KR20070113499A - A method for inhibiting angiogenesis using dkk1 - Google Patents

Abstract

A DKK1(Dickkopf-1) protein or a DNA encoding the same are provided to inhibit angiogenesis effectively in an umbilical vein endothelial cell, a DKK1-overexpressed cell line and a DKK1-transduced mouse, thereby being widely used for developing a therapeutic agent for preventing or treating diseases caused by the angiogenesis. A method for inhibiting angiogenesis comprises a step of treating a tissue of mammals excluding human with a DKK1 protein or a DNA encoding the same. A pharmaceutical composition for treating and preventing diseases caused by angiogenesis comprises a DKK1 protein or a DNA encoding the same as an effective ingredient, wherein the DNA encoding the DKK1 protein is provided by being included in a non-viral vector or a viral-vector.

Description

A method for inhibiting angiogenesis using DKK1

Figure 1a is a diagram showing the morphological differentiation process of human umbelical vein endothelial cells (HUVEC) on Matrigel divided into three stages,

Figure 1b is a diagram showing the expression patterns of the DKK1 gene in each stage of morphogenesis of human umbilical vein endothelial cells,

Figure 2a is a diagram showing the expression and purification of DKK1 recombinant protein,

Figure 2b is a diagram showing the inhibition of tube formation in human umbilical vein endothelial cells, depending on the concentration of the DKK1 recombinant protein,

Figure 3a is a diagram confirming the production of DKK1 gene overexpression and suppression cell lines using lentivirus,

Figure 3b is a diagram comparing the degree of tube formation of DKK1 gene overexpression and inhibitory cell line,

Figure 4a is a schematic diagram of the vector for the production of DKK1 transduced mouse,

Figure 4b is a diagram confirming the DKK1 transduction of the mouse through DNA amplification,

Figure 4c is a diagram comparing the size of normal mice and DKK1 transduced mice,

5 is a diagram showing the change in the degree of vascular germination of aortic ring tissue of normal mice and DKK1 transduced mice,

Figure 6a is a diagram comparing the degree of angiogenesis in the embryo (embryo) of normal mice and DKK1 transduced mice,

Figure 6b is a diagram comparing the degree of angiogenesis of the head in the embryo (embryo) of normal mice and DKK1 transgenic mice,

FIG. 6C is a diagram comparing fluorescence staining of the degree of development of the aorta of the spine in the embryos of normal mice and DKK1 transduced mice. FIG.

Figure 6b is a diagram comparing the degree of development of the side chain artery of the spine in the embryo (embryo) of normal mice and DKK1 transduced mouse by comparison.

The present invention relates to a method of inhibiting angiogenesis using DKK1 (Dickkopf-1), which is known as a co-receptor inhibitor of Wnt proteins.

Angiogenesis refers to the process by which new blood vessels are formed, which rarely occurs in normal living conditions, but is an essential process in embryogenesis, corpus luteum formation or wound healing. In particular, angiogenesis is known to play an important role when tumors metastasize to other sites (Folkman J and Klagsburn M, Science , 235 (4787), pp442-447, 1987).

The angiogenesis process is generally caused by stimulation of angiogenesis factors, resulting in the formation of lumen due to the degradation of the basal membrane, proliferation, proliferation, and differentiation of vascular endothelial cells due to proteases. Consists of being generated.

However, there are diseases caused by angiogenesis that is not controlled autonomously and grows pathologically. Diseases related to angiogenesis in pathological conditions include hemangioma, angiofibroma, angioplasty and cardiovascular diseases such as atherosclerosis, angiogenesis, and edema sclerosis. Eye diseases caused by angiogenesis include corneal graft angiogenesis and angiogenesis. Glaucoma, diabetic retinopathy, corneal disease caused by neovascularization, spot degeneration, pterygium, retinal degeneration, posterior capsular fibrosis, granular conjunctivitis and the like. Chronic inflammatory diseases such as arthritis, psoriasis, capillary dilatation, purulent granulomas, seborrheic dermatitis, dermatological diseases such as acne, Alzheimer's and obesity are also associated with angiogenesis, and cancer growth and metastasis necessarily depends on angiogenesis (D'Amato RJ and Adamis AP, Ophthalmology , 102 (9), pp1261-1262, 1995; Arbiser JL, J. Am . Acad . Dermatol ., 34 (3), pp486-497, 1996; O'Brien KD et al. Circulation , 93 (4), pp 672-682, 1996; Hanahan D and Folkman J, Cell , 86 (3), pp 353-364, 1996).

Angiogenesis, in particular, plays an important role in the growth and metastasis of cancer cells. Tumors are supplied with nutrients and oxygen for growth and proliferation through neovascularization, and new blood vessels that penetrate the tumor give metastasis by allowing metastatic cancer cells to enter the blood circulation (Folkman and Tyler). , Cancer Invasion and metastasis , Biologic mechanisms and Therapy (SB Day ed.) Raven press, New York, pp 94-103, 1977; Polverini PJ, Crit . Rev. Oral Biol . Med . , 6 (3), pp230-247, 1995). The main cause of death of cancer patients is metastasis, and the current metastasis of cancer does not contribute to the survival of cancer patients.

Arthritis, a representative disease of inflammatory diseases, is caused by autoimmune abnormalities, but as the disease progresses, chronic inflammation in the synovial cavity between joints induces angiogenesis and destroys cartilage. In other words, synovial cells and vascular endothelial cells proliferate in the synovial cavity with the help of inflammation-induced cytokines, forming articular pannus, a layer of connective tissue that occurs in the cartilage as the angiogenesis progresses, destroying cartilage that acts as a cushion. (Koch AE et al., Arthritis Rheum ., 29 (4), pp471-479, 1986; Stupack DG et al., Braz . J. Med . Biol. Res ., 32 (5), pp573-581, 1999 Koch AE, Arthritis Rheum ., 41 (6), pp 951-962, 1998).

Many ocular diseases, which cause millions of blindness worldwide each year, are also caused by angiogenesis (Isner JM and Asahara T, J. Clin . Invest ., 103 (9), pp1231-1236, 1999). For example, diseases such as macular degeneration, diabetic retinopathy, retinopathy of premature infants, neovascular glaucoma and corneal disease caused by neovascularization are the diseases caused by neovascularization (Adamis AP). et al., Angiogenesis , 3 (1), pp 9-14, 1999). Among them, diabetic retinopathy is a complication of diabetes, in which capillaries in the retina invade the vitreous body and eventually become blind.

Psoriasis, which is characterized by red spots and skin, is also a chronic proliferative disease of the skin. It does not heal and involves pain and malformations. In normal cases, keratinocytes proliferate once a month, whereas psoriasis patients proliferate at least once a week. Angiogenesis is bound to occur because a lot of blood is required for this rapid proliferation (Folkman J, J. Invest . Dermatol ., 59 (1), pp 40-43, 1972).

Obesity and adipose tissue can also be controlled by the lower vasculature, and treatment with anti-angiogenic inhibitors results in dose-dependent weight loss and fat cell tissue reduction (Rupnick MA et al. al., Proc . Natl . Acad . Sci. USA , 99 (16), pp10730-5, 2002).

In recent years, blood loss and inflammation in Alzheimer's brain suggest that angiogenesis inducers are expressed and angiogenesis occurs actively, resulting in the deposition of beta amyloid and secretion of neurotoxic peptides. Drugs that inhibit angiogenesis may be able to prevent and treat Alzheimer's disease (Vagnucci AH Jr. et al., Lancet, 361 (9357), pp605-608, 2003).

New blood vessel formation requires many genetic changes during endothelial differentiation. Recently, researches on regulating angiogenesis by discovering genes that regulate differentiation of vascular endothelial cells and regulating these genes have been actively conducted. In addition, therapies that inhibit angiogenesis of tumors have attracted a lot of attention in recent years in that they have a wide effective range, low toxicity, and no resistance in the body upon direct endothelial administration. However, in the case of the existing angiogenesis inhibitors, various side effects such as damage to the surrounding normal vascular tissues have been reported, and in some cases a disadvantage that may not show the therapeutic effect of angiogenesis. Therefore, if a method is developed that can minimize side effects and effectively treat angiogenesis, it is expected to provide an electricity in the treatment of various diseases causing angiogenesis such as malignant tumors. Until now, no results have been shown. Thus, there is a continuing need to develop methods for treating angiogenesis safely and effectively.

DKK1 is a kind of Dickkopf, a Wnt inhibitory protein, and was first known as an important protein involved in hair formation of amphibians ( Xenopus ) (Glinka A et al., Nature , 391 (6665), pp357-362, 1998). DKK1 has two characteristic cysteine-rich domains and is divided into linkages of various lengths. Specific cysteine-2 moieties are highly conserved among these Dickkopf families and contain 10 conserved cysteine amino acids (Krupnik VE et al., Gene , 238 (2), pp301-313, 1999).

To date, DKK1's function has only been reported to degenerate neurons in the brain of Alzheimer's patients, to inhibit melanocyte growth and differentiation, and to regulate the cell cycle of stem cells. No research reports have been taught or described (Caricasole A et al., J. Neurosci . , 24 (26), pp6021-6027, 2004; Yamaguchi Y et al, J. cell Biol . , 165 (2), pp275 -85, 2004; Gregory CA. et al, J. Biol . Chem . , 278 (30), pp28067-28078, 2003).

Therefore, the present inventors have diligently researched a gene that regulates vascular endothelial cell differentiation process and identified a function to develop a method that can safely and effectively treat angiogenesis. As a result, the DKK1 protein, known as a co-receptor inhibitor of Wnt protein The present invention was completed by confirming that it can be effectively used in the treatment of diseases related to angiogenesis by effectively inhibiting angiogenesis when it is treated in vivo or transfected or transduced with DNA encoding the DKK1 protein.

An object of the present invention is to provide a method for inhibiting angiogenesis using DKK1 (Dickkopf-1) protein or DNA encoding the same.

In order to achieve the above object, the present invention provides a method for inhibiting angiogenesis, comprising the step of processing a DNA encoding DKK1 (Dickkopf-1) protein or DKK1 protein in tissues of mammals except humans.

DNA encoding the DKK1 (Dickkopf-1) protein of the present invention is characterized in that it is delivered into the tissue using a non-viral vector or viral vector.

Non-viral vectors of the invention are characterized by comprising plasmids which can be expressed in animal cells.

Viral vectors of the invention are characterized by comprising adenovirus vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors or herpes simplex virus vectors.

The present invention also provides a pharmaceutical composition for the prevention and treatment of diseases caused by angiogenesis, including DKK1 protein or DNA encoding the same as an active ingredient.

Diseases caused by angiogenesis of the present invention are angiogenesis-mediated hemangioma, hemangiofibroma, angioplasty, arteriosclerosis, angiogenesis, edema sclerosis, corneal graft angiogenesis, neovascular glaucoma, diabetic retinopathy, cornea caused by neovascularization Disease, degeneration of spots, pterygium, retinal degeneration, posterior capsular fibrosis, granulomatous conjunctivitis, degenerative plaque, retinopathy of premature infants, arthritis, psoriasis, capillary dilatation, purulent granulomas, Alzheimer's disease, obesity, seborrheic dermatitis, acne and solid cancer It is characterized in that the disease caused by one or more angiogenesis selected from the group consisting of cancer diseases.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for inhibiting angiogenesis comprising the step of processing a DNA encoding a DKK1 (Dickkopf-1) protein having the amino acid sequence of SEQ ID NO: 1 or a DKK1 protein, the nucleotide sequence of SEQ ID NO: 2 encoding the same. However, DKK1 of the present invention is not limited to the above sequence, and includes all mammalian DKK1.

The DKK1 protein includes DKK1 protein or recombinant DKK1 protein extracted from mammalian tissue.

The DKK1 protein of the present invention and the DKK1 gene encoding the same can be obtained as follows.

Complementary DNA obtained by isolating and reverse transcripting total RNA of vascular endothelial cells as a template can be used to obtain amplified DKK1 gene by polymerization using DKK1 primers, preferably the primers of SEQ ID NOs: 5 and 6.

The obtained DKK1 gene was treated with restriction enzymes, preferably EcoRI and XhoI, to clone the plasmid for protein purification, preferably pcDNA-His, to obtain a plasmid, and the plasmid was expressed as an expression cell, preferably a human kidney cell. 293 cells (ATCC, USA) are transformed with gene transfer reagents, preferably Lipofectamin (Invitrogen, USA). Thereafter, only the transformed cells are isolated using a selective transforming cell separation reagent, preferably G418 (Sigma, USA), and then cultured in large quantities, and the culture medium is a column, preferably a nickel column (Ni-NTA agarose, Qiagen, USA) to purify the secreted DKK1 protein into the medium and confirm the expression of recombinant DKK1 protein.

When DKK1 protein obtained by the above method was treated to human umbilical vein endothelial cells, DKK1 overexpressing cell line transfected with DNA encoding DKK1 (Dickkopf-1) protein inhibited vascular formation, and DKK1 ( The aortic ring tissue of mice transduced with the DNA encoding Dickkopf-1) protein inhibited vascular sprouting, and the embryo of the transduced mouse suppressed angiogenesis and thus exhibited excellent angiogenesis inhibitory activity. Could be confirmed.

The present invention also provides a pharmaceutical composition for the prevention or treatment of diseases caused by angiogenesis, which contains an DKK1 protein that inhibits angiogenesis obtained by the above method or an DNA encoding the same as an active ingredient.

Compositions comprising a protein of the present invention or DNA encoding the same may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Compositions comprising the protein of the present invention or DNA encoding the same, oral formulations, external preparations, suppositories, and sterile injectable solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. Formulated in the form of can be used. Carriers, excipients and diluents which may be included in the composition comprising the extract of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

A vector capable of inserting the DNA to continuously supply the DNA encoding the DKK1 (Dickkopf-1) protein, which is an active ingredient of the present invention, to the affected area may include an adenovirus vector and an adeno-associated virus. associated virus), retrovirus vector, lentivirus vector, herpes simplex virus vector or plasmid that can be expressed in animal cells.

Dosage forms of the composition containing the protein or DNA encoding the same can be used for any of the agents conventionally used in injections. For example, the substrate may be distilled water, sodium chloride solution, mixtures of sodium chloride and mineral salts or the like, solutions of mannitol, lactose, dextran, glucose, etc., amino acid solutions such as glycine, arginine, organic acid solutions or salt solutions and glucose solutions. Mixing and similar solutions. Injectables may also be prepared as solutions, suspensions or colloidal solutions by adding osmotic pressure regulators, pH regulators, vegetable oils such as sesame oil or soybean oil, or surfactants such as lecithin, nonionic surfactants, etc., in the usual manner. Can be. Such injectables may be prepared in powder form, lyophilized form and dissolved immediately before use.

The composition containing the protein or DNA encoding the same according to the present invention as an active ingredient may be dissolved in a pre-sterilized substrate if necessary immediately before gene treatment in the solid phase, or may be used as it is without additional treatment in the liquid phase.

Preferred dosages of the protein of the present invention or DNA encoding the same may vary depending on the condition and weight of the patient, the severity of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the amount of 0.0001 to 1000 mg / kg may be administered once to several times per week, or once via a catheter after surgery, locally, at the site of disease caused by angiogenesis. It can be absorbed at the target point. The dosage can be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect. The dosage does not limit the scope of the invention in any aspect.

The protein of the present invention or DNA encoding the same can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

Below, The invention is illustrated in detail by the following reference and experimental examples.

However, the following Reference Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Reference Examples and Experimental Examples.

Reference Example  1. Human Umbilical Vein Endothelial Cells  ( HUVEC Culture

Vascular cells were isolated from the mother's umbilical vein isolated from Yonsei University College of Gynecology. After washing the vessels with cord buffer (0.2% glucose phosphate buffered saline), about 5 ml of 0.2% type I collagenase (type I collagenase, Sigma-Aldrich Co., MO, USA) was injected into the vein. It was left to stand at 37 degreeC for 5 minutes. At room temperature, 20 ml of cord buffer was placed in a vein, and the vascular cells separated from the other side were collected. Once again, only the cord buffer was injected into the blood vessel and reacted at 37 ° C., and the umbilical cord endothelial cells (HUVEC; human umbelical vein) were collected. endothelial cells) were collected and washed and injected into T75 flasks for tissue culture coated with 0.1% gelatin. Culture medium EGM ^TM -2 complete medium by using a ^(TM EGM -2 complete medium, Cambrex, MD, USA) incubated at 37 ℃, 5% CO ₂ incubator and remove the trypsin -EDTA solution when the confluent state cells Were isolated and 3-6 generation cells were used for the experiment.

Reference Example  2. Recombinant human DKK1  Protein acquisition

Total RNA from vascular endothelial cells was isolated using TRIZOL reagent (Invitrogen, USA) and reverse transcribed using oligo (dT) primer and reverse transcriptase (Promega, USA), followed by polymerase (Stratagen, USA) Using the following conditions, in detail, pre-denaturation for 5 minutes at 94 ℃, denaturation for 30 seconds at 94 ℃, DKK1 primers of SEQ ID NO: 5 and 6 for 30 seconds at 50 ℃ (annealing), the polymerization was repeated 30 times in one extension of 72 seconds at 72 ℃, cloned into a pcDNA-His vector, a protein purification plasmid by treating EcoRI and XhoI to the amplified DKK1 gene Plasmids were transformed into 293 cells (ATCC, USA), which are human kidney cells, using lipofectamine (Lipofectamin, Invitrogen, USA). Only transformed cells were isolated using G418 (Sigma, USA), a selective transformed cell separation reagent, and cultured in large quantities. The culture medium was applied to a nickel column (Ni-NTA agarose, Qiagen, USA) and secreted into the medium. DKK1 protein was purified to confirm the expression of recombinant DKK1 protein (see FIG. 2A).

Reference Example  3. Lentivirus  Vector DKK1  Overexpression and Inhibition Cell Line Construction

A plasmid prepared by cloning a human DKK1 gene into a lentiviral vector was obtained from vector Korea (vectorcorea, Chungcheongnam-do, Korea Republic) obtained from DKK1 overexpression and suppression cell line transfected into a virus producing cell line, and prepared in Reference Example 1. After 48 hours of addition to human umbilical vein endothelial cells, RNA was isolated from these cells and subjected to the following reverse transcription and polymerization reaction to confirm DKK1 gene expression. Total RNA was isolated using trizol reagent (TRIzol reagent, Invitrogen, USA), followed by reverse transcription using oligo (dT) primer and reverse transcriptase (Promega, USA), followed by polymerase (Stratagen, USA). The following conditions, in detail, pre-denaturation for 5 minutes at 94 ℃, denaturation for 30 seconds at 94 ℃, annealing primers for 30 seconds at 50 ℃, extended for 30 seconds at 72 ℃ (extension) was repeated once, the polymerization was repeated a total of 30 times, the results are shown in Figure 3a.

As shown in Figure 3a, it was confirmed that the overexpression or expression suppression cell line of DKK1 using a lentiviral system was well prepared.

Reference Example  4. DKK1  Transduction Mouse Construction

In order to analyze the effects of DKK1 gene on angiogenesis in vivo , mice overexpressed only in blood vessels were constructed using Tie2 transcriptional regulatory sites activated only in vascular endothelial cells (Schlaeger TM et al., Proc . Natl. Acad. Sci. USA, 94 (7), 3058-3063, 1997). As shown in the schematic diagram of FIG. 4A, the mouse DKK1 gene of SEQ ID NO: 4 was inserted into a transduction vector (pSP vector, Clontech, USA) after treatment with restriction enzymes HindIII and NotI (NEB, UK). Cloned (see FIG. 4A). The plasmid was linearized by treatment with restriction enzyme SalI (NEB, UK), and DNA fragments were isolated and isolated from mice (C57BL6, Orient, Korea) that promoted ovulation with gonadotropin-releasing hormone (Gonadotropin, Sigma, USA). The eggs were transduced and transduced, fertilized and implanted into surrogate mother mice (ICR, Orient, Korea). Twenty-one days later, the tail of the mouse was cut and treated with proteinase K (proteinase K, Sigma, USA) to separate DNA, and then amplified using DKK1 primers of SEQ ID NOs. , (Denatured for 30 seconds at 94 ℃, binding for 30 seconds at 55 ℃, extended for 30 seconds at 72 ℃) x 30 times, and extended for 10 minutes at 72 ℃ to confirm the transduced mice (see Figure 4b).

As shown in Figure 4c, it was confirmed that the size is significantly different between normal mice and DKK1 transduced mice, it can be assumed that the result is due to abnormal development of blood vessels.

Experimental Example  One. Umbilical Vein Endothelial Cells During the differentiation process DKK1 Manifestations of

250 μl of 10 mg protein / ml concentration of Matrigel (Matrigel, Collaborative Biomedical Products, USA) was placed in a 16 mm diameter cell culture well and polymerized at 37 ° C. for 30 minutes. Human umbilical vein endothelial cells of Reference Example 1 were treated with 20% (v / v) fetal bovine serum (FBS; fetal bovine serum, Hyclone, USA), 100 units / ml penicillin (penicillin, Invitrogen, USA), 10 µg / ml strepto M199 growth medium (invitrogen, including mycine (streptomycin, Invitrogen, USA), 3ng / ml fibroblast growth factor (bFGF; basic fibroblast growth factor, Upstate Biotechnology, USA), 5 units / ml heparin (heparin, Sigma, USA) USA) and obtained by treatment with trypsin, which was then resuspended in the growth medium, spread on the prepared Matrigel layer at a concentration of 2 x 10 ⁵ cells / well and induced differentiation (see FIG. 1A). The differentiation process was divided into three stages according to the degree of differentiation of cells, in detail, the first stage of differentiation initiation stage used as a control group, the second stage of initiation of blood vessel formation, and the stage of complete differentiation Three steps. RNA was isolated from the cells of each step using TRIZOL solution (Invitrogen, USA), and reverse transcription and amplification were performed using the primers of SEQ ID NOs: 5 and 6 as shown in the polymerization conditions of Reference Example 4, and then the expression pattern of the DKK1 gene was confirmed. (See FIG. 1B).

As shown in Figure 1b, the DKK1 gene showed a significant decrease in its expression during the tube formation process, and the experimental results indicate that DKK1 may act as a negative regulator of tube formation.

Experimental Example  2. Umbilical Vein Endothelial Cells  Affecting tube formation DKK1 Effect

250 μl of 10 mg protein / ml concentration of Matrigel (Matrigel, Collaborative Biomedical Products, USA) was placed in a 16 mm diameter cell culture well and polymerized at 37 ° C. for 30 minutes. Human umbilical vein endothelial cells of Reference Example 1 were treated with 20% (v / v) fetal bovine serum (FBS; fetal bovine serum, Hyclone, USA), 100 units / ml penicillin, 10 μg / ml streptomycin. , 3ng / ml fibroblast growth factor (bFGF; basic fibroblast growth factor, Upstate Biotechnology, USA), cultured in M199 growth medium (Invitrogen, USA) containing 5 units / ml heparin and obtained by treatment with trypsin, The matrigel layer prepared by floating again with the growth medium was spread at a concentration of 2 x 10 ⁵ cells / well and induced differentiation. After incubation for 20 hours with addition of DKK1 at concentrations of 50 ng / ml and 100 ng / ml simultaneously with differentiation induction, the formed tubular formation was analyzed by optical imaging (ZEISS, Germany) and analyzed (see FIG. 2b). ). At this time, the experimental group was not treated with DKK1 as a control group.

As shown in Figure 2b, when compared with the control group DKK1 treatment group was found to significantly reduce the area and connection of the tube formed in proportion to the increase in the concentration of DKK1 treatment.

Experimental Example  3. DKK1  Effects on Tube Formation in Overexpressed or Expression Inhibited Cell Lines DKK1 Effect

250 μl of 10 mg protein / ml concentration of Matrigel (Matrigel, Collaborative Biomedical Products, USA) was placed in a 16 mm diameter cell culture well and polymerized at 37 ° C. for 30 minutes. 20% (v / v) fetal bovine serum (FBS; fetal bovine serum, Hyclone, USA), 100 units / ml penicillin (penicillin, Invitrogen, USA), 10 μg / ml streptococcus Cultured in M199 growth medium (Invitrogen, USA) containing mycin (streptomycin, Invitrogen, USA), 3 ng / ml fibroblast growth factor (bFGF; basic fibroblast growth factor, Upstate Biotechnology, USA), 5 units / ml heparin and trypsin After obtained by treatment, it was suspended again in the growth medium and spread on the matrigel layer prepared at a concentration of 2 × 10 ⁵ cells / well and incubated for 9 hours, the tube formation formed was taken by optical imaging technique (See Figure 3b). At this time, the experimental group infected with the lentiviral was used as a control.

As shown in FIG. 3B, tube formation was inhibited in the cell line overexpressing DKK1, and tube formation was rather promoted in the cell line in which DKK1 expression was suppressed.

Experimental Example  4. DKK1  Effect on Vascular Sprouting in Aortic Ring Tissues in Transduced Mice DKK1 Effect

Atrial ring tissue obtained by cutting 1 mm of arterial blood vessels produced along 6-week-old normal mice (C57BL6, Orient, Gyeonggi-do, Korea) and the DKK1-transduced mouse prepared in Reference Example 4 was obtained. Place in a 48-well of plate coated with [mu] l, again cover with 40 [mu] l of Matrigel and seal, then add vascular endothelial cell culture medium (SFM, Invitrogen, USA) to each well to a final volume of 200 [mu] l, 5 After one day, the number of germinated blood vessels (sprout) extending from each ring was measured, and the degree of vascular germination between the DKK1 treated group and the control group was shown in FIG. 5 (see FIG. 5). The degree of germination was expressed by dividing the blood vessels into 5 sites and scoring the points by 5 points if they germinated and 5 points if they did not germinate.

As shown in FIG. 5, it was confirmed that vascular germination in aortic annulus tissues obtained from mice that DKK1 was overexpressed in blood vessels was significantly suppressed compared to normal mice.

Experimental Example  5. DKK1  Embryo of transgenic mouse ( embryo ) On angiogenesis DKK1 Effect

The von Willebrand factor, a protein that expresses only vascular endothelial cells after 9-10 days of gestation, takes out embryos of normal and DKK1 transduced embryos and soaks in 4% paraformaldehyde for one day. After staining with an antibody of [von Willebrand Factor (vWF)] (Chemicon, USA), the development of blood vessels was observed in FIG. 6 (Sadler JE, J. Thromb . Haemost . , 3 (8), pp1702-1709, 2005).

As shown in Figure 6a, it was shown that the development of blood vessels is suppressed in the embryo of the mouse transfected with DKK1.

For further confirmation, the blood vessels generated in the head of the embryo were magnified (x200) using an optical microscope (Olympus, Japan) (see FIG. 6B). It was confirmed that angiogenesis in the head region of the embryo transduced with DKK1 was significantly suppressed compared to the angiogenesis of the normal embryo.

In order to confirm the effect of DKK1 on the inhibition of angiogenesis of other sites, the development degree of the spinal aorta and the side chain artery of the abdomen prepared as described above was fluorescently stained as follows. The embryos were dehydrated for one day in 30% sucrose solution and then frozen at -70 ° C, the frozen tissue was cut into 10 μm thick and attached to slides and blocked with goat serum for 2 hours before vonville Antibodies of von Willebrand Factor (vWF) were used as primary antibodies and treated for one day at 4 ° C. The next day, with green phosphors for vascular cell staining and red phosphors for perivascular smooth muscle cell staining The secondary antibody was treated for 1 hour, covered with a cover glass, and observed under a fluorescence microscope, as shown in Figs. 6c and 6d, as shown in Figs. 6c and 6d. And it was confirmed that the development of the side chain artery is suppressed.

As described and demonstrated in detail above, the present invention provides a method of inhibiting angiogenesis using DKK1. The method for inhibiting angiogenesis using DKK1 of the present invention comprises transfecting or transducing and expressing the mammalian tissue using DNA encoding the DKK1 protein or DKK1 (Dickkopf-1) protein. Since DKK1 effectively inhibits angiogenesis, the present invention may be widely used for the development of therapeutic agents for the prevention and treatment of diseases caused by angiogenesis.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION, YONSEI UNIVERSITY <120> A METHOD FOR INHIBITING ANGIOGENESIS USING DKK1 <130> DIF / 2006-05-0012 / SY <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 266 <212> PRT <213> Human DKK1 <400> 1 Met Met Ala Leu Gly Ala Ala Gly Ala Thr Arg Val Phe Val Ala Met   1 5 10 15 Val Ala Ala Ala Leu Gly Gly His Pro Leu Leu Gly Val Ser Ala Thr              20 25 30 Leu Asn Ser Val Leu Asn Ser Asn Ala Ile Lys Asn Leu Pro Pro Pro          35 40 45 Leu Gly Gly Ala Ala Gly His Pro Gly Ser Ala Val Ser Ala Ala Pro      50 55 60 Gly Ile Leu Tyr Pro Gly Gly Asn Lys Tyr Gln Thr Ile Asp Asn Tyr  65 70 75 80 Gln Pro Tyr Pro Cys Ala Glu Asp Glu Glu Cys Gly Thr Asp Glu Tyr                  85 90 95 Cys Ala Ser Pro Thr Arg Gly Gly Asp Ala Gly Val Gln Ile Cys Leu             100 105 110 Ala Cys Arg Lys Arg Arg Lys Arg Cys Met Arg His Ala Met Cys Cys         115 120 125 Pro Gly Asn Tyr Cys Lys Asn Gly Ile Cys Val Ser Ser Asp Gln Asn     130 135 140 His Phe Arg Gly Glu Ile Glu Glu Thr Ile Thr Glu Ser Phe Gly Asn 145 150 155 160 Asp His Ser Thr Leu Asp Gly Tyr Ser Arg Arg Thr Thr Leu Ser Ser                 165 170 175 Lys Met Tyr His Thr Lys Gly Gln Glu Gly Ser Val Cys Leu Arg Ser             180 185 190 Ser Asp Cys Ala Ser Gly Leu Cys Cys Ala Arg His Phe Trp Ser Lys         195 200 205 Ile Cys Lys Pro Val Leu Lys Glu Gly Gln Val Cys Thr Lys His Arg     210 215 220 Arg Lys Gly Ser His Gly Leu Glu Ile Phe Gln Arg Cys Tyr Cys Gly 225 230 235 240 Glu Gly Leu Ser Cys Arg Ile Gln Lys Asp His His Gln Ala Ser Asn                 245 250 255 Ser Ser Arg Leu His Thr Cys Gln Arg His             260 265 <210> 2 <211> 801 <212> DNA <213> Human DKK1 <400> 2 atgatggctc tgggcgcagc gggagctacc cgggtctttg tcgcgatggt agcggcggct 60 ctcggcggcc accctctgct gggagtgagc gccaccttga actcggttct caattccaac 120 gctatcaaga acctgccccc accgctgggc ggcgctgcgg ggcacccagg ctctgcagtc 180 agcgccgcgc cgggaatcct gtacccgggc gggaataagt accagaccat tgacaactac 240 cagccgtacc cgtgcgcaga ggacgaggag tgcggcactg atgagtactg cgctagtccc 300 acccgcggag gggacgcggg cgtgcaaatc tgtctcgcct gcaggaagcg ccgaaaacgc 360 tgcatgcgtc acgctatgtg ctgccccggg aattactgca aaaatggaat atgtgtgtct 420 tctgatcaaa atcatttccg aggagaaatt gaggaaacca tcactgaaag ctttggtaat 480 gatcatagca ccttggatgg gtattccaga agaaccacct tgtcttcaaa aatgtatcac 540 accaaaggac aagaaggttc tgtttgtctc cggtcatcag actgtgcctc aggattgtgt 600 tgtgctagac acttctggtc caagatctgt aaacctgtcc tgaaagaagg tcaagtgtgt 660 accaagcata ggagaaaagg ctctcatgga ctagaaatat tccagcgttg ttactgtgga 720 gaaggtctgt cttgccggat acagaaagat caccatcaag ccagtaattc ttctaggctt 780 cacacttgtc agagacacta a 801 <210> 3 <211> 272 <212> PRT <213> Mouse DKK1 <400> 3 Met Met Val Val Cys Ala Ala Ala Ala Val Arg Phe Leu Ala Val Phe   1 5 10 15 Thr Met Met Ala Leu Cys Ser Leu Pro Leu Leu Gly Ala Ser Ala Thr              20 25 30 Leu Asn Ser Val Leu Ile Asn Ser Asn Ala Ile Lys Asn Leu Pro Pro          35 40 45 Pro Leu Gly Gly Ala Gly Gly Gln Pro Gly Ser Ala Val Ser Val Ala      50 55 60 Pro Gly Val Leu Tyr Glu Gly Gly Asn Lys Tyr Gln Thr Leu Asp Asn  65 70 75 80 Tyr Gln Pro Tyr Pro Cys Ala Glu Asp Glu Glu Cys Gly Ser Asp Glu                  85 90 95 Tyr Cys Ser Ser Pro Ser Arg Gly Ala Ala Gly Val Gly Gly Val Gln             100 105 110 Ile Cys Leu Ala Cys Arg Lys Arg Arg Lys Arg Cys Met Arg His Ala         115 120 125 Met Cys Cys Pro Gly Asn Tyr Cys Lys Asn Gly Ile Cys Met Pro Ser     130 135 140 Asp His Ser His Phe Pro Arg Gly Glu Ile Glu Glu Ser Ile Ile Glu 145 150 155 160 Asn Leu Gly Asn Asp His Asn Ala Ala Ala Gly Asp Gly Tyr Pro Arg                 165 170 175 Arg Thr Thr Leu Thr Ser Lys Ile Tyr His Thr Lys Gly Gln Glu Gly             180 185 190 Ser Val Cys Leu Arg Ser Ser Asp Cys Ala Ala Gly Leu Cys Cys Ala         195 200 205 Arg His Phe Trp Ser Lys Ile Cys Lys Pro Val Leu Lys Glu Gly Gln     210 215 220 Val Cys Thr Lys His Lys Arg Lys Gly Ser His Gly Leu Glu Ile Phe 225 230 235 240 Gln Arg Cys Tyr Cys Gly Glu Gly Leu Ala Cys Arg Ile Gln Lys Asp                 245 250 255 His His Gln Ala Ser Asn Ser Ser Arg Leu His Thr Cys Gln Arg His             260 265 270 <210> 4 <211> 818 <212> DNA <213> Mouse DKK1 <400> 4 atgatggttg tgtgtgcagc ggcagctgtc cggttcttgg ccgtgtttac aatgatggct 60 ctctgcagcc tccctctgct aggagccagt gccaccttga actcagttct catcaattcc 120 aacgcgatca agaacctgcc cccaccgctg ggtggtgctg gggggcagcc gggctctgct 180 gtcagtgtgg cgccgggagt tctctatgag ggcgggaaca agtaccagac tcttgacaac 240 taccagccct acccttgcgc tgaagatgag gagtgcggct ctgacgagta ctgctccagc 300 cccagccgcg gggcagccgg cgtcggaggt gtacagatct gtctggcttg ccgaaagcgc 360 aggaagcgct gcatgaggca cgctatgtgc tgccccggga actctgcaaa aatggaatat 420 gcatgccctc tgaccacagc cattttcctc gaggggaaat tgaggaaagc atcattgaaa 480 accttggtaa tgaccacaac gccgccgcgg gggatggata tcccagaaga accacactga 540 cttcaaaaat atatcacacc aaaggacaag aaggctccgt ctgcctccga tcatcagact 600 gtgccgcagg gctgtgttgt gcaagacact tctggtccaa gatctgtaaa cctgtcctta 660 aagaaggtca ggtgtgcacc aagcacaaac ggaaaggctc ccacgggctg gagatattcc 720 agcgctgtta ctgcggggaa ggcctggctt gcaggataca gaaagatcac catcaagcca 780 gcaattcttc taggctccac acctgccaga gacactaa 818 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human DKK1 forward primer <400> 5 atgatggctc tgggcgcagc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human DKK1 reverse primer <400> 6 ttagtgtctc tgacaagtgt 20 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> Mouse DKK1 forward primer <400> 7 atgatggttg tgtgtgcagc ggc 23 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Mouse DKK1 reverse primer <400> 8 ttagtgtctc tggcaggtg 19

Claims (7)

A method of inhibiting angiogenesis, comprising treating DKK1 (Dickkopf-1) protein or DNA encoding the same to tissue of a mammal other than human. A pharmaceutical composition for the prevention and treatment of diseases caused by angiogenesis, including DKK1 protein as an active ingredient. Pharmaceutical composition for the prevention and treatment of diseases caused by angiogenesis comprising DNA encoding the DKK1 protein as an active ingredient. The pharmaceutical composition according to claim 3, wherein the DNA encoding the DKK1 protein is provided in a non-viral vector or a viral vector. 5. The pharmaceutical composition of claim 4, wherein the non-viral vector comprises a plasmid that can be expressed in animal cells. The pharmaceutical composition according to claim 4, wherein the viral vector comprises an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a lentiviral vector or a herpes simplex virus vector. According to claim 2, The disease caused by angiogenesis is angiogenesis-mediated hemangioma, angiofibroma, angioplasty, arteriosclerosis, angiogenesis, edematous sclerosis, corneal graft angiogenesis, neovascular glaucoma, diabetic retinopathy, neovascularization Corneal disease caused by blood vessels, degeneration of spots, pterygium, retinal degeneration, posterior capsular fibrosis, granular conjunctivitis, degenerative group, retinopathy of premature infants, arthritis, psoriasis, capillary dilatation, purulent granulomas, Alzheimer's disease, obesity, seborrheic dermatitis, acne And a pharmaceutical composition for the prevention or treatment of diseases caused by one or more angiogenesis selected from the group consisting of cancer diseases including solid cancer.

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