COMPOSITION FOR PREVENTING OR TREATING PERIPHERAL VASCULAR DISEASE USING HEPATOCYTE GROWTH FACTOR AND STROMAL CELL DERIVED FACTOR 1A

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating peripheral vascular disease, the composition comprising, as an active ingredient: (a) hepatocyte growth factor (HGF) or an isoform thereof, and stromal cell derived factor 1α (SDF-1α); or (b) a polynucleotide encoding the HGF and a polynucleotide encoding the SDF-1α. The peripheral vascular disease (for example, ischemic limb disease) can be more effectively prevented or treated through the significant promotion of vascular endothelial cell migration and angiogenesis in the case of singly using the composition of the present invention than in the case of using HGF, an isoform thereof, SDF-1α or a polynucleotide codes a protein thereof.

Claims

1. A pharmaceutical composition for preventing or treating a peripheral artery disease (PAD), the pharmaceutical composition comprising: as active ingredients, (a) hepatocyte growth factor (HGF) or an isoform thereof, and stromal cell derived factor 1α (SDF-1α); or (b) a polynucleotide encoding HGF and a polynucleotide encoding SDF-1α.

2. The composition of claim 1, wherein the HGF comprises the amino acid sequence of SEQ ID NO: 1.

3. The composition of claim 1, wherein the isoform of the HGF comprises full-length HGF (flHGF) and deleted variant HGF (dHGF).

4. The composition of claim 3, wherein the full-length HGF comprises the amino acid sequence of SEQ ID NO: 2 and the deleted variant HGF comprises the amino acid sequence of SEQ ID NO: 3.

5. The composition of claim 1, wherein the SDF-1α comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 8.

6. The composition of claim 1, wherein the polynucleotide encoding HGF comprises a sequence corresponding to exons 1 to 4 of human HGF gene, intron 4 of human HGF gene or a fragment sequence thereof, and a sequence corresponding to exons 5 to 18 of human HGF gene.

7. The composition of claim 6, wherein the polynucleotide encoding HGF includes the nucleotide sequence of SEQ ID NO: 5.

8. The composition of claim 1, wherein the polynucleotide encoding SDF-1α a comprises the nucleotide sequence of SEQ ID NO: 6.

9. The composition of claim 1, wherein the peripheral artery disease is ischemic limb disease.

10. The composition of claim 1, wherein each of the polynucleotides is naked DNA or a nucleotide contained in a gene delivery system.

11. The composition of claim 10, wherein the gene delivery system is a vector.

12. The composition of claim 11, wherein the vector is a plasmid.

13. The composition of claim 12, wherein the plasmid is pCK.

14. A method for preventing or treating a peripheral artery disease, the method comprising a step of administering a composition to a subject in need thereof, the composition comprising, as active ingredients, (a) hepatocyte growth factor (HGF) or an isoform thereof, and stromal cell derived factor 1α (SDF-1α); or (b) a polynucleotide encoding HGF and a polynucleotide encoding SDF-1α.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0073] FIG. 1 illustrates an effect of the use of HGF and SDF-1α on the cell migration of HUVECs.

[0074] FIGS. 2a and 2b illustrate an effect of the use of HGF and SDF-1α on angiogenesis.

[0075] FIG. 3 illustrates an effect of the use of pCK-HGF and pCK-SDF-1α on the hindlimb conditions of hindlimb ischemia mouse models.

MODE FOR CARRYING OUT THE INVENTION

[0076] Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

EXAMPLES

Example 1

Experimental Method for Investigating Effect of Combination of HGF and SDF-1α on Cell Migration of Human Umbilical Vein Endothelial Cells (HUVECs)

[0077] HUVECs, which were obtained by taking only the endothelial cells of the vein from the human umbilical cord, making the cells into single cells, and culturing the single cells, were purchased from Lonza.

[0078] In order to investigate the effects of HGF (SEQ ID NO: 1, R&D systems Cat No. 294-HG-025/CF, USA) and SDF-1α (SEQ ID NO: 4, R&D systems Cat No. 350-NS-010/CF, USA) on the cell migration of HUVECs, the transwell (Corning, cat#3422) was coated with 1% gelatin, and then the cells were seeded at 2×10.sup.4 cells per well. After the incubation for one hour to allow the cells to adhere, the experimental groups were organized as follows (50 ng/ml HGF; 50 ng/ml SDF-1α; 25 ng/ml HGF+25 ng/ml SDF-1α). The respective experimental groups were treated with corresponding proteins for 2 hours, and in order to measure the degree of cell migration, the cells were stained with crystal violet, and the number of migrated cells in the transwell was measured using a microscope.

[0079] As a result, the treatment with 50 ng/ml HGF alone increased cell migration by 1.8-fold compared with the control, and the treatment with 50 ng/ml SDF-1α alone increased cell migration by 1.5-fold compared with the control. The co-treatment with HGF and SDF-1α at 25 ng/ml each increased cell migration by 2.5-fold compared with the control, showing a better effect on cell migration compared with the treatment with HGF and SDF-1α alone (FIG. 1).

Example 2

Investigation on Effect of Combination of HGF and SDF-1α on Angiogenesis in Matrigel Plug Assay

[0080] The effect of HGF and SDF-1α on angiogenesis was investigated using Matrigel plug assay.

[0081] Five-week-old C57BL/6 mice were divided as follows to organize experimental groups (PBS; 300 ng HGF; 150 ng HGF+150 ng SDF-1α). Here, 1 unit of heparin was added to 400 μl Matrigel Matrix (Corning, cat #356231) and proteins corresponding to each experimental group were added. The resulting matrigel mixture was subcutaneously injected into the mouse abdomen. After 7 days, the mice were sacrificed and the transplanted matrigel matrix was isolated. In order to quantify the degree of angiogenesis, the level of hemoglobin contained in each matrigel was measured by Drabkin's assay.

[0082] As a result, the group added with 300 ng HGF increased the level of hemoglobin by about 1.8-fold compared with the group added with PBS, and the group added with HGF and SDF-1α at 150 ng each increased the level of hemoglobin by about 2.3-fold compared with the group added with PBS, showing the improvement in the degree of angiogenesis compared with the administration with HGF alone (FIGS. 2a and 2b).

Example 3

Investigation on Effect of Administration with HGF and SDF-1α in Hindlimb Ischemia (HLI) Mouse Model

Example 3-1

Preparation of Plasmid DNA

[0083] Preparation of pCK-HGF Plasmid

[0084] Prior to the experiment on the hindlimb ischemia mouse models, the plasmid DNA to be used was prepared in the following manner. The pCK vector is constructed such that a subject to be expressed is regulated under enhancer/promoter of the human cytomegalovirus (hCMV), and the pCK vector is disclosed in detail in Lee et al., Biochem. Biophys. Res. Commun. 272:230 (2000) and WO 2000/040737. The pCK-HGF plasmid used in the present invention was prepared by inserting, into the pCK vector, a hybrid gene (i.e., HGF-X7 gene; SEQ ID NO: 5) in which a fragment sequence of intron 4 of the human HGF gene is inserted between exons 4 and 5 of the human HGF gene according to the method disclosed in WO 2003/078568.

[0085] Preparation of pCK-SDF-1α

[0086] On the basis of the gene information of human SDF-1α (NCBI Reference Sequence: NM_199168.3), gene synthesis was conducted by adding the NheI and NotI restriction enzyme sequences to both ends of the gene. The synthesized human SDF-1α fragment was inserted into the pCK vector using NheI and NotI. The sequence of the human SDF-1α gene inserted into the pCK vector is the same as that of SEQ ID NO: 6.

Example 3-2

Preparation of Hindlimb Ischemia (HLI) Mouse Models and Administration of Plasmid DNA

[0087] The HLI mouse model is the most representative mouse model to mimic human critical limb ischemia (CLI) [1, 2]. The method of producing the mouse model is as follows. Seven-week-old male Balb/c mice were anesthetized with a mixture of zoletil and rumpun, and the skin of the thigh was incised about 1 cm. After that, the position of the femoral artery inside the thigh was found to tightly bind a length of about 1 cm of the artery using 6-0 thickness of thread, and the tissue therebetween was cut out to remove the blood vessel. This method can induce ischemic conditions by removing blood vessels descending below the thigh. At the same time as the HLI induction, the plasmid DNA to be evaluated was administered to the muscle near the removed blood vessel. After that, the incision was sutured well and the mice were observed to recover from the anesthesia.

[0088] The HLI mouse models were organized into 6 mice per group, and each of the following plasmids was administered: 200 μg pCK; 200 μg pCK-HGF; 200 μg pCK-SDF-1α; 200 μg pCK-HGF+200 μg pCK-SDF-1α. At 3, 10, 17, 22, and 27 days after administration, the hindlimb conditions were observed, scored according to a predetermined criteria, and quantified. The criteria used here are as follows [3]: 0=normal state; 1=toenail necrosis; 2=toe necrosis; 3=foot tissue necrosis

[0089] As a result, after the HLI induction, the group administered with pCK showed that the average hindlimb conditions began to deteriorate gradually, and the score after about two weeks increased to about 1.83. The group administered with pCK-HGF showed that the score increased to 0.66-0.83 over time. Meanwhile, the group co-administered with pCK-HGF and pCK-SDF-1α showed that the score after the HLI induction was maintained at zero (FIG. 3).

[0090] Resultantly, it was verified through examples 1-3 above that the effect of promoting the migration and angiogenesis of vascular endothelial cells and the treatment effect for an ischemic limb disease were more remarkable when HGF and SDF-1α were co-administered or the polynucleotides encoding HGF and SDF-1α respectively were co-administered rather than when the respective proteins or polynucleotides were administered alone.

REFERENCES

[0091] 1. Limbourg, A., et al., Evaluation of postnatal arteriogenesis and angiogenesis in a mouse model of hind-limb ischemia. Nat Protoc. 4(12): p. 1737-46, 2009. [0092] 2. Couffinhal, T., et al., Mouse model of angiogenesis. Am J Pathol. 152(6): p. 1667-79, 1998. [0093] 3. Clayton, J. A., D. Chalothorn, and J. E. Faber, Vascular endothelial growth factor-A specifies formation of native collaterals and regulates collateral growth in ischemia. Circ Res. 103(9): p. 1027-36, 2008.

[0094] Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.