WO2015025956A1 - Pharmaceutical composition for treating myocardial damage, pharmaceutical composition for preventing myocardial damage, pharmaceutical composition for treating heart failure, pharmaceutical composition for preventing heart failure, method for treating or preventing myocardial damage or heart failure, mfg-e8, uses of mfg-e8, and method for screening compounds for treating or preventing myocardial damage or heart failure - Google Patents

Abstract

MFG-E8 is produced by myofibroblasts appearing in an infarct region. The production of MFG-E8 is regulated by TGF-β/SRF signals. Myofibroblasts ingest dead cells through MFG-E8, inducing an anti-inflammatory response due to phagocytosis by such macrophages. Consequently, in MFG-E8-deficient mice, many more apoptotic cells remain in an infarct region of the heart without undergoing phagocytosis, causing excessive inflammation in said region. As a result, the survival rate of MFG-E8-deficient mice after myocardial infarction decreases significantly. Conversely, the removal of apoptotic cells is facilitated by injecting MFG-E8 into the infarct region, alleviating inflammation in the infarct region, resulting in a marked improvement in cardiac function after a myocardial infarction. These findings indicate that MFG-E8 may provide a novel treatment target for the treatment of myocardial infarctions.

Description

Pharmaceutical composition for treating myocardial disorder, pharmaceutical composition for preventing myocardial disorder, pharmaceutical composition for treating heart failure, pharmaceutical composition for preventing heart failure, method for treating or preventing myocardial disorder or heart failure, use of MFG-E8, MFG-E8 And a method for screening a compound for treating or preventing myocardial injury or heart failure

The present invention relates to a pharmaceutical composition for treating or preventing myocardial injury or heart failure, a method for treating or preventing myocardial injury or heart failure, MFG-E8, MFG for the manufacture of a medicament for treating or preventing myocardial injury or heart failure. -Use of E8 and methods of screening for compounds that treat or prevent myocardial injury or heart failure.
This application claims priority based on US Provisional Application No. 61 / 868,599 filed in the United States on August 22, 2013, the contents of which are incorporated herein by reference.

The heart contributes to blood circulation throughout the body by constantly contracting and expanding. In addition, the heart supplies itself with blood through the coronary arteries. Myocardial infarction is a state in which blood is not supplied downstream because the coronary artery is occluded by arteriosclerosis or the like. Lifestyle-related diseases such as diabetes and hyperlipidemia are known as risk factors for myocardial infarction, and the number of patients is expected to increase with an aging society in the future. The first treatment of myocardial infarction is reopening of the coronary artery by surgery, and at the same time, a method of removing the thrombus caused by the thrombolytic agent is used. Patients will continue to take antiplatelet drugs to prevent reocclusion. However, 3-10% of patients subsequently develop a rupture of the left ventricular free wall, often death. Heart rupture is only about 15% of the total cause of death after myocardial infarction, but there are no drugs to treat and prevent heart rupture, and it is said that the treatment of myocardial infarction is well established. It is difficult (see Non-Patent Document 1).

After myocardial infarction, an inflammatory reaction is first caused with myocardial necrosis. In the inflammatory phase, events such as leukocyte migration, extracellular matrix protein degradation, and dead cell removal occur. In the case of humans, the period from the onset of myocardial infarction to about the fourth day is regarded as the inflammatory stage. Thereafter, the repair reaction is promoted as the inflammatory response is attenuated. In the repair phase, events such as fibroblast development, tissue fibrosis, and angiogenesis occur. The repair period is about 2 weeks after the onset of myocardial infarction in humans. Cardiac rupture is thought to occur frequently 1 to 4 days after the onset of myocardial infarction, and there are many reports of relationships with inflammatory and repair responses, so understanding of inflammatory and repair responses after myocardial infarction is important. It is considered important in treatment (see Non-Patent Document 2).

Various proteins are produced after myocardial infarction, and they are deeply involved in inflammatory and repair reactions (see Non-Patent Document 3). When focusing on the secreted protein, for example, Thrombopondin1 (TSP-1), which is observed in the early stage after myocardial infarction, has enhanced inflammation after infarction and higher mortality in the deficient mice than in wild-type mice ( Non-patent document 4). From this, it is considered that TSP-1 has an action of reducing inflammation.
In addition, Periostin produced from the early stage to the late stage after myocardial infarction has a delayed development of myofibroblasts after infarction and a high mortality in the deficient mice compared to wild-type mice (see Non-Patent Documents 5 and 6). ). From this, it is considered that Periostin contributes to the repair reaction via myofibroblasts.
Thus, it is thought that various proteins involved in inflammation and repair are produced after myocardial infarction, but how each protein is involved in inflammation or the response to which protein is fibrosis or angiogenesis There is still insufficient understanding as to whether this is causing a repair response.

Milk fat global epidermal growth factor 8 (hereinafter referred to as “MFG-E8”) is a secreted protein that is produced by macrophages of several tissues (see Non-Patent Document 7). Recently, it has been reported that MFG-E8 is an important molecule that promotes phagocytosis of apoptotic cells by macrophages (see Non-Patent Document 8). It is known that apoptotic cells expose phosphatidylserine (PS) on their surface (see Non-Patent Documents 9 and 10). MFG-E8 is considered to promote phagocytosis by binding this PS to integrin αvβ3 or αvβ5 present on the surface of phagocytic cells and bridging them (see Non-Patent Document 8).

Apoptotic cells are normally phagocytically removed. However, if it remains without being removed for some reason, the membrane structure that has been maintained until then collapses and shifts to a state called late necrosis. In late-stage necrotic cells, various factors present in the cells are released to the outside of the cells to cause inflammation. From these, antibodies against self are produced (see Non-Patent Documents 9 to 11).
Very recently, not only immunocompetent cells such as macrophages and microglia, but also cells that were previously thought to have no phagocytic ability, are responsible for the phagocytosis of dead cells under certain circumstances. It has become clear that it contributes to maintaining homeostasis. For example, it has been reported that Sertoli cells that exist as a support for sperm cells in the testis phagocytose sperm cells that have undergone apoptosis (see Non-Patent Document 12). Inhibition of phagocytosis of dead cells by Sertoli cells was observed to cause abnormal spermatogenesis, and phagocytosis by Sertoli cells was considered important in the testis. It has also been reported that phagocytosis of apoptotic neuron progenitor cells by neuronal progenitor cells is involved in the progression of neurogenesis in the brain (see Non-Patent Document 13). In this report, it has been observed that normal neurogenesis does not progress when phagocytosis by neuronal progenitor cells is inhibited.
During myocardial infarction, it is known that apoptosis occurs not only in cardiomyocyte necrosis in the infarcted area but also in the vicinity of the infarcted area (see Non-Patent Documents 14 to 16).

As described above, a treatment method for myocardial infarction has not been established yet, and an effective treatment method is required.
The present invention has been made in view of the above circumstances, and is a pharmaceutical composition for treating or preventing myocardial injury or heart failure, a method for treating or preventing myocardial injury or heart failure, MFG-E8, treating or preventing myocardial injury or heart failure. It is an object of the present invention to provide a method for screening a compound for treating or preventing myocardial injury or heart failure, and the use of MFG-E8 for the manufacture of a medicament for prevention.

Although it is known that many cell deaths occur around the myocardial infarction region, there are few reports on the removal of these dead cells. Therefore, the present inventors focused on MFG-E8, which has been reported to be involved in the mechanism of removing phagocytosis of apoptotic cells, and involved the involvement of MFG-E8 in phagocytosis of dead cells during myocardial infarction, We examined the involvement in heart conditions. As a result, it was newly found that myofibroblasts phagocytose dead cells during myocardial infarction, and that MFG-E8 expressed by resident fibroblast-derived myofibroblasts is involved in phagocytosis of dead cells. I found it. Further, it has been found that administration of MFG-E8 contributes to the improvement of cardiac function after myocardial infarction, and the present invention has been completed.

That is, the pharmaceutical composition for treating or preventing myocardial injury or heart failure according to the present invention, the method for treating or preventing myocardial injury or heart failure, MFG-E8, and the manufacture of a medicament for treating or preventing myocardial injury or heart failure The method of screening for a compound for treating or preventing myocardial injury or heart failure and the use of MFG-E8 are [1] to [11] below.
[1] A pharmaceutical composition for treating myocardial injury, comprising MFG-E8 as an active ingredient.
[2] A pharmaceutical composition for preventing myocardial injury, comprising MFG-E8 as an active ingredient.
[3] A pharmaceutical composition for treating heart failure, comprising MFG-E8 as an active ingredient.
[4] A pharmaceutical composition for preventing heart failure, comprising MFG-E8 as an active ingredient.
[5] A method for treating or preventing myocardial injury or heart failure, comprising a step of administering MFG-E8.
[6] MFG-E8 for use as a therapeutic or prophylactic agent for myocardial injury or heart failure.
[7] Use of MFG-E8 for the manufacture of a medicament for treating or preventing myocardial injury or heart failure.
[8] A screening method for a compound for treating myocardial injury or heart failure, using MFG-E8 expression induction as an index.
[9] A method for screening a compound for preventing myocardial injury or heart failure using MFG-E8 expression induction as an index.
[10] A method for screening a compound for treating myocardial injury or heart failure using the MFG-E8 bioassay system.
[11] A method for screening a compound for preventing myocardial injury or heart failure using the MFG-E8 bioassay system.

According to the present invention, MFG-E8 can be used to effectively treat or prevent myocardial injury such as myocardial infarction or heart failure caused by myocardial infarction.

In Experimental Example 1, it is the figure which showed the expression level of MFG-E8 protein computed from the result of the Western blot. In Experimental Example 2, it is the fluorescence-staining photograph figure which examined the expression of MFG-E8 in an immune cell in a mouse | mouth heart tissue. In Experimental Example 2, it is the fluorescence-staining photograph figure which confirmed the expression of MFG-E8 in the fibroblast (vimentin positive) in a mouse | mouth heart tissue. In Experimental Example 2, it is a fluorescence-stained photograph figure which confirmed the expression of MFG-E8 in the myofibroblast (alpha SMA positive) in a mouse | mouth heart tissue. In Experimental Example 2, it is the fluorescence-staining photograph figure which confirmed the expression of MFG-E8 in the myofibroblast in a mouse | mouth heart tissue. It is a schematic diagram which shows the cut surface at the time of producing the infarct area | region periphery section | slice in Experimental example 4. FIG. In Experimental Example 4, it is a photograph figure which shows the fluorescence staining result of MFG-E8 in the infarct area | region surrounding tissue of a mouse | mouth heart tissue. In Experimental Example 4, it is a photograph figure which shows the fluorescence dyeing | staining result of MFG-E8 etc. in the infarction area | region surrounding mouse heart tissue. In Experimental Example 5, it is a photograph figure which shows the fluorescence dyeing | staining result in the mouse | mouth heart tissue before and behind myocardial infarction. In Experimental Example 6, it is a figure which shows the result of having measured the expression level of MFG-E8 etc. after TGF-β1 stimulation over time in rat fibroblasts. In Experimental Example 6, it is a figure which shows the result of having measured the expression level of MFG-E8 etc. when TGF-β1 and CCG1423 are used in rat fibroblasts. In Experimental Example 6, it is a figure which shows the result of having measured the expression level of MFG-E8 etc. when TGF-β2 and siSRF are used in a human cell line. In Experimental Example 7, it is a figure which shows the result of having measured the expression level of MFG-E8 etc. in a mouse | mouth myofibroblast when CCG-1423 is used. In Experimental Example 7, it is a figure which shows the result of having measured the expression level of MFG-E8 etc. in a mouse | mouth myofibroblast when siRNA is used. In Experimental Example 8, it is a figure which shows the result of having confirmed the peripheral blood component after bone marrow transplantation. In Experimental Example 8, it is a figure which shows the result of having examined the expression of MFG-E8 in the bone marrow origin myofibroblast by fluorescent staining. In Experimental Example 8, it is a figure which shows the result of having confirmed the peripheral blood component after bone marrow transplantation. In Experimental Example 8, it is a figure which shows the result of having examined the expression of MFG-E8 in the bone marrow origin myofibroblast by fluorescent staining. In Experimental Example 8, it is a figure which shows the result of having examined the expression of MFG-E8 in a FSP-1 (S100A4) positive myofibroblast by fluorescent staining. In Experimental example 9, it is a figure which shows the 28-day survival rate in the wild type mouse | mouth or MFG-E8 deficient mouse | mouth after a myocardial infarction operation. In Experimental example 10, it is a figure which shows the HE dyeing | staining section | slice of the heart tissue after a myocardial infarction in a MFG-E8 deficient mouse. FIG. 10 is a graph showing the thickness of the left ventricular wall after myocardial infarction in MFG-E8-deficient mice in Experimental Example 10. In Experimental example 10, it is a figure which shows the picrosirius red dyeing | staining section | slice of the heart tissue after a myocardial infarction in a MFG-E8 deficient mouse. In Experimental Example 10, it is a figure which shows the amount of collagen deposition in the heart tissue after myocardial infarction in an MFG-E8 deficient mouse. In Experimental Example 11, it is a figure which shows the fluorescence dyeing | staining of the heart tissue after the myocardial infarction in a MFG-E8 deficient mouse. In Experimental Example 11, it is a figure which shows the amount of myofibroblasts of the heart tissue after myocardial infarction in the MFG-E8 deficient mouse. In Experimental example 12, it is a figure which shows the echocardiographic evaluation result of the 3rd day after myocardial infarction operation. In Experimental Example 12, it is a figure which shows the echocardiographic evaluation result of the 28th day after myocardial infarction operation. In Experimental example 12, it is a figure which shows the cardiac hemodynamic evaluation result on the 3rd day after myocardial infarction operation. In Experimental Example 12, it is a figure which shows the cardiac hemodynamic evaluation result of the 28th day after myocardial infarction operation. In Experimental example 12, it is a figure which shows the organ weight and the body weight measurement result on the 3rd day after myocardial infarction operation. In Experimental example 12, it is a figure which shows the organ weight and the body weight measurement result on the 28th day after myocardial infarction operation. In Experimental example 13, it is the figure which carried out the fluorescence observation of the apoptotic cell taken in into the myofibroblast of a wild type mouse after myocardial infarction. In Experimental Example 13, fluorescence observation was performed on apoptotic cells incorporated into myofibroblasts of MFG-E8-deficient mice after myocardial infarction. In Experimental Example 13, fluorescence observation of apoptotic cells in heart tissue of wild-type mice or MFG-E8-deficient mice after myocardial infarction was performed. In Experimental example 13, it is a figure which shows the result of having calculated the ratio of the apoptotic cell per total number of cells in a visual field after myocardial infarction. In Experimental example 13, it is a figure which shows a mode that the myofibroblast has taken in the apoptotic cell. In Experimental example 13, it is the figure which showed a mode that the myofibroblast of a wild type or MFG-E8 deficient mouse took in an apoptotic cell. In Experimental Example 13, it is a figure which shows the relationship examination result of recombinant MFG-E8 addition amount and myofibroblast phagocytic ability. In Experimental example 14, it is a figure which shows the fraction of a macrophage or a non-white blood cell. In Experimental example 14, it is the result of having confirmed the integrin alpha v expression in a macrophage or a non-white blood cell. In Experimental example 14, it is the fluorescence-staining photograph figure which confirmed the integrin (beta) 5 expression in a macrophage. In Experimental example 14, it is the result of confirming integrin αvβ3 expression in the CD11b positive fraction. In Experimental example 14, it is the result of having confirmed the integrin αvβ3 expression in the CD11b negative fraction. In Experimental example 14, it is the result of confirming integrin αvβ3 expression in the F4 / 80 positive fraction. In Experimental example 14, it is the result of confirming integrin αvβ3 expression in the αSMA positive fraction. In Experimental Example 15, the ratio of macrophages was confirmed in wild-type mice or MFG-E8-deficient mice after myocardial infarction. In Experimental Example 15, the ratio of leukocytes, granulocytes, monocytes or macrophages was confirmed in wild-type mice or MFG-E8-deficient mice after myocardial infarction. In Experimental Example 15, the ratios of macrophages and neutrophils were confirmed in wild-type mice or MFG-E8-deficient mice after myocardial infarction. In Experimental Example 16, it is a figure which shows the result of having investigated the expression change of the inflammation related factor after a myocardial infarction treatment, and the fibrosis related factor in a wild type mouse | mouth and a MFG-E8 deficient mouse. In Experimental example 17, it is a figure which shows the IL-6 production amount change of a myofibroblast by an apoptotic cell phagocytosis. In Experimental example 17, it is a figure which shows the TGF- (beta) production amount change of a myofibroblast by an apoptotic cell phagocytosis. In Experimental Example 18, MFG-E8 positive myofibroblasts were confirmed in the infarct region of a human myocardial infarction patient. In Experimental Example 19, it is a figure which shows the administration site | part in MFG-E8 administration with respect to the heart after a myocardial infarction. In Experimental Example 19, it is a figure which shows the infarct area | region and risk area | region after MFG-E8 administration after myocardial infarction. In Experimental example 19, it is the figure which quantified the difference of the infarction area | region and risk area | region by MFG-E8 administration after myocardial infarction. In Experimental Example 20, the effect of MFG-E8 administration on the inflammation at the infarct site after myocardial infarction was examined from the expression levels of IL-1β and MIP-2. In Experimental Example 20, the influence of the dose of MFG-E8 on the inflammation at the infarct site after myocardial infarction was examined from the expression levels of IL-1β and MIP-2. In Experimental example 21, it is the photograph figure which confirmed the influence of MFG-E8 administration with respect to the number of apoptotic cells of a heart tissue using fluorescent staining. In Experimental Example 21, the effect of MFG-E8 administration on the number of apoptotic cells after myocardial infarction was quantified based on a photograph. In Experimental Example 22, the effect of MFG-E8 administration on cardiac function after myocardial infarction was examined over time. In Experimental Example 22, the effect of MFG-E8 administration on cardiac function and heart weight after myocardial infarction was examined 10 weeks after administration. In Experimental Example 23, it is the tissue-stained photograph figure which examined the influence of MFG-E8 administration with respect to the fibrosis of the heart after a myocardial infarction 10 weeks after administration. In Experimental Example 23, the effect of MFG-E8 administration on the fibrosis of the heart after myocardial infarction is quantified based on a photograph.

The present invention will be described in detail below. In the present specification, the pharmaceutical composition for treating myocardial injury, the pharmaceutical composition for preventing myocardial injury, the pharmaceutical composition for treating heart failure and the pharmaceutical composition for preventing heart failure are collectively referred to as “treatment of myocardial injury or heart failure or Sometimes referred to as “preventive pharmaceutical composition”.

<Pharmaceutical composition for treatment or prevention of myocardial injury or heart failure>
The pharmaceutical composition for treating or preventing myocardial injury or heart failure of the present invention contains MFG-E8 as an active ingredient.
That is, the pharmaceutical composition of the present invention containing MFG-E8 as an active ingredient can be administered orally or parenterally (eg, nasal, pulmonary, intestinal, transdermal, subcutaneous, intravenous, intramuscular). Or myocardial injury or heart failure can be treated or prevented by administration to animals other than humans.

The amount of MFG-E8 contained in the pharmaceutical composition as an active ingredient is not particularly limited as long as it is an effective amount capable of producing an effect, and the target disease type and condition; target animal species and their age , Sex, body weight; can be determined appropriately according to the dosage form described below. For example, in the range of 0.001 to 10 mg / kg, preferably 0.05 to 1 mg / kg, more preferably 0.05 to 0.5 mg / kg, this is divided into once or several times a day. Can be appropriately determined according to the use form and dosage form.

By using the pharmaceutical composition of the present invention for patients after the onset of myocardial injury or heart failure, a therapeutic effect can be achieved. Moreover, a prophylactic effect can be produced by using the pharmaceutical composition of the present invention for a subject who has not developed myocardial injury or heart failure, particularly a subject who has a risk of myocardial injury or heart failure.

The pharmaceutical composition of the present invention can be used after preparing an appropriate dosage form according to the administration route. Specific examples of the dosage form include injections, transdermal preparations, suppositories, powders, tablets, capsules, granules, capsules, patches, ointments, haptics, aerosols, and the like.

The pharmaceutical composition of the present invention can contain MFG-E8, which is an active ingredient, and other additives and drugs. Specific examples of the other additive include one or more known and commonly used excipients, binders, disintegrants, lubricants and the like. The other drug is not particularly limited as long as it does not inhibit the action of the active ingredient of the present invention. One or two kinds of known drugs that can be used for treatment or prevention of myocardial injury or heart failure. The above can be selected as appropriate.

<Method for treating or preventing myocardial injury or heart failure>
The method for treating or preventing myocardial injury or heart failure of the present invention comprises the step of administering MFG-E8.
In the method of the present invention, the subject to which MFG-E8 is administered is not particularly limited as long as it is a subject requiring treatment or prevention. Specifically, humans or non-human animals can be mentioned.
In the method of the present invention, the timing of administration of MFG-E8 is not particularly limited, and if it is intended for therapeutic effect, it is preferably performed after the onset of myocardial injury or heart failure, with the aim of preventing effect. If so, it is preferably performed at any time before the onset of myocardial injury or heart failure. In particular, MFG-E8 contributes to the recovery of cardiac function by contributing to phagocytosis of dead cells after myocardial infarction, and therefore, as a treatment after myocardial injury such as myocardial infarction, and prevention of heart failure related to myocardial infarction. It is preferably used after the onset of myocardial infarction.

In the method of the present invention, the method for administering MFG-E8 is not particularly limited and may be oral or parenteral (for example, nasal, pulmonary, enteral, transdermal, subcutaneous, intravenous) , Intramuscular).

In the method of the present invention, MFG-E8 may be administered alone or in any dosage form together with other additives and drugs. Examples of the additive, drug, and dosage form include those described above.
The dose of MFG-E8 is not particularly limited as long as it is an effective amount capable of exerting an effect. The type and condition of the target disease; the target animal species, its age, sex, and body weight; It can be determined appropriately according to the shape and the like. For example, in the range of 0.001 to 10 mg / kg, preferably 0.05 to 1 mg / kg, more preferably 0.05 to 0.5 mg / kg, this is divided into once or several times a day. Are preferably administered.

<MFG-E8>
The MFG-E8 of the present invention is for use as a therapeutic or prophylactic agent for myocardial injury or heart failure.
The method of using (administering) MFG-E8 is the same as in the above embodiment.

<Use of MFG-E8>
The use of MFG-E8 of the present invention is for the manufacture of a medicament for treating or preventing myocardial injury or heart failure.
Examples of the medicament for treating or preventing myocardial injury or heart failure include the aforementioned pharmaceutical composition.

<Method of screening for compound for treating or preventing myocardial injury or heart failure>
The compound screening method for treating or preventing myocardial injury or heart failure according to the present invention uses a system for examining the induction of MFG-E8 expression or a bioassay system for MFG-E8. There are no particular limitations on the system for examining the induction of MFG-E8 expression or the bioassay system for MFG-E8, and the methods used in the examples described later can be used.
Specifically, for example, in an animal or wild-type animal in which a candidate substance is knocked out, an assay relating to cardiac findings, cardiac function or prognosis after the occurrence of myocardial infarction; Assay for changes in apoptotic cell phagocytosis of myofibroblasts when a purified or artificially produced candidate substance is added; in a myocardial infarction animal model, cardiac findings after administration of a candidate substance after the occurrence of myocardial infarction; Assay for cardiac function or prognosis; Binding assay between candidate substance and apoptotic cells; Binding assay between candidate substance and cells expressing αvβ3 integrin; Promoter region of MFG-E8 gene was isolated, and Luciferase gene was linked downstream Examples include a reporter and a Luciferase assay using the reporter.

Next, the present invention will be described in more detail with reference to examples and the like, but the present invention is not limited to these examples. All animal experiments below were conducted according to various animal experiment guidelines at Kyushu University.

The reagents and antibodies used in the following studies, and their suppliers are shown below.
Wild-type C57BL / 6J mouse: obtained from Nippon Claire Co., Ltd.
MFG-E8 gene knockout (deficient) mice: Individuals assigned by Professor Shigekazu Nagata (Kyoto University Graduate School of Medicine, Department of Biomedical Sciences, Department of Biomedical Chemistry) were bred in the animal breeding room (SPF). A thing was used.
Anti-MFG-E8 antibody: MBL
Anti-GAPDH antibody: Santacruz
Anti-αSMA antibody: Thermo Fisher Scientific
Anti-CD68 antibody: Serotec
Anti-Gr-1 antibody: BioLegend
Anti-vimentin antibody: BD Biosciences
PE-conjugated anti-integrin αv antibody: BioLegend
FITC-conjugated anti-integrin β3 antibody: BioLegend
FITC-conjugated anti-integrin β5 antibody: eBioscience
Anti-CD45 antibody: BioLegend
FITC-conjugated anti-CD45.1 antibody: BioLegend
APC-conjugated anti-CD45.2 antibody: BioLegend
Alexa fluor 488-conjugated anti-CD45.2 antibody: BioLegend
APC-conjugated anti-CD11b antibobdy: BioLegend
FITC-conjugated anti-CD11b antibobdy: BioLegend
PerCP / Cy5.5-conjugated anti-F4 / 80 antibody: BioLegend
APC-conjugated anti-Ly-6G antibody: BioLegend
APC-conjugated anti-CD86 antibody: BioLegend
FITC-conjugated anti-CD206 antibody: BioLegend
HRP-conjugated anti-hamster IgG: manufactured by Immuno Research Laboratoties
Cy3-conjugated anti-hamster IgG: manufactured by Immuno Research Laboratoties
Alexa fluor 488-conjugated anti-mouse IgG: manufactured by invitrogen
Alexa fluor 488-conjugated anti-rat IgG: manufactured by invitrogen
Alexa fluor 488-conjugated anti-rabbit IgG: manufactured by invitrogen
Alexa fluor 647-conjugated anti-mouse IgG: manufactured by invitrogen
CF405S-conjugated anti-mouse IgG: Biotium
Rat MFG-E8 Probe: Assay ID Rn00563082_m1; manufactured by Applied Biosystems
Mouse MFG-E8 Probe: Assay ID Mm00500549_m1; manufactured by Applied Biosystems
Mouse Acta2_Probe: Assay ID Hs00426835_g1; manufactured by Applied Biosystems
Rat Acta2_Probe: Assay ID Rn01759928_g1; Applied Biosystems
Rat Adam12_Probe: Assay ID Rn01759928_g1; manufactured by Applied Biosystems
Mouse Srf_Probe: Assay ID Mm00491032_m1; manufactured by Applied Biosystems
Eukaryotic 18S-rRNA_probe: Assay ID Hs03003631_g1; manufactured by Applied Biosystems

In the following experimental examples, the results are based on experiments conducted under the same independent condition of at least 3 cases, and all are expressed as mean value ± standard error. A comparison between two groups is performed using unpaired t test, and a comparison between multiple groups is performed using Student-Newman-Keuls test by One way-ANOVA. There is a significant difference when the P value is less than 5%. It was judged.

[Reference Example 1] Preparation of myocardial infarction model mouse The myocardial infarction model mouse used in the present specification was prepared as follows.
First, 8-10 week old male wild-type C57BL / 6J mice or MFG-E8 gene knockout mice (hereinafter referred to as “MFG-E8 deficient mice”, “MFG-E8 KO mice”, and “KO mice”) Prepared.) To this mouse, somnopentyl injection (50 mg / kg sodium pentobarbital) was intraperitoneally administered and then fixed to the operating table in the supine position. After shaving the neck and chest, the midline of the neck was incised under a surgical microscope to expose the trachea. The cannula was inserted into the trachea, and artificial respiration was performed at a tidal volume of 0.5 cc and a respiration rate of 120 times / min. The myocardial infarction was performed by exposing the heart through an incision between the second ribs on the left side of the radius and ligating the left anterior descending coronary artery with a 6 mm silk blade suture. Thereafter, the incision site was sutured with a suture. Mice subjected to the above treatment were designated as a myocardial infarction (MI) group, and mice treated similarly except for ligation of the coronary artery were designated as a sham treatment (control) group.
After the myocardial infarction operation, mouse heart tissue and the like were collected after a predetermined number of days, and various examinations shown below were performed.

[Experimental Example 1] Measurement of MFG-E8 protein expression level Western blotting was performed using mouse hearts collected on days 0, 2, 4, 7 and 28 after myocardial infarction. Then, the expression level of MFG-E8 protein was measured.
Specifically, 1 ml of RIPA buffer (50 mM Tris-Cl pH 8.0, 150 mM NaCl, 1.0% Nonidet P-40, 0.5% sodium deoxycholate) and protease inhibition cocktail stored at −80 ° C. Homogenization was performed until the tissue became uniform (Nacalai tesque). The supernatant obtained by centrifuging the homogenized solution was subjected to protein quantification using BIO-RAD Protein Assay (manufactured by BIO-RAD), followed by 2 × SDS sample buffer (100 mM Tris-Cl pH 6.8, 20%). Glycerol, 2% SDS, 0.04% Bromophenol Blue) and boiled at 95 ° C. for 5 minutes. The obtained sample was electrophoresed by SDS PAGE Gel at 20-30 μg per well, and then transferred to a PVDF membrane (Amersham Hybond-P). After incubating the PVDF membrane in a 5% BSA / TBS-T (20 mM Tris-Cl pH 7.4, 137 mM NaCl, 0.2% Tween-20) solution for 1 hour, PVDF membrane in various TBS-T solutions containing antibodies The membrane was left at 4 ° C. overnight. The next day, the plate was washed 3 times for 10 minutes with the TBS-T solution, and incubated for 1 hour in the TBS-T solution containing various HRP-labeled secondary antibodies. Thereafter, the plate was washed with TBS-T solution three times for 10 minutes, and Western Lightening Plus (manufactured by PerkinElmer) or Supersignal West Pico (Thermo Scientific). 　 The product was made to emit light. After exposure and development on a FUJI Medical X-ray film (manufactured by FUJIFILM), the image was captured, and the density of the band was quantitatively measured using a Scion Image.
Thereafter, the results of correcting and calculating the expression level of MFG-E8 protein using GAPDH protein as an internal standard are shown in FIG. In FIG. 1, error bars indicate standard errors (SEM), and n = 3.
MFG-E8 has two types of splicing variants, and it has been reported that only long form is involved in promoting phagocytosis of dead cells (see Non-Patent Document 8). Therefore, in this study, quantification was performed only for the band (L) corresponding to the long form of MFG-E8.

As is apparent from the results shown in FIG. 1, in the myocardial infarction (MI) treatment group, a marked increase in the expression of MFG-E8 protein was observed on the second day after the treatment, compared with the control (sham) group. Expression was highest on day. In both the MI and sham groups, expression of MFG-E8 was also observed in the chronic inflammatory phase (about 25 days after the treatment) after the acute inflammatory phase (about 0 to 4 days after the treatment). It was. Therefore, it was revealed that the expression level of MFG-E8 protein increases in the heart after myocardial infarction.

[Experimental Example 2] Identification of cells producing MFG-E8 Using mouse heart tissue collected 3 days after myocardial infarction, identification of cells producing MFG-E8 was attempted by fluorescent staining.
Specifically, the extracted mouse heart was embedded in Surgipath FSC 22 (manufactured by Leica) and frozen in liquid nitrogen to obtain a frozen specimen. The frozen specimen was sliced to 4 μm with a cryostat (model CM1100; manufactured by Leica) and air-dried for 1 hour. The frozen sections were fixed with -20 ° C Cold-acetone or 4% PFA for 10 minutes and incubated with 5% BSA / PBS for 1 hour. Then, it was left overnight at 4 ° C. in 5% BSA / PBS containing a primary antibody described later against various proteins. The next day, the cells were incubated for 1 hour in 5% BSA / PBS containing various fluorescently labeled secondary antibodies against the primary antibody. Then, it was sealed with a Vectashield Hard Set Mounting Medium with DAPI (manufactured by Vector Laboratories) or FluorSave ^™ Reagent (manufactured by Calbiochem), and observed and imaged with a fluorescence microscope. The results are shown in FIGS. 2, 3A and 3B. In this study, the control group (sham treatment group) is not used.

FIG. 2 shows the results of examining the expression of MFG-E8 in immune cells by co-staining of immune cell markers (CD68, Gr-1) and MFG-E8. CD68 is a marker for monocytes or macrophages, and Gr-1 is a marker for granulocytes such as neutrophils. As a result of co-staining (Overlay), it was found that immune cells expressing CD68 and Gr-1 hardly express MFG-E8.
3A and 3B show the results of examining the expression of MFG-E8 in myofibroblasts by co-staining with myofibroblast markers and MFG-E8. Vimentin (FIG. 3A) or αSMA (α-smooth muscle actin; FIG. 3B) was used as a fibroblast marker. As a result of co-staining (Overlay), it was found that both vimentin-positive fibroblasts and αSMA-positive fibroblasts expressed and produced MFG-E8.

[Experimental Example 3] Examination of the origin of myofibroblasts producing MFG-E8 Using mouse heart tissue collected on the first day after myocardial infarction, myofibroblasts producing MFG-E8 by fluorescence staining The origin was examined.
Specifically, the frozen section was fluorescently stained in the same manner as in Experimental Example 2 except that the tissue on the first day after the procedure was used and the antibody was changed, and observation and imaging were performed with a fluorescent microscope. The results are shown in FIG.
In FIG. 4, the upper row shows the results of co-staining with Vimentin and MFG-E8, which are fibroblast markers including undifferentiated fibroblasts, and the lower row shows αSMA and MFG, which are markers of differentiated myofibroblasts. -Results of co-staining with E8. In both the upper and lower rows, the rightmost row shows the result of triple staining by adding DAPI as a nuclear marker in addition to co-staining of these markers.
From this result, it was confirmed that not only myoma fibroblasts expressing αSMA but also a part of fibroblasts expressing Vimentin, which seems to be in the process of differentiation into myofibroblasts, express MFG-E8. did it.
There are at least three types of myofibroblasts derived from bone marrow, endothelial cells (endothelial-mesenchymal transition), and resident fibroblasts (differentiation). From the results shown in FIG. 4, it was confirmed that at least cardiac resident fibroblast-derived myofibroblasts express and produce MFG-E8.

[Experimental Example 4] Examination of expression distribution of MFG-E8 around the infarct site Examination of MFG-E8 expression distribution around the myocardial infarction site using mouse heart tissue collected 3 days after myocardial infarction went.
Specifically, the frozen section was fluorescently stained and observed and imaged with a fluorescence microscope in the same manner as in Experimental Example 2 except that the preparation method of the specimen and the section was changed. In this examination, the cut surface of the section is as shown in FIG. FIG. 6 shows the result of co-staining of MFG-E8 and nucleus, and FIG. 7 shows the result of staining or co-staining of MFG-E8, αSMA, and nucleus.
From the results of FIGS. 6 to 7, it was confirmed that MFG-E8 was highly expressed in myofibroblasts in the infarct region.

[Experimental Example 5] Examination of expression distribution of MFG-E8 before and after myocardial infarction operation Using mouse heart tissue before myocardial infarction operation and mouse heart tissue collected on the 3rd day after myocardial infarction operation, The expression distribution was examined.
Specifically, in the same manner as in Experimental Example 2, frozen sections were prepared and MFG-E8 and αSMA were fluorescently stained, and observed and imaged with a fluorescence microscope. The results are shown in FIG.
In FIG. 8, the upper part shows the result in the mouse heart tissue before the myocardial infarction operation, and the lower part shows the result in the mouse heart tissue on the third day after the myocardial infarction operation. As is clear from the results of FIG. 8, MFG-E8 is hardly expressed in the mouse heart tissue before the myocardial infarction, whereas MFG-E8 is highly expressed in the mouse heart tissue after the myocardial infarction. I found out. In the mouse heart tissue after myocardial infarction, high expression of MFG-E8 was observed particularly at the infarct site.

[Experimental Example 6] Examination on induction of expression of MFG-E8 and the like using TGF-β According to the experimental example described above, myofibroblasts producing MFG-E8 may originate from resident fibroblasts. Was considered high. Since resident fibroblasts do not express MFG-E8, it is considered that MFG-E8 is expressed along with differentiation into myofibroblasts. Thus, changes in the expression level of MFG-E8 and the like were examined using TGF-β, which is known to be involved in the induction of differentiation from fibroblasts to myofibroblasts.
In addition, serum response factor (SRF), a transcription factor downstream of TGF-β, and myocardin-related transcription factor (MRTF), a cofactor of SRF, are also used to express genes characteristic of myofibroblasts such as αSMA. It is known to be involved. Therefore, in the presence of CCG1423, an inhibitor of SRF and MRTF, changes in the expression level of MFG-E8 and the like by TGF-β stimulation, and expression of MFG-E8 and the like by TGF-β stimulation when siRNA for SRF is used. The amount change was also examined.

(1) Induction of expression of MFG-E8 and the like in rat cardiac fibroblasts by stimulation with TGF-β1 Specifically, 10 ng / mL TGF was isolated from resident fibroblasts isolated from rat neonatal heart. Stimulation was performed by adding -β1 to the medium. After stimulation, fibroblasts at 24 hours, 48 hours, and 72 hours were collected.
In some samples, CCG1423 was added to the medium at a concentration of 1 μM or 10 μM immediately before stimulation with 10 ng / mL of TGF-β1, and collected by culturing for 24 hours.

RNA was purified from the collected cells using RNeasy Micro Kit (Qiagen) and RNeasy Mini Kit (Qiagen). The concentration of the obtained RNA was determined by measuring absorbance and used for real-time PCR.
The primers and TaqMan probe used for quantification of the sample mRNA were those designed by Primer Express Software (manufactured by Applied Biosystems). For the measurement, based on the protocol of One-Step PrimeScript ™ RT-PCR Kit (manufactured by TAKARA), a reverse transcription reaction was performed at 42 ° C. for 10 minutes. After denaturation of the reverse transcriptase at 95 ° C. for 10 seconds, the reaction was performed at 60 ° C. for 35 seconds. The extension reaction was performed 40 cycles. Analysis was performed using 18S rRNA as an internal standard.

FIG. 9A shows the results of examining the temporal changes in the expression levels of Acta2, Adam12 and Mfg-e8 by TGF-β1. In addition, FIG. 9B shows the results relating to the expression level of MFG-E8 when CCG1423 was used in combination with TGF-β1 expression induction for 24 hours (24 hours). The result shown in the figure is each expression level corrected by 18S rRNA which is an internal standard. Acta2 is Smooth Muscle Actin, and Adam12 is a protease having a disintegrin domain and a metalloprotease domain, and is a protease that is considered to be involved in the TGF-β signal, and is a marker molecule for myofibroblasts.
From the results of FIG. 9A, it was found that Acta2, Adam12, and Mfg-e8 are induced to be expressed by TGF-β1. Similarly, when the fibroblasts were stimulated using monosodium ureate (MSU), peroxyredoxin (Prx), high mobility group box protein-1 (HMGB1), ATP, etc., the induction of MFG-E8 expression was induced. Not recognized (not shown). MSU, Prx, and HMGB1 are all known to be representative damage-related molecular patterns (DAMPs) released from cells due to tissue injury such as during myocardial infarction.

Further, from the results of FIG. 9B, the expression induction of Acta2 and Mfg-e8 by stimulation with TGF-β1 is canceled depending on its concentration by treatment with CCG1423, and the expression levels of Acta2 and MFG-E8 are significantly reduced. It could be confirmed.

(2) Induction of expression of MFG-E8 etc. by stimulation with TGF-β2 in human umbilical vein endothelial cell line Specifically, 10 ng / mL TGF-β2 was added to the medium for human umbilical vein endothelial cells (HUVEC) And stimulated. Cells were collected 72 hours after stimulation, and real-time PCR was performed in the same manner as in (1) above. In some samples, siRNA was used to silence the expression of the SRF gene with respect to HUVEC before TGF-β2 stimulation. The results are shown in FIG. 9C. The result shown in the figure is each expression level corrected by 18S rRNA which is an internal standard.
From the results of FIG. 9C, it was confirmed that the expression induction of Acta2, Mfg-e8 and Srf by TGF-β2 stimulation was canceled by siSRF, and the expression levels of Acta2, Mfg-e8 and SRF were reduced.

From the above results, the expression of MFG-E8 and the like is enhanced by differentiating fibroblasts into myofibroblasts by the action of TGF-β, and MFG-E8 and the like in myofibroblasts and human cell lines. It was suggested that expression is dependent on downstream factors and / or cofactors of TGF-β.

[Experimental Example 7] Examination of expression level of MFG-E8 and the like in myofibroblasts derived from myocardial infarction mice (1) Examination using CCG1423 Using myofibroblasts from myocardial infarction model mice, depending on the presence or absence of CCG1423, Changes in the expression levels of various factors such as MFG-E8 were examined.
Specifically, first, the mouse heart extracted on the third day after the myocardial infarction operation was divided into 5-6 pieces after removing the atrium. Thereafter, the heart pieces are shaken at 37 ° C. for 10 minutes in Collagenase A solution (0.1% Collagenase A, 0.1% BSA, 0.1% 4-hydroxylic acid / PBS) 3 to 5 times. Myofibroblasts were isolated. The isolated cells were cultured overnight in DMEM (10% FBS, 50 U / ml penicillin / streptomycin), and those stuck to the plate were used as isolated myofibroblasts.
The isolated myofibroblasts were collected for 24 hours in the presence or absence of CCG1423 and then collected by a conventional method.
Using the recovered myofibroblasts, the expression levels of Acta2 and Mfg-e8 were measured by real-time PCR in the same manner as in Experimental Example 6. The results are shown in FIG. 10A.

(2) Examination using siSRF In the same manner as in (1) above, for myofibroblasts isolated from mice 3 days after myocardial infarction, gene expression was suppressed using siRNA against the SRF gene. After culturing, the cells were collected by a conventional method.

From the results shown in FIG. 10A, it was confirmed that the use of CCG1423 significantly decreased the expression of Acta2 and Mfg-e8 depending on its concentration.
From the results shown in FIG. 10B, it was also confirmed that the expression of Acta2, Mfg-e8 and Srf was significantly reduced by using siSRF.
From the above results, it was reconfirmed that the expression of MFG-E8 in myofibroblasts depends on SRF and MRTF.
The expression levels of CTGF (Connective Tissue Growth Factor) and MRTF-A in mouse myofibroblasts using CCG1423, siSRF, or siMRTF-A were similarly examined using primers for each target. The addition of CCG1423 significantly reduced the expression of CTGF. Moreover, the expression of CTGF and MRTF-A decreased when any siRNA was used (results not shown).

[Experimental Example 8] Examination of MFG-E8 production by bone marrow-derived myofibroblasts or endothelial cell-derived myofibroblasts As described above, it was confirmed that myofibroblasts express MFG-E8. In addition to cells derived from resident fibroblasts, there are cells derived from bone marrow and endothelial-mesenchymal transition cells. Therefore, an examination was conducted as to whether myofibroblasts derived from bone marrow or endothelial-mesenchymal transition cells produce MFG-E8.

(1) Examination of bone marrow-derived myofibroblasts Specifically, first, mice having haplotypes different from Ly5 (CD45) antigen were used (obtained from Sankyo Lab Service).
First, γ-ray irradiation (10 Gy) was performed using a gamma cell 40 on MFG-E8 KO mice having a haplotype of Ly5.2, and this was used as a recipient. Thereafter, a wild-type mouse having a Ly5.1 haplotype was used as a donor, and bone marrow from which bone marrow fluid was collected from the tibia and femur of the donor mouse was subjected to bone marrow transplantation by intravenous injection through the recipient's fundus vein. Two weeks after transplantation, cells in mouse peripheral blood were stained with FITC-anti-CD45.1 antibody and APC-anti-CD45.2 antibody, and chimerism check was performed using FACS. As a result, it was confirmed that in the bone marrow transplanted mouse, 98.6% of all bone marrow cells present in the peripheral blood vessels were occupied by donor-derived cells (FIG. 11A).
Thereafter, myocardial infarction was performed in the same manner as in Reference Example 1 using the bone marrow transplanted mouse. FIG. 11B shows the result of co-staining using mouse heart tissue collected on the third day after myocardial infarction in the same manner as in Experimental Example 2.
As shown in FIG. 11B, bone marrow-derived cells expressing CD45 (CD45.1) were found in the mouse heart tissue 3 days after myocardial infarction. However, almost no expression of MFG-E8 was observed in these bone marrow-derived cells expressing CD45.

In the same manner as described above, the bone marrow of MFG-E8 KO mice having Ly5.2 haplotype was transplanted to wild-type mice having Ly5.1 haplotype by a conventional method, and myocardial infarction was performed.
When bone marrow-derived cells were confirmed using peripheral blood of bone marrow transplanted mice, 99.61% of all bone marrow cells present in peripheral blood vessels were occupied by donor-derived cells in bone marrow transplanted mice. This was confirmed (FIG. 12A).
Co-staining was performed in the same manner as in Experimental Example 2 using mouse heart tissue collected on the third day after myocardial infarction. The result is shown in FIG. 12B.
As shown in FIG. 12B, bone marrow-derived cells expressing CD45 (CD45.2) were found in the mouse heart tissue 3 days after myocardial infarction. However, CD45 expression and MFG-E8 expression were not colocalized.

(2) Examination of Endothelial Mesenchymal Converted Cell-Derived Myofibroblasts Using the mouse heart tissue prepared in the same manner as in Reference Example 1 and collected 3 days after myocardial infarction, vimentin, S100A4, and MFG- Co-staining was performed using an antibody against E8. The S100A4 antibody is an antibody that detects FSP-1 (Fibroblast-Specific Protein-1), which is a marker of endothelial-mesenchymal transition (EMT) cells.
As shown in FIG. 13, it was confirmed that a part of myofibroblasts expressing vimentin was derived from endothelial mesenchymal transition cells expressing FSP-1 (S100A4). It was also confirmed that many myofibroblasts expressing vimentin express MFG-E8. However, as is apparent from the results of co-staining, myofibroblasts derived from endothelial-mesenchymal transition cells that express FSP-1 did not express MFG-E8.

From the results of (1) and (2) above, it was suggested that bone marrow-derived cells and endothelial mesenchymal transition cells are not involved in MFG-E8 production.

[Experimental Example 9] Examination of survival rate after myocardial infarction due to MFG-E8 deficiency Using wild-type mice and MFG-E8 KO mice, myocardial infarction was performed in the same manner as in Reference Example 1 above, and 28 days after the operation. Observations were made. A graph of the survival rate for 28 days after the treatment is shown in FIG.
The survival rate in the wild-type control group (non-treated group) and MFG-E8 KO mouse control group (non-treated group) was 100%, whereas the survival rate of wild-type mice after myocardial infarction was 71.4%. Yes, the survival rate of MFG-E8 KO mice after myocardial infarction was 26.3%. When the dead mice were dissected, intrapleural hemorrhage and left ventricular free wall cracking were observed, and the cause of death was cardiac rupture due to left ventricular free wall rupture in all mice. Many cardiac ruptures were observed on the 4th to 5th day after myocardial infarction.
From the above results, it was found that MFG-E8 deficiency increases the risk of cardiac rupture after myocardial infarction and significantly decreases the survival rate.

[Experimental example 10] Examination of cardiac findings after myocardial infarction due to MFG-E8 deficiency In the above experimental example 9, all the causes of death after myocardial infarction were due to cardiac rupture, and thus the patient was removed 3 days after myocardial infarction. Tissue staining of heart sections was performed using a mouse heart.
(1) HE staining Specifically, a paraffin section of a mouse heart was used, and paraffin was removed with xylene and ethanol, followed by washing with running water for 3 minutes. Thereafter, the mixture was incubated for 5 minutes in Mayer's hematoxylin solution and washed with running water for 10 minutes. Further, the mixture was incubated in eosin Y solution for 5 minutes, washed with light water, dehydrated with ethanol and transparent with xylene, and encapsulated with biolite.
When the heart tissue after HE staining was observed, as shown in FIG. 15A, the left ventricular wall of the MFG-E8 KO mouse (KO MI) after the myocardial infarction was compared with the left ventricular wall of the wild type mouse. Its thickness was significantly reduced. FIG. 15B is a graph in which the thickness of the left ventricular wall is digitized. As is clear from FIG. 15B, the left ventricular wall thickness is significantly thinner in the myocardial infarction group (MI) than in the non-surgery group (sham), and the MFG-E8 KO mouse is treated. The group had thinner walls than the wild-type mouse treatment group.

(2) Picrosirius red staining It is known that fibrosis occurs after myocardial infarction to repair the infarcted site. Therefore, in order to evaluate the degree of fibrosis, picrosirius red staining capable of staining collagen was performed.
Specifically, paraffin sections of mouse heart were used, and paraffin was removed with xylene and ethanol, followed by incubation in picric acid saturated 0.1% Direct Red 80 solution for 60 minutes. Thereafter, it was washed with 0.01N HCl, dehydrated with ethanol and transparent with xylene, and sealed with biolite.
When the heart tissue after Picrosirius red staining was observed, as shown in FIG. 15C which is a stained section image and FIG. 15D in which the amount of collagen in FIG. 15C was quantified, significant deposition of collagen was recognized by myocardial infarction treatment. It was. On the other hand, in MFG-E8 KO mice after myocardial infarction, collagen deposition was attenuated as compared to wild-type mice after myocardial infarction.

The above results suggest that the MFG-E8 deficiency attenuates the repair reaction at the infarct site after myocardial infarction.

[Experimental Example 11] Examination by fluorescence staining of heart tissue after myocardial infarction due to MFG-E8 deficiency Wild type or MFG-E8 KO mouse heart tissue on day 3 after myocardial infarction and untreated wild Using the type or MFG-E8 KO mouse heart tissue, αSMA fluorescence staining was performed in the same manner as in Experimental Example 3 and the like. The photograph figure of a result is shown to FIG. 16A, and the graph which digitized the result of FIG. 16A is shown to FIG. 16B.
From the above results, it was confirmed that the proportion of αSMA positive cells showing myofibroblasts necessary for the repair response after infarction was significantly reduced by MFG-E8 deficiency.

[Experimental Example 12] Evaluation of cardiac function and cardiac morphology after myocardial infarction (1) Echocardiography Nemi XG general-purpose ultrasound image in wild type or MFG-E8 KO mice on days 3 and 28 after myocardial infarction Echocardiographic evaluation was performed using a diagnostic device (SSD-580A; Toshiba Medical Systems Corporation).
Somnopentyl injection (50 mg / kg pentobarbital sodium) was administered into the abdominal cavity of the mouse, and then fixed to the operating table in the supine position. A 14-MHz transducer was lightly applied to the left anterior chest wall to obtain a two-dimensional parasternal left ventricular short-axis cross section. The transducer was moved and an M-mode image was recorded from a short-axis image with the largest left ventricular cavity. The left ventricular end diastolic diameter (LVIDd) and end systolic diameter (LVIDs) were measured according to the M-mode method according to the American College of Echocardiography leading-edge to leading-edge. The end diastole was when the left ventricular expansion diameter was the largest, and the end systolic diameter was when the movement of the left ventricular rear wall was the largest. The ejection fraction (EF) was calculated using the Pombo method.

First, since the heart rate (HR) was not significantly different in each group, it was considered that the influence of the heart rate on each parameter was the same for any group.
In the myocardial infarction treatment group, a decrease in IVSTd and EF and an increase in LVIDs were observed. This result was the same on the third day (FIG. 17A) and the 28th day (FIG. 17B).

(2) Hemodynamics In the wild-type or MFG-E8 KO mice on the 3rd and 28th day after the myocardial infarction operation, the somnopentyl injection solution (50 mg / kg pentobarbital sodium) was administered into the abdominal cavity of the mouse, and the supine position And fixed to the operating table, and the neck was shaved and incised. Thereafter, a 1.4-Fr micropressure measurement catheter (model SPR-671; Millar) was inserted from the right carotid artery and advanced into the left ventricle to measure the left ventricular pressure. Analysis of left ventricular hemodynamic parameters was performed with Digi-Med Heart Performance Analyzer (Model 410; Micro-Med) and Digi-Med System Integrator (Model 210; Micro-Med) connected to a catheter. The results on the third day after the treatment are shown in FIG. 18A, and the results on the 28th day after the treatment are shown in FIG. 18B.

First, since the heart rate (HR) was not significantly different in each group, it was considered that the influence of the heart rate on each parameter was the same for any group.
In the myocardial infarction (MI) group, end diastolic pressure (EDP), pressure drop time constant (tau), pressure differential maximum value (dP / dt Max) as an index of contractility, and pressure as an index of relaxation ability All parameters of the differential minimum value (dP / dt Min) were significantly worse compared to the non-sham group.
In particular, regarding EDP on the third day after the treatment, significant deterioration was observed in the MFG-E8 KO MI group compared to the wild-type MI group.

(3) Organ Weight and Body Weight Heart weight and lung weight were measured in wild-type or MFG-E8 KO mice on day 3 and day 28 after myocardial infarction. The results are shown in FIG. 19A (day 3) and FIG. 19B (day 28). The heart weight was significantly increased in the myocardial infarction (MI) group compared to the non-sham group (sham).

[Experimental Example 13] Examination of the effect of MFG-E8 on apoptosis during myocardial infarction (1) TUNEL method around the infarcted area and fluorescent staining with αSMA Heart section around the infarcted area of the mouse 3 days after myocardial infarction Were stained with TUNEL method to detect apoptosis, or αSMA.
Specifically, the excised heart was embedded in Surgipath FSC 22 (manufactured by Leica) and frozen in liquid nitrogen was used as a frozen specimen. The frozen specimen was sliced to 4 μm with a cryostat and air-dried for 1 hour. Frozen sections were fixed for 10 minutes at -20 ℃ cold-acetone, subsequent work in accordance with the ApopTag ^R Fluorescein In Situ Apoptosis Detection Kit protocol. The result of the wild type mouse is shown in FIG. 20A, and the result of the MFG-E8 KO mouse is shown in FIG. 20B.
As shown in FIGS. 20A and 20B, in the wild type, TUNEL-positive apoptotic cells were incorporated into αSMA-positive myofibroblasts (FIG. 20A). On the other hand, in MFG-E8 KO mice, although TUNEL-positive apoptotic cells were observed, they were not inside αSMA-positive myofibroblasts but on the surface and outside of myofibroblasts (FIG. 20B). ).

(2) Fluorescent staining with the TUNEL method and DAPI in the boundary region between the infarcted site and the non-infarcted site 2
Heart sections of mice 3 days after myocardial infarction were fluorescently stained with the TUNEL method for detecting apoptosis or DAPI for staining nuclei as described above. After myocardial infarction, it has been reported that apoptosis occurs mainly in the boundary region between the infarcted site and the non-infarcted site. A photograph of the results (upper part: wild type, lower part: KO) is shown in FIG. 21A, and the ratio of apoptotic cells in the total cells in the visual field (TUNEL + cells / DAPI%) is shown in FIG. 21B.
As a result, as shown in FIGS. 21A and 21B, in the MFG-E8 KO mouse, a large number of apoptotic cells remained without being phagocytosed as compared with the wild type.

(3) Examination of Apoptotic Cell Phagocytosis Using Recombinant MFG-E8 Preparation of Apoptotic Cells First, a thoracic line was extracted from a C57BL / 6J female 4-8 week old mouse. The excised chest line was crushed by rubbing two glass slides and filtered using a mesh filter. The collected thymocytes were centrifuged, suspended in serum-free DMEM, and cell count was performed. After preparation to 1 × 10 ⁷ cells / ml, 1 μM Celltracker ^™ Green CMFDA was added at a ratio of 1 μl / 10 ml and cultured at 37 ° C. for 30 minutes. After incubation, 5 times cooled 10% FBS / DMEM was added and left on ice for 5 minutes to stabilize the fluorescent label. Thereafter, washing was performed, and cell counting was performed again. After preparation to 1.2 × 10 ⁷ cells / ml, 1 μl / 1 ml of 10 mM dexamethasone was added, and apoptosis was induced by incubation at 37 ° C. for 4 hours. After incubation, washing was performed using 10% FBS DMEM replacement, and the obtained cells were used as apoptotic cells.

Preparation of Recombinant mMFG-E8 A retrovirus that expresses FLAG-mouse MFG-E8 was produced using PLAT-E cells, and the gene was introduced into NIH3T3 cells. FLAG-MFG-E8 stably expressing NIH3T3 cells were cultured at 37 ° C. for 72 hours. Since MFG-E8 is a secreted protein, the cultured cell supernatant was collected, and impurities were removed by centrifugation and filtration.
Protein purification from the above culture supernatant was performed at 4 ° C. ANTI-FLAG M2-Affinity Gel (SIGMA) was buffer-substituted 3 times with PBS and then packed in a Poly-Prep chromatography column (BIO-RAD) to prepare a column bed. After washing the column with PBS, the collected cell supernatant was added to adsorb FLAG-MFG-E8. After washing with PBS, an eluate (10 mM Tris pH 7.5, 5M LiCl) was added to elute FLAG-MFG-E8. In order to concentrate the eluted fraction, it was added to Amicon Ultra-4 (MILLIPORE), centrifuged at 3500 rpm at 4 ° C., PBS was added for buffer exchange, and the mixture was centrifuged again. The concentrated protein solution was finally sterilized by filtration using ULTRA FREE-MC (MILLIPORE).

Evaluation of phagocytic ability of myofibroblasts Myofibroblasts isolated from wild-type or MFG-E8 KO mice on day 3 after myocardial infarction were seeded on CC2-coated 4-well chamber slide and muscle Apoptotic thymocytes fluorescently labeled with respect to fibroblast 1 were added at a ratio of 10 and incubated at 37 ° C. for 90 minutes. Thereafter, the cells were washed with PBS to remove apoptotic cells that were not phagocytosed. Fibroblasts were fixed with 4% PFA, permeabilized with 0.1% Triton-X, stained with anti-MFG-E8antibody, and observed with a confocal microscope. Phagocytosis was evaluated as the number of apoptotic cells phagocytosed per myofibroblast as a phagocytosis index. The evaluation was performed on 10 fields or more and 150 cells or more.

First, when apoptotic cells incorporated into the cells under phase difference observation were confirmed, they could be identified as dark images (center of FIG. 22). Analysis of fluorescence (left in FIG. 22) and phase difference (right in FIG. 22) showed that myofibroblasts incorporated apoptotic cells into the cells (FIG. 22, arrows).
Next, as described above, the number of apoptotic cells taken up per myofibroblast (Phagocytosis index) was quantitatively analyzed based on the photograph shown in FIG. 23A. As a result, compared with the wild type, MFG-E8 KO significantly reduced the phagocytic ability of myofibroblasts (FIGS. 23A-B). When recombinant MFG-E8 was added to myofibroblasts derived from this MFG-E8 KO mouse, the phagocytic ability was significantly increased depending on the amount added (FIG. 23B).
From the above results, it was found that myofibroblasts phagocytose dead cells (apoptotic cells) in an MFG-E8-dependent manner at the infarct region boundary after myocardial infarction.

[Experimental Example 14] Identification of integrin αvβ3-expressing cells around the myocardial infarction site It is known that MFG-E8 binds to integrin αvβ3 or integrin αvβ5 on the cell surface. Therefore, cells were collected from the heart excised from the mouse 3 days after myocardial infarction using an enzyme reaction to examine whether integrin αvβ3 or integrin αvβ5 was expressed on the cell surface.

(1)
Specifically, hearts were removed from wild-type mice or MFG-E8 KO mice 3 days after myocardial infarction, and the atrium was removed and divided into 5-6 pieces. Thereafter, the heart pieces were shaken at 37 ° C. for 10 minutes in Dispase II solution (2.4 U / ml Dispase II, 0.25% trypsin-EDTA / PBS) for 3 to 5 times to obtain single cells. Released. The isolated cells were incubated at 4 ° C. for 10 minutes in Mouse BD Fc Block-containing FACS buffer (2% FBS, 0.05% NaN ₂ / PBS), and then various antibodies were incubated at 4 ° C. for 1 hour in FACS buffer. By doing so, the surface antigen was stained. For intracellular antigen staining, cells were isolated, fixed with 4% PFA / PBS at 4 ° C. for 10 minutes, and permeabilized with 0.01% Saponin / PBS at 4 ° C. for 10 minutes.

As shown in FIG. 24A, F4 / 80-positive macrophage cells (FIG. 24A, upper part) and CD11b-negative non-white blood cells (FIG. 24A, lower part) were fractionated, and integrin expression was confirmed.
As a result, it was confirmed that integrin αv was expressed in both macrophages and non-white blood cells (FIG. 24B).

(2)
Using the macrophage positive cells fractionated in (1) above, fluorescent staining was performed to examine the expression of integrin β5. Macrophage cells were collected and fractionated from heart pieces at the site of infarction and a site separated from the infarction (non-infarcted site; Remote area).
As a result, it was confirmed that many vimentin-positive fibroblasts expressed integrin β5 at the infarct site (upper part of FIG. 24C). On the other hand, vimentin positive cells at the non-infarcted site did not express integrin β5 (lower part of FIG. 24C).

(3)
Next, CD11b positive or negative cells, F4 / 80 positive cells, and αSMA positive cells were examined in the same manner as (1) except that only wild-type mice were used.
As a result, integrin αv expression was confirmed in CD11b positive cells, but integrin β3 and β5 expression could not be confirmed (FIG. 25A). On the other hand, in the CD11b negative cells, the expression of integrin αv and integrin β3 was confirmed (FIG. 25B).
As a more detailed cell marker, the integrin expression of the F4 / 80 positive fraction, which is a macrophage marker, was observed, but the integrin αv expression was also observed in the macrophage, but the integrin β3 and β5 expression could not be confirmed. (FIG. 25C).
Examples of CD11b negative cells present in the heart after myocardial infarction include myofibroblasts. Thus, when integrin expression was observed in the αSMA positive fraction that is a marker of myofibroblasts, the expression of both integrin αv and β3 could be confirmed in this fraction (FIG. 25D).
From the above results, it was shown that the action point of MFG-E8 is not leukocytes such as macrophages but myofibroblasts.

[Experimental Example 15] Examination of proportions of various cells in mice after myocardial infarction In the same manner as in Experimental Example 14, the proportions of various cells in wild-type or KO mice after myocardial infarction were examined.
As a result, CD86-positive M1 macrophages were significantly higher in KO mice than wild-type mice, whereas CD206-positive M2 macrophages were significantly lower in proportion (FIG. 26A).
In addition, CD45 positive leukocytes and CD11b positive granulocytes, monocytes or macrophages were significantly higher in KO mice than in wild type mice (FIG. 26B).
Furthermore, the proportion of F4 / 80 positive macrophages was significantly higher in KO mice than in wild type mice. There was no difference in Ly-6G positive neutrophils (FIG. 26C).
From the above results, it was considered that in MFG-E8 KO mice, the contents flowed out from dead cells that remained without phagocytosis, and as a result, many macrophages and the like flowed into the infarcted region.

[Experimental Example 16] Examination of mRNA expression changes of cytokines, chemokines and the like due to MFG-E8 deficiency After myocardial infarction, inflammation is caused by myocardial necrosis. In the inflammatory phase, the expression of inflammatory cytokines such as TNF-α and substrate proteolytic enzymes such as MMP-9 is induced, and the necrotic tissue is actively removed. Inflammation is an important event that leads to the subsequent activation of infarct site repair, but excessive inflammation conversely promotes excessive destruction of tissue and consequently attenuates the repair response. Therefore, suppressing excessive inflammation that occurs after myocardial infarction is important in preventing heart rupture. Therefore, the expression of various cytokines and chemokines after myocardial infarction was examined.

Specifically, first, in the same manner as in the above experimental example, only the infarct region of the heart was extracted from wild type and MFG-E8 KO mice on the third day after myocardial infarction, and total RNA was extracted, and real-time RT-PCR was performed. The method was used to quantify the expression of cytokines, chemokines and other mRNAs associated with inflammation or fibrosis. The sequences of the primers and TaqMan probe used are shown in Table 1. Moreover, the measurement result after correction | amendment by GAPDH is shown in FIG.

From the above results, it was found that all measured inflammatory or anti-inflammatory cytokines, chemokines, etc. (TNF-α, IL-1β, IL-6, TGF-β, IL-10, MIP, at the infarct site of the wild-type mouse heart -2, MMP-9, CTGF, ColA1) expression was increased. Regarding TNF-α, IL-1β, IL-6, MIP-2, and MMP-9, the increase in expression at the infarct site was further enhanced by MFG-E8 deficiency (FIG. 27).
This result suggests that MFG-E8 suppresses inflammation at the infarct site by promoting phagocytosis of apoptotic cells.

[Experimental Example 17] Study on cytokine production of myofibroblasts by phagocytosis of apoptotic cells It is known that macrophages induce anti-inflammatory cytokine production when phagocytosing apoptotic cells. Therefore, it was next examined whether or not MFG-E8-positive myofibroblasts have anti-inflammatory properties by phagocytosing apoptotic cells.

Specifically, fibroblasts collected from the heart were induced to differentiate by culturing for 48 hours in the presence of TGF-β1 (1 ng / ml) to obtain myofibroblasts.
The obtained myofibroblasts were seeded on a 6-well plate, apoptotic thymocytes were added at a ratio of 10 to myofibroblasts 1, and incubated at 37 ° C. for 2 hours. Thereafter, the cells were washed with PBS to remove apoptotic cells that were not phagocytosed. Next, the myofibroblasts after phagocytosis were stimulated with lipopolysaccharide (LPS; 1 μg / ml), and after culturing for 12 hours, myofibroblasts were collected by a conventional method.
Using the collected myofibroblasts, total RNA was extracted in the same manner as in Experimental Example 16 and the like, and mRNA expression of IL-6 and TGF-β was quantified using a real-time RT-PCR method. 28A and 28B show the measurement results after correction by GAPDH.

The above results revealed that, like macrophages, this myofibroblast is more likely to produce anti-inflammatory cytokine TGF-β by phagocytosis of apoptotic cells (FIG. 28B). On the other hand, it became clear that TNF-α, IL-1β and IL-6, which are inflammatory cytokines, are more difficult to produce (FIG. 28B; the results of TNF-α and IL-1β are not shown). )

[Experimental Example 18] Confirmation of MFG-E8-positive myofibroblasts in the infarct region of human myocardial infarction Patients were subjected to immunostaining using a mouse anti-human MFG-E8 antibody on heart sections of patients suffering from myocardial infarction. went. Heart sections were provided by Professor Masahiko Kuroda, Department of Molecular Pathology, Tokyo Medical University, and MFG-E8 antibody was provided by Professor Shigekazu Nagata, Kyoto University.
Specifically, a section of a human heart fixed with formalin was prepared and treated for 10 minutes at 121 ° C. in a 10 mM NaCitrate (pH 6.0) solution to activate the antigen. Thereafter, 0.3% H ₂ O ₂ / methanol solution was treated for 10 minutes to quench endogenous peroxidase. Subsequently, blocking with 10% normal goat serum / PBS was performed for 10 minutes. Thereafter, the above mouse anti-human MFG-E8 antibody (200-fold dilution) was reacted at room temperature for 1 hour. Secondary antibody was detected by DAB color development using DAKO LSAB2 kit according to the instructions (DAKO DAB color development kit). As a result, no MFG-E8 positive cells were observed in the non-infarcted area, but cells expressing MFG-E8 were observed in the vicinity of the infarcted area (FIG. 29). Further examination using serial sections revealed that MFG-E8 positive cells were SMA positive myofibroblasts (not shown).

From the results of the above Experimental Examples 1 to 18, it became clear for the first time that myofibroblasts phagocytose dead cells during myocardial infarction. In addition, no molecule involved in phagocytosis of dead cells during myocardial infarction has been found so far, but the present inventors have now found that MFG-E8 is involved for the first time.
Also, only resident fibroblast-derived myofibroblasts express MFG-E8; myofibroblasts, like macrophages, have anti-inflammatory properties after phagocytosis of dead cells; It was revealed that myofibroblasts trained with MFG-E8 were also observed at actual human myocardial infarction sites.

[Experimental Example 19] Examination of the effect of MFG-E8 administration on the heart after myocardial infarction (1) MFG-E8 administration into mouse myocardium As in Reference Example 1 above, 8-week-old C57BL / 6J wild-type male Mice were subjected to myocardial infarction. At that time, after ligating the left anterior hind limb of the left coronary artery with a 6 mm silk blade suture, MFG-E8 / PBS (1.6 μg or 3.2 μg) or PBS was added to the left and right of the ischemic myocardium downstream from the ligature. 10 μl each was administered. MFG-E8 used for administration was obtained by sterilizing the recombinant MFG-E8 produced in Experimental Example 13 using ULTRA FREE-MC (manufactured by Millipore). For administration, a 29G needle (BD Rhodos ™; 326631) was used. After administration, it was confirmed that there was no bleeding or solution leakage, and the incision was sutured with a suture. FIG. 30A shows a schematic diagram of the treatment. The ligation point is ●, and the MFG-E8 administration point is □.

(2) Confirmation of infarct area and risk area After the myocardial infarction operation and MFG-E8 administration in the model mouse of (1) above, the heart was excised after perfusion with 1% Evans blue staining solution. The isolated heart was treated with 1% TTC staining solution. A photograph of the heart after TTC staining is shown in FIG. 30B. The Evans blue non-stained part corresponds to a risk area (AAR), and the TTC stained part corresponds to an infarcted area. FIG. 30C shows the result of measuring the size of the infarct region and the size of the risk region, and quantifying the ratio of the infarct region to the risk region or the ratio of the risk region to the left ventricle.
As a result of FIGS. 30B to 30C, no difference was observed between the MFG-E8 administration group and the non-administration group.

[Experimental Example 20] Examination of the effect of MFG-E8 administration on inflammation at the infarct site after myocardial infarction In the same manner as in Experimental Example 19, myocardial infarction and MFG-E8 administration were performed. In some samples, the administered MFG-E8 was 1.6 μg or 3.2 μg. On the 4th day after the treatment and administration, the heart was removed from the mouse, and the heart was divided into an infarcted site and a non-infarcted site. Thereafter, total RNA was recovered from each site by the same method as described above, and the expression levels of IL-1β and MIP-2 were examined by real-time RT-PCR.
FIG. 31A shows the results of the expression levels of IL-1β and MIP-2 when 1.6 μg of MFG-E8 was administered, corrected by GAPDH. As a result, increased expression levels of IL-1β and MIP-2 were observed at the infarct site. It was also found that this increase in expression was significantly suppressed in the MFG-E8 administration group.
In addition, FIG. 31B shows the results of the expression levels of IL-1β and MIP-2 when 1.6 μg or 3.2 μg of MFG-E8 was corrected with 18S rRNA. As a result, it became clear that the inflammation after the infarction was suppressed depending on the concentration of MFG-E8 to be administered.

[Experimental Example 21] Examination of the effect of MFG-E8 administration on apoptosis after myocardial infarction Myocardial infarction and MFG-E8 administration were performed in the same manner as in Experimental Example 19 (PBS administration group: N = 7, MFG-E8 administration) Group: N = 7). In some samples, the concentration of MFG-E8 administered was 3.2 μg. Three days after the treatment and administration, the heart was removed from the mouse, and the left ventricle of the heart was selectively fluorescently stained by the same method as in Experimental Example 13 and the like. The results are shown in FIG. 32A.
As shown in FIG. 32A, the proportion of TUNEL positive cells showing apoptotic cells decreased in the group administered MFG-E8 after myocardial infarction. As a result of measuring and calculating the proportion of apoptotic cells in the total cells in the visual field (TUNEL positive cells / DAPI%), it was confirmed that the number of apoptotic cells was significantly reduced (FIG. 32B).

[Experimental Example 22] Examination of influence of MFG-E8 administration on cardiac function after myocardial infarction In the same manner as in Experimental Example 19, myocardial infarction was performed and 3.2 μg of MFG-E8 was administered. Echocardiographic evaluation was performed over time in the same manner as in Experimental Example 13 from 2 to 10 weeks after the myocardial infarction operation and MFG-E8 administration.
As a result, there was no significant difference in heart rate (HR) in all groups (not shown), and the effect of heart rate on each parameter was affected by any group (PBS administration group: N = 10, MFG-E8 administration group). : N = 9) is considered to be the same. At all time points, in the MFG-E8 administration group, the decrease in cardiac function after myocardial infarction was improved (FIG. 33).
FIG. 34 shows the results of the 10th week. In the 10th week, cardiac function and hemodynamics were also examined with a cardiac catheter (PBS administration group: N = 7, MFG-E8 administration group: N = 7), and MFG-E8 administration also improved cardiac function by this method. It became clear that an improvement was seen (FIG. 34).
Further, FIG. 34 also shows the results of measuring the ratio of heart weight (HW) to body weight (BW) at 10 weeks (PBS administration group: N = 10, MFG-E8 administration group: N = 9). . As a result, the heart weight ratio was significantly decreased by MFG-E8 administration.

[Experimental Example 23] Examination of the effect of MFG-E8 administration on cardiac fibrosis after myocardial infarction In the same manner as in Experimental Example 19, myocardial infarction was performed and 3.2 μg of MFG-E8 was administered (PBS administration group: N = 6, MFG-E8 administration group: N = 5). At 10 weeks after myocardial infarction and MFG-E8 administration, the heart was removed, sections were prepared, and Masson trichrome staining was performed. The resulting stained photograph is shown in FIG. 35A. Moreover, the result of having calculated the infarct area | region with respect to the left ventricle area | region is shown to FIG. 35B.
As a result, it was confirmed that administration of MFG-E8 decreased the fibrosis region for the entire left ventricle.

From the results of Experimental Examples 19 to 23, it became clear for the first time that administration of MFG-E8 after myocardial infarction can significantly improve inflammation and cardiac function at the infarct site after myocardial infarction.
Since MFG-E8 contributes greatly in the event of removal of dead cells after myocardial infarction, which has not been targeted by drugs so far, it can be a new drug discovery seed. MFG-E8 is considered to have a high possibility of becoming a new treatment method including the combined use with an existing treatment method.

The preventive pharmaceutical composition and the method for treating or preventing myocardial injury or heart failure according to the present invention are very useful for the prevention or treatment of myocardial injury or heart failure, and can be used in the pharmaceutical or medical industry.

Claims (11)

1. A pharmaceutical composition for treating myocardial injury, comprising MFG-E8 as an active ingredient.
2. A pharmaceutical composition for preventing myocardial injury, comprising MFG-E8 as an active ingredient.
3. A pharmaceutical composition for treating heart failure, comprising MFG-E8 as an active ingredient.
4. A pharmaceutical composition for preventing heart failure, comprising MFG-E8 as an active ingredient.
5. A method of treating or preventing myocardial injury or heart failure, comprising a step of administering MFG-E8.
6. MFG-E8 for use as a therapeutic or prophylactic agent for myocardial injury or heart failure.
7. Use of MFG-E8 for the manufacture of a medicament for treating or preventing myocardial injury or heart failure.
8. A method for screening a compound for treating myocardial injury or heart failure, using MFG-E8 expression induction as an index.
9. A method for screening a compound for preventing myocardial injury or heart failure, using MFG-E8 expression induction as an index.
10. A method for screening a compound for treating myocardial injury or heart failure using an MFG-E8 bioassay system.
11. A method for screening a compound for preventing myocardial injury or heart failure using an MFG-E8 bioassay system.

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