MIN, Jeong Ki (#106-302, Grand Apt. Seoksa-dong, Chuncheon-si, Gangwon-do 200-765, KR)
YUN, Chae Ok (#301, Bien-Etro 86-5, Banpo-dong, Seocho-gu, Seoul 137-040, KR)
KIM, Young Myeong (#105-503, Hyundai 1-cha Apt. Toegye-dong, Chuncheon-si, Gangwon-do 200-753, KR)
KWON, Young Guen (#101-102, Dogok-rexle Apt. 527, Dogok-dong, Gangnam-gu, Seoul 135-506, KR)
MIN, Jeong Ki (#106-302, Grand Apt. Seoksa-dong, Chuncheon-si, Gangwon-do 200-765, KR)
YUN, Chae Ok (#301, Bien-Etro 86-5, Banpo-dong, Seocho-gu, Seoul 137-040, KR)
KIM, Young Myeong (#105-503, Hyundai 1-cha Apt. Toegye-dong, Chuncheon-si, Gangwon-do 200-753, KR)
| Claims
[I] A method for inhibiting angiogenesis in a mammal, comprising the step of administering an effective amount of DKKl (Dickkopf-1) protein or DKKl protein-encoding DNA to mammal.
[2] The method according to claim 1, wherein said DNA is administered using by viral vector or non- viral vector.
[3] The method according to claim 2, wherein said viral vector is adenovirus vector, adeno-associated virus vector, retrovirus vector, lentivirus vector or herpes simplex virus vector.
[4] The method according to claim 2, wherein said non- viral vector comprises the plasmid which can be expressed in animal cells.
[5] The method according to claim 1, wherein said DKKl protein is represented by
SEQ ID NO: 1.
[6] The method according to claim 1, wherein said DNA is represented by SEQ ID
NO: 2.
[7] A use of a DKKl protein or DKKl protein-encoding DNA for manufacture of medicines employed for treating or preventing the diseases caused by the angiogenesis.
[8] A pharmaceutical composition comprising DKKl protein or DKKl protein- encoding DNA as an active ingredient in an effective amount to treat and prevent the diseases caused by angiogenesis.
[9] The pharmaceutical composition of according to claim 8, wherein said DKKl protein is represented by SEQ ID NO: 1.
[10] The pharmaceutical composition of according to claim 8, wherein said diseases caused by angiogenesis is hemangioma, hemangiofibroma, vascular malformation, arteriosclerosis, vascular adhesion, edematous sclerosis, corneal graft neovascularization, neovascular glaucoma, diabetic retinopathy, corneal neovascularization, macular degeneration, pterygium, retinal degeneration, retrolental fibroplasias, trachoma, arthritis, psoriasis, telangiectasia, pyogenic granuloma, seborrhoic dermatitis, acne, alzheimer s disease, obesity or tumor.
[II] A method for treating or preventing the diseases caused by the angiogenesis, wherein said method comprises administering a therapeutically effective amount of a DKKl protein or DKKl protein-encoding DNA into the mammal suffering with the diseases caused by angiogenesis.
[12] A health care food comprising a DKKl protein or DKKl protein-encoding DNA as an active ingredient for preventing and improving the diseases caused by the angiogenesis. [13] The health care food according to claim 12, wherein said health food is provided as pill, powder, granule, tablet, chewing table, capsule or beverage type. |
Description
A METHOD FOR INHIBITING ANGIOGENESIS USING DKKl AND COMPOSITION COMPRISING THE SAME
Technical Field
[1] The present invention relates to a method for inhibiting angiogenesis using by DKKl protein and the composition comprising the same.
[2]
Background Art
[3] Angiogenesis is a process by which new capillary blood vessels are formed. This process rarely occurs under normal biological condition but it always accompanied by embryogenesis, corpus luteum formation and wound healing. Particularly, angiogenesis plays an important role in tumor metastasis (Folkman J and Klagsburn M, Science, 235(4787). pp.442-447, 1987).
[4] Angiogenesis procedure consists of four steps, i.e., the 1 st step is the dissociation of capillary basal lamina by the action of protease enzyme caused by the stimulation of angiogenic factors, the 2" step is the migration and proliferation of blood endothelial cells, the 3 r step is the formation of capillary tubes due to the differentiation of blood endothelial cell, and the 4 step is the reconstruction of new capillary blood vessels.
[5] There have been reported till now that lots of pathological diseases are caused by the disorder of angiogenesis control or by rampant capillary growth, for example, an- giogenesis-involved diseases such as hemangioma, hemangiofibroma, vascular malformation, and so on; cardiovascular diseases such as arteriosclerosis, vascular ad hesion, edematous sclerosis and so on; ophthalmic diseases such as corneal graft neovascularization, neovascular glaucoma, diabetic retinopathy, corneal neovascularization, macular degeneration, pterygium, retinal degeneration, retrolental fibroplasias, trachoma and so on; chronological inflammatory disease such as arthritis and so on; dermatological disease such as psoriasis, telangiectasia, pyogenic granuloma, seborrhoic dermatitis, acne and so on; alzheimer's disease, obesity and tumor etc (D'Amato RJ and Adamis AP, Ophthalmology, 102(91. pp.1261- 1262, 1995; Arbiser JL, J. Am. Acad. Dermatol, 34(3). pp.486-497, 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).
[6] Angiogenesis is accompanied with many genetic changes during the differentiation of vascular endothelial cell. Accordingly, recently, many researches have been focused on the regulation of angiogenesis through the control of differentiation-controlling genes and a method for inhibiting angiogenesis. However, it has been reported that
already-known inhibitors of angiogenesis have various side effects such as the damage of vessel tissues, limit to obtain desired efficacy etc. Accordingly, there has been still needed to develop new method for treating angiogenesis with safe and desirable efficacy till now.
[7] DKKl, a repressor protein of Wnt protein is one of the Dickkopf family members and has been firstly reported that it plays an important role in forming the head of amphibian, Xenopus (Glinka A et al., Nature, 391(6665 * ). pp.357-362, 1998). It has two specific cysteine-rich domains and is divided into various lengths of connection regions. Particularly, the protein belonged to Dickkopf family highly conserves cystein-2 region between the family members as well as 10 cysteines (Krupnik VE et al., Gene, 238m. pp.301-313, 1999).
[8] There have been reported that DKKl is involved with the degeneration of neuronal cells in brain, repression of the growth and differentiation of melanocytes, and regulation of cell cycle etc (Caricasole A et al., J. Neurosci., 24(26). pp.6021-6027, 2004; Yamaguchi Y et al., /. cell Biol, 165(21 pp.275-85, 2004; Gregory CA. et al., J. Biol. Chem., 278(301 pp.28067-28078, 2003).
[9] However, there has been not reported or disclosed on the inhibiting angiogenesis- inhibiting activity of DKKl in any of above cited literatures, the disclosures of which are incorporated herein by reference.
[10]
Disclosure of Invention Technical Problem
[11] Accordingly, the present inventors of the present invention have intensively studied to find several differentiation-control genes in endothelial cell and new effective method for inhibiting angiogenesis finally, they have found that DKKl shows potent inhibiting effect on angiogenesis and thus the protein could be useful in treatment and prevention of angiogenesis-involved diseases.
[12]
Technical Solution
[13] In accordance with the present invention, the present invention provides a method for inhibiting angiogenesis in a mammal, comprising the step of administering to mammal an effective amount of DKKl (Dickkopf- 1) protein or DKKl protein-encoding DNA.
[14] The term "DKKl protein-encoding DNA" disclosed herein is administered to mammal using by viral vectors or non- viral vectors.
[15] The term "non-viral vectors" disclosed herein comprises the plasmid which can be expressed in animal cells.
[16] The term "viral vectors" disclosed herein comprises adenovirus vector, adeno-
associated virus vector, retrovirus vector, lentivirus vector or herpes simplex virus vector.
[17]
[18] It is another object of the present invention to provide a pharmaceutical composition comprising DKKl protein or DKKl protein-encoding DNA as an active ingredient in an effective amount to treat and prevent the diseases caused by angiogenesis.
[19] It is the other object of the present invention to provide a use of a DKKl protein or
DKKl protein-encoding DNA for manufacture of medicines employed for treating or preventing the diseases caused by the angiogenesis.
[20] It is the other object of the present invention to providea method for treating or preventing the diseases caused by the angiogenesis, wherein said method comprises administering a therapeutically effective amount of a DKKl protein or DKKl protein- encoding DNA into the mammal suffering with the diseases caused by angiogenesis.
[21]
[22] Also, it is the other object of the present invention to provide a health care food composition comprising DKKl protein or DKKl protein-encoding DNA as an active ingredient in an effective amount to prevent and alleviate the diseases caused by angiogenesis.
[23] The term "disease caused by angiogenesis" disclosed herein comprises hemangioma, hemangiofibroma, vascular malformation, arteriosclerosis, vascular adhesion, edematous sclerosis, corneal graft neovascularization, neovascular glaucoma, diabetic retinopathy, corneal neovascularization, macular degeneration, pterygium, retinal degeneration, retrolental fibroplasias, trachoma, arthritis, psoriasis, telangiectasia, pyo genie granuloma, seborrhoic dermatitis, acne, alzheimer's disease, obesity or tumor in human or mammal.
[24] The term "DKKl protein" disclosed herein comprises the amino acid represented by
SEQ ID NO: 1.
[25] The term "DKKl protein-encoding DNA" disclosed herein comprises the gene represented by SEQ ID NO: 2.
[26] Above described DKKl sequences is not limited to the DKKl sequences of mammal but comprise all the DKKl in mammals.
[27] Above described DKKl protein comprises the DKKl protein isolated from the tissues of mammal and recombinant DKKl proteins.
[28]
[29] The inventive DKKl protein and the gene encoding the same may be prepared in accordance with the following preferred embodiment.
[30]
[31] Hereinafter, the present invention is described in detail.
[32]
[33] For the present invention, the above-described DKKl protein and the gene encoding the same can be prepared by following procedure:
[34] The entire RNA purified from HUVEC is reverse-transcribed to obtain complimentary DNA at the 1 st step; PCR was performed to obtain complimentary DNA as a template and DKKl primers, preferably, the DKKl primers represented by SEQ ID NO: 5 and SEQ ID NO: 6 and amplified DKKl genes at the 2 nd step; the DKKl genes prepared from 2 ™ step are treated with restriction enzyme, preferably EcoRI and Xhol, cloned into protein purifying plasmid, preferably pcDNA-His vector, to obtain plasmid at the 3 r step; the plasmid is transformed with expression cell lines, preferably, human renal cell line 293 (ATCC, USA) using gene delivery reagent, preferably, Lipofectamin (Invitrogen, USA) at the 4 step; the transformed cells are selected using by selective-transforming cell isolating reagents, preferably, G418 (Sigma, USA) and mass-cultured in medium at the 5 step; and the medium is performed to column chromatography, preferably, nickel column (Ni-NTA agarose, Qiagen, USA) to purify DKKl proteins secreted from the medium and the expression of recombinant DKKl protein is confirmed at the 6 step.
[35] The DKKl proteins prepared by the above-described step inhibit the tube formation in HUVEC and DKKl over-expressed cell lines transformed with DKKl protein- encoding DNA, the blood vessel sprouting in arterial circle tissues of transformed mice with DKKl protein encoding DNA, and the vascular development in the embryo of the mice. Therefore, it has been confirmed that the proteins of the present invention showed potent angiogenesis-inhibiting activity. Additionally, not only full-length of the DKKl but also fragment of DKKl show similar angiogenesis-inhibiting activity each other.
[36]
[37] It is another object of the present invention to provide a pharmaceutical composition comprising DKKl protein or DKKl protein-encoding DNA prepared by the above- described preparation method as an active ingredient in an effective amount to treat and prevent the diseases caused by angiogenesis.
[38] It is the other object of the present invention to provide a use of a DKKl protein or
DKKl protein-encoding DNA prepared by the above-described preparation method for manufacture of medicines employed for treating or preventing the diseases caused by the angiogenesis.
[39] It is the other object of the present invention to provide a method for treating or preventing the diseases caused by the angiogenesis, wherein said method comprises administering a therapeutically effective amount of a DKKl protein or DKKl protein- encoding DNA prepared by the above-described preparation method into the mammal
suffering with the diseases caused by angiogenesis.
[40]
[41] In accordance with another aspect of the present invention, there is provided a use of a DKKl or DKKl protein-encoding DNA prepared by the above described preparation method for inhibition of angiogenesis.
[42]
[43] The inventive composition for treating the disease caused by the angiogenesis may comprise the above-described DKKl protein or DKKl protein-encoding DNA as 0.1 ~ 50% by weight based on the total weight of the composition.
[44]
[45] The inventive composition may additionally comprise conventional carrier, adjuvants or diluents in accordance with a using method well known in the art. It is preferable that said carrier is used as appropriate substance according to the usage and application method, but it is not limited. Appropriate diluents are listed in the written text of Remington's Pharmaceutical Science (Mack Publishing co, Easton PA).
[46]
[47] Hereinafter, the following formulation methods and excipients are merely exemplary and in no way limit the invention.
[48]
[49] The composition according to the present invention can be provided as a pharmaceutical composition containing pharmaceutically acceptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient, for example, human or mammal such as mouse, rat, livestock, etc by employing any of the procedures well known in the art.
[50] Pharmaceutical formulations containing present composition may be prepared in any form, such as oral dosage form (powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule), or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like), or injectable preparation (solution, suspension, emulsion).
[51]
[52] For example, the compositions of the present invention can be dissolved in oils,
propylene glycol or other solvents that are commonly used to produce an injection. Suitable examples of the base or carrier in the injection include various salt mixture such as physiological saline, inorganic salt or the mixture thereof; sugar solution such as mannitol, lactose, dextran etc; amino acid such as glycine, arginine etc; polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, organic acid solution, salt solution, or the mixture thereof etc, but are not limited to them. The injectable preparation of the present invention may be prepared by adding conventional additives in injection, for example, osmotic controller, pH controller, vegetable oil, lecithin, surfactant such as non-ionic surfactant to the above-described base in order to make appropriate formulation such as solution, suspension, colloidal solution etc. In case of solid composition of the present invention, the composition is dissolved in sterilized base prior to use in genetic therapy and liquid composition of the present invention may be used directly without particular treatment.
[53]
[54] For topical administration, the composition of the present invention can be formulated in the form of ointments and creams.
[55]
[56] The composition of the present invention in pharmaceutical dosage forms may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active ingredients.
[57]
[58] The DKKl protein-encoding DNA disclosed herein to be supplied into the affected part may be used in the inserted form of vectors, for example adenovirus vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, herpes simplex virus vector or plasmid expressed in mammal cells.
[59]
[60] The desirable dose of the inventive composition varies depending on the condition such as age, sex etc and the weight of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to topically administer into the affected part at the amount ranging from 0.001 to 100mg/kg, preferably, 0.1 to 100mg/kg by weight/week one or more than once a week of the inventive composition of the present invention through catheter after surgery. The dose may be administered in single or divided into several times per day or week.
[61]
[62] It is still another object of the present invention to provide a health care food comprising DKKl protein or DKKl protein-encoding DNA as an active ingredient for
preventing and improving diseases caused by the angiogenesis.
[63]
[64] The above-described composition therein can be added to food, additive or beverage, wherein, the amount of the above described protein or DNA in food or beverage may generally range from about 0.01 to 95w%, preferably 1 to 80w% of total weight of food for the health care food composition.
[65]
[66] The present invention provides a composition of the health care beverage comprising a DKKl protein or DKKl protein-encoding DNA for preventing and alleviating the disease caused by the angiogenesis in mammal.
[67]
[68] To develop for health care food, examples of addable food comprising the above- described protein or DNA of the present invention are various food, beverage, gum, vitamin complex, health improving food and the like, and can be used as powder, granule, tablet, chewing tablet, capsule or beverage etc.
[69]
[70] Inventive composition of the present invention has no toxicity and adverse effect therefore they can be used with safe.
[71]
[72] The above-described composition therein can be added to food, additive or beverage, wherein, the amount of the above-described DKKl protein or DKKl protein-encoding DNA in food or beverage may generally range from about 0.01 to 80w/w%, preferably 0.01 to 15w/w% of total weight of food for the health food composition and 0.02 to 5g, preferably 0.3 to Ig on the ratio of 100ml of the health care beverage composition.
[73]
[74] Providing that the health care beverage composition of present invention contains the above-described DKKl protein or DKKl protein-encoding DNA as an essential component in the indicated ratio, there is no particular limitation on the other liquid component, wherein the other component can be various deodorant or natural carbohydrate etc such as conventional beverage. Examples of aforementioned natural carbohydrate are monosaccharide such as glucose, fructose etc; disaccharide such as maltose, sucrose etc; conventional sugar such as dextrin, cyclodextrin; and sugar alcohol such as xylitol, and erythritol etc. As the other deodorant than aforementioned ones, natural deodorant such as taumatin, stevia extract such as levaudioside A, gly- cyrrhizin et al., and synthetic deodorant such as saccharin, aspartam et al., may be useful favorably. The amount of above described natural carbohydrate is generally ranges from about 1 to 20 g, preferably 5 to 12 g in the ratio of 100 Cl of present beverage composition.
[75]
[76] The other components than aforementioned composition are various nutrients, a vitamin, a mineral or an electrolyte, synthetic flavoring agent, a coloring agent and improving agent in case of cheese chocolate et al., pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent used in carbonate beverage et al. The other component than aforementioned ones may be fruit juice for preparing natural fruit juice, fruit juice beverage and vegetable beverage, wherein the component can be used independently or in combination. The ratio of the components is not so important but is generally range from about 0 to 20 w/w % per 100 w/w % present composition. Examples of addable food comprising aforementioned protein or DNA therein are various food, beverage, gum, vitamin complex, health improving food and the like.
[77]
[78] The inventive composition may additionally comprise one or more than one of organic acid, such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid; phosphate, such as phosphate, sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate; natural anti-oxidants, such as polyphenol, catechin, α- tocopherol, rosemary extract, vitamin C, green tea extract, licorice root extract, chitosan, tannic acid, phytic acid etc.
[79]
[80] The above-described inventive DKKl protein may be 20 to 90 % high concentrated liquid, power, or granule type.
[81]
[82] Similarly, the above-described DKKl protein or DKKl protein-encoding DNA can comprise additionally one or more than one of lactose, casein, dextrose, glucose, sucrose and sorbitol.
[83]
[84] Inventive DKKl protein or DKKl protein-encoding DNA of the present invention has no toxicity and adverse effect therefore; they can be used with safe.
[85]
[86] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.
[87] The present invention in more specifically explained by the following examples.
However, it should be understood that the present invention is not limited to these examples.
Advantageous Effects
[89] As described in the present invention, the DKKl showed potent inhibitory effect on the tube formation of HUVEC, sprouting of arterial circle tissues and vascular development of mice embryo. Therefore, it can be used as the therapeutics or functional health food for treating and preventing diseases caused by the angiogenesis. Brief Description of the Drawings
[90] The above and other objects, features and other advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;
[91]
[92] Fig. 1 shows the differentiation procedure of the HUVEC (Human umbilical vein endothelial cell) on Matrigel,
[93] Fig. 2 represents the expression of DKKl gene on differentiation of the HUVEC according to each stage,
[94] Fig. 3 presents the confirmed result of the expression and purification of DKKl recombinant,
[95] Fig. 4 shows the inhibition of tube formation on HUVEC according to the concentration of treated DKKl recombinant,
[96] Fig. 5 represents the produced result of DKKl expression cell line and DKKl repression cell line using by lentiviruses,
[97] Fig. 6 shows the comparison result of the tube formation on DKKl expression and repression cell lines,
[98] Fig. 7 presents the vector diagram for producing DKKl -transgenic mouse,
[99] Fig. 8 represents the confirmed result of DKKl -transgenic mouse by DNA amplification,
[100] Fig. 9 shows the comparison results of mouse size of normal and DKKl -transgenic mouse,
[101] Fig. 10 presents the repression of the sprouting of endothelial cells from aorta of DKKl -transgenic mouse,
[102] Fig. 11 represents the comparison results of angiogenesis in embryo of normal mouse and DKKl -transgenic mouse,
[103] Fig. 12 shows the comparison results of angiogenesis in the head region of embryo,
[ 104] Fig .13 presents the growth of descending aorta in embryo of normal mouse and DKKl -transgenic mouse,
[105] Fig. 14 presents the growth of segmental vessel in embryo of normal mouse and DKKl -transgenic mouse. Best Mode for Carrying Out the Invention
[106] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.
[107] The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.
[108]
Mode for the Invention
[109] The following Reference Example, Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.
[HO]
[111] Reference Example 1. Cultivation of HUVEC
[112]
[113] HUVECs (Human umbilical vein endothelial cell) were isolated from the umbilical cords obtained from gynecology department of Yonsei University Hospital according to following process. After washing veins with Cord buffer (0.2% glucose phosphate buffered saline), 5D of 0.2% type I collagenase (Sigma-Aldrich Co., MO, USA) was added to the veins and the veins were left alone at 37°C for 5 min. After adding 2OD of cord buffer to veins at room temperature, the vein cells separated from the opposite end were collected. The cord buffer was added to the veins again to react at 37°C. The collected human umbilical vein endothelial cells (HUVECs) was washed and poured to the coated T75 flask used for tissue culture with 0.1% of gelatin. The cells were cultured in EGM™-2 complete medium (Cambrex, MD, USA) in 5% CO 2 culture incubator at 37°C and when the cells became to confluent phase, the cells were separated from trypsin-EDTA solution. The cells of 3-4 passage obtained from the above process were used in the experiment.
[114]
[115] Reference Example 2. Preparation of the recombinant protein of human DKKl
[116]
[117] Total RNA was isolated from vascular endothelial cells using TRIzol reagent
(Invitrogen, USA), performed to reverse transcription using by oligo (dT) primer and reverse transcriptase (Promega, USA). Polymerization Reaction (PCR cycles) was performed 30 times using by polymerase (Stratagen, USA) as follows; pre- denaturation at 94°C for 5min; denaturation at 94°C for 30sec; annealing at 50 0 C for 30sec using by DKKl primers represented by SEQ ID NO: 5 and 6; extension at 72°C for 30sec. After cloning the amplified DKKl gene into pcDNA-His vector by treating with EcoRI and Xhol, the cloned plasmid was transformed into human embryonic
kidney cell line 293 (ATCC, USA) using by Lipofectamin (Invitrogen, USA). The transformed cells were selected using by G418 reagent (Sigma, USA), a selective transformation cell isolating agent and selected cells were cultured in medium on a large scale. The secreted DKKl proteins in the medium were purified with nickel column (Ni-NTA agarose, Qiagen, USA) and the expression of recombinant DKKl protein was confirmed ( See, Fig. 3).
[118]
[119] Reference Example 3. Preparation of DKKl over-expressed and repressed cell lines using by lentivirus vector
[120]
[121] The DKKl-recombinated viruses obtained from the over-expressed and repressed cell lines of DKKl prepared by transforming cloned plasmid to virus production cell lines using by lentivirus, were purchased from Macrogen Inc (South Korea). 48hours after the addition of DKKl virus to HUVECs prepared in Reference Example 2, the isolated RNA from the cells was performed to reverse-transcription and polymerization to confirm the expression phase of DKKl mRNA as follows: total RNA was isolated using by TRIzol reagent (Invitrogen, USA), performed to reverse transcription using by oligo (dT) primer and following PCR cycles were repeated 30 times using by reverse transcriptase (Stratagen, USA); pre-denaturation at 94°C for 5min using by polymerase (Stratagen, USA), denaturation at 94°C for 30sec, annealing at 50°C for 30sec using by primers and extension at 72°C for 30sec.
[122] As shown in Fig. 5, it has been confirmed that the over-expressed cell lines and repressed cell lines of DKKl were well-produced.
[123]
[ 124] Reference Example 4. Preparation of DKKl-transgenic mouse
[125]
[126] The DKKl over expression mouse was prepared using the Tie2 transcription regulatory region activated in only vascular endothelial cells to determine the effect of DKKl gene on angiogenesis in vivo (Schlaeger TM et al., Proc. Natl. Acad. ScL USA, 94(7). pp.3058-3063, 1997).
[127] As shown in Fig. 7, mouse DKKl gene represented by SEQ ID NO: 4 was treated with Hindm and NotI(NEB, England) and cloned into Psp vector (Clontech, USA). The cloned plasmid was treated with SaII(NEB, England) to prepare DNA fragments and the prepared DNA fragments were injected into the ovules isolated from the mouse (C57BL6, Orient Inc, Korea) of which ovulation had been stimulated by gonadotropin releasing-hormone (Sigma, USA) to induce transduction, and then, the DKKl-transgenic ovules were implanted on surrogate mother mouse after fertilization. The tail of the mouse born after the 21 st fertilization was cut, treated with proteinase K
(Sigma, USA) to isolate DNA, and the isolated DNA was amplified by PCR [(pre-denaturation at 94°C for 5min, denaturation at 94°C for 30sec, annealing at 55°C for 30sec and extension at 72°C for 30sec)x30 cycles and post-extension at 72°C for lOmin].
[128] As shown in Figs. 8 and 9, it has been confirmed that the transduction on mouse was well-processed and the difference between the size of normal and DKKl -transgenic mice is significant, of which result is attributed to the abnormal development of blood vessels.
[129]
[130] Experimental Example 1. DKKl expression spectra during the differentiation of human umbilical vein endothelial cells
[131]
[132] 250D of Matrigel (Collaborative Biomedical Products, USA; density: 1OD proteins/D) was added to the well plates with the diameter of 16D and performed to polymerization at 37°C for 30min. The HUVECs prepared in Reference Example 1 were cultured in M 199 growth medium (Invitrogen, USA) containing 20 % (v/v) fetal bovine serum (FBS, Hyclone, USA), 100 units/D of penicillin (Invitrogen, USA), 10D/D of streptomycin (Invitrogen, USA), 3ng/D of bFGF (basic fibroblast growth factor; Upstate Biotechnology, USA) and 5 units/D of heparin (Sigma, USA) and trypsin was added th ereto to obtain cultured cells. The cells were suspended in the growth medium and spread onto Matrigel layer in the concentration of 2x10 5 cells/well to induce the differentiation of cells ( See, Fig. 1).
[133] As shown in Fig. 1, differentiation consists of 3 steps; the 1 st step is the beginning of differentiation used as control group, the 2 nd step is the formation of blood vessel-like structure due to cell transfer and the 3 step is the completion of blood vessel-like structure formation.
[134] After isolating RNA from the cells in each step using by TRIZOL solution
(Invitrogen, USA), the isolated RNA was performed to reverse transcription using by the primers represented by SEQ ID NO: 5 and SEQ ID NO: 6, and amplification according to the process disclosed in Reference Example 4 ( See Fig. 2).
[135] As shown in Fig. 2, it has been confirmed that the expression of DKKl genes was significantly reduced during the tube formation, of which result confirmed that the DKKl may function as a negative regulator of tube formation.
[136]
[137]
[138] Experimental Example 2. The effect of DKKl on the tube formation in human umbilical vein endothelial cells
[139]
[140] 250D of Matrigel (Collaborative Biomedical Products, USA; density: 1OD proteins/D) was added to the well plates with the diameter of 16D and performed to polymerization at 37°C for 30min. The HUVECs prepared in Reference Example 1 were cultured in M 199 growth medium (Invitrogen, USA) containing 20 % (v/v) fetal bovine serum (FBS, Hyclone, USA), 100 units/D of penicillin (Invitrogen, USA), 10D/D of streptomycin (Invitrogen, USA), 3ng/D of bFGF (basic fibroblast growth factor; Upstate Biotechnology, USA) and 5 units/D of heparin (Sigma, USA) and trypsin was added thereto to obtain cultured cells. The cells were suspended in the growth medium and spread onto Matrigel layer in the concentration of 2x10 cells/well to induce the differentiation of cells ( See, Fig. 4). 50ng/D and lOOng/D of DKKl was treated thereto and then the cells were cultured for 20 hours. The rate of tube formation was measured by an optical microscopy (ZEISS, Germany) and the group which was not treated with DKKl was regarded as a negative control group.
[141] As shown in Fig. 4, the result demonstrates that the area and connection rate of formed tubes in DKKl treatment group were significantly reduced according to the treated concentration of DKKl treated group compared with negative control group.
[142]
[143]
[144] Experimental Example 3. The effect of DKKl on the tube formation in DKKl over-expressed and repressed cell lines
[145]
[146] 250D of Matrigel (Collaborative Biomedical Products, USA; density: 1OD proteins/D) was added to the well plates with the diameter of 16D and performed to polymerization at 37°C for 30min. TheDKKl over-expressed and repressed cell lines prepared in Reference Example 3 were cultured in M 199 growth medium (Invitrogen, USA) containing 20 % (v/v) fetal bovine serum (FBS, Hyclone, USA), 100 units/D of penicillin (Invitrogen, USA), 10D/D of streptomycin (Invitrogen, USA), 3ng/D of bFGF (basic fibroblast growth factor; Upstate Biotechnology, USA) and 5 units/D of heparin (Sigma, USA) and trypsin was added thereto to obtain cultured cells. The cells were suspended in the growth medium and spread onto Matrigel layer in the concentration of 2x10 cells/well to induce the differentiation of cells ( See. Fig. 6).
[147] The rate of tube formation was measured by an optical microscopy (ZEISS,
Germany) and the group which was infected with lentivirus was regarded as a negative control group.
[148] As shown in Fig. 6, the tube formation in DKKl over-expressed cell line was repressed whereas that in DKKl repressed cell line was stimulated.
[149]
[150]
[151] Experimental Example 4. The effect of DKKl on the sprouting of endothelial cells from aorta of DKKl-transgenic mouse
[152]
[153] The aorta which had been isolated from the back region of DKKl-transgenic mouse prepared in Reference Example 4 and 6-weeks-old normal mouse were cut into the size of lmm and the arterial circle tissues were laid on 48-well plates coated with 11OD of matrigel. The well was sealed again with 4OD of Matrigel and HUVEC culture medium (SFM, Invitrogen, USA) was added to each well to the extent the final volume of each well reached to 200D. After 5 days, the number of sprout formed from each circle was counted and the rate of sprout in DKKl-transgenic mouse group was compared with that in control group ( See. Fig. 10). The rate of sprout was scored by dividing the sprout into five parts according to following criteria; 5 points was assigned in case that all the 5 parts were sprouted, 0 points was assigned in case that none was sprouted.
[154] As can be seen in Fig. 10, the result demonstrates that the sprouting of the arterial circle tissues in DKKl-transgenic mouse was significantly repressed compared with that in normal mouse.
[155]
[156]
[157] Experimental Example 5. The effect of DKKl on the development of blood vessel in the embryo of DKKl-transgenic mouse
[158]
[159] The embryos delivered from the 9 to 10 pregnant normal and DKKl-transgenic mice were fixed with 4% paraformaldehyde for a day and stained with the antibody of von Willebrand Factor (V wf) (Chemicon, USA) specifically expressed only on vascular endothelial cells to observe the development of blood vessel by the method disclosed in literature (Sadler J. E., J. Thromb. Haemost., 3£8), ppl702-1709, 2005).
[160] As can be seen in Fig. 11, the result demonstrates that the development of blood vessel in the embryo of DKKl-transgenic mice was generally repressed.
[161] To confirm further, the formed vessels of the head region of embryo were magnified (x200) using by optical microscope (Olympus, Japan). As can be seen in Fig. 12, the result demonstrates that the angiogenesis of the embryo head region of DKKl-transgenic mice was significantly repressed compared with that of normal mice.
[162] To verify the effect of DKKl on the angiogenesis of other regions, the development of descending aorta and segmental vessel in the embryos prepared by the above- described process was determined as follows. The embryos were frozen at -70 0 C after the dehydration with 30% sucrose solution for a day and the frozen tissues were cut into a thickness of 1OD to adhere on slide. The tissue was performed to blocking with goat serum for 2hrs and antibodies against vWF and α-SMA were treated therewith at
4°C for a day as primary antibodies. The next day, the secondary antibody attached with green fluorescent reagent for staining endothelial cell and red fluorescent reagent for staining smooth muscle cells neighboring with the vessels was treated therewith for I hour and then the cells were observed by a fluorescence microscope.
[163] As shown in Fig. 13 and 14, the result demonstrates that the development of descending aorta and segmental vessel in the embryos of DKKl -transgenic mice was significantly repressed.
[164]
[165]
[166] Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows.
[167]
[168] Preparation of injection
[169] DKKl protein lOOmg
[170] Sodium metabisulfite3.0mg
[171] Methyl parabenθ.8mg
[172] Propyl parabenO.1 mg
[173] Distilled water for injectionoptimum amount
[174] Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2D ample and sterilizing by conventional injection preparation method.
[175]
[176] Preparation of powder
[177] DKKl protein 500mg
[178] Corn Starch lOOmg
[179] Lactose lOOmg
[180] TalclOmg
[181] Powder preparation was prepared by mixing above components and filling sealed package.
[182]
[183] Preparation of tablet
[ 184] DKKl protein 200mg
[185] Corn StarchlOOmg
[186] Lactose lOOmg
[187] Magnesium stearate optimum amount
[188] Tablet preparation was prepared by mixing above components and entabletting.
[189]
[190] Preparation of capsule
[191] DKKl protein 1 OOmg
[192] Lactose50mg
[193] Corn starch50mg
[194] Talc2mg
[195] Magnesium stearateoptimum amount
[196] Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method. [197]
[198] Preparation of liquid
[199] DKKl protein lOOOmg
[200] Sugar 2Og
[201] Polysaccharide20g
[202] Lemon flavor20g
[203] Liquid preparation was prepared by dissolving active component, and then filling all the components in IOOOD ample and sterilizing by conventional liquid preparation method. [204]
[205] Preparation of health food
[206] DKKl protein lOOOmg
[207] Vitamin mixtureoptimum amount
[208] Vitamin A acetate70mg
[209] Vitamin El. Omg
[210] Vitamin B 0.13mg
[211] Vitamin B 2 0.15mg
[212] Vitamin B60.5mg
[213] Vitamin B 120.2mg
[214] Vitamin ClOmg
[215] BiotinlOmg
[216] Amide nicotinic acid 1.7mg
[217] Folic acid50mg
[218] Calcium pantothenic acid 0.5mg
[219] Mineral mixtureoptimum amount
[220] Ferrous sulfatel.75mg
[221] Zinc oxide0.82mg
[222] Magnesium carbonate25.3mg
[223] Monopotassium phosphatel5mg
[224] Dicalcium phosphate 55mg
[225] Potassium citrate90mg
[226] Calcium carbonate lOOmg
[227] Magnesium chloride24.8mg
[228] The above mentioned vitamin and mineral mixture may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention.
[229]
[230] Preparation of health beverage
[231] DKKl protein lOOOmg
[232] Citric acidlOOOmg
[233] Oligosaccharide lOOg
[234] Apricot concentration2g
[235] Taurinelg
[236] Distilled water900D
[237] Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85 0 C for 1 hour, filtered and then filling all the components in IOOOD ample and sterilizing by conventional health beverage preparation method.
[238]
[239] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.
[240]
Industrial Applicability
[241] As described in the present invention, the DKKl showed potent inhibitory effect on the tube formation of HUVEC, sprouting of arterial circle tissues and vascular development of mice embryo. Therefore, it can be used as the therapeutics or functional health food for treating and preventing diseases caused by the angiogenesis. Sequence Listing
[242] SEQ ID NO: 1 is amino acid sequence of human DKKl , SEQ ID NO:2 is DNA sequence of human DKKl, SEQ ID NO:3 is amino acid sequence of mouse DKKl, SEQ ID NO:4 is DNA sequence of mouse DKKl, SEQ ID NO:5 is forward primer sequence of human DKKl, SEQ ID NO:6 is reverse primer sequence of human DKKl, SEQ ID NO:7 is forward primer sequence of mouse DKKl, and SEQ ID NO:8 is reverse primer sequence of mouse DKKl.