KR20110117601A - Method for screening a compound associated with angiogenesis - Google Patents

Abstract

The present invention provides a method for searching for compounds associated with angiogenesis.

Description

Method for screening a compound associated with angiogenesis

The present invention relates to a method for searching for compounds associated with angiogenesis.

Low-density lipoprotein receptor-related protein 6: LRP6 is a protein encoded by the LRP6 gene in humans. The gene and amino acid sequences of LRP6 are known. For example, in humans and mice, gene and amino acid sequences are represented by NM_002336 and NP_002327; And NM_008514 and NP_032540. LDL receptors are transmembrane surface protein involved in receptor mediated endocytosis of lipoproteins and protein ligands. LRP6, together with the receptor or Frizzled, acts as a coreceptor for Wnt, delivering a conventional Wnt / beta-catenin signaling cascade. Through interaction with conventional Wnt / beta-catenin signaling cascades, LRP6 is known to be involved in the differentiation, proliferation, migration and development of many cancer types. LRP6 is subjected to gamma-secretase dependent regulated intramembrane proteolysis (RIP), but the exact location of the cleavage site is unknown.

DKK2, an inhibitor of the Wnt protein, has been reported to act as an inhibitor or promoter of Wnt signaling. DKK2 is divided into two cysteine-rich domains and linkage regions of varying lengths. In particular, DKK2 belonging to the Dickkopf family has well preserved cysteine-2 regions between the family members as well as 10 cysteines. DKK2 has been reported to be closely associated with the differentiation of osteoclasts.

Angiogenesis is the process by which new capillaries are formed. This process plays an important role in embryonic formation, corpus luteum formation, wound healing and tumor metastasis. Angiogenesis has been reported to be regulated by various stimulatory and inhibitory factors, such as growth factors, cytokines and lipid metabolism factors. Angiogenic stimulators can be classified into several factors, such as cell growth inducers, cytokines with immunological activity, hormones and lipid products, and the like. However, the stimulator has a problem in clinical application because it acts on vascular endothelial cells as well as other neighboring cells.

Promoting angiogenesis can be used for therapeutic purposes. For example, angiogenic factors can be administered to a subject to promote the formation of blood vessels, thereby treating the ischemic disease of the subject. The angiogenic factors include vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), developmentally regulated developmental endothelial locus-1 (Del-1), hepatocyte growth factor (HGF), platelet derived Endothelial cell growth factor (PD-EGF), angiopoietin and FGF.

However, it has not been reported that fusion with DKK2 or its Fc can promote angiogenesis. In addition, it has not been disclosed that the DKK2 or its Fc and fusion are involved in angiogenesis through LRP6-mediated signaling. In particular, it has not been disclosed that the DKK2 or its Fc and fusions are involved in angiogenesis through LRP6-mediated signaling, independent of WNT / Frizzled mediated signaling.

One embodiment is to provide a method for searching for compounds associated with angiogenesis.

One embodiment of the invention comprises contacting DKK2 with a cell in the presence of a test compound; Detecting the interaction of DKK2 with LRP6; And determining from the detected interactions whether the test compound is a candidate compound associated with angiogenesis.

The method comprises contacting DKK2 with cells in the presence of a test compound. The contact can be by incubating in a liquid medium capable of maintaining the growth of the cells. The contact can be made, for example, by incubating in a liquid medium capable of maintaining the growth of the cells. The medium may be a known medium depending on the cell selected.

The contact may be in the presence of an inhibitor of WNT / Frizzled mediated signaling. By doing so, it is possible to search for substances involved in LRP6 mediated signaling independently of WNT / Frizzled mediated signaling. By searching for a substance involved in LRP6 mediated signaling, a substance involved in angiogenesis can be searched. The inhibitor may be one that can inhibit the interaction of WNT / Frizzled. For example, it may be an antibody or a ligand that specifically binds to at least one of WNT and Frizzled. Or, it may be a decoy compound that specifically binds to at least one of WNT and Frizzled to inhibit the interaction. For example, the decoy compound is a group consisting of soluble Frizzled related protein (sFRP), for example sFRP-1, sFRP-2, sFRP-3, sFRP-4 and sFRP-5 (R & D Systems). It may be selected from.

The contact may be made under conditions that promote a change in the shape of the cell. Conditions for promoting morphological changes of the cells can be used methods known in the art. For example, the contact may be by culturing the cells on a Matrigel-coated medium that promotes morphological changes of the cells. Matrigel is a trade name for gelatinous protein mixtures secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells and is available from BD Biosciences. This mixture is similar to the complex extracellular environment found in many tissues. In this specification, the term "Matrigel" is used interchangeably with gelatinous protein mixture. In the course of the experiment, matrigel is distributed in a cold state (4 ° C.) on a plastic tissue culture container, and when incubated at 37 ° C., the matrigel protein self-assembles to form a thin film covering the container surface. Cells cultured on Matrigel show complex cell behavior that is not observed under laboratory conditions. For example, endothelial cells produce sophisticated spiderweb-like networks on Matrigel coated surfaces but not on plastic surfaces. This network strongly suggests a microvascular capillary system that fills living tissue with blood.

The test compound may include any compound as long as it is a compound to be tested. The test compound may be a single molecule or a polymer. The test compound may be, for example, chemically synthesized or biosynthesized molecule. The biosynthesized molecule can be a protein, nucleic acid or sugar.

The cell may be a cell expressing LRP6. The cell may be an endothelial cell, for example HUVEC. Low-density lipoprotein receptor-related protein 6: LRP6 is a protein encoded by the LRP6 gene in humans. The gene and amino acid sequences of LRP6 are known. For example, in humans and mice, gene and amino acid sequences are represented by NM_002336 and NP_002327; And NM_008514 and NP_032540. LDL receptors are transmembrane surface protein involved in receptor mediated endocytosis of lipoproteins and protein ligands. LRP6, together with the receptor or Frizzled, acts as a coreceptor for Wnt, delivering a conventional Wnt / beta-catenin signaling cascade. Through interaction with conventional Wnt / beta-catenin signaling cascades, LRP6 is known to be involved in the differentiation, proliferation, migration and development of many cancer types. LRP6 is subjected to gamma-secretase dependent regulated intramembrane proteolysis (RIP), but the exact location of the cleavage site is unknown.

The method includes detecting the interaction of DKK2 with LRP6. The interaction can be detected by known methods. The interaction may be to measure the direct binding of DKK2 and LRP6, or to measure the complex formed by the interaction. For example, the detection of the interaction can be made by measuring the amount of DKK2-LRP6 complex. If the amount of the complex is measured, it may be determined that there is a lot of interaction, and if the amount of the complex is measured, it may be determined that the interaction is low.

In addition, the interaction may be to measure the signaling result downstream of the signaling caused by the interaction of DKK2 and LRP6. In this case, when the signal transmission result is increased, it may be determined that the interaction is increased. The downstream signaling result may be one or more selected from the group consisting of APC activation, Asef2 activation and Cdc42 activation. We found that DKK2 interacts with LRP6 to activate Cdc42, via APC / Asef2, independent of Frizzled mediated signaling.

APC (adenomtous polyposis coli) protein is a protein encoded by the APC gene in humans. Mutations in the APC gene can cause colorectal cancer. APC is classified as a tumor suppressor gene. Human APC protein has the amino acid sequence (2843aa) of NCBI Reference Sequence: NP_000029 and may be encoded by the nucleotide sequence of NCBI Reference Sequence: NM_000038. In mice, the APC protein has an amino acid sequence of NCBI Reference Sequence: XP_622559 (2634aa) and may be encoded by the nucleotide sequence of NCBI Reference Sequence: XM_620559. APC proteins are involved in the typical Wnt pathway. A typical Wnt pathway is when the Wnt protein binds to the cell surface receptors of the Frizzled family, which activates the Dishevelled family protein and consequently changes the amount of β-catenin that reaches the nucleus. It is a series of events that take place. Deschveld (DSH) is a key component of membrane-bound Wnt receptor complexes and, when activated by Wnt binding, inhibits a second complex of proteins including axin, GSK-3 and protein APC. The axin / GSK-3 / APC complex usually promotes the proteolysis of β-catenin intracellular signaling molecules. When this "β-catenin destruction complex" is inhibited, the pool of β-catenin in the cytoplasm is stabilized, some β-catenin can enter the nucleus and interact with TCF / LEF family transcription factors Thereby promoting specific gene expression.

Asef2 (APC-stimulated guanine nucleotide exchange factor 2) is an Asef1 homolog with similar N-terminal APC binding regions (ABR) and Src-homology 3 (SH3) domains. Asef1 and Asef2 exchange activity is Cdc42 specific. Asef2's ABR does not function independently, but acts in tandem with the SH3 domain and binds to APC. Asef2 activation requires APC to release ABRSH3 from the C-terminal tail. Asef2 is Genbank accesion No. It may have an amino acid sequence encoded by AK055770.

Thus, the detection of the interaction may be to measure the interaction of Asef2 with APC in the cell. Detection of the interaction may be measuring the amount of APC-Asef2 complex in the cell. Isolation of proteins or protein complexes is known in the art. For example, salting out, precipitation, centrifugation, filtration, electrophoresis, size exclusion chromatography, ion exchange chromatography and affinity chromatography using affinity molecules such as antibodies or ligands can be used. It is also known in the art to identify isolated proteins and their complexes. For example, electrophoresis, western blotting, or chemiluminescent staining may be included. In this case, when the amount of the complex measured is large, it is determined that the interaction of APC and Asef2 is large, and when the amount of the measured complex is small, it may be determined that the interaction of APC and Asef2 is small. In addition, when the interaction between the APC and Asef2 is large, it may be determined that the interaction between DKK2 and LRP6 is large, and when the interaction between the APC and Asef2 is small, it may be determined that the interaction between DKK2 and LRP6 is small.

In addition, the detection of the interaction may be to measure Cdc42 activation in the cell. Detection of the interaction may comprise the step of specifically precipitating activated Cdc42 in the cell. Measurement of Cdc42 activation can be made, for example, by isolating Cdc42 (GTP-Cdc42) to which GTP is bound in cells. Methods of isolating GTP-Cdc42 are known to those skilled in the art. For example, the detection of this interaction involves incubating the cell lysate with a fusion protein (GST-Pak1-PBD) of the PBD domain of GST and human p21-activated kinase 1 (Pak1) to complex Cdc42 with GST-Pak1-PDB. Obtaining; Reacting the incubated sample with glutathione immobilized on a solid medium to separate the substance bound to glutathione; And reacting the separated substance with an anti-Cdc42 antibody to separate the substance that binds to the anti-Cdc42 antibody. The method may be performed by a method of measuring activated Cdc42 in a cell. Methods for measuring activated Cdc42 can be made using commercially available kits, such as the cdc42 Activation kit (Product #: EKS-440: Stressgen Bioreagents). The GST-Pak1-PDB fusion protein has a molecular weight of about 35 kDa, and the step of separating the substance binding to the anti-Cdc42 antibody may be performed by western blotting. In western blotting the cdc42 signal is located at about 24 kDa.

Cdc42 belongs to the Rho-family of small GTP binding proteins. Cdc42 includes Cdc42Hs and G25k and promotes actin polymerization to form phylopodia. Philopodia is mainly found in motility cells and neuronal growth cones. Activation and inactivation of Cdc42 is regulated by GEP (guanine nucleotide exchanger) and GAP (GTPase activating protein), respectively. Activated Cdc42 (GTP binding form) interacts with various downstream effectors, including Pak1. Binding of Cdc42 to the p21-binding domain (PBD) inhibits the intrinsic and GAP-activated GTPase activity of Cdc42. Therefore, PBD of Pak1 can be used as a probe to specifically isolate the activation or CTP form of Cdc42.

The method includes determining from the detected interaction whether the test compound is a candidate compound associated with angiogenesis. The determination may comprise determining the test compound as a compound associated with angiogenesis when the interaction is increased relative to a control that contacts DKK2 with the cells in the absence of the test compound. As used herein, the term “candidate compound associated with angiogenesis” is one or more selected from the group consisting of compounds that promote angiogenesis, compounds that promote phylopodia motility of endothelial cells, and compounds that promote angiogenic germination. It is interpreted to include. In addition, the term "associated with angiogenesis" is interpreted to include one or more selected from the group consisting of promoting angiogenesis, promoting phylopodia motility of endothelial cells, and promoting angiogenic germination. do.

Another embodiment of the invention comprises contacting DKK2 with LRP6 in the presence of a test compound; Detecting the interaction of DKK2 with LRP6; And determining from the detected interactions whether the test compound is a candidate compound associated with angiogenesis.

The method comprises contacting DKK2 with LRP6 in the presence of a test compound. The contact can be made using LRP6 separated from the cell membrane. The contact may be in a liquid medium known to be suitable for the combination of LRP6 and DKK2 to occur. For example, the liquid medium may be a buffer. The contact can be made in the absence of WNT and / or Frizzled.

The method includes detecting the interaction of DKK2 with LRP6. The interaction can be detected by known methods. The interaction may be to measure the direct binding of DKK2 and LRP6, or to measure the complex formed by the interaction. For example, the detection of the interaction can be made by measuring the amount of DKK2-LRP6 complex. If the amount of the complex is measured, it may be determined that there is a lot of interaction, and if the amount of the complex is measured, it may be determined that the interaction is low.

The method includes determining from the detected interaction whether the test compound is a candidate compound associated with angiogenesis.

The determination may comprise determining the test compound as a compound associated with angiogenesis when the interaction is increased relative to a control that contacts DKK2 with LRP6 in the absence of the test compound.

In the present embodiment, unless otherwise stated, for the same terms, "contacting DKK2 with a cell in the presence of a test compound, said other embodiment; detecting the interaction of said DKK2 with LRP6; and Determining whether the test compound is a candidate compound associated with angiogenesis from the detected interactions.

According to the method for searching for compounds associated with angiogenesis of the present invention, it is possible to efficiently search for compounds associated with angiogenesis.

1 shows the results of measurement of mRNA and protein levels of DKK1 and DKK2 by RT-PCR (left) and Western blotting (right) at the same time (0.5, 8 and 12 hours) during morphology on Matrigel Drawing.
FIG. 2 shows the results of measurement of human DKK1 and DKK2 promoter activity by luciferase reporter assay.
3 is a diagram showing the effect of DKK1 on tube formation by HUVEC (A) and the effect of DKK1 on proliferation of HUVEC (B).
4 is a diagram showing the effect of DKK2 on tube formation by HUVEC (A) and the effect of DKK2 on proliferation of HUVEC (B).
5 to 7 show reversible expression changes of DKK1 and DKK2 during in vitro morphological changes.
8 to 10 show the effects of angiogenic mediator and microenvironment on the expression of DKK1 and DKK2.
FIG. 11 shows the role of DKK1 during in vitro conformation of HUVECs.
12 shows the role of DKK2 during in vitro conformation of HUVECs.
FIG. 13 shows the effect of recombinant DKK1, DKK2 and fusions of these with Fc on the formation of EC tube-like structures.
14 shows the effect of knockdown of DKK1 on the proliferation of HUVECs on gelatin-coated plates.
15 is a diagram showing the effect of DKK2 on angiogenesis measured by the in vivo Matrigel plug assay.
Figure 16 shows the effect of DKK2 on angiogenesis measured by mouse corneal pocket assay.
Figure 17 shows the effect of recombinant DKK2 protein on vessel recruitment in Matrigel plugs.
18 shows the results of analyzing the gene constructs used to prepare EC-specific DKK2 Tg mice and the prepared EC-specific DKK2 Tg mice.
19 and 20 show that angiogenesis of the retina is increased in DKK2 Tg mice.
21 shows aortic segments harvested from wild type and DKK2 Tg mice (n = 7 per group).
Figure 22 shows isolectin B4 staining of the whole-loaded P12 retina, showing the results of quantifying the vessel branching point in the ganglion layer (1st).
Fig. 23 shows the retina of the DKK2 Tg mouse at 10 weeks.
24 to 26 show that DKK2 promotes angiogenesis to improve tissue recovery in animal models with hind limb ischemia and myocardial infarction.
27 is an image showing that the cardiac function is effectively improved after DKK2 injection.
FIG. 28 shows the effects of DKK1 and DKK2 on the expression of VEGF isoform and GFAP.
29 and 30 show that DKK2 increases phyllopodia protrusions in a Cdc42 dependent manner.
30 shows the effect of DKK2 on phyllophodia expansion in HUVEC.
FIG. 31 shows changes in expression of Cdc42 during morphology change. FIG.
32 shows the effect of DKK2 on phyllophodia expansion.
33-37 show that DKK2-induced Cdc42 activation requires LRP6-mediated APC / Asef2 signaling.
FIG. 38 shows the effects of APC and Asef2 on DKK2-induced EC morphological changes.
39 shows a lentiviral vector map.
40 shows a pGL3-Basic Vector vector map.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

We found that DKK1 and DKK2, known as WNT antagonists that bind to LDL receptor-related protein 5/6 (LRP 5/6) and inhibit the β-catenin signaling pathway, are morphogenetic differentiation on Matrigel. It was observed that the cells were fractionally expressed in endothelial cells (EC).

Using biochemistry and transgenic animal analysis, we found that DKK1 and DKK2 play opposite roles in the regulation of angiogenesis. Surprisingly, DKK2 has been shown to significantly improve tissue regeneration through functional neovascularization in ischemic animal models. It also provides novel mechanistic insights of DKK2 in promoting angiogenic germination.

Method and material

Unless otherwise stated, the following examples were conducted using the following methods and materials.

(1) cell culture

Human umbilical cord vein endothelial cells (HUVECs) were previously isolated from umbilical veins by collagenase treatment and used from 2-4 passages (Jaffe, EA et al. 1973. J). Clin Invest 52: 2745-2756). HUVECs were cultured in EC basal medium (EBM) or EC growth medium (ECM, Clonetics) supplemented with 3% FBS.

(2) 2D Matrigel  In vitro morphology and in vitro oligonucleotides Microarray

In vitro morphological changes were analyzed as previously known (Min, JK et al., 2007, Blood 109: 1495-1502). Briefly, 800 μl of growth factor-reduced Matrigel (BD Biosciences) was transferred to 60 mm tissue culture wells and polymerized at 37 ° C. for 30 minutes. HUVECs were plated on layers of Matrigel at a density of 1 × 10 ⁶ cells / plate and incubated for the indicated times (0.5, 8, and 18 hours). At each time, total RNA was isolated and hybridized to HG-U133A 2.0 microarray (54,675 genes; Affymetrix Santa Clara, CA, USA). Standard protocols for sample preparation and microarray processing are available from Affymetrix. Expression data were analyzed using Microarray Suite Version 5.0 (Affymetrix).

(3) EC  Proliferation assay

HUVECs were seeded at a density of 2 × 10 ⁴ cells / well in gelatin coated 24-well plates. After 48 hours or 72 hours, cells were washed twice with M199 and incubated for 6 hours in M199 containing 0.5 μCi / ml of [ ³ H] thymidine (Amersham, Aylesbury, United Kingdom). High molecular weight DNA was precipitated using 10% trichloroacetic acid at 4 ° C. for 30 minutes. After washing twice with iced water, ³ H-radioactivity was solubilized in 0.2 N NaOH / 0.1% SDS and determined by a liquid scintillation counter.

(4) in vivo Matrigel  Plug analysis

Matrigel plug analysis was performed as previously known. Briefly, 0.6 ml Matrigel containing DKK2, DKK1, VEGF + DKK1 and 10 units of heparin in a given amount was injected subcutaneously in 7 week old C57BL / 6 mice (Orient Co. Seoul, Korea). The injected Matrigel quickly formed a single, solid gel plug. After 5 days the skin of the mice was easily pulled back to expose the Matrigel plug, which remained intact. Angiogenesis was quantified by measuring hemoglobin by the Drabkin method using Drabkin reagent kit 525 (Sigma, St Louis, MO). Hemoglobin concentrations were calculated with known amounts of hemoglobin analyzed in parallel. To identify infiltrating ECs, immunohistochemistry was performed using anti-CD-31 antibodies (BD Biosciences, San Jose, CA).

(5) Mouse Corneal Pocket Analysis

8-week-old C57BL6 mice were anesthetized using zoletil 50 (15 mg / kg) and lumpoon (5 mg / kg). Ten minutes later, alkanes were dropped into the eyes.

Using a Grafe cataract knife, a micropocket was made in the cornea. Sucrose octasulfate-aluminum complex (Sigma) coated with poly-2-hydroxyethyl methacrylate (Sigma P3932) containing 200 ng VEGF (KOMA Biotech., Cat no.K0921632) and 1 μg DKK2-Fc (self-produced) Micro pellets (0.35 × 0.35 mm) of S0652) were implanted into the corneal micropockets. The pellet was placed 0.6-0.8 mm from the corneal limbus. After implantation tardomyocel-L oint was applied to the eye. Seven days after the implantation, the eyes were examined under a microscope. Cryosections were stained with anti-CD31 (BD Pharmingen ^™ ) and anti-NG2 (Millipore).

(6) aortic ring analysis ( aortic ring assay )

Aorta were collected from 6-8 week old C57BL / 6 wild type (WT) and DKK2 transgenic (Tg) mice. 48 well plates were coated with 100 μL Matrigel and gelled, then the rings were placed in the wells and sealed in place by overlay of 40 μL Matrigel.

EC growth medium (EGM) was added to the wells in a final volume of 200 μL of human endothelial serum-free medium (Invitrogen). On day 5, cells were fixed and stained with isorectin B4. The analysis results were scored with double blind from 0 (weakest positive) to 5 (strongest positive). Each data point was analyzed six times. Endothelial germination was imaged over time on an Olympus IX81-ZDC inverted fluorescence microscope.

(7) Analysis of mouse retinal vasculature

Vascular pattern of the dissected mouse retina was analyzed according to a modified method of previously reported content (Gerhardt, H. et al. 2003. J Cell Biol 161: 1163-1177).

Mice were sacrificed by ketamine injection and the eyes enucleated in PBS. The eyes were fixed for 1 h at 4 ° C. in 4% PFA-PBS, pH 7.4. The retina is cut as previously known (Kang, Y. et al., PLoS One 4: e4275), fixed in 70% ethanol overnight at 4 ° C., washed with PBS and permeabilized with PBS containing 1% Triton-X-100 for 1 hour. The retina was then incubated for 4 hours at 37 ° C. in blocking solution and overnight at 4 ° C. in Alexa 488-conjugated isolectin GS-IB4 solution (Molecular Probes). After washing five times in PBS containing 1% Triton-X-100, the retina was mounted flat on the slide using a fluorescence mounting medium. Flat mounted retinas were analyzed by fluorescence microscopy using an Olympus IX81-ZDC inverted fluorescence microscope and a confocal fluorescent microscope (Carl Zeiss LSM 510 META).

(8) Cold slice Immunofluorescence  dyeing

Tissues were fixed overnight in 4% PFA-PBS (pH 7.4) at 4 ° C. and washed with PBS at room temperature. Next, the tissues were incubated overnight in a 15% sucrose solution at 4 ° C. and transferred to 30% sucrose at 4 ° C. before allowing the tissues to settle. All fixation, washes and incubations were smoothed. Tissues were allowed to sit in O.C.T. Penetrated in embedding medium (Tissue Tek) and frozen. The tissue was transferred to an embedding mold filled with O.C.T. The mold was cooled in liquid nitrogen. After freezing the material, the tissue was wrapped in aluminum foil and stored at -70 ° C. Sections (8-50 μm thick) were cut at −20 ° C. and stored at −70 ° C. until slides were needed. Sections were prefixed with acetone for 30 minutes at −70 ° C. and then simply dried to remove acetone.

OCT was removed with water. Sections were incubated in blocking solution for 4 hours at 37 ° C. or overnight at 4 ° C. and then overnight in primary antibody at 4 ° C. After washing five times with 0.3% Triton-X-100 in PBS for 15 minutes each, the sections were incubated in secondary antibody overnight at 4 ° C. Prior to washing, the sections were treated with DAPI (1 g / ml) and then washed at least 5 times with 0.3% Triton-X-100 in PBS for 15 minutes each. All antibodies were dissolved in antibody diluent (Dako). Sections were analyzed by fluorescence microscopy using an Olympus IX81-ZDCinverted fluorescence microscope or a confocal microscope (Carl Zeiss, LSM 510 META).

(9) mouse hind limb ischemia model

BalB / cAnNCriBgi-nu Nude Male Mice Charles River Japan Inc. It was purchased from (Yokohama, Japan). All mice were 7-8 weeks old (15-20g) at the time of experiment. Hind limb ischemia was induced by ligation and excision of the right femoral artery and vein under ketamine-xylazine anesthesia. For angiogenic potential studies, mice were divided into three groups after ischemia induction. Mice were saline containing PBS (20 μl with 0.9% NaCl) alone, saline solution containing DKK2-Fc (20 μl of 600 ng / μl), or vascular endothelial growth factor (VEGF). The solution (20 μl of 150 ng / μl) was injected intramuscularly.

10 NIR  Neon Imaging

The inventors of the present invention, Kang et al PLoS One As described by 4: e4275, a customized optical system for NIR fluorescence imaging was used. In short, the system uses a custom 830-nm bandpass filter (Asahi Spectra USA, Torrance, CA, USA) and a 760nm light emitting diode array (SMC760; Marubeni America, Sunnyvale, CA, USA). CCD digital camera (PIXIS 1024; Princeton instruments, Princeton, NJ, USA) is adopted. For time series ICG imaging, mice in ketamine-xylazine anesthesia were injected intravenously with a vein bolus of ICG (400 ml / l; 0.1 ml; Sigma, St. Louis, MO, USA). ICG fluorescence images were obtained in the dark for 10 minutes at 1 second intervals immediately after injection. After continuous imaging, perfusion maps and necrosis probability were obtained using customized and computer programs.

(11) myocardial infarction model and experimental procedure

Myocardial infarction (MI) was induced in 8-week-old Sprague-Dawley male rats (Song, H. et al., Stem Cells 25: 1431-1438). Rats are anesthetized with ketamine (10 mg / kg) and xylazine (5 mg / kg), and surgical occlusion of the left anterior descending coronary artery is followed by a 6-0 silk suture. suture) (Johnson & Johnson, Belgium).

After 60 minutes of occlusion, the ligated myocardium was reperfused and 3 μg of DKK2-Fc protein was Hamilton syringe with 30-gauge needles in three locations from the injured region to the border. syringe, Hamilton Co., Reno, NV, USA). Throughout the operation, rats were ventilated with 95% O ₂ and 5% CO ₂ using a Harvard ventilator. Six animals per group were used for morphological analysis one week after surgery. For functional studies, we performed echocardiography at 3 weeks. To determine the infarct size, the heart was removed and 1% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma, St. Louis, MO, USA) solution for 20 minutes at 37 ° C., Perfusion to pH 7.4 and then fixed in 10% PBS-buffered formalin overnight at 2-8 ° C.

(12) Rat  Histology and Immunohistochemistry of Myocardial Infarction Model

The heart was excised and fixed in 10% PBS-buffered formalin for 24 hours and embedded in paraffin. Sections (5 μm thick) were mounted on gelatin-coated glass slides to allow the use of other stains on the sections. After paraffin removal and dehydration, the tissue sections were stained with Masson's trichrome for analysis of fibrosis. For histological analysis, the RTU VECTASTATIN Universal Quick kit (Vector Laboratories, Burlingame, Calif., USA) was used for paraffin sections. Sections were paraffin removed, dehydrated and washed with PBS. Antigen retrieval was performed by sonication for 10 minutes in 10 mM sodium citrate, pH 6.0. Sections were incubated in 3% H ₂ O ₂ to inhibit intrinsic peroxidase activity. Sections were blocked in 2.5% normal horse serum and incubated in primary antibody (CD31). Biotinylated pre-specific general secondary antibodies and streptabin / peroxidase complex reagents were stained with antibodies using a DAB substrate kit (Vector Laboratories). Used for heart slices. Sections were counterstained with 1% methyl green, dehydrated in 100% N-butanol, ethanol, and xylene and loaded into Vecta Mount Medium (Vector Laboratories).

(13) organization TUNEL  analysis

TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis is a method of detecting DNA fragmentation by labeling the ends of nucleic acids. TUNEL analysis was performed according to the manufacturer's instructions (Chemicon International Inc. Temecula, CA, USA). Positive control samples were prepared from these sections by treating normal heart sections with DNase I (10 U / ml, 10 minutes at room temperature). Sections were pretreated with 3.0% H ₂ O ₂ , reacted with TdT enzyme for 1 hour at 37 ° C. and incubated in deoxygenin-conjugated nucleotide substrates at 37 ° C. for 30 minutes. Nuclei showing DNA fragmentation were visualized by addition of 3,3-diaminobenzidine (DAB) (Vector Laboratories, Burlingame, CA, http://www.vectorlab.com) for 5 minutes. Finally, sections were counterstained with methyl green and analyzed by light microscopy. For each group, 6 sections were prepared and 10 different regions were observed per section (x200).

(14) echocardiography

Transthoracic echocardiographic studies were performed using a GE Vivid Seven ultrasound machine (GE Medical System, Salt Lake City, UT, USA) equipped with a 10.0 MHz transducer. performed by a cardiologist. Rats received a general anesthetic and placed in the left lateral decubitus position. An echo transducer was placed on the left hemithorax and short axis views were recorded. Two-dimensional images were obtained at the midpapillary level (Song, H. et al. Stem Cells 25: 1431-1438). M-mode tracing of LV contraction was obtained at the same level as the shortened view. LV end diastolic diameter (LVEDD) and LV end systolic diameter (LVESD) were measured by M-mode tracing.

Percent fractional shortening (% FS) was calculated using (LVEDD-LVESD) / LVEDDx100 (%). LVEDV is calculated as 7.0xLVEDD ³ /(2.4+LVEDD), LVESV is calculated as 7.0xLVESD ³ /(2.4+LVESD), and the LV ejection fraction (EF) is EF (%) = (LVEDV-LVESV) Calculated as / LVEDVx100. Two images per view were obtained and each parameter was measured from three consecutive beats per image. Six values of each parameter were obtained and averaged. Ecocardiograms were digitized, stored and analyzed with EchoPAC with custom 2-D strain rate imaging software. Three or more images were obtained in the shortened view and the parameters were measured from three consecutive bits in each image.

(15) RT - PCR  analysis

Total RNA was isolated from HUVEC using TRIzol reagent kit. Different amounts of total RNA (0.5-5 μg) were amplified by RT-PCR and the correlation between the amount of RNA used and the level of PCR product obtained from target mRNA and internal standard (GAPDH) mRNA was investigated. Briefly, 42 in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl ₂ , 10 mM DTT, and 1 mM dNTPs using 200 U reverse transcriptase and 500 ng oligo (dT) primers. CDNA was synthesized from target RNA for 1 hour at < RTI ID = 0.0 > The reaction was stopped by heating at 70 ° C. for 15 minutes. Next, a total of 1 μl of cDNA mixture was used for enzyme amplification. PCR was performed using 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl ₂ , 0.2 mM dNTPs, _2.5 U Taq in a DNA thermal cycler (model PTC-200; MJ Research). In DNA polymerase (Promega), and 0.1 μM of each primer for the target gene, it was performed according to the following conditions: 5 minutes at 94 ° C for the first cycle, 30 seconds from the second cycle, 30 seconds at 55 ° C annealing And 25 cycles of 30 seconds at 72 ° C. extension. Final extension was 10 minutes at 72 ° C.

The following primers are designed for RT-PCR and are available from Bioneer Inc. It was synthesized by (Seoul, Korea) (Table 1).

Target Primer Pair Name SEQ ID NO: mDKK2 mDKK2_A 5 and 6 mDKK2 mDKK2_B 7 and 8 mGAPDH mGAPDH 9 and 10 DKK1 DKK1 11 and 12 DKK2 DKK2 13 and 14 KREMEN2 KREMEN2 15 and 16 LRP6 LRP6 17 and 18 LRP5 LRP5 19 and 20 APC APC 21 and 22 Asef2 Asef2 23 and 24 GAPDH GAPDH 25 and 26

(16) Cdc42  analysis

Cdc42 activity was measured using a CDC42 activation kit (Upstate, CA) according to the manufacturer's instructions (Upstate Biotechnology, Inc.).

HUVECs were washed once with ice-cold PBS and 25 mM Hepes, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl ₂ , 1 mM EDTA, 10% glycerol, 1 mM Na ₃ VO ₄ , 10 Lysis buffer was dissolved at 4 ° C. for 15 minutes with μg / ml aprotinin, 10 μg / ml leupeptin, and 25 mM NaF. Insoluble material was removed by centrifugation.

5 μg of PAK1-agarose beads that specifically bind to active Cdc42 were added to the cell lysate and incubated at 4 ° C. for 1 hour. Agarose beads were washed three times with lysis buffer and boiled in 2 × Laemmli sample buffer. Samples were partitioned by SDS-PAGE and immunoblotted using anti-Cdc42 antibody.

(17) Animal Research

All animals were maintained in air laminar flow cabinets under specific pathogen-free conditions. All facilities have been approved by the Association of Assessment and Accreditation of Laboratory Animal Care (AAALAC), and all animal experiments have established established institutional guidelines for the Animal Core Facility at Yonsei University College of Medicine. It was performed according to institutional guidelines.

(18) statistical analysis

Data are presented as mean ± SD or ± SE. Statistical comparisons between groups were performed using the Student t test after one-way ANOVA.

Example  1: Endothelial morphology changing DKK1  And DKK2 Manifestations of

In order to identify the factors acting at the sprouting front, we used gene microarrays to isolate genes that reversibly change expression during in vitro proliferation and morphology change of EC. The gene microarray used was HG-U133A 2.0 microarray (54,675 genes; Affymetrix Santa Clara, CA, USA).

1-4 show that DKK1 and DKK2 are expressed in vitro during endothelial morphological changes in vitro to separately regulate angiogenesis.

1 shows the results of measurement of mRNA and protein levels of DKK1 and DKK2 by RT-PCR (left) and Western blotting (right) at the same time (0.5, 8 and 12 hours) during morphology on Matrigel Drawing.

FIG. 2 shows the results of measurement of human DKK1 and DKK2 promoter activity by luciferase reporter assay. In the luciferase reporter assay, luciferase activity shows DKK1 promoter and DKK2 promoter activity. HUVECs cultured in gelatin-coated plates were transfected with pGL3 luciferase reporter vectors and the transfected cells were transferred to gelatin-coated plates (gelatin) and Matrigel-coated plates (morphogenesis) 6 hours after transfection. In FIG. 2, pGL3 represents HUVECs transduced with the control vector pGL3. Here, pGL3 is a commercially available pGL3-Basic Vector (Promega Corporation, Madison, Wi., USA), which is a vector without the promoters of DKK1 and DKK2 introduced. 40 shows a pGL3-Basic Vector vector map. SEQ ID NO: 27 shows the nucleotide sequence of a pGL3-Basic Vector. DKK1 and DKK2 represent HUVECs transduced with a vector into which the pGL3 DKK1 promoter and the DKK2 promoter were introduced upstream of the luc gene of the pGL3-Basic Vector. Data represent mean ± SD (** p <0.01).

3 is a diagram showing the effect of DKK1 on tube formation by HUVEC (A) and the effect of DKK1 on proliferation of HUVEC (B). In FIG. 3A, HUVECs stably expressing eGFP and control (CTL) shRNA, eGFP and DKK1 shRNA or DKK1 and control (CTL) shRNA were established using lentiviruses (Macrogen Inc, South Korea). Each gene was introduced through a respective lentiviral vector. As the lentiviral vector, one having a structure in which a gene corresponding to the DKK2 locus was introduced in pLentiM1.2 / DKK2 (9053 bp) having the map disclosed in FIG. 39 was used. Here shRNA (small hairpin RNA) is a sequence of RNA that can make a tight hairpin turn used to silence gene expression through RNA interference. shRNAs are cut by cellular machinery into siRNAs, which are combined with RNA-induced silencing complexes (RISCs) to bind and cleave mRNA. The control shRNA used a sequence that is not complementary to a transcript of another gene as a random sequence, and the DKK1 shRNA has a sequence complementary to mRNA encoded from the DKK1 gene. Thus, HUVECs stably expressing DKK1 shRNA are cells in which the expression of the DKK1 gene is silenced. Stable transducers were plated on Matrigel-coated plates at a density of ^1.5 × 10 ⁵ cells / well and incubated for 18 hours. Capillary-like networks completely differentiated into tube-like structures were quantified using Image-Pro Plus software. In 3B, the proliferation index of HUVECs transfected with eGFP or DKK1 was examined via [ ³ H] thymidine integration assay.

4 is a diagram showing the effect of DKK2 on tube formation by HUVEC (A) and the effect of DKK2 on proliferation of HUVEC (B). At 4A, HUVECs stably expressing eGFP, DKK2, control (CTL) shRNA, and DKK2 shRNA were established using lentiviruses (Macrogen Inc, Korea). The lentiviral vector was used in pLentiM1.2 / DKK2 (9053 bp) with the map disclosed in FIG. 39, with a similar structure except for DKK2. Here shRNA (small hairpin RNA) is a sequence of RNA that can make a tight hairpin turn used to silence gene expression through RNA interference. shRNAs are cut by cellular machinery into siRNAs, which are combined with RNA-induced silencing complexes (RISCs) to bind and cleave mRNA. The control shRNA was used as a random sequence that was not complementary to the transcripts of other genes, and the DKK2 shRNA had a sequence complementary to mRNA encoded from the DKK2 gene. Thus, HUVECs stably expressing DKK2 shRNA are cells in which the expression of the DKK1 gene is silenced. Stable transducers were plated on Matrigel-coated plates at a density of ^1.5 × 10 ⁵ cells / well and incubated for 18 hours. Capillary-like networks completely differentiated into tube-like structures were quantified using Image-Pro Plus software. At 4B, the proliferation index of HUVECs transfected with eGFP or DKK2 was examined via [ ³ H] thymidine integration assay. Data represent mean ± SD (** p <0.01, *** p <0.001, ns: not significant).

As shown in Figures 1 to 4, interestingly, the decope members DKK1 and DKK2 showed opposite expression changes during morphological changes of HUVECs. DKK1 was rapidly downregulated when inducing morphological changes on 2D Matrigel, while DKK2 was upregulated during induction of morphological changes.

5 to 7 show reversible expression changes of DKK1 and DKK2 during in vitro morphological changes.

5 is a diagram showing the process of in vitro endothelial morphology change of HUVEC in 2D Matrigel. Micrographs of HUVECs were obtained at different time points (S1: 0.5, S2: 8 and S3: 18 hours).

Figure 6 HUVECs growing on gelatin were plated on Matrigel-coated plates and incubated for 8 hours. Next, the cells were replated into gelatin-coated plates and allowed to proliferate for a designated time (18 hours and 36 hours).

FIG. 7 shows the results of measuring mRNA and protein levels of DKK1 and DKK2 by RT-PCR (left) and Western blotting (right) during the experiments of FIGS. 5 and 6. G: gelatin, M: Matrigel, G18: 18 hours after replating on gelatin, G36: 36 hours after replating on gelatin.

As shown in FIGS. 5-7, the opposite expression changes of DKK1 and DKK2 indicate that they are reversibly and regulated at the transcriptional and translational levels during morphological conditions on Matrigel and during proliferation on gelatin.

We further investigated the regulation of DKK1 and DKK2 gene expression. 8 to 10 show the effects of angiogenic mediator and microenvironment on the expression of DKK1 and DKK2.

Figure 8 shows angiogenesis mediators (VEGF, HGF, EGF, IFG, bFGF and PDGF) (A), hypoxic conditions (B) and microenvironment (gelatin, Matrigel, fibronectin, laminin, or collagen) (C) are DKK1 and DKK2 It is a figure which shows the effect on the expression of. In FIG. 8A, HUVEC is expressed in VEGF (20 ng / ml), HGF (20 ng / ml), EGF (20 ng / ml), IGF (50 ng / ml), bFGF (20 ng / ml), or PDGF (50 ng). / ml) for 8 hours. In FIG. 8B, HUVECs were incubated for a specified time under hypoxia or normal oxygen conditions. In FIG. 8C, HUVECs were plated on gelatin (G)-, Matrigel (M)-, fibronectin (F)-, laminin (L)-, or gelatin (C) -coated plates and incubated for 8 hours. MRNA levels of DKK1 and DKK2 were measured by RT-PCR.

9 is a diagram showing the effect of laminin on the expression of DKK1 and DKK2. In FIG. 9, HUVECs were plated at a density of 1 × 10 ⁶ cells / well on Matrigel (M) -coated plates or Matrigel and laminin (L) -mixture coated plates and incubated for 0.5 or 8 hours. 9A shows micrographs and FIG. 9B shows mRNA levels of DKK1 and DKK2 measured by RT-PCR.

10 shows the effect of cell density on the expression of DKK1 and DKK2 in Matrigel-coated plates of HUVEC. In FIG. 10, HUVECs were plated on Matrigel-coated plates at the specified density and incubated for 0.5 or 8 hours. 10A shows micrographs and FIG. 10B shows mRNA levels of DKK1 and DKK2 measured by RT-PCR.

As shown in FIGS. 8-10, none of the angiogenic factors or hypoxic conditions tested affected DKK1 and DKK2 expression (FIGS. 8A and B). However, DKK2 was sufficiently induced by laminin recognition, the major component of Matrigel in EC, and did not require morphological changes of EC (8A, 9A and 9B). Unexpectedly, downregulation of DKK1 correlated with the induction of morphological changes in EC (FIGS. 10A and 10B) and of specific extracellular matrix (ECM) components. It was not significantly affected by recognition (FIG. 8C).

Example  2: in vitro during angiogenesis DKK1  And DKK2 Separate roles in

In this example we determined the role of DKK1 and DKK2 in vitro angiogenesis.

FIG. 11 shows the role of DKK1 during in vitro conformation of HUVECs. In FIG. 11, HUVECs stably expressing eGFP and control (CTL) shRNA, eGFP and DKK1 shRNA, or DKK1 and control (CTL) shRNA were established by lentiviruses. Here, the DKK1 shRNA and the control shRNA are as described with reference to FIG. 3. FIG. 11A shows the results of DKK1 mRNA and protein levels measured by RT-PCR (top) and Western blotting (bottom). In FIG. 11A, results are shown after stable transducers are plated at a density of ^1.5 × 10 ⁵ cells / well on gelatin-coated plates (Gelatin) or Matrigel-coated plates (Morphogenesis) and incubated for 18 hours. FIG. 11B shows micrographs taken after stable transducers were plated at a density of ^1.5 × 10 ⁵ cells / well on Matrigel-coated plates and incubated for 18 hours.

12 shows the role of DKK2 during in vitro conformation of HUVECs. In FIG. 12, HUVECs stably expressing eGFP, DKK2, control (CTL) shRNA, or DKK2 shRNA were established by lentiviruses. Here, the DKK2 shRNA and the control shRNA are as described with reference to FIG. 4. 12A shows the results of DKK2 mRNA and protein levels measured by RT-PCR (top) and western blotting (bottom). 12A is the result after stable transducers are plated at a density of ^1.5 × 10 ⁵ cells / well on gelatin-coated plates (Gelatin) or Matrigel-coated plates (Morphogenesis) and incubated for 18 hours. 12B shows micrographs taken after stable transducers were plated at a density of ^1.5 × 10 ⁵ cells / well on Matrigel-coated plates and incubated for 18 hours.

FIG. 13 shows the effect of recombinant DKK1, DKK2 and fusions of these with Fc on the formation of EC tube-like structures. In FIGS. 13A and B, HUVEC ( ⁵ × 10 ⁵ ) was recombinant native DKK1 (100 ng / ml), Fc-fusion (DKK1-Fc) (100 ng / ml), recombinant natural DKK2 (100 ng) on Matrigel. / ml), DKK1 (100 ng / ml) or Fc-fusion (DKK1-Fc) (DKK2-Fc) (100 ng / ml) or VEGF (20 ng / ml) in SEQ ID NO: 3 ( Incubated with 100 ng / ml). Figure 13A shows representative endothelial tubes as micrographs taken 18 hours after incubation. FIG. 13B is a diagram showing the results of quantifying capillary-like networks completely differentiated into tube-like structures by Image-Pro Plus software. Data are mean ± SD; ** p <0.01 or *** p <0.001. FIG. 13C is a diagram showing the result of staining with Coomassie Blue by recombinant SDS (DKK1, DKK2) or Fc-fusion (DKK1-Fc, DKK2-Fc) by SDS-PAGE.

14 shows the effect of knockdown of DKK1 on the proliferation of HUVECs on gelatin-coated plates. In FIG. 14, the proliferation index of HUVECs stably transduced by control or DKK1-specific shRNA was examined via [ ³ H] thymidine integration assay.

As described above, the formation of EC tube-like structures on 2D Matrigel was inhibited by sustained expression of DKK1 or by treatment with recombinant DKK1 protein during morphology of in vitro HUVEC (FIG. 3A, FIG. 11). And FIG. 13). In contrast, overexpression of DKK2 or recombinant DKK2 protein treatment enhanced the robustness of EC tube-like structures, and knockdown of DKK2 during morphology inhibited robustness (FIGS. 4A, 12 and 13). In addition, a fusion of DKK2 and Fc, for example, a fusion of DKK2 and Fc having the amino acid sequence of SEQ ID NO: 3, was used in spontaneous DKK2 despite being used at a lower molar concentration than DKK2, i.e., DKK2. The unexpected effect of forming a similar tube-like structure when used was shown (FIGS. 13A and B).

Interestingly, EC proliferation on gelatin was markedly inhibited by DKK1 overexpression and increased by DKK1 shRNA transduction (FIG. 3B and FIG. 14). Overexpression of DKK2 did not significantly affect EC proliferation on gelatin (FIG. 4B).

These results indicate that DKK1 and DKK2 function inversely during angiogenesis.

Example  3: in vivo DKK2 To promote angiogenesis

The inventors confirmed that DKK2 is a proangiogenic factor in vivo. To assess the effects of DKK1 and DKK2 on angiogenesis in vivo, Matrigel implant assays were used in mice ("(4) In vivo Matrigel Plug Assay" and "(5) Mice in Methods and Materials). Corneal pocket analysis ". In this example, DKK1-Fc fusion and DKK2-Fc fusion (SEQ ID NO: 3) were used as one form of DKK1 and DKK2, respectively.

15 is a diagram showing the effect of DKK2 on angiogenesis measured by the in vivo Matrigel plug assay. In FIG. 15, Matrigel plugs treated with VEGF (200 ng) and DKK2-Fc (1 μg) were excised from mice 5 days after injection (n = 5 per group). Size bar: 100 μm. FIG. 15A shows CD31 staining and FIG. 15B quantifies the hemoglobin concentration in the Matrigel plug. White arrows indicate CD31-stained vessels.

Figure 16 shows the effect of DKK2 on angiogenesis measured by mouse corneal pocket assay. FIG. 16A shows the results of corneal angiogenic responses induced by VEGF (200 ng) -containing micropellets, or DKK2-Fc (1 μg) -containing micropellets. Asterisk (*) indicates the inserted micropellet. Fig. 16C shows the results of staining the CD31 and NG2 cold sections of the eye. Green: CD31-positive, red: NG2-positive, blue: DAPI. Here, CD31-positive cells represent vascular endothelial cells, NG2-positive cells represent perivascular cells, and DAPI staining is nuclear staining. FIG. 16B shows the pericyte coverage calculated as the ratio of NG2 staining area to CD31 staining area. Here, the ratio of NG2 / CD31 represents the degree of covering vascular endothelial cells. Data is mean ± SD (*** p <0.001).

Figure 17 shows the effect of recombinant DKK2 protein on vessel recruitment in Matrigel plugs. C57BL / 6 mice were injected with 0.6 ml Matrigel containing VEGF (200 ng) and DKK2-Fc (1 μg) as shown (n = 5 per group). After 5 days, mice were sacrificed and Matrigel plugs were excised and removed. 17 shows a photograph of the cut plug.

As shown in Figures 15-17, the DKK2-Fc fusion or VEGF-containing Matrigel plugs were red in color, indicating neovascularization (Figure 17). The color of the Matrigel plug co-treated with VEGF and DKK1-Fc fusions was similar to the control treated plug (FIG. 17). Quantification of hemoglobin and CD31 staining of Matrigel plugs consistently confirmed that DKK2-Fc fusions significantly increased angiogenesis, while DKK1-Fc fusions inhibit VEGF-induced angiogenesis compared to control treatment. (Figures 15 A and B).

We further investigated the structural features of DKK2-induced blood vessels in a mouse corneal pocket assay. The growth of VEGF-induced blood vessels is relatively fast, as known, but the structure appears to be disassembled and leaky (FIG. 16A) (Cao, R. et al., 2003. Nat Med 9: 604-613). In contrast, DKK2 induced well-organized vasculature networks by distinct vascular tree-like structures (FIG. 16A). Consistently, DKK2-induced blood vessels showed more area of surrounding cells on EC, as compared to VEGF-stimulated capillaries, which play an important role in blood vessel growth and stability (FIGS. 16B and C).

Example  4: DKK2 Tg In mice Angiogenesis  Germination and Philopodia  Motility dynamics ) increase

To further investigate the angiogenic function of DKK2 in vivo, we generated transgenic (Tg) mice expressing mouse DKK2 (mDKK2) under the control of an EC-specific Tie2 promoter / enhancer (FIG. 18).

18 shows the results of analyzing the gene constructs used to prepare EC-specific DKK2 Tg mice and the prepared EC-specific DKK2 Tg mice. 18A shows a gene construct configured to specifically express DKK2 in endothelial cells. FIG. 18B shows the results of confirming the presence of the transgene for founder mice by Western blotting. FIG. Three DKK2 founder mice per group were identified. FIG. 18C extracts total RNA from P12 mouse retina and shows primer set A (DKK2 cDNA-specific primer sets: SEQ ID NOs: 5 and 6) and primer set B (Tg-specific primer sets: SEQ ID NOs: 7 and 8). It is a figure which showed the result which confirmed that Tg-specific DKK2 expression occurs by RT-PCR used. 18D is a diagram showing the results of real-time RT-PCR using primer set A. FIG.

Of the three Tg mouse lines produced, we were unable to detect significant developmental abnormalities in the embryonic and adult stages. However, in some cases, DKK2 Tg embryos showed somewhat higher capillary densities at age E9.5 and higher compared to wild-type littermates.

Because retinal blood vessels are well identified for their growth rate and patterning during early development, we analyzed the phenotype of DKK2 Tg mice in the retina (see "(7) Analysis of Mouse Retinal Vasculature" in Methods and Materials). .

19 and 20 show that angiogenesis of the retina is increased in DKK2 Tg mice. 19A and D show the results of staining the whole-mounted P4 retina of isolectin B in wild type mice or DKK2 Tg mice. In 19A, the dashed white line indicates the extent of vasodilation. Scale bar: 100 μm. In 19D, white dots represent phylopodia extending from the tip cell. 19B is the result of quantification of blood vessel density, 19C is the germination length, 19E is the tip cell number, 19F is the phyllopodia number.

FIG. 20 shows the results of staining whole-mounted P12 retinas with isolelectin B in wild type mice or DKK2 Tg mice. FIG. 20A is a diagram showing the result of staining with isolectin B. FIG. Scale bar: 100 μm. 20B shows the result of quantifying the blood vessel density of the ganglion (ganglion (1st)), 30C represents the blood vessel density of the outer nuclear (3rd) cell layer.

21 shows aortic sections harvested from wild-type and DKK2 Tg mice (n = 7 per group) (see (6) Aortic Ring Analysis of Materials and Methods). Fig. 21A shows the results of phase contrast microscopy of endothelial germination forming branching cords from the edge of the aortic section. FIG. 21C shows the results of capturing the dynamic movement of endothelial germination from the edge of the aortic section as a real time video. Arrows indicate Philopodia. 21B shows germination scores and 21D quantifies tip cell number. Germination scores ranged from 0 (weakest positive) to 5 (strongest positive). Data represent mean ± SD (* p <0.05, *** p <0.001).

FIG. 22 shows isolectin B4 staining of the whole-loaded P12 retina, showing the results of quantifying the vascular branch points in the ganglion layer (1st).

Fig. 23 shows the retina of the DKK2 Tg mouse at 10 weeks. 23A shows retinal vascular staining of 10 week old wild type (n = 15) and DKK2 (n = 9) Tg mice. Flat mounted retinas were analyzed by confocal fluorescence microscopy (LSM 510 META). Size bar: 200 μm. 23B and 23C show the relative blood vessel density (B) and branch number (C) of Tg mouse retinas for wild type. Density and number of branches were measured by Multi Guage V2.2. Data is mean ± SD (*** p <0.001).

As noted above, at P4, DKK2 Tg mice showed increased vasculature density and germination length in retinal vessels compared to wild-type mates (FIG. 19, A-C). The number of tip cells and phyllophodia was consistently increased in DKK2 Tg mice (Figure 19, D-F). In the P12 retina, the vein density and branching of the ganglion layer (first layer) as well as the germinated plexus in the deeper layer (third layer) were significantly increased in DKK2 Tg mice (FIG. 20, AC, and 22). In addition, retinal vessel density increase was maintained until 10 weeks after birth (FIG. 23).

In addition, consistently, in vitro ( ex vivo ) In aortic ring analysis, mean length and branch number of endothelial germination were increased in DKK2 Tg aorta compared to wild type cohort (FIGS. 21A and B). In a time-lapse video microscopy recorded over 9 hours, the aorta of DKK2 Tg mice showed high motility phyllopodia protrusion of the tip cells (FIGS. 21, C and D).

Example  5: in ischemia and myocardial infarction animal models DKK2 To promote angiogenesis

We investigated the effect of topical infusion of DKK2 protein on therapeutic angiogenesis in a mouse model with hind limb ischemia.

Hind limb ischemia was induced by ligation and excision of the right femoral artery and vein, leading to a severe vascular perfusion defect.

24 to 26 show that DKK2 promotes angiogenesis to improve tissue recovery in animal models with hind limb ischemia and myocardial infarction. In this example, DKK2-Fc fusion (SEQ ID NO: 3) was used as a form of DKK2.

FIG. 24 shows the effect of DKK2-Fc fusion on blood perfusion and necrosis probability in ischemic hind limbs of mice. As shown in FIG. 24, intramuscular injection of DKK2-Fc fusion increased blood perfusion in the ischemic hind limbs of mice and reduced the probability of necrosis. Figure 24 quantifies blood perfusion rate and necrosis probability (poor perfusion rate: 0-30% / min, moderate perfusion rate: 30-100% / min, good perfusion rate : 100 <% / min).

FIG. 25 shows an increase in myocardial infarction repair after DKK2-Fc fusion injection in a rat myocardial infarction model. A and B show the results validated by TTC staining one week after myocardial infarction, and injection of the DKK2-Fc fusion into the myocardial infarction model reduced LV infarct size. C and D show representative images taken from Masson's trichrome-stained section (muscles are stained red and collagen is stained blue). E and G show the results of staining microvessels with CD31 on cardiac muscle tissue (E) and TUNEL analysis (G). F and H are the results of quantitative analysis of microvascular (F) and TUNEL analysis (H). The yellow dotted line represents the infarct region. Blue arrows indicate microvascular and apoptotic cells, respectively.

FIG. 26 shows that cardiac function is effectively improved by DKK2-Fc fusion injection. Cardiac function was measured by two-dimensional conventional variables: FS and LVEF measurements 3 weeks after DKK2-Fc fusion injection in myocardial infarction rats. FS (%) = [(LVEDD-LVESD) / LVEDD] × 100 (%). LVEDV = 7.0 × LVEDD3 / (2.4 + LVEDD), LVESV = 7.0 × LVESD3 / (2.4 + LVESD), and LVEF (%) = (LVEDV-LVESV) / LVEDV × 100 (* p <0.05, ** p <0.01 , *** p <0.001). W: wild type, S: control (Sham), V: VEGF, D2: DKK2-Fc fusion.

27 is an image showing that the cardiac function is effectively improved after injection of the DKK2-Fc fusion. The image shows dilator function (A) and diastolic function (B). Cardiac function was measured by two-dimensional typical variables: fractional shortening (FS) and LV injection fraction (EF) three weeks after DKK2-Fc fusion injection in myocardial infarction rats.

As a result, intramuscular injection of the DKK2-Fc fusion significantly reduced necrosis in ischemic mice compared to ischemic mice injected with PBS (FIGS. 24, A and B). Indocyanine green (ICG) imaging significantly improved blood perfusion on postoperative 3 days in ischemic mice compared to PBS-injected mice (FIGS. 24, A and B). This is consistent with the hypothesis that reduced necrosis is due to improved angiogenesis in the ischemic hind limb.

We further investigated the therapeutic effects of DKK2-Fc fusions in rat models with myocardial infarction. One week after myocardial infarction and DKK2-Fc fusion protein injection, the size of the left ventricular (LV) infarct was assessed in the DKK2 administered group and the control group (sham).

As a result, administration of the DKK2-Fc fusion protein significantly reduced the infarct size and the degree of fibrosis in the infarct region in the DKK2-Fc fusion administration group compared to the control group (FIG. 25, AD). Compared to the control group, the mean microvascular number per field was higher in the infarcted heart in the DKK2-Fc fusion group (FIG. 25, E and F), and the incidence of TUNEL-positive cardiomyocytes was significantly reduced. (FIG. 25, G and H).

Cardiac function in DKK2-Fc fusion protein-administered myocardial infarction animals was measured by transthoracic echocardiography. Echocardiographic data showed that DKK2-Fc fusion increased LV fractional shortening (FS) and LV ejection fraction (EF) in the DKK2-Fc fusion group compared to the control group ( 26, and 27 and Table 2).

Wild type (WT) Control (sham) VEGF DKK2-Fc fusion LVEDD (mm) 6.24 ± 0.41 7.87 ± 0.50 6.95 ± 0.38 6.38 ± 0.33 LVESD (mm) 3.15 ± 0.11 5.88 ± 0.42 4.83 ± 0.49 3.39 ± 0.23 FS (%) 49.15 ± 1.83 25.31 ± 0.56 30.62 ± 3.20 46.89 ± 0.84 LVEF (%) 79.61 ± 1.55 48.31 ± 1.15 56.68 ± 4.96 77.28 ± 1.06

Table 2 shows the effective improvement of cardiac function by DKK2-Fc fusion injection. Administration of the DKK2-Fc fusion or VEGF further improved systolic performance and cardiac dimensions as compared to the control. However, LV end diastolic diameter (LVEDD) and LV end systolic diameter (LVESD) were smaller in the DKK2-Fc fusion group compared to the VEGF group (FIG. 27 and Table 2). ).

The DKK2-Fc fusion group experienced an additional increase in systolic performance compared to the VEGF group (16.27% increase in% FS and 20.5% increase in% EF) (FIG. 26, and FIG. 27 and Table 1).

Example  6: Cdc42  Through activation DKK2  Induced EC of Philopodia  Protrusion (filopodial extrusion )

The present inventors investigated the mechanism of action of DKK2 in the vasculature. Previous studies have shown that tip cell filopodial extension in the retina that occurs is dependent on astrocyte-derived VEGF-A, which acts directly on the tip cell. However, the levels of glial fibrillary acidic protein (GFAP) and VEGF-A mRNA, as well as the relative levels of VEGF-A splice isoforms, remained unchanged in DKK2 Tg mice compared to wild-type mice. (Fig. 28). This suggests that DKK2 acts directly on the EC.

28 is a diagram showing the effects of DKK1 and DKK2 on the expression of VEGF isoform and GFAP. RT-PCR analysis detected VEGF isoform and GFAP of the retina at P4.

The tips of the sprouts consist of cells with high mobility along with many phyllophodia and lamellipodia-like processes conferred by the action of small GTPases such as Cdc42. Therefore, we hypothesized that DKK2 could stimulate tip cell phyllopodia expansion by regulating the activation of Cdc42.

29 and 30 show that DKK2 increases phyllopodia protrusions in a Cdc42 dependent manner. 29A shows the results of analyzing HUVECs stably expressing eGFP, DKK1 or DKK2 for Cdc42 activity. Introduction of the eGFP, DKK1 or DKK2 gene was introduced via a lentiviral vector as described in FIG. 3. 29B shows the results of incubating the stable transductors for 2 hours on Matrigel-coated plates (morphogenesis) and measuring Cdc42 activity. Cdc42 activity measurement was performed using a commercially available Cdc42 activation kit according to the manufacturer's instructions. The kit was confirmed by Western blotting the total amount of Cdc42 separated into a kit measuring GTP-Cdc42.

30 shows the effect of DKK2 on phyllophodia expansion in HUVEC. In Figures 30A and 30B, HUVECs were transiently transduced with eGFP controls or expression vectors encoding eGFP and Cdc42 (N17). In the case of eGFP, pGL3 vector as described in FIG. 2 was used, and in the case of Cdc42 (N17), the lentiviral vector described in FIG. 3 was used. Here, eGFP-Cdc42 (N17) indicates that the eGFP is fused to the N terminus of Cdc42 (N17), a dominant negative mutant of human Cdc42. Cdc42 (N17) is a substitution of asparagine (N) with threonine (T), the amino acid number 17 of human Cdc42, and inhibits the normal activation of Cdc42.

After 24 hours, these cells were plated on Matrigel-coated plates and incubated with PBS or DKK2-Fc fusions (1.5 μg / ml) for 2 hours. Next, micrographs were taken (A) and the number of phyllopodia was quantified (B). Arrows indicate Philopodia expansion. Data is mean ± SD (** p <0.01). 30C shows the results of incubating HUVECs stably expressing eGFP or DKK2 with sFRP (200 ng / ml) for 24 hours and measuring Cdc42 activity.

FIG. 31 shows changes in expression of Cdc42 during morphology change. FIG. In FIG. 31, HUVECs were plated on Matrigel-coated plates (morphogenesis) and incubated for a designated time. Next, Cdc42 activity was measured.

32 shows the effect of DKK2 on phyllophodia expansion. In FIG. 32, HUVECs were transiently transduced with eGFP controls or expression vectors encoding dominant negative mutants of eGFP and Cdc42 (Cdc42 N17). In the case of eGFP, the pGL3 vector as described in FIG. 2 was used, and in the case of Cdc42 (N17), the lentiviral vector described in FIG. 3 was used. Transduction with pGL3 vectors was done by incubating the lipofectamine and expression vectors for 30 minutes and then adding them to the cells in the medium. After 24 hours, cells were plated on Matrigel-coated plates and incubated with PBS or DKK2-Fc fusion protein (1.5 μg / ml) for 2 hours. Next, micrographs were taken. White and black arrows indicate Philopodia expansion. Yellow dotted circle indicates the transduced cells.

In HUVECs cultured on gelatin, overexpression of DKK2 induced Cdc42 activation, increasing the amount of GTP-Cdc42, but not overexpression of DKK1 (FIG. 29A). During EC morphology, Cdc42 spontaneously activated in EC plated on Matrigel and its activation was somewhat enhanced in DKK2-expressing ECs, but markedly decreased in DKK1-expressing cells (FIGS. 29B and 31). In addition, phyllopodia overhang was significantly increased after 2 hours by DKK2-Fc fusion protein treatment compared to the control, and the dominant negative form of Cdc42 blocked DKK2-Fc fusion protein induced phyllopodia expansion (FIG. 30, A and B, and FIG. 32). These results suggest that DKK2 can promote angiogenesis, at least in part, by imparting the dynamics of the tip cell structure, requiring Cdc42 activation and phylopodia overhang.

To identify the mechanism that supports DKK2-mediated Cdc42 activation in the EC, we determined whether the WNT / Frizzled complex or LRP5 / 6 is involved in signaling events induced by DKK2. Treatment of soluble Frizzled related protein (sFRP) (R & D Systems: sFRP-1), ie, decoy antagonists of WNT, did not inhibit DKK2-mediated Cdc42 activation (FIG. 30C).

33-37 show that DKK2-induced Cdc42 activation requires LRP6-mediated APC / Asef2 signaling.

FIG. 33 shows the results of transducing HUVECs stably expressing eGFP or DKK2 with control siRNA (CTL), LRP5-specific siRNA or LRP6-specific siRNA. Herein, the control siRNA, LRP5-specific siRNA and LRP6-specific siRNA each have a ... sequence ( sequence desired ). HUVECs stably expressing eGFP or DKK2 were prepared as described in FIG. 4, and siRNA transduction was performed by incubating with lipofectamine for 30 minutes and then adding to cells in the medium.

33A shows the results of measuring Cdc42 activity using Western blotting after 60 hours. Cells were plated at a density of ^1.5 × 10 ⁵ cells / well on Matrigel-coated plates and incubated for 18 hours. 33B and 33C show micrographs of HUVECs transduced with LRP6-specific siRNA (B) or LRP5-specific siRNA (C), and capillary-like networks were measured with Image-Pro Plus software. Indicates. Data is mean ± SD (*** p <0.001, ns; not significant).

Fig. 34 shows the expression of APC / Asef2 in HUVECs stably expressing DKK2. HUVECs stably expressing DKK2 were prepared as described in FIG. 4. 34A shows the results of co-immunoprecipitation for APC and Asef2 for HUVECs stably expressing eGFP or DKK2. Cell lysates were prepared from cells overexpressing eGFP or DKK2, immunoprecipitated with anti-APC antibody and probed with anti-Asef2 or anti-APC antibody. 34B shows the results of transient transduction of cells expressing eGFP or DKK2 with control siRNA or LRP6-specific siRNA. After culturing the cells for 60 hours, the cell lysates were co-immunoprecipitated. Here, the control siRNA was used as a random sequence that is not complementary to the transcripts of other genes, and the LRP6-specific siRNA has a sequence complementary to the mRNA of the LRP6 gene. In addition, siRNA transduction was done by incubating itself with lipofectamin for 30 minutes and then adding to the cells in the medium.

35 shows the results of transient transduction of cells expressing eGFP or DKK2 with control siRNA, APC-specific siRNA or Asef2-specific siRNA. HUVECs stably expressing eGFP or DKK2 were prepared as described in FIG. 4, and siRNA transduction was performed by incubating with lipofectamine for 30 minutes and then adding to cells in the medium. Here, the control siRNA used a sequence that is not complementary to the transcripts of other genes as a random sequence, and APC-specific or Asef2-specific siRNAs have sequences complementary to mRNAs of the APC gene and Asef2 gene, respectively. Figure 35A shows the result of measuring the Cdc42 activity by Western blotting after incubating the transduced HUVEC cells for 60 hours. 35B and 35C plate cells transiently transduced with APC-specific siRNA (B) or Asef2-specific siRNA (C) at a density of ^1.5 × 10 ⁵ cells / well on Matrigel-coated plates, 18 The results of incubation for time are shown. Capillary-like networks were measured with Image-Pro Plus software.

36 and 37 show that DKK2 receptor is LRP6 in HUVEC. In FIG. 36A, mRNA levels of DKK1, DKK2, LRP5, and LRP6 were measured by RT-PCR at 0.5 h, 8 h and 18 h during EC morphology change. 36B and 36C show the results of transient transduction of HUVECs with control siRNA (CTL), LRP5-specific siRNA (B), or LRP6-specific siRNA (C). Transduction was done by incubating the siRNA itself with lipofectamin for 30 minutes and then adding it to the cells in the medium. After 40 hours (for mRNA) or 60 hours (for protein), mRNA and protein levels were measured by RT-PCR (top) and Western blotting (bottom). In FIG. 37, HUVECs stably expressing eGFP or DKK2 are transiently transduced with LRP5 specific siRNA (B) or LRP6 specific siRNA (A). Transduction was done by incubating the siRNA itself with lipofectamin for 30 minutes and then adding it to the cells in the medium. Cells were plated at a density of ^1.5 × 10 ⁵ cells / well on Matrigel-coated plates and incubated for 18 hours. This is the result of taking a fine picture.

FIG. 38 shows the effects of APC and Asef2 on DKK2-induced EC morphological changes. In Figures 38A and C, the results show that HUVECs are transiently transduced with control siRNA (CTL), APC-specific siRNA, or Asef2-specific siRNA. After 40 hours (relative to mRNA) or 80 hours (relative to protein), mRNA and protein levels were measured by RT-PCR (top) and Western blotting (bottom). 38B and D show the results of the transient transduction of HUVECs stably expressing eGFP or DKK2 with control siRNA (CTL), APC-specific siRNA, or Asef2-specific siRNA. After 60 hours of incubation, cells were plated at a density of ^1.5 × 10 ⁵ cells / well on Matrigel-coated plates, incubated for 18 hours, and micrographs taken.

Surprisingly, knockdown experiments of LRP5 and LRP6 revealed that LRP6, but not LRP5, was importantly involved in DKK2-mediated Cdc42 activation even though both LRPs were expressed in EC (FIGS. 33A and 36A-C). Consistently, siRNA specific for LRP6 completely blocked DKK2-induced EC morphology, but siRNA for LRP5 had no significant effect (FIGS. 33 B and C, and FIGS. 37 A and B). These results suggest that DKK2, via LRP6, activates the Cdc42 signaling pathway independent of WNT / Frizzled signaling.

GEF activity of the APC-stimulated-exchange-factor (Asef) 2, where the adenomatous polyposis coli protein (APC) is a guanine-nucleotide exchange factor (GEF) specific for Cdc42 It has recently been reported to promote.

Since APC is one of the components acting downstream of LRP6, it was assumed that DKK2 promoting LRP6 would induce APC / Asef2 complex formation and consequently induce Cdc42 activation. Indeed, APC and Asef2 interactions were markedly increased in ECs expressing DKK2 and blocked by LRP6 siRNA (FIGS. 34A and B). In addition, knockdown of APC or Asef2 inhibited Cdc42 activation and EC morphological changes induced by DKK2 (FIGS. 35 A-C and 38). Taken together, these results indicate that the angiogenic action of DKK2 is mediated through its cognate receptor LRP6 (ECRP) in the EC, and at least the APC / Asef2 complex is located downstream of LRP6 inducing Cdc42 activation and tip cell motility stimulation. Imply that.

<110> Theragen Bio Inc. <120> Method for screening a compound associated with angiogenesis <130> PN090808 <150> US61 / 326,509 <151> 2010-04-21 <160> 27 <170> KopatentIn 1.71 <210> 1 <211> 259 <212> PRT <213> Homo sapiens <400> 1 Met Ala Ala Leu Met Arg Ser Lys Asp Ser Ser Cys Cys Leu Leu Leu   1 5 10 15 Leu Ala Ala Val Leu Met Val Glu Ser Ser Gln Ile Gly Ser Ser Arg              20 25 30 Ala Lys Leu Asn Ser Ile Lys Ser Ser Leu Gly Gly Glu Thr Pro Gly          35 40 45 Gln Ala Ala Asn Arg Ser Ala Gly Met Tyr Gln Gly Leu Ala Phe Gly      50 55 60 Gly Ser Lys Lys Gly Lys Asn Leu Gly Gln Ala Tyr Pro Cys Ser Ser  65 70 75 80 Asp Lys Glu Cys Glu Val Gly Arg Tyr Cys His Ser Pro His Gln Gly                  85 90 95 Ser Ser Ala Cys Met Val Cys Arg Arg Lys Lys Lys Arg Cys His Arg             100 105 110 Asp Gly Met Cys Cys Pro Ser Thr Arg Cys Asn Asn Gly Ile Cys Ile         115 120 125 Pro Val Thr Glu Ser Ile Leu Thr Pro His Ile Pro Ala Leu Asp Gly     130 135 140 Thr Arg His Arg Asp Arg Asn His Gly His Tyr Ser Asn His Asp Leu 145 150 155 160 Gly Trp Gln Asn Leu Gly Arg Pro His Thr Lys Met Ser His Ile Lys                 165 170 175 Gly His Glu Gly Asp Pro Cys Leu Arg Ser Ser Asp Cys Ile Glu Gly             180 185 190 Phe Cys Cys Ala Arg His Phe Trp Thr Lys Ile Cys Lys Pro Val Leu         195 200 205 His Gln Gly Glu Val Cys Thr Lys Gln Arg Lys Lys Gly Ser His Gly     210 215 220 Leu Glu Ile Phe Gln Arg Cys Asp Cys Ala Lys Gly Leu Ser Cys Lys 225 230 235 240 Val Trp Lys Asp Ala Thr Tyr Ser Ser Lys Ala Arg Leu His Val Cys                 245 250 255 Gln lys yle             <210> 2 <211> 3659 <212> DNA <213> Homo sapiens <400> 2 cgggagcccg cggcgagcgt agcgcaagtc cgctccctag gcatcgctgc gctggcagcg 60 attcgctgtc tcttgtgagt caggggacaa cgcttcgggg caactgtgag tgcgcgtgtg 120 ggggacctcg attctcttca gatctcgagg attcggtccg gggacgtctc ctgatcccct 180 actaaagcgc ctgctaactt tgaaaaggag cactgtgtcc tgcaaagttt gacacataaa 240 ggataggaaa agagaggaga gaaaagcaac tgagttgaag gagaaggagc tgatgcgggc 300 ctcctgatca attaagagga gagttaaacc gccgagatcc cggcgggacc aaggaggtgc 360 ggggcaagaa ggaacggaag cggtgcgatc cacagggctg ggttttcttg caccttgggt 420 cacgcctcct tggcgagaaa gcgcctcgca tttgattgct tccagttatt gcagaacttc 480 ctgtcctggt ggagaagcgg gtctcgcttg ggttccgcta atttctgtcc tgaggcgtga 540 gactgagttc atagggtcct gggtccccga accaggaagg gttgagggaa cacaatctgc 600 aagcccccgc gacccaagtg aggggccccg tgttggggtc ctccctccct ttgcattccc 660 acccctccgg gctttgcgtc ttcctgggga ccccctcgcc gggagatggc cgcgttgatg 720 cggagcaagg attcgtcctg ctgcctgctc ctactggccg cggtgctgat ggtggagagc 780 tcacagatcg gcagttcgcg ggccaaactc aactccatca agtcctctct gggcggggag 840 acgcctggtc aggccgccaa tcgatctgcg ggcatgtacc aaggactggc attcggcggc 900 agtaagaagg gcaaaaacct ggggcaggcc tacccttgta gcagtgataa ggagtgtgaa 960 gttgggaggt attgccacag tccccaccaa ggatcatcgg cctgcatggt gtgtcggaga 1020 aaaaagaagc gctgccaccg agatggcatg tgctgcccca gtacccgctg caataatggc 1080 atctgtatcc cagttactga aagcatctta acccctcaca tcccggctct ggatggtact 1140 cggcacagag atcgaaacca cggtcattac tcaaaccatg acttgggatg gcagaatcta 1200 ggaagaccac acactaagat gtcacatata aaagggcatg aaggagaccc ctgcctacga 1260 tcatcagact gcattgaagg gttttgctgt gctcgtcatt tctggaccaa aatctgcaaa 1320 ccagtgctcc atcaggggga agtctgtacc aaacaacgca agaagggttc tcatgggctg 1380 gaaattttcc agcgttgcga ctgtgcgaag ggcctgtctt gcaaagtatg gaaagatgcc 1440 acctactcct ccaaagccag actccatgtg tgtcagaaaa tttgatcacc attgaggaac 1500 atcatcaatt gcagactgtg aagttgtgta tttaatgcat tatagcatgg tggaaaataa 1560 ggttcagatg cagaagaatg gctaaaataa gaaacgtgat aagaatatag atgatcacaa 1620 aaagggagaa agaaaacatg aactgaatag attagaatgg gtgacaaatg cagtgcagcc 1680 agtgtttcca ttatgcaact tgtctatgta aataatgtac acatttgtgg aaaatgctat 1740 tattaagaga acaagcacac agtggaaatt actgatgagt agcatgtgac tttccaagag 1800 tttaggttgt gctggaggag aggtttcctt cagattgctg attgcttata caaataacct 1860 acatgccaga tttctattca acgttagagt ttaacaaaat actcctagaa taacttgtta 1920 tacaataggt tctaaaaata aaattgctaa acaagaaatg aaaacatgga gcattgttaa 1980 tttacaacag aaaattacct tttgatttgt aacactactt ctgctgttca atcaagagtc 2040 ttggtagata agaaaaaaat cagtcaatat ttccaaataa ttgcaaaata atggccagtt 2100 gtttaggaag gcctttagga agacaaataa ataacaaaca aacagccaca aatacttttt 2160 tttcaaaatt ttagttttac ctgtaattaa taagaactga tacaagacaa aaacagttcc 2220 ttcagattct acggaatgac agtatatctc tctttatcct atgtgattcc tgctctgaat 2280 gcattatatt ttccaaacta tacccataaa ttgtgactag taaaatactt acacagagca 2340 gaattttcac agatggcaaa aaaatttaaa gatgtccaat atatgtggga aaagagctaa 2400 cagagagatc attatttctt aaagattggc cataacctgt attttgatag aattagattg 2460 gtaaatacat gtattcatac atactctgtg gtaatagaga cttgagctgg atctgtactg 2520 cactggagta agcaagaaaa ttgggaaaac tttttcgttt gttcaggttt tggcaacaca 2580 tagatcatat gtctgaggca caagttggct gttcatcttt gaaaccaggg gatgcacagt 2640 ctaaatgaat atctgcatgg gatttgctat cataatattt actatgcaga tgaattcagt 2700 gtgaggtcct gtgtccgtac tatcctcaaa ttatttattt tatagtgctg agatcctcaa 2760 ataatctcaa tttcaggagg tttcacaaaa tggactcctg aagtagacag agtagtgagg 2820 tttcattgcc ctctataagc ttctgactag ccaatggcat catccaattt tcttcccaaa 2880 cctctgcagc atctgcttta ttgccaaagg gctagtttcg gttttctgca gccattgcgg 2940 ttaaaaaata taagtaggat aacttgtaaa acctgcatat tgctaatcta tagacaccac 3000 agtttctaaa ttctttgaaa ccactttact acttttttta aacttaactc agttctaaat 3060 actttgtctg gagcacaaaa caataaaagg ttatcttata gtcgtgactt taaacttttg 3120 tagaccacaa ttcacttttt agttttcttt tacttaaatc ccatctgcag tctcaaattt 3180 aagttctccc agtagagatt gagtttgagc ctgtatatct attaaaaatt tcaacttccc 3240 acatatattt actaagatga ttaagactta cattttctgc acaggtctgc aaaaacaaaa 3300 attataaact agtccatcca agaaccaaag tttgtataaa caggttgcta taagcttggt 3360 gaaatgaaaa tggaacattt caatcaaaca tttcctatat aacaattatt atatttacaa 3420 tttggtttct gcaatatttt tcttatgtcc acccttttaa aaattattat ttgaagtaat 3480 ttatttacag gaaatgttaa tgagatgtat tttcttatag agatatttct tacagaaagc 3540 tttgtagcag aatatatttg cagctattga ctttgtaatt taggaaaaat gtataataag 3600 ataaaatcta ttaaattttt ctcctctaaa aactgaaaaa aaaaaaaaaa aaaaaaaaa 3659 <210> 3 <211> 474 <212> PRT <213> Artificial Sequence <220> <223> DKK2-Fc fusion protein <400> 3 Ser Phe Phe Phe Leu Phe Glu Lys Arg Lys Leu Asn Ser Ile Lys Ser   1 5 10 15 Ser Leu Gly Gly Glu Thr Pro Gly Gln Ala Ala Asn Arg Ser Ala Gly              20 25 30 Met Tyr Gln Gly Leu Ala Phe Gly Gly Ser Lys Lys Gly Lys Asn Leu          35 40 45 Gly Gln Ala Tyr Pro Cys Ser Ser Asp Lys Glu Cys Glu Val Gly Arg      50 55 60 Tyr Cys His Ser Pro His Gln Gly Ser Ser Ala Cys Met Val Cys Arg  65 70 75 80 Arg Lys Lys Lys Arg Cys His Arg Asp Gly Met Cys Cys Pro Ser Thr                  85 90 95 Arg Cys Asn Asn Gly Ile Cys Ile Pro Val Thr Glu Ser Ile Leu Thr             100 105 110 Pro His Ile Pro Ala Leu Asp Gly Thr Arg His Arg Asp Arg Asn His         115 120 125 Gly His Tyr Ser Asn His Asp Leu Gly Trp Gln Asn Leu Gly Arg Pro     130 135 140 His Thr Lys Met Ser His Ile Lys Gly His Glu Gly Asp Pro Cys Leu 145 150 155 160 Arg Ser Ser Asp Cys Ile Glu Gly Phe Cys Cys Ala Arg His Phe Trp                 165 170 175 Thr Lys Ile Cys Lys Pro Val Leu His Gln Gly Glu Val Cys Thr Lys             180 185 190 Gln Arg Lys Lys Gly Ser His Gly Leu Glu Ile Phe Gln Arg Cys Asp         195 200 205 Cys Ala Lys Gly Leu Ser Cys Lys Val Trp Lys Asp Ala Thr Tyr Ser     210 215 220 Ser Lys Ala Arg Leu His Val Cys Gln Lys Ile Leu Glu Ser Arg Leu 225 230 235 240 Val Pro Arg Gly Ser Ala Ser Asp Lys Thr His Thr Cys Pro Pro Cys                 245 250 255 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro             260 265 270 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys         275 280 285 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp     290 295 300 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 305 310 315 320 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu                 325 330 335 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn             340 345 350 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly         355 360 365 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu     370 375 380 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 385 390 395 400 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn                 405 410 415 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe             420 425 430 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn         435 440 445 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr     450 455 460 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 4 <211> 1638 <212> DNA <213> Artificial Sequence <220> <223> DKK2-Fc fusion gene <400> 4 agtctttttt ttttcttttc gagaaaagga aactcaactc catcaagtcc tctctgggcg 60 gggagacgcc tggtcaggcc gccaatcgat ctgcgggcat gtaccaagga ctggcattcg 120 gcggcagtaa gaagggcaaa aacctggggc aggcctaccc ttgtagcagt gataaggagt 180 gtgaagttgg gaggtattgc cacagtcccc accaaggatc atcggcctgc atggtgtgtc 240 ggagaaaaaa gaagcgctgc caccgagatg gcatgtgctg ccccagtacc cgctgcaata 300 atggcatctg tatcccagtt actgaaagca tcttaacccc tcacatcccg gctctggatg 360 gtactcggca cagagatcga aaccacggtc attactcaaa ccatgacttg ggatggcaga 420 atctaggaag accacacact aagatgtcac atataaaagg gcatgaagga gacccctgcc 480 tacgatcatc agactgcatt gaagggtttt gctgtgctcg tcatttctgg accaaaatct 540 gcaaaccagt gctccatcag ggggaagtct gtaccaaaca acgcaagaag ggttctcatg 600 ggctggaaat tttccagcgt tgcgactgtg cgaagggcct gtcttgcaaa gtatggaaag 660 atgccaccta ctcctccaaa gccagactcc atgtgtgtca gaaaattctc gagtctagac 720 tggtgccgcg cggcagcgct agcgacaaaa ctcacacatg cccaccgtgc ccagcacctg 780 aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga 840 tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa gaccctgagg 900 tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg 960 aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact 1020 ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca gcccccatcg 1080 agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc 1140 catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct 1200 atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga 1260 ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg 1320 acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc 1380 acaaccacta cacgcagaag agcctctccc tgtccccggg taaatgaggt accggccggc 1440 catttaaata caggcccctt ttcctttgtc gatatcatgt aattagttat gtcacgctta 1500 cattcacgcc ctcctcccac atccgctcta accgaaaagg aaggagttag acaacctgaa 1560 gtctaggtcc ctatttattt tttttaatag ttatgttagt attaagaacg ttatttatat 1620 ttcaaatttt cagggtgg 1638 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mKK2_A primer <400> 5 cagagatggg atgtgttgcc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mDKK2_A primer <400> 6 cctgatggag cactggtttg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mDKK2_B primer <400> 7 gatgggtttt gttgtgctcg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mDKK2_B primer <400> 8 atgtttcagg ttcaggggga 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mGAPDH primer <400> 9 cgccacagtt tcccggaggg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mGAPDH primer <400> 10 ccctccaaaa tcaagtgggg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DKK1 primer <400> 11 cctggagtgt aagagctttg 20 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> DKK1 primer <400> 12 ccaagagatc cttgcgtt 18 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DKK2 primer <400> 13 tagagattga gtttgagcct 20 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> DKK2 primer <400> 14 aaagggtgga cataagaaa 19 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> KREMEN 2 primer <400> 15 caccgactgt gaccagat 18 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> KREMEN2 primer <400> 16 gagtagatga cgccctgag 19 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRP6 primer <400> 17 gtccttccac tcataggtca 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRP6 primer <400> 18 gtgggtagag gtgatgagaa 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRP5 primer <400> 19 gggtggtgtc tattttgtgt 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRP5 primer <400> 20 ccggaatgtt tgaagagtag 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> APC primer <400> 21 caggcaaaac aaaatgtggg 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> APC primer <400> 22 cgcttaggac tttgggttcc 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Asef2 primer <400> 23 tctactcggg ggagctgact 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Asef2 primer <400> 24 cctgcttcct ctgcagttca 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH primer <400> 25 cgccacagtt tcccggaggg 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH primer <400> 26 ccctccaaaa tcaagtgggg 20 <210> 27 <211> 4818 <212> DNA <213> Artificial Sequence <220> <223> pGL3 basic vector <400> 27 ggtaccgagc tcttacgcgt gctagcccgg gctcgagatc tgcgatctaa gtaagcttgg 60 cattccggta ctgttggtaa agccaccatg gaagacgcca aaaacataaa gaaaggcccg 120 gcgccattct atccgctgga agatggaacc gctggagagc aactgcataa ggctatgaag 180 agatacgccc tggttcctgg aacaattgct tttacagatg cacatatcga ggtggacatc 240 acttacgctg agtacttcga aatgtccgtt cggttggcag aagctatgaa acgatatggg 300 ctgaatacaa atcacagaat cgtcgtatgc agtgaaaact ctcttcaatt ctttatgccg 360 gtgttgggcg cgttatttat cggagttgca gttgcgcccg cgaacgacat ttataatgaa 420 cgtgaattgc tcaacagtat gggcatttcg cagcctaccg tggtgttcgt ttccaaaaag 480 gggttgcaaa aaattttgaa cgtgcaaaaa aagctcccaa tcatccaaaa aattattatc 540 atggattcta aaacggatta ccagggattt cagtcgatgt acacgttcgt cacatctcat 600 ctacctcccg gttttaatga atacgatttt gtgccagagt ccttcgatag ggacaagaca 660 attgcactga tcatgaactc ctctggatct actggtctgc ctaaaggtgt cgctctgcct 720 catagaactg cctgcgtgag attctcgcat gccagagatc ctatttttgg caatcaaatc 780 attccggata ctgcgatttt aagtgttgtt ccattccatc acggttttgg aatgtttact 840 acactcggat atttgatatg tggatttcga gtcgtcttaa tgtatagatt tgaagaagag 900 ctgtttctga ggagccttca ggattacaag attcaaagtg cgctgctggt gccaacccta 960 ttctccttct tcgccaaaag cactctgatt gacaaatacg atttatctaa tttacacgaa 1020 attgcttctg gtggcgctcc cctctctaag gaagtcgggg aagcggttgc caagaggttc 1080 catctgccag gtatcaggca aggatatggg ctcactgaga ctacatcagc tattctgatt 1140 acacccgagg gggatgataa accgggcgcg gtcggtaaag ttgttccatt ttttgaagcg 1200 aaggttgtgg atctggatac cgggaaaacg ctgggcgtta atcaaagagg cgaactgtgt 1260 gtgagaggtc ctatgattat gtccggttat gtaaacaatc cggaagcgac caacgccttg 1320 attgacaagg atggatggct acattctgga gacatagctt actgggacga agacgaacac 1380 ttcttcatcg ttgaccgcct gaagtctctg attaagtaca aaggctatca ggtggctccc 1440 gctgaattgg aatccatctt gctccaacac cccaacatct tcgacgcagg tgtcgcaggt 1500 cttcccgacg atgacgccgg tgaacttccc gccgccgttg ttgttttgga gcacggaaag 1560 acgatgacgg aaaaagagat cgtggattac gtcgccagtc aagtaacaac cgcgaaaaag 1620 ttgcgcggag gagttgtgtt tgtggacgaa gtaccgaaag gtcttaccgg aaaactcgac 1680 gcaagaaaaa tcagagagat cctcataaag gccaagaagg gcggaaagat cgccgtgtaa 1740 ttctagagtc ggggcggccg gccgcttcga gcagacatga taagatacat tgatgagttt 1800 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct 1860 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt 1920 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc 1980 tacaaatgtg gtaaaatcga taaggatccg tcgaccgatg cccttgagag ccttcaaccc 2040 agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt 2100 ctttatcatg caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc tcgctcactg 2160 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 2220 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 2280 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 2340 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 2400 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 2460 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 2520 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 2580 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 2640 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 2700 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 2760 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 2820 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 2880 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 2940 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 3000 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 3060 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 3120 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 3180 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 3240 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 3300 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 3360 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 3420 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 3480 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 3540 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 3600 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 3660 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 3720 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 3780 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 3840 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 3900 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 3960 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 4020 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgcgccct 4080 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg 4140 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg 4200 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac 4260 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct 4320 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt 4380 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt 4440 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt 4500 ttaacaaaat attaacgctt acaatttgcc attcgccatt caggctgcgc aactgttggg 4560 aagggcgatc ggtgcgggcc tcttcgctat tacgccagcc caagctacca tgataagtaa 4620 gtaatattaa ggtacgggag gtacttggag cggccgcaat aaaatatctt tattttcatt 4680 acatctgtgt gttggttttt tgtgtgaatc gatagtacta acatacgctc tccatcaaaa 4740 caaaacgaaa caaaacaaac tagcaaaata ggctgtcccc agtgcaagtg caggtgccag 4800 aacatttctc tatcgata 4818

Claims (17)

Contacting DKK2 with cells in the presence of a test compound;
Detecting the interaction of DKK2 with LRP6; And
Determining from the detected interactions whether the test compound is a candidate compound associated with angiogenesis. The method of claim 1, wherein the contacting is in the presence of an inhibitor of WNT / Frizzled mediated signaling. The method of claim 2, wherein the inhibitor is a soluble Frizzled related protein (sFRP). The method of claim 1, wherein said contact is made under conditions that promote morphology change of the cell. The method of claim 1, wherein the contacting is by culturing the cells on a Matrigel-coated medium that promotes morphological changes of the cells. The method of claim 1, wherein the cell is a cell expressing LRP6. The method of claim 1, wherein the cells are vascular endothelial cells. The method of claim 1, wherein the detection of the interaction measures the amount of DKK2-LRP6 complex. The method of claim 1, wherein the detection of the interaction measures the interaction of Asef2 with APC in the cell. The method of claim 1, wherein the detection of the interaction measures the amount of APC-Asef2 complex in the cell. The method of claim 1, wherein the detection of the interaction measures Cdc42 activation in the cell. The method of claim 1, wherein the detecting of the interaction comprises specifically depositing activated Cdc42 in the cell. The method of claim 1, wherein the detection of the interaction comprises incubating the cell lysate with a fusion protein (GST-Pak1-PBD) of the PBB domain of GST and human p21-activated kinase 1 (Pak1). Obtaining a complex of;
Reacting the incubated sample with glutathione immobilized on a solid medium to separate the substance bound to glutathione; And
And reacting the isolated substance with an anti-Cdc42 antibody to separate the substance that binds to the anti-Cdc42 antibody. The method of claim 1, wherein the determination comprises determining the test compound as a compound associated with angiogenesis when the interaction is increased relative to a control that contacts DKK2 with the cell in the absence of the test compound. Contacting DKK2 with LRP6 in the presence of a test compound;
Detecting the interaction of DKK2 with LRP6; And
Determining from the detected interactions whether the test compound is a candidate compound associated with angiogenesis. The method of claim 15, wherein the detection of the interaction measures the amount of DKK2-LRP6 complex. The method of claim 15, wherein said determining comprises determining said test compound as a compound associated with angiogenesis when said interaction is increased relative to a control contacting DKK2 with LRP6 in the absence of a test compound.

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