KR101518369B1 - CD11b+CX3CR1+ cells and a composition for preventing or treating ischemic disease comprising the same - Google Patents

Abstract

The present invention relates to CD11b ⁺ CX3CR1 ⁺ cells having angiogenesis-promoting function and uses thereof. More specifically, the present invention relates to a CD11b ⁺ CX3CR1 ⁺ cell and a composition for promoting angiogenesis comprising the same. The present invention also relates to a composition for preventing or treating ischemic diseases comprising the above cells.

Description

CD11b + CX3CR1 + cells and a composition for preventing or treating ischemic diseases comprising the same,

The present invention relates to CD11b ⁺ CX3CR1 ⁺ cells having angiogenesis-promoting function and uses thereof. More specifically, the present invention relates to a CD11b ⁺ CX3CR1 ⁺ cell and a composition for promoting angiogenesis comprising the same. The present invention also relates to a composition for preventing or treating ischemic diseases comprising the above cells.

Angiogenesis is a process in which new blood vessels are generated in existing blood vessels and provides blood vessels supplying nutrients, growth factors, and oxygen necessary for hypoxic or ischemic tissues and organ metabolism due to blood circulation disorders, It is known to functionally improve related tissue damage and injury.

Excessive angiogenesis is a major cause of disease exacerbation, but the formation of blood vessels is also a cause of serious disease. For example, a placenta with uneventful angiogenesis is an important cause of abortion, and in the case of necrosis, ulceration and ischemia due to the formation of blood vessels, tissue or organ dysfunction may be caused or even cause death. In addition, diseases such as arteriosclerosis, myocardial infarction, ischemic stroke and angina are caused by poor blood supply. Accordingly, attempts have been actively made to apply drugs and techniques for inducing or promoting angiogenesis to the treatment of diseases dependent on angiogenesis, such as ischemic diseases.

Vascular endotherial growth factor (VEGF), a well-known angiogenesis promoting factor, has been used as a treatment for various human diseases such as myocardial infarction, cerebral ischemic injury, limb ischemia and wound healing (J Folkman, Annu. Rev. Med. 57: 1-18, 2006; JM Isner & T Asahara, J. Clin. Invest. 103: 1231-1236, 1999). In addition, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), developmentally regulated endothelial locus-1 (Del-1), hepatocyte growth factor (HGF) angiogenic factor itself such as platelet-derived endothelial cell growth factor, angiopoietin, transforming growth factor (TGF), and epidermal growth factor (EGF) Or a gene for producing the same is administered to an ischemic site to promote the production of collateral vessels to treat ischemic diseases or to treat severe ischemic diseases in which percutaneous arterial plasty or arterial bypass can not be performed However, since these factors are difficult to separate and purify as proteins and are expensive, they are used in clinical applications (see, for example, There is fear. Recently, there has been an increased interest in research for the development and establishment of new angiogenesis agents.

The present inventors have found that CD11b ⁺ CX3CR1 ⁺ cells isolated from mouse ischemia-induced muscle tissue induce angiogenesis through interaction with Fkn, activate vascular endothelial cell migration, infiltration and tubular formation associated with angiogenesis, And the present invention has been completed.

One object of the present invention is to provide a cell comprising an immunophenotype of CD11b ⁺ and CX3CR1 ⁺ and exhibiting angiogenesis-promoting function.

It is another object of the present invention to provide a composition for promoting angiogenesis comprising the cell.

It is another object of the present invention to provide a pharmaceutical composition for preventing or treating ischemic diseases comprising the above cells.

It is another object of the present invention to provide a method for promoting angiogenesis comprising the step of administering an effective amount of said cells to an individual.

Another object of the present invention is to provide a method for preventing or treating ischemic diseases comprising administering an effective amount of said cells to a subject.

It is known that monocyte / myeloid lineage cells can secrete growth factors that promote angiogenesis and form blood vessels to increase blood flow to damaged tissue. However, it is unclear whether the specific subpopulations of monocyte / myeloid lineages involved in neovascularization or their mechanism of action are presently known. To date, several monocyte / myeloid lineages have been proposed.

In the previous study, when inducing ischemia in the muscle of C57BL / 6 mice, we found that CD11b ⁺ Gr1 ^dim cells, which are known as myeloid derived suppressor cells, increase in ischemic muscle, It has been confirmed that the blood flow in the ischemic muscle is remarkably increased by separating and injecting again. As a result of further studies, it was confirmed that CD11b ⁺ Gr1 ^dim cells were monocyte / macrophage lineage cells including CX3CR1 ⁺ , CCR2 ⁺ , CCR5 ⁺ , and CXCR4 ⁺ monocytes. In particular, CD11b ⁺ CX3CR1 ^{+ &} Lt ^{; / RTI &} gt ^; cells.

To investigate the relationship between CD11b ⁺ CX3CR1 ⁺ cells and angiogenesis, the present inventors investigated the relationship between CD11b ⁺ CX3CR1 ⁺ cells and angiogenesis using a hypoechoic model of C57BL / 6 mouse, The pattern of CD11b ⁺ CX3CR1 ⁺ cell number change was analyzed and changes in blood flow when CD11b ⁺ CX3CR1 ⁺ cells were injected into ischemic muscle and changes in blood flow when CD11b ⁺ CX3CR1 ⁺ cells were pretreated were analyzed.

As a result, ischemia induced by 4 to CD11b ⁺ CX3CR1 ⁺ cells gradually decreased while the number increased rapidly in the ischemia induction on day 14 there was confirmed that the cell count is reduced to roughly before ischemia induction, this CD11b ⁺ CX3CR1 ⁺ cell number of vessels through It was found that the regeneration was remarkably increased at the time when the regeneration occurred vigorously. In addition, the expression of Fkn (fractalkine; CX3CL1), which is known to affect CX3CR1 ⁺ cell migration, is similar to that of CD11b ⁺ CX3CR1 ⁺ cell number, and the expression is highest at 4 days after induction of ischemia (Figs. 1A to 1C).

In addition, when the CD11b ⁺ CX3CR1 ⁺ cells isolated from the mouse ischemic muscle were injected directly into the ischemic muscle, the blood flow was gradually increased over time. These blood flow increasing effects were observed in CD11b ^- CX3CR1 ^- cells or CD11b ⁺ CX3CR1 ^- cells. Furthermore, a significant increase in blood flow was observed in the effective dose range even in the group injected with Fkn protein alone associated with the migration of these cells, confirming that CD11b ⁺ CX3CR1 ⁺ cells play important roles in new blood vessel formation after ischemic injury (Figs. 2A and 2B).

In addition, when the function of CD11b ⁺ CX3CR1 ⁺ cells or Fkn was blocked by pretreating neutralizing anti-CX3CR1 antibody or neutralizing anti-Fkn antibody in mice, the function of improving the blood flow of CD11b ⁺ CX3CR1 ⁺ cells was deteriorated, and the reaction of CX3CR1 and Fkn And plays an important role in the generation of new blood vessels (Fig. 3). Further, after the ischemia induction during formation of new blood vessels CD11b ⁺ CX3CR1 ⁺ cells go to the ischemic site, and by confirming via immunofluorescence that the new vascular structures in these areas are formed (Fig. 4), CD11b ⁺ CX3CR1 ⁺ cells new It is concluded that the effect of improving blood flow is promoted by promoting angiogenesis.

Thus, the present inventors have completed the present invention in which CD11b ⁺ CX3CR1 ⁺ cells are provided as agents for treating angiogenesis promoting agents and ischemic diseases.

Hereinafter, the present invention will be described in more detail.

In one embodiment, the invention relates to a cell comprising an immunophenotype of CD11b ⁺ and CX3CR1 ⁺ and exhibiting angiogenesis-promoting function.

In another aspect, the present invention relates to a composition for promoting angiogenesis comprising cells comprising an immunophenotype of CD11b ⁺ and CX3CR1 ⁺ .

In another embodiment, the present invention relates to a method for promoting angiogenesis, comprising the step of administering an effective amount of the cell to an individual.

In another aspect, the present invention relates to a pharmaceutical composition for the treatment of ischemic diseases comprising cells comprising an immunophenotype of CD11b ⁺ and CX3CR1 ⁺ .

In another aspect, the present invention relates to a method for preventing or treating an ischemic disease comprising administering an effective amount of the cell to an individual.

We isolated the CD11b ⁺ CX3CR1 ⁺ cells in the leg muscles that induced ischemia by dissecting the femoral artery of C57BL / 6 mice and then examined the surface antigens for more precise confirmation of the cells of this lineage . As a result, the CD11b ⁺ CX3CR1 ⁺ cells were ^positive for bone marrow cells (CD45) and macrophages (F4 / 80), whereas endothelial cells (VEGFR-2), dendritic cells (CD11c), NK cells (FIG. 1d), CD11b ⁺ CX3CR1 ⁺ cells migrating into ischemic muscle were monocyte / macrophage lineage cells. These cells correspond to functional cells that the present inventors first isolate and provide.

Thus, preferably, the cell of the invention is a cell comprising an immunophenotype of CD11b ⁺ and CX3CR1 ⁺ .

More preferably, the cell of the invention is a cell that additionally comprises one or more immunophenotypes selected from CD45 ⁺ , VEGFR2 ^- , CD11c ^- , NK1.1 ^- and CD3 ^- in addition to the immunophenotype of CD11b ⁺ and CX3CR1 ⁺ have.

Furthermore, cells having additional immunophenotypic or morphological characteristics added thereto may be included in the scope of the cells of the present invention as long as they exhibit the characteristics of the above-described immunophenotypes and exhibit angiogenesis promoting action.

The cells of the present invention are obtained by artificially cutting the lower abdominal aorta of a mouse and isolating muscle cells, but the method of obtaining the same is not limited thereto and can be obtained from ischemic tissues or blood of various mammals including mice, Can be obtained in large quantities by in vitro culture, differentiation and / or amplification using knowledge known in the art. For example, ischemic tissues are treated with collagenase and / or proteolytic enzymes to dissociate the tissues and then subjected to Ficoll-Hypaque separation, moidfied Ficoll-Hypaque separation, 3% gelatin separation, Percoll separation, panning, immunomagnetic cell sorting, fluorescence activated cell sorting, and the like. The above-described methods are merely representative examples, and the cells of the present invention can be obtained by various methods utilizing knowledge known in the art.

Since the cell of the present invention has angiogenesis promoting action, it can be used as an angiogenesis promoter, and further can be used for the prevention or treatment of various diseases requiring angiogenesis.

Ischemic diseases are the representative diseases requiring angiogenesis. Ischemic disease refers to a disease in which blood is partially deficient due to various causes such as arterial stenosis or contraction, thrombosis or embolism. Examples of ischemic diseases include heart failure, hypertensive heart disease, arrhythmia, congenital heart disease, myocardial infarction, stroke, peripheral vascular disease, angina pectoris, cerebrovascular dementia, coronary artery insufficiency, cerebral insufficiency and vascular insufficiency. And all ischemic diseases requiring angiogenesis may be included in the scope of the present invention.

As used herein, the term "prophylactic " means any action that promotes angiogenesis upon administration of the composition of the present invention, or any act that inhibits or delays the growth, spread or recurrence of an angiogenesis dependent disease.

As used herein, the terms " treatment "and" improvement "refer to all actions in which the administration of the composition of the invention improves or alleviates an angiogenesis dependent disease.

The term "administering" as used herein is meant to provide any desired composition of the invention to an individual by any suitable method.

As used herein, the term "individual" refers to an animal that has suffered from a disease in which angiogenesis-dependent diseases can be improved by administering the composition of the present invention, preferably a vertebrate animal, more preferably a mammalian animal, , Dog, cat, goat, pig, rat, guinea pig, hamster, chimpanzee or gorilla.

As used herein, the term "effective amount " means an amount sufficient to prevent or treat a disease, including the type of disease, severity, activity of the drug, sensitivity to the drug, The duration of treatment, factors including drugs used simultaneously, and other factors well known in the medical arts.

The composition of the present invention may be administered orally or parenterally as a cell therapy agent and may be administered orally or parenterally in the form of intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine injection, May be administered by intrathoracic injection and may be administered directly to the disease site.

Preferably, the pharmaceutical composition according to the present invention may be administered in the form of an injection in a site where the angiogenesis of the mammal is to be induced by mixing with the composition for injection, and the injection composition is preferably an isotonic aqueous solution or suspension, The pharmaceutical compositions may be sterilized or contain adjuvants such as preservatives, stabilizers, wetting agents, emulsifier solution boosters, salts for controlling osmotic pressure and / or buffers, or other therapeutically useful substances. In addition, the pharmaceutical composition according to the present invention can be formulated in the form of an oral preparation, an external preparation, a suppository or the like according to a conventional method.

In addition, the composition of the present invention can be administered directly or indirectly with a pharmaceutically acceptable carrier, such as transplantation through a surgical site at a site requiring repair or expansion, delivery using a catheter, a matrix, or an injection needle have. Pharmaceutically acceptable carriers include suitable organic or organic carrier materials which do not deleteriously react with the cells, compositions or components of the present invention, and may be water, salt solutions (Ringer's solution), alcohols, oils, gelatin, lactose, Carbohydrates such as amylose or starch, fatty acid esters, hydroxymethylcellulose, and polyvinylpyrrolidone. These preparations may be sterilized and, where appropriate, be mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifying agents, salts for osmotic pressure, buffers and coloring agents. Pharmaceutical carriers suitable for use in the present invention are known in the art.

In addition, the composition of the present invention may optionally be administered together with other beneficial drugs or biological molecules (growth factors, nutritional factors, etc.). Biomolecules that may be administered together may include anti-apoptotic agents, anti-inflammatory agents, immunomodulators, anti-proliferators, corticosteroids, antibodies, anti-thrombotic agents, anti-oxidants, or topical anesthetics.

To the cell 1 X 10 ⁴ to about ¹⁰ X 10 10 cells / kg, preferably a pharmaceutically effective amount is 2.5 X 10 ⁷ to about 5 X 10 ⁷ dog, and more preferably, but can use a 2.5 X 10 ⁷ cells / kg, For example, 2 X 10 ⁹ to 4 X 10 ⁹ on the basis of 70 kg of an adult. However, it may vary depending on the weight, age, sex, health condition, diet, administration period, administration method, elimination rate, severity of disease, etc. of a particular patient. The pharmaceutical composition of the present invention may be administered once a day or once a week depending on the half-life period. The dose is not intended to limit the scope of the invention in any way.

Since the cell of the present invention has angiogenesis promoting action, it can be used as an angiogenesis promoter, and further can be used for the prevention or treatment of various diseases requiring angiogenesis.

FIG. 1A shows the results of comparing intracellular CD11b ⁺ CX3CR1 ⁺ cell fractions before induction of ischemia in mice and after induction of ischemia 4 days later.
FIG. 1B shows changes in intracellular CD11b ⁺ CX3CR1 ⁺ cell numbers over time after induction of islet ischemia in mice.
Figure 1c shows the expression of Fkn in the muscle over time after induction of islet ischemia in mice.
Figure 1d shows the results of surface antigen analysis of CD11b ⁺ CX3CR1 ⁺ cells isolated from ischemic muscle.
FIG. 2A shows the results of blood flow comparison over time according to cell injection.
FIG. 2B shows the results of blood flow comparison over time according to Fractalkine protein injection.
FIG. 3 shows the result of blood flow comparison after injection of neutralizing anti-Fkn antibody and anti-CX3CR1 antibody after induction of lower limb ischemia.
Figure 4 shows the CD11b ⁺ CX3CR1 ⁺ cell distribution in ischemic muscle after ischemic induction.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Mouse ischemic induction and Muscle cell  detach

Following the regulations of the Catholic University Animal Ethics Committee, ischemia was induced by removing the veins and arteries from the thighs of 10-12 week old C57BL / 6 mice. Single cells were isolated from ischemic muscle using 0.2% collagenase (210 U / mg; Worthington, Lakewood, NJ) and distearate (0.95 U / mg; Invitrogen, Grand Island, NY). For FACS analysis, the separated cells were reacted with APC-conjugated anti-mouse CD11b, biotinylated anti-mouse CX3CR1 antibody for 30 minutes at 4 ° C. Thereafter, the cells were further reacted with FITC-conjugated streptavidin (Molecular Probes, Eugene, OR) for 30 minutes. CD11b ⁺ CX3CR1 ⁺ cells were analyzed and isolated using a flow cytometer (MoFlo ™ XDP Cell Sorter (Beckman Coulter, Brea, Calif.)).

Example  2. FACS  Analysis and real-time polymerase reaction

Surface antigens of CD11b ⁺ CX3CR1 ⁺ cells were analyzed by FACS, and Fkn expression in tissues was examined using real time PCR.

The expression levels of these proteins were measured by using F4 / 80, VEGFR2, NK1.1 antibody (eBiosciences, San Diego, Calif.) Or CD45, CD3 and CD11c antibody (BD Pharmigen, San Jose, CA) in isolated CD11b ⁺ CX3CR1 ⁺ Were investigated using flow cytometry. (MoFlo ™ XDP Cell Sorter)

The expression of Fkn in the tissues was measured by using Fkn primer (forward: GGA CAG GAC CTC AGT CCA GA, reverse direction: TCG GGG ACA GGA GTG ATA AG) using a device of MyiQ Real Time PCR Detection Systems (Bio-Rad, Hercules, Canada) Bio-Rad IQ5 software was used for measurement and analysis.

Example  3. Isolate from ischemic muscle CD11b ⁺ CX3CR1 ⁺ Cell and Fkn And blood vessel formation

CD11b ⁺ CX3CR1 ⁺ , CD11b ⁺ CX3CR1 ^- and CD11b ^- CX3CR1 ^- cells isolated from ischemic muscle were injected into the ischemic muscle of the mice 48 hours after induction of ischemia (5X10 ⁵ cells / mouse). The mouse recombinant protein Fkn (R & D System, Minneapolis, Minn.) Was dissolved in 0.5, 1, 5 ug of PBS in 500 μl of PBS and injected into the ischemic muscle for 5 days every 100 μl immediately after ischemic induction.

To block the function of CX3CR1 or Fkn, anti-CX3CR1 neutralizing antibody and anti-Fkn neutralizing antibody were used. 0.5 μg of anti-CX3CR1 neutralizing antibody (TP501, Torrey Pines Biolabs, San Diego, Calif.) Was injected into the ischemic muscle for 5 days each day. The same amount of control IgG as the anti-Fkn neutralizing antibody (TP233, Torrey Pines Biolabs) was also injected in the same manner. We used a laser Doppler perfusion scanner (LDPI, Moore instruments, Devon, UK) to measure the blood flow in the ischemic muscle immediately after ischemic induction.

Example  4. Organization Immunofluorescent staining

The muscle was isolated from the ischemic legs at 0, 1, 4, 7, 14, 21 and 28 days after ischemia induction and embedded in OCT compound (Tissue-Tek, Sakura Finetek Co., Tokyo, Japan) And attached to a glass. After immobilization in 4% paraformaldehyde for 15 minutes, the cells were washed with PBS and reacted with 10% normal horse serum for 1 hour to prevent nonspecific reaction. Each tissue was immunoreacted with a rabbit anti-CX3CR1 antibody, rabbit anti-Fkn antibody for 16 hours at 4 ° C, washed three times, and stained with biotinylated anti-rabbit antibody (Vector Labs, Burlingame, CA) The reaction was allowed to proceed at room temperature for 1 to 2 hours. After secondary reaction with Cy3-streptavidin (Jacson Labs, Bar Harbor, Maine), the cells were washed and then reacted with anti-CD11b antibody and reacted with FITC-conjugated antibody at room temperature for 1 hour. The nuclei were stained with diamidino-2-phenylindole (DAPI, Sigma) and then observed with a Zeiss fluorescence microscope.

Example  5. Statistical Analysis

The results of all experiments were expressed as mean ± SD. Differences in cell populations after ischemic injury were analyzed by unpaired t-test. The repeated measures ANOVA was used to analyze the difference in the results between the various groups. P value was 0.05 or less.

Experiment result

1. Ischemic injury in ischemic muscle CD11b ⁺ CX3CR1 ⁺ Number of cells remarkably Increase .

Ischemic muscle tissue was detached from the C57BL / 6 mouse ischemia-repertory model and the change of CD11b ⁺ CX3CR1 ⁺ cell number was measured using a flow cytometer. As a result, the percentage of CD11b ⁺ CX3CR1 ⁺ cells in muscle was 0.1 ± 0.04% of total cells before surgery and increased to 4.05 ± 0.07% after 4 days of induction of ischemia (n = 5, p <0.0001) . In addition, the number of CD11b ⁺ CX3CR1 ⁺ cells isolated from ischemic muscle increased 180 times after surgery 4 days after surgery (n = 5, 0.01 ± 0.02X10 ⁵ / g in preoperative tissue, 1.84 ± 0.9 in tissue 4 days after surgery) ^{X10 5 / g; p = 0.03} ), 14 days is reduced to a similar level as before surgery (tissue from ^{0.24 ± 0.33X10 5 / g),} CD11b + CX3CR1 + cell counts increased significantly in time vigorously occurs the revascularization (Fig. 1B).

The expression level of Fkn (Fractalkine), which affects CD11b ⁺ CX3CR1 ⁺ cell migration, was analyzed using real - time PCR. Fkn mRNA expression was highest at day 4 after arterial incision (Fig. 1c), indicating that increased Fkn expression in ischemic muscle was associated with an increase in CD11b ⁺ CX3CR1 ⁺ cell number.

CX3CR1 surface antigens are cell surface antigens that are expressed variously in macrophages, monocytes, endothelial cells, dendritic cells, NK cells and T cells. In order to determine what kind of cells CD11b ⁺ CX3CR1 ⁺ cells are, CD11b ⁺ CX3CR1 ⁺ cells in the muscle were isolated and examined for surface antigens. CD11b ⁺ CX3CR1 ⁺ cells were positive for endothelial cells (VEGFR-2), dendritic cells (CD11c), NK cells (NK1.1) and T cells (CD3) (Fig. 1d), CD11b ⁺ CX3CR1 ⁺ cells migrating into ischemic muscle were monocyte / macrophage lineage cells.

2. Separated from muscle CD11b ⁺ CX3CR1 ⁺ Cells Fkn  If the protein is injected into the ischemic muscle, Increase .

And CD11b ^{- -} CX3CR1 ^- muscle-derived CD11b ⁺ CX3CR1 ⁺ cells CD11b ⁺ CX3CR1 ^+, CD11b ⁺ CX3CR1 on the fourth day ischemic muscle after ischemia induction to see if increasing the angiogenesis to separate the cells (control) in the ischemic muscles The changes of blood flow were measured by LDPI (Laser Doppler Perfusion Image). As a result, the blood flow (78.03 ± 3.16%, day 28) of the mice injected with CD11b ⁺ CX3CR1 ⁺ cells was compared with the group injected with CD11b ^- CX3CR1 ^- cells (38.99 ± 0.26%, 28 days) (34.26 + 4.76%; p = 0.001; n = 5). CD11b ⁺ CX3CR1 ^- cells (50.8 ± 2.5%, 28 ^{days; n = 5) - CX3CR1 -} CD11b in the group injected with as compared to the group who underwent only cells or surgery but have some blood flow increases, the injection of CD11b ⁺ CX3CR1 ⁺ cells Compared with one group (P = 0.001) (Fig. 2a). These results suggest that CD11b ⁺ CX3CR1 ⁺ cells play important role in new blood vessel formation after ischemic injury.

In addition, if the blood flow is increased by direct injection of Fkn protein, which induces CX3CR1 ⁺ cells after ischemic injury, it is thought that this can be used more easily in clinical practice. And the blood flow of the ischemic leg was measured. On the 28th day after ischemia, 0.5 ㎍ of Fkn protein injected group showed no increase in blood flow (40.54 ± 2.34%, 28.6 days, 37.69 ± 4.85%, 28 days) compared to the PBS - injected control group, but 1 ㎍ and 5 ㎍ Fkn In the injected group, the blood flow was increased to 51.72 ± 0.4% and 77.83 ± 11.99% on the 28th day after ischemia, respectively, compared with the normal leg, and the blood flow was significantly increased compared with the control group (P = 0.01 , P = 0.012) (Figure 2b).

3. CX3CR1 and Fkn Blocking of the function of the blood flow is deteriorated.

To investigate the relationship between Fkn-CX3CR1 response and angiogenesis, neutralizing anti-CX3CR1 antibody, neutralizing anti-Fkn antibody or control IgG was injected into the muscle of C57BL / 6 mice inducing ischemia and LDPI was measured. On the 28th day, blood flow in the muscle was measured as 25.94 ± 4.89% and 29.41 ± 4.59% in the muscle of the mice injected with neutralizing anti-CX3CR1 antibody or neutralizing anti-Fkn antibody (p = 0.037 and p = 0.01, respectively) Of LDPI was significantly lower than that of 46.25 ± 3.23% (FIG. 3). These results suggest that the interaction of Fkn-CX3CR1 contributes to the formation of new blood vessels in the ischemic muscle.

4. CD11b ⁺ CX3CR1 ⁺ Cells migrate to ischemic muscle.

To investigate the role of CD11b ⁺ CX3CR1 ⁺ cells in the formation of new blood vessels after induction of ischemia, immunofluorescent staining was performed using anti-CD11b antibody (green) and anti-CX3CR1 antibody (red) in ischemic muscle. In normal tissues, a small number of CD11b ⁺ CX3CR1 ⁺ cells were distributed only around the extracellular blood vessels (arrow; yellow; FIG. 4A). On the fourth day of ischemia, a large number of cells migrated into the ischemic muscle (Fig. 4B), and many of them were CD11b ⁺ CX3CR1 ⁺ cells (arrow; yellow; Fig. On the 7th day of ischemia, the damaged muscle cells were completely removed, and several vessel structures were formed inside the ischemic muscle (Fig. 4D). After 3 weeks of ischemia, the newly formed blood vessels were composed of CX3CR1 ⁺ cells (arrow; red color; Fig. 4E), and when observed at 200x magnification, CD11b ⁺ CX3CR1 ⁺ cells were located at regular intervals (Arrow; yellow; Fig. 4F). After the ischemic injury, CD11b ⁺ CX3CR1 ⁺ cells migrated to the ischemic site and were found to be involved in the formation of new vascular structures at this site.

Claims (9)

A cell comprising an immunophenotype of CD11b ⁺ , CX3CR1 ⁺ , CD45 ⁺ , F4 / 80 ⁺ , VEGFR2 ^- , CD11c ^- , NK1.1 ^- and CD3 ^- and exhibiting angiogenesis promoting function. delete ^{CCD11b +, CX3CR1 +, CD45 +} , F4 / 80 +, VEGFR2 -, CD11c -, NK1.1 - and CD3 ^- angiogenesis promoting composition for containing cells, including immune phenotype. delete The composition for promoting angiogenesis according to claim 3, further comprising an Fkn (fractalkine) protein. A cell comprising an immunophenotype of CD11b ⁺ , CX3CR1 ⁺ , CD45 ⁺ , F4 / 80 ⁺ , VEGFR2 ^- , CD11c ^- , NK1.1 ^- and CD3 ^- . delete 7. The method of claim 6, wherein the ischemic disease is selected from the group consisting of heart failure, hypertensive heart disease, arrhythmia, congenital heart disease, myocardial infarction, stroke, peripheral vascular disease, angina pectoris, cerebrovascular dementia, coronary artery insufficiency, &Lt; / RTI &gt; or a pharmaceutically acceptable salt thereof, for the prophylaxis or treatment of ischemic diseases. The pharmaceutical composition for preventing or treating ischemic diseases according to claim 6, further comprising an Fkn (fractalkine) protein.

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