KR20140094462A - Pharmaceutical composition comprising CD31-ITIM polypeptide or nucleic acids encoding the same for promoting blood vessel formation - Google Patents

Abstract

Disclosed is a pharmaceutical composition to promote angiogenesis comprising CD31-ITIM protein or a gene thereof. According to the present invention, the pharmaceutical composition promotes angiogenesis, thereby effectively being used to prevent or treat symptoms due to ischemia, or various diseases; for example, tissue damage, cerebral infarction, stroke, reperfusion injury, myocardial infarction, congestive heart failure, peripheral vascular occlusion, cardiac hypertrophy, low cardiac contraction, low cardiac dilatation, maladjusted cardiomegaly, systolic heart failure, diastolic heart failure, high blood pressure heart failure, arterial/mitral valve diseases, pulmonary valve disease, or ischemic colitis, due to ischemia.

Description

A pharmaceutical composition for accelerating angiogenesis comprising a CD31-ITIM polypeptide or a gene encoding the CD31-ITIM polypeptide, wherein the pharmaceutical composition comprises CD31-ITIM polypeptide or nucleic acid encoding the same,

The present invention relates to the development of therapeutic agents for diseases associated with the regulation of angiogenesis using proteins.

Ischemia is an ischemic cardiovascular disease that is rarely recognized as having ischemic blood due to severe ischemia. Methods for treating ischemic cardiovascular diseases include cell therapy and pharmacological methods.

Cell therapy methods mainly use adult stem cells or progenitor cells capable of tissue regeneration. For example, there are attempts to regenerate blood vessels and muscles using bone marrow derived stem cells. However, there are difficulties in practical use due to insufficient understanding of methods for selecting stem cells suitable for cell and tissue regeneration constituting adult stem cells. For example, when whole bone marrow-derived stem cells that are not fractionated are used, calcification of the myocardium may be induced.

As a pharmacological method, Cilostazol, which increases cAMP in platelets by selective inhibition of phosphodiesterase III and exhibits an action of inhibiting platelet aggregation, may be mentioned. Smooth muscle cell proliferation inhibitory action, and triglyceride lowering action. In addition, other neuronal cell death prevention effects have been revealed, and these actions are used as clinical therapeutic agents for actual ischemic diseases.

Atherosclerosis is a pathological condition that develops into ischemic vascular diseases such as myocardial infarction, stroke, and vascular death, and treatment is very important because it can lead to death even in severe cases. Clopidogrel is a thienopyridine-based anti-platelet aggregation inhibitory drug that is used to treat atherosclerosis by blocking platelet adhesion and aggregation by blocking the binding of fibrinogen by blocking the ADP receptor on platelets.

U.S. Published Patent Application No. 2010/0119490 relates to the use of CD31-expressing cells as therapeutic agents, and discloses methods for using CD31-expressing cells for tissue regeneration.

Korean Patent Publication No. 2007-0054140 is related to cord blood-derived multipotent stem cells and a cell therapy agent for treating ischemic necrosis disease containing the same, and provides a cell therapy agent for treating ischemic necrosis disease caused by obstructive arterial disease.

There is a need to develop various therapeutic agents other than drugs used in addition to conventional cell therapy agents or low molecular compounds.

The present invention seeks to provide a therapeutic agent for diseases requiring angiogenesis.

In one aspect, the disclosure provides one or more CD31-ITIM polypeptides for promoting angiogenesis, or a gene encoding the same. In one embodiment according to the present disclosure, a plurality of CD31-ITIM polypeptides may be included, particularly 1 to 4, and the polypeptide may be phosphorylated at a specific residue. The angiogenesis to which the peptide of the present invention is effective includes angiogenesis and / or angiogenesis.

In another aspect, the present invention provides a method of producing a polypeptide comprising the steps of providing a gene encoding a polypeptide according to the present invention, a prokaryotic or eukaryotic cell expression vector comprising said gene, and a bacterial, yeast or mammalian cell such as E. coli transformed with said vector do.

Promoting angiogenesis by the peptides according to the present invention relates to the prevention or treatment of an ischemic disease or condition, wherein the ischemic disease or condition is selected from the group consisting of tissue damage due to ischemia, stroke, stroke, reperfusion injury, myocardial infarction, Including cardiac hypertrophy, cardiac hypertrophy, cardiac hypertrophy, cardiac hypertrophy, maladaptive cardiac insufficiency, systolic heart failure, diastolic heart failure, hypertensive heart failure, arterial and vascular disease, pulmonary artery disease cardiovascular, or ischemic enteritis But is not limited thereto.

In another aspect, the invention provides a composition for promoting angiogenesis, comprising a CD31-ITIM protein or a gene encoding the same, and in one embodiment is used for the treatment of ischemic conditions or diseases. Various derived CD31-ITIM may be used, for example, the CD31-ITIM protein or the gene encoding it is derived from a mammal, particularly a human. The ischemic disease or condition occurs in muscle, brain, kidney, large intestine, or heart, and the ischemic symptom or disease is selected from the group consisting of ischemic tissue damage, cerebral infarction, stroke, reperfusion injury, myocardial infarction, Cardiac hypertrophy, arterial and vascular disease, pulmonary arterial disease, cardiovascular disease, or ischemic enteritis, but not limited to, cardiomyopathy, cardiomyopathy, cardiac hypertrophy, hypothyroidism, hypothyroidism, maladaptation equipment, systolic heart failure, diastolic heart failure, It is not.

The CD31-ITIM of the present invention, or a gene encoding the same, or a pharmaceutical composition containing the CD31-ITIM can induce angiogenesis and induce various diseases requiring promotion of angiogenesis, for example, ischemic diseases caused by muscle, brain, heart, For example, tissue damage caused by ischemia, cerebral infarction, stroke, reperfusion injury, myocardial infarction, congestive heart failure, peripheral vascular occlusion, cardiac hypertrophy, hypochondroplasia, hypotonia, maladaptive device, systolic heart failure, diastolic heart failure , Hypertensive heart failure, arterial and vascular disease, pulmonary arterial disease cardiovascular, or ischemic intestinal cardiovascular regeneration or recovery.

Figure 1 shows the result of analysis of the effect of rhCD31 (ITIM) on angiogenesis using HUVEC (GFP-HUVEC) using fluorescence microscopy.
FIG. 2 shows the results of analysis of migration of HUVEC using rhythm 31 (ITIM) in a Scratch wound healing assay (in vitro). FIG. 2a shows a photograph taken at 6 hours after treatment of rhCD31, And the wound size was determined at 6 hours after treatment.
FIG. 3 shows the results of analysis of gene expression of molecules known to be important for angiogenesis by quantitative RT-PCR (qRT-PCR).

The present invention is based on the discovery that the ITIM of CD31 or PECAM-1, known as endothelial cell markers, is angiogenic.

CD31 or PECAM-1 (Platelte Endothelial Cell Adhesion Molecule-1) is a 130 kDa type I transmembrane glycoprotein belonging to the immunoglobulin superfamily. It is a cell-cell junction of endothelial cells and a monocyte, granulocyte, natural killer cell, It is known to be expressed on the surface of hematopoietic cells such as naive T cells and platelets. The amino terminus is involved in the kinase-dependent binding necessary for cell-cell interaction into an immunoglobulin-like domain. The ITIM (immunoreceptor tyrosine inhibitory motif) present in the cell functions as a scaffold for collecting signaling molecules such as tyrosine phosphatase, It mediates the inhibitory function of proteins. In general, it was used as an endothelial cell marker in tissue sections, but its function of promoting angiogenesis is not known.

Thus, in one aspect, the present invention relates to a polypeptide for promoting angiogenesis comprising one or more CD31-ITIM polypeptides.

As described above, the CD31-ITIM of the present invention is a motif that forms the carboxy terminal present in the cell of CD31. NP_000433, and ITIM corresponds to amino acid residues 621-738. More than one ITIM may be used for purposes of this application. In one embodiment according to the present application, there are 1 to 4, especially 2. Each ITIM motif can be linked using known peptides or non-peptide linkages. Two or more ITIM motifs can be connected in two or more.

Further, the ITIM motifs herein may be phosphorylated at one or more tyrosine residues. For example, 690 and / or 713 positions may be phosphorylated based on the sequence disclosed in NP_000433. Upon phosphorylation, SHP-2 and Gab1 are recruited, which activates Erk and amplifies the lower signal (Ref: Peter J. Newman and Debra K. Newman, ATVB, 2003 Jun 1; 23 (6): 953-64 ).

The above-mentioned sequences are illustrative, and the CD31-ITIM polypeptide or the gene encoding the CD31-ITIM polypeptide is included in the scope of the present invention as well as various mammalian-derived and functionally equivalent variants thereof, including human beings, which exhibit the effects according to the present invention. The CD31 amino acid sequence that can be used in the present invention includes, for example, GenBank number NP_000433 in the case of a human origin, NC_000017 in a nucleic acid sequence, and mutants which are functionally equivalent thereto. ITIM is an intracellular region of CD31 (amino acid residues 621-738) present at the carboxy terminus of the CD31. The CD31 or ITIM polypeptide herein has at least 85% homology with the ITIM amino acid sequence set forth in GenBank No. NP_000433.

The CD31-ITIM herein may include one or more ITIM molecules, one in one embodiment, two in another, and three in another embodiment.

The term "functionally equivalent variant" refers to a variant of wild-type protein or its gene sequence derived from a specific species, but having the ability to promote angiogenesis. Thus, for example, the CD31-ITIM of the present invention or the gene encoding it may be derived from human-derived GenBack NO. Lt; RTI ID = 0.0 > NP-000433 < / RTI > Mutants, derivatives, alleles, variants and variants encoding a protein consisting of an amino acid sequence in which one or more amino acids in the amino acid sequence are substituted, deleted, added and / or inserted, And homologues.

As such, as long as a gene encoding a protein having an amino acid sequence in which at least one amino acid is substituted, deleted and / or added in the amino acid sequence encoding the wild-type CD31-ITIM protein also encodes a protein having an equivalent function, . Furthermore, for example, there is a case where the base sequence mutation does not involve a mutation of the amino acid in the protein (axial shift), and such a degenerative mutant is also included in the gene of the present invention.

The proteins used in the present invention can be prepared using methods known in the art. For example, a plasmid containing the corresponding gene encoding the protein may be transferred to prokaryotic or eukaryotic cells such as insect cells and mammalian cells for overexpression, followed by purification. For example, the plasmid can be used by cloning the gene into a vector such as pET28b (Novagen), transferring it to a cell line, and then purifying the expressed protein by a separation method known in the art. The purification method is usually performed by centrifuging a cell lysate or an in vitro translation product to remove cell debris and the like, and thereafter precipitating, dialysis, various column chromatography, for example, ion exchange chromatography, gel- HPLC, reversed-phase HPLC, SDS-PAGE for Fragment, and affinity column. Affinity columns can be made, for example, using anti-screening protein antibodies.

The peptide of the present invention may be used as a therapeutic peptide, and the therapeutic peptide may be a protein or an analogue thereof positively affecting the function of an individual, and may be used to reduce, inhibit, alleviate, alleviate, or stabilize the occurrence or progression of a disease, . In one embodiment, the polypeptide of the invention is effective in promoting angiogenesis and may be used in the treatment of a variety of ischemic diseases resulting from decreased blood flow.

Accordingly, in another aspect, the present invention relates to a composition for promoting angiogenesis comprising a peptide according to the present invention and / or a gene encoding the same. The composition of the present invention is effective for promoting angiogenesis and can be used for the treatment of various ischemic diseases caused by reduction of blood flow.

As used herein, " angiogenesis " includes both " vasculogenesis " and " angiogenesis ". Angiogenesis is characterized by the differentiation of endothelial cell precursors into mature endothelial cells and the assembly of these cells to form new blood vessels, as occurs in the formation of primary vascular grafts in early embryos. Angiogenesis is caused by the growth and branching of blood vessels that already exist. In another aspect, it may be derived from an already existing blood vessel, or it may be stem cells, blood vessel cells or other progenitor cells. Methods for measuring angiogenesis are well known and include, for example, the number of non-segmented blood vessel segments (number of segments per unit area), functional blood vessel density (total length of perfused blood vessels per unit area), vessel diameter, or vessel volume density The total vessel volume calculated on the basis of the diameter and length of each segment per unit area).

As used herein, the term " treatment " is a concept involving inhibiting, eliminating, alleviating, ameliorating, improving and / or preventing a disease or condition or condition due to a disease.

As used herein, the term " ischemic symptom " includes various symptoms due to oxygen deficiency due to insufficient blood circulation in which blood supply to the tissue is stopped due to bleeding, embolism and infarction, . As used herein, the term " ischemic disease " refers to a disorder or a disease caused by ischemic tissue or organs, and may occur in any organ including various tissues such as muscle tissue, brain, heart, kidney, and large intestine. For example, cerebral ischemia leads to diseases such as stroke and reperfusion injury. In the case of intestine, decreases in blood flow to the large intestine may cause ischemic colitis, and cardiac ischemia may lead to myocardial infarction and congestive heart failure.

In one embodiment, the ischemic disease is ischemic heart disease, including cardiovascular tissue damage due to ischemia. Ischemic heart disease is a heart disease caused by a decrease in blood flow to the myocardium due to atherosclerosis of the coronary artery. Clinically, for example, myocardial infarction, angina pectoris, congestive heart failure, peripheral vascular occlusion, But are not limited to, cardiac insufficiency, low cardiac relaxation, maladaptive cardiac output, systolic heart failure, diastolic heart failure, hypertensive heart failure, arterial and vascular disease, and pulmonary valve disease. The composition comprising CD31-ITIM may be used to promote angiogenesis of cardiac tissue or to enhance cardiac function.

In other embodiments, the ischemic disease is ischemic brain disease, including, but not limited to, stroke, reperfusion injury, and ischemic diseases. The composition comprising CD31-ITIM may be used to promote brain tissue angiogenesis or to enhance brain function.

As used herein, the term " therapeutically effective amount " means the amount needed to relieve, or treat, a patient ' s symptoms, condition or disease. The effective amount of CD31-ITIM contained in the composition of the present invention may vary depending on the route of administration, the age, body weight, underlying disease, general health condition, and the like of the patient, and will ultimately be determined at the discretion of the medical practitioner.

The composition comprising the ITIM gene of the present invention or a protein encoded thereby may contain the active ingredient in an amount of 0.0001 to 50% by weight based on the total weight of the active ingredient.

Compositions comprising the ITIM gene of the present invention or a protein encoded thereby can be administered parenterally, for example, intravenously, intramuscularly, or directly into a lesion at the time of clinical administration. Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA) according to the degree, type or effective ingredient of each disease.

The composition of the present invention may be prepared and administered in various parenteral formulations. In the case of formulation, in addition to the above-described effective ingredients, one or more pharmaceutically acceptable carriers may be further included.

The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and one or more of these components. , A buffer solution, a bacteriostatic agent, and the like may be added. The parenteral preparation can be formulated into injectable formulations such as, for example, aqueous solutions, suspensions, emulsions and the like by additionally adding a diluent, a dispersant, a surfactant, a binder and a lubricant, Target organ specific antibodies or other ligands may be used in conjunction with the carrier.

The daily dose of the therapeutic agent is about 0.005 to 10 mg / kg, preferably 0.05 to 1 mg / kg, and the dosage is appropriately selected depending on the patient's body weight, age, sex, health condition, diet, administration time, The severity of the disease, and the like, and may vary depending on the condition of the patient and the severity of the disease.

Furthermore, the compositions of the present invention can be used alone or in combination with methods for the treatment of diseases associated with ischemia, or using surgery, hormonal therapy, drug therapy and biological response modifiers.

Hereinafter, the present invention will be described more specifically with reference to the following examples, but the scope of the present invention is not limited thereto.

Example 1. Fabrication of HUVEC expressing GFP

Lentiviral vector PLL3.7 system (Addgene plasmid 11795, USA) expressing GPF (Green Fluorescence Protein) was transduced into HUVEC (Human Umbilical Vein Endothelial Cell, Lonza, USA) Respectively. Specifically, the method for producing virus from lentiviral vector was carried out by using ViraPower ™ Lentiviral Packaging Mix (Invitrogen), and after 24 hours, 24 hours after the exchange of the culture medium (DMEM, 10% FBS) And then centrifuged at 25,000 rpm for 90 minutes at 4 ° C to obtain virus. The thus obtained virus was transfected into HUVEC to prepare GFP-expressing HUVEC. For the transduction method, the previously obtained lentivirus was used at a concentration of about ⁵ × 10 ⁵ pfu / ml. The culture medium was EGM-2MV (Lonza), treated with 10 ug / ml of polybrene, and incubated at 37 ° C and 5% CO ₂ for 18 hours.

HUVEC was cultured in EGM-2MV medium in a culture dish coated with 1.5% gelatin (Sigma-Aldrich, USA).

Example 2. rhCD31  ( ITIM ) On blood vessel formation

The effect on angiogenesis was performed using GFR Matrigel (BD Bioscience, USA) according to the manufacturer's method. In summary, semi-solid polymerization was carried out by coating Matrigel in 35 mm conical dishes (ibidi Inc.) on ice at approximately 80 μl, followed by incubation at 37 ° C, 5% CO ₂ for 30 minutes. Subsequently, approximately 3 x 104 GFP-HUVEC cells cultured in EBM-2 (Endothelial basal medium-2) containing 1% FBS were added to the dish. Bovine serum albumin (BSA) and rhCD31 (ITIM) molecules were treated with 200 ng / ml of each, followed by incubation at 37 ° C and 5% CO ₂ for 24 hours. The rhCD31 (ITIM) protein was purchased from PROSPEC. The tube formation of HUVEC (GFP-HUVEC) was then compared via fluorescence microscopy (Olympus IX2, 40 magnification).

The results are shown in FIG. Tube formation was significantly increased in rhCD31 treated cells compared to cells treated with BSA as a control. This indicates that the rhCD31 (ITIM) protein can promote angiogenesis.

Migration was assessed using Scratch wound healing assay (in vitro). Specifically, HUVEC was seeded on a 6-well plate and scratched after 24 hours. Then, rhCD31 (100, 200 ng / ml) was treated with EBM-2 medium containing 5% FBS and Thymidine (1 mM). After six hours, the wound size was quantified and migration was evaluated. The results are shown in Fig. FIG. 2A is a photograph of rhCD31 taken 6 hours after treatment, and FIG. 2B is a result of quantifying wound size 6 hours after rhCD31 treatment. As shown in the figure, migration was increased in the rhCD31 treated condition.

Example 3. Analysis of Expression Patterns of Molecules Important for Angiogenesis

Expression patterns of molecules important for angiogenesis were analyzed by reverse transcription - polymerase chain reaction.

10% FBS media control to cultured HUVEC in EBM-2 (BSA) containing and rhCD31 (ITIM), rhCD31 (extra membrane fragment) 4 hours of incubation the at 200ng / ml after treatment 37 ℃, 5% CO _2, respectively Respectively. The RNAs from each experimental group were extracted using RNeasy Mini Kit (Qiagen, USA) according to the manufacturer's instructions and cDNA synthesis kit (Takara) was used according to the manufacturer's instructions. Using the primers described in the following Table 1, gene expression of molecules known to be important for the production of blood light was analyzed by PCR. The results are shown in FIG. The expression of VEGF, HGF, Ang2, and IL-10 was increased in HUVEC treated with rhCD31 (ITIM) protein as well as the expression of integrin genes such as Integrin alpha 5, Integrin beta 3 and Integrin beta 8 Expression also increased.

Gene name Primer sequence Tm (占 폚) Product length ( bp ) IL10 FW
(Foward) 5 '- GCCTAACATGCTTCGAGATC-3' 58 206 RV
(Reverse) 5 '- TGATGTCTGGGTCTTGGTTC-3' VEGF FW 5 '-GCCAGCACATAGGAGAGATGAG-3' 60 161 RV 5 '-AACGTACACGCTCCAGGACTTA 3' HGF FW 5 '-CTGGTTCCCCTTCAATAGCA-3' 60 165 RV 5 '-CTCCAGGGCTGACATTTGAT-3' Ang2 FW 5 '-AGTCATCCAACACCGAGAAGAT-3' 60 317 RV 5 '-CCGAATCCTTTGTGCTAAAATC-3' Integrin a5 FW 5 '- TGCAGTGTGAGGCTGTGTACA-3' 60 88 RV 5 '- GTGGCCACCTGACGCTCT-3' Integrin b3 FW 5 '-CCGTGACGAGATTGAGTCA-3' 60 132 RV 5 '-AGGATGGACTTTCCACTAGAA-3' Integrin b8 FW 5 '- AATTTGGTAGTGGAAGCCTATC-3' 60 146 RV 5 '- GTCACGTTTCTGCATCCTTC-3' GAPDH FW 5 ' -GAGTCAACGGATTTGGTCGT-3 ' 60 185 RV 5 '-GACAAGCTTCCCGTTCTCAG-3'

Taken together, the results of FIGS. 1 and 2 show that HUVEC treated with rhCD31 (ITIM) not only increased the angiogenic gene but also increased the actual tube formation. This indicates that rhCD31 (ITIM) protein can promote angiogenesis.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (7)

At least one CD31-ITIM polypeptide or a gene encoding said polypeptide.
The pharmaceutical composition for promoting angiogenesis according to claim 1, wherein 1-4 CD31-ITIM polypeptides or genes are contained.
The pharmaceutical composition for promoting angiogenesis according to claim 1, wherein the CD31-ITIM polypeptide is phosphorylated.
2. The pharmaceutical composition according to claim 1, wherein said CD31-ITIM polypeptide is derived from mammals.
The pharmaceutical composition for accelerating angiogenesis according to claim 1, wherein the angiogenesis comprises angiogenesis or angiogenesis, or angiogenesis and angiogenesis.
The pharmaceutical composition for promoting angiogenesis according to claim 1, wherein said angiogenesis promotion is prevention or treatment of ischemic diseases or symptoms.
7. The method of claim 6, wherein the ischemic disease or condition is selected from the group consisting of ischemic tissue damage, cerebral infarction, stroke, reperfusion injury, myocardial infarction, congestive heart failure, peripheral vascular occlusion, cardiac hypertrophy, hypothyroidism, A medicament for promoting angiogenesis, which is a non-adaptive heart unit, systolic heart failure, diastolic heart failure, hypertensive heart failure, arterial and vascular disease, pulmonary artery disease cardiovascular, or ischemic enteritis.

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