting of diabetic ulcers, necrosis, ischemic disease, obstructive vascular disease, cardiovascular disease and ischemia. A method for promoting angiogenesis comprising administering a transformed vascular endothelial progenitor cell according to any one of claims 1 to 4 to a site in need of angiogenesis in a mammal, except for humans.

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