JP7466547B2 - Screening method for vascular endothelial cell activator - Google Patents

Description

Related Applications:
This specification includes the contents described in the specification of Japanese Patent Application No. 2019-147942 (filed on August 9, 2019), which is the priority basis of this application.
Technical field:
The present invention relates to a method for screening for a substance that controls vascular endothelial cell activation, and more particularly to a method for screening for a substance that promotes or inhibits vascular endothelial cell activation using the amount of VEGF uptake into vascular endothelial cells as an index.

Transplantation of bone marrow mononuclear cells (BM-MNCs) is known to activate angiogenesis in ischemic tissues. BM-MNCs contain a relatively high percentage of hematopoietic stem cells and can be easily prepared from autologous tissues, making them a suitable source of cell therapy and frequently used in experimental treatments of ischemic diseases such as limb ischemia, myocardial infarction, and cerebral infarction. However, it remains unclear how BM-MNCs activate angiogenesis.

Initially, differentiation of hematopoietic stem cells contained in BM-MNCs into endothelial cells was proposed as the fundamental mechanism, but subsequent studies have revealed that only a very small number of transplanted cells differentiate into endothelial cells, and this is not the fundamental mechanism. Secretion of multiple growth factors and exosomes has also been proposed, but no clear evidence has been presented. Furthermore, it is known that the survival period of cells after transplantation is relatively short, and it is thought that secretion of growth factors and exosomes is not the fundamental mechanism for the strong angiogenic activation observed for a long period after BM-MNC transplantation.

Vascular endothelial growth factor (VEGF) is one of the most important angiogenesis-promoting factors. VEGF activates tyrosine kinase by binding to the VEGF receptor present on the cell surface, transmits a signal into the cell, and changes the function and structure of the cell. A method for screening endothelial cells with high angiogenesis ability using the expression ratio of VEGF receptors VGFR1 and VGFR2 as an index (Patent Document 1) and a method for screening substances with angiogenesis-promoting activity using phosphorylation of VEGFR1 and migration of the cells as an index using a cultured vascular endothelial cell line expressing VEGFR1 on the cell surface have been reported (Patent Document 2). In addition, a method for evaluating angiogenesis ability by measuring the blood concentrations of VEGF and endostatin has been reported (Patent Document 3). As described above, angiogenesis-promoting ability has been measured in the past by the concentration of VEGF and the dynamics of the VEGF receptor, but the idea of directly measuring the angiogenesis-promoting ability of a candidate substance by the amount of VEGF uptake by vascular endothelial cells, as in the present invention, was completely lacking.

JP 2005-065697 A JP 2007-244273 A WO2002/031497

The objective of the present invention is to apply the mechanism of action of angiogenesis induced by BM-MNC transplantation to establish a method for screening small molecular weight substances that can replace its function, and to provide new candidate therapeutic agents for ischemic diseases including cerebral infarction, myocardial infarction, and limb ischemia, as well as diseases in which angiogenesis is involved in the progression of the pathology, such as cancer, inflammatory diseases, and rheumatism.

Conventionally, BM-MNCs have been thought to promote angiogenesis by secreting various growth factors related to the promotion of angiogenesis. The present inventors have discovered that BM-MNCs do not promote intravascular neovascularization by secreting various growth factors related to the promotion of angiogenesis, but rather that BM-MNCs act on vascular endothelial cells and promote the uptake of angiogenesis-promoting factors into vascular endothelial cells, thereby promoting angiogenesis. They have also discovered that it is possible to search for new candidates for therapeutic agents for ischemic diseases by screening for low molecular weight compounds that have the same effect as BM-MNCs, using as an indicator the change in the amount of uptake of VEGF or other angiogenesis-promoting factors into vascular endothelial cells.

That is, the present invention relates to the following (1) to (13).
(1) A method for screening for a substance that promotes or inhibits vascular endothelial cell activation, comprising the steps of:
1) culturing vascular endothelial cells with a test substance in the presence of labeled VEGF;
2) determining the amount of VEGF taken up into vascular endothelial cells;
3) A step of evaluating the effect of the test substance on activating vascular endothelial cells based on the amount of VEGF taken up into the vascular endothelial cells.
The label may be a fluorescent label, an enzyme label, or a radioactive label, preferably a fluorescent label.
(2) The method according to (1), wherein the amount of VEGF taken up into vascular endothelial cells is determined by a change in a signal derived from labeled VEGF in vascular endothelial cells before and after incubation with a test substance.
(3) The method according to (1), wherein the amount of labeled VEGF taken up by vascular endothelial cells is determined by a change in the signal derived from the labeled VEGF in the culture supernatant before and after incubation with a test substance.
(4) The method according to any one of (1) to (3), further comprising a step of comparing the change in the amount of VEGF uptake into vascular endothelial cells caused by the test substance with the change in the amount of VEGF uptake into vascular endothelial cells caused by a standard substance.
(5) The method according to any one of (1) to (4), wherein in step 3), if the amount of VEGF uptake into the vascular endothelial cells is increased, the test substance is evaluated as being useful as a substance that promotes vascular endothelial cell activation, and if the amount of VEGF uptake into the vascular endothelial cells is decreased, the test substance is evaluated as being useful as a substance that inhibits vascular endothelial cells.
(6) The method according to any one of (1) to (5), further comprising a step of determining the concentration in the culture supernatant of one or more cytokines selected from the group consisting of angiopoietin 2, endoglin, IGFBP (insulin-like growth factor binding protein), TNFα, and TGFα.
(7) The method according to (6), wherein the cytokine comprises at least angiopoietin 2.
(8) The method according to (6) or (7), in which if culturing with a test substance increases the amount of VEGF taken up by vascular endothelial cells and decreases the cytokine concentration in the culture supernatant, the test substance is evaluated as useful as a candidate substance for promoting vascular endothelial cell activation, and if it decreases the amount of VEGF taken up by vascular endothelial cells and increases the cytokine concentration in the culture supernatant, the test substance is evaluated as useful as a substance for inhibiting vascular endothelial cell activation.
(9) The method according to any one of (1) to (8), wherein the vascular endothelial cell activation promoter is a candidate for a therapeutic drug for ischemic disease, and the vascular endothelial cell activation inhibitor is a candidate for an angiogenesis inhibitor.
In other words, the methods (1) to (8) can be used as screening methods for ischemic disease therapeutic drugs or angiogenesis inhibitors. Examples of the ischemic disease include limb ischemia, ischemic heart disease including myocardial infarction, angina pectoris, and heart failure, and ischemic cerebrovascular disorders such as cerebral infarction. Angiogenesis inhibitors are useful for treating diseases in which angiogenesis is involved in the progression of the pathology (angiogenic diseases), such as cancers such as solid tumors and hemangiomas, chronic rheumatoid arthritis, Crohn's disease, Behcet's disease, psoriasis, atherosclerosis, diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, and age-related macular degeneration.
(10) A method for screening a therapeutic agent for ischemic disease , comprising the steps of:
1) culturing vascular endothelial cells with a test substance in the presence of labeled VEGF;
2) determining the amount of VEGF taken up into vascular endothelial cells;
3) selecting the test substance as a candidate for a therapeutic agent for ischemic disease when the amount of VEGF uptake into the vascular endothelial cells is increased.
(11) A method for screening an angiogenesis inhibitor, comprising the steps of:
1) culturing vascular endothelial cells with a test substance in the presence of labeled VEGF;
2) determining the amount of VEGF taken up into vascular endothelial cells;
3) selecting the test substance as an angiogenesis inhibitor when the amount of VEGF taken up by the vascular endothelial cells is reduced.
(12) The method according to any one of (1) to (11), wherein the label is a fluorescent label.
(13) The method according to any one of (1) to (12), wherein the vascular endothelial cells are human vascular endothelial cells.

To date, methods are known for evaluating the angiogenesis activation effect using the expression of VEGF receptors and the amount of VEGF secreted as indicators (see above), but no method is known for evaluating the activation effect of vascular endothelial cells using the promotion of VEGF uptake as an indicator.

According to the present invention, it is possible to screen known low molecular weight compounds for substances that have the effect of activating vascular endothelial cells through a mechanism similar to that of BM-MNC, making it possible to replace the function of BM-MNC in the body with low molecular weight compounds. A screening system that uses the amount of intracellular uptake of labeled VEGF as an indicator enables high-throughput screening of substances that promote or inhibit vascular endothelial cell activation. The identified test substances are also advantageous from the standpoint of drug delivery, as their target is vascular endothelial cells.

[FIG. 1] FIG. 1 shows the uptake of vascular endothelial growth factor (VEGF) into human umbilical vein endothelial cells (HUVECs) co-cultured with BM-MNCs.
(A) A significant decrease in VEGF concentration in the medium was observed by co-culture with BM-MNC. In contrast, non-contact co-culture with BM-MNC using cell culture inserts did not decrease VEGF levels. (B) HUVEC and BM-MNC could be clearly distinguished by anti-CD31 and anti-CD45 antibodies. (C-G) Streptavidin-Allophycocyanin (APC) uptake into HUVEC was not observed (C). Compared to HUVEC culture alone (D), VEGF uptake into HUVEC was observed to be elevated after BM-MNC co-culture (E). Blockade of BM-MNC gap junctions with 1-octanol (F) or carbenoxolone (G) abolished the co-culture effect. (H-J) Streptavidin-APC binding to BM-MNC was not observed (H). When BM-MNCs were cultured alone (I) or cocultured with HUVECs (J), little VEGF uptake into BM-MNCs was observed. *p<0.01 versus medium, **p<0.01 versus medium, n=3 for each group (A).
[FIG. 2] FIG. 2 shows the transfer of BCECF (2',7'-Bis(carboxyethyl)-4 or 5-carboxyfluorescein) from BM-MNC to HUVEC via gap junctions in vitro.
(A-D) FACS analysis of naive (A) and HUVECs (A-C) cocultured with BCECF-labeled BM-MNCs (B). BCECF-positive HUVECs were observed after coculture with BCECF-labeled BM-MNCs. BCECF transfer was blocked when cocultured with cell culture inserts (C) or when the gap junction uncoupler 1-octanol was applied (D). (E, F) HUVECs were stained with Alexa Fluor® 488 fluorescently labeled antibody (hereafter Alexa488) alone (E) or with anti-Cx37 (connexin 37) antibody and Alexa488 (F). Little Cx37 expression was observed in HUVECs. (G, H) HUVECs stained with isotype control antibody (G) or anti-Cx43 (connexin 43) antibody (H). Cx43 expression was observed in HUVEC. (I, J) BM-MNCs stained with Alexa488 alone (I) or anti-Cx37 antibody and Alexa488 (J). Cx37 expression was observed in BM-MNC. (K, L) BM-MNCs stained with isotype control antibody (K) or anti-Cx43 antibody (L). Cx43 expression was observed in HUVEC.
FIG. 3 shows the transfer of BCECF from BM-MNC to endothelial cells via gap junctions in vivo.
(A-D) BCECF-positive BM-MNCs transplanted 5 min after intravenous injection into the brain after cerebral infarction. BCECF-positive cells (A) were observed in the brain microvasculature (B). Overlay of the images demonstrates BCECF migration from BM-MNCs to endothelial cells through the CD31-positive endothelial cell membrane (C, D). (E, F) Connexin 37 (Cx37) expression was not observed in the contralateral healthy cerebral cortex (E), but was clearly expressed in the ipsilateral side 48 h after induction of cerebral infarction (F). (G, H) Connexin 43 (Cx43) expression was barely observed in the healthy cortex (G), but increased expression was observed after cerebral infarction (H). (I-L) Accumulation of Cx37 (J) accompanied by infiltration of BCECF (J) was observed in endothelial cells (K, L). (M-P) Accumulation of Cx43 (M) accompanied by infiltration of BCECF (N) was observed in endothelial cells (O, P). Scale bars, 20 μm (A), 5 μm (D, I), 40 μm (E).
FIG. 4 shows that ultrastructural changes in endothelial cells suggest a decrease in autophagy in BM-MNC transplanted animals.
(A) Normal capillaries consisting of endothelial cells, basal layer, pericytes, and astrocytes (astrocyte foot processes) in a non-infarcted mouse. (B, C) Representative photographs of capillaries in a lesioned area. A significant number of autophagosome-like vacuoles were observed in endothelial cells of PBS-treated mice (B). In contrast, several autophagosome-like vacuoles with attached pericytes were observed in endothelial cells of BM-MNC-treated mice 72 hours after induction of cerebral infarction, despite the disappearance of astrocytes (astrocyte foot processes) (C). (D, E) A significant number of autophagosome-like vacuoles were observed in endothelial cells of PBS-treated mice (D). In contrast, few autophagosome-like vacuoles were observed in endothelial cells of BM-MNC-treated mice (E). (F, G) Photographs of capillaries in the uninjured contralateral cortex. Autophagosome-like vacuoles were observed in some endothelial cells of PBS-treated mice (F). In contrast, no autophagosome-like vacuoles were observed in endothelial cells of BM-MNC-treated mice (G). (H) Quantitative analysis confirmed that BM-MNC transplantation suppressed the formation of autophagosome-like vacuoles in the relevant areas. Scale bar, 2 μm (A), p<0.01 versus PBS, n=5 in each group (H).
[Figure 5] Figure 5 shows the expression of autophagy marker LC3 in endothelial cells after cerebral infarction.
(A) Most CD31-positive endothelial cells in the lesion area express LC3 in both PBS- and BM-MNC-treated mice. (B) In the perilesional area, LC3-positive endothelial cells were rarely observed in BM-MNC-treated mice, whereas most endothelial cells were LC3-positive in PBS-treated mice. (C) In the uninjured contralateral cortex, some LC3-positive endothelial cells were observed in PBS-treated mice, whereas no LC3-positive endothelial cells were observed in BM-MNC-treated mice. Scale bar, 20 μm (A).
[FIG. 6] FIG. 6 shows Hif-1α expression after BM-MNC transplantation.
(A-C) Little expression of Hif-1α was observed in the uninjured brain (A) and in the post-stroke area 3 hours (B) or 48 hours (C) after induction of brain injury. (D) However, increased expression of Hif-1α was observed in capillary-like structures 1 hour after BM-MNC transplantation. (E-H) BCECF signal (E) colocalized with Hif-1α expression in CD31-positive endothelial cells (G, H). Scale bars, 40 μm (A), 10 μm (E).
[Figure 7] Figure 7 shows the changes in concentrations of various cytokines in the HUVEC culture supernatant.
HUVEC cultured alone (PBS), co-cultured with BM-MNC (BM-MNC). The graph shows the change (pM) in cytokine concentration in the supernatant over 6 hours of culture. (A) Angiopoetin-2, (B) Edoglin, (C) TGFα, (D) IGFBP-1, (E) TNFα.
[FIG. 8] FIG. 8 shows the promoting effect of VEGF uptake by HUVEC (human umbilical vein endothelial cells) by low molecular weight compounds in comparison with a standard substance.
(A) Fluorescence background, (B) standard/no test substance, (C) standard substance (Caffeic acid), (D) test substance 1 (4-0-Caffeoylquinic acid), (E) test substance 2 (Chicoric acid).

The present invention relates to a method for screening for substances that promote or inhibit vascular endothelial cell activation using VEGF uptake in vascular endothelial cells as an indicator, in particular for substances that have the effect of controlling vascular endothelial cell activation through a mechanism similar to that of BM-MNC.

1. Step 1) "Vascular endothelial cells" by culturing vascular endothelial cells together with a test substance in the presence of labeled VEGF
The "vascular endothelial cells" used in the present invention are not particularly limited as long as they are culturable vascular endothelial cells that express a receptor for VEGF, but are preferably mammalian-derived cells, and more preferably human-derived cells. Examples of human vascular endothelial cells include human umbilical vein endothelial cells (HUVEC), human brain microvascular endothelial cells (HBMEC), human umbilical artery endothelial cells (HUAEC), human aortic endothelial cells (HAoEC), human coronary artery endothelial cells (HCAEC), human pulmonary artery endothelial cells (HPAEC), human saphenous vein endothelial cells (HSaVEC), human dermal vascular endothelial cells (HDBEC), human dermal microvascular endothelial cells (HDMEC), human uterine microvascular endothelial cells (HUtMEC), human pulmonary microvascular endothelial cells (HPMEC), human cardiac microvascular endothelial cells (HCMEC), and human dermal microlymphatic endothelial cells (HDLEC), and any of these can be used as commercially available cultured cell lines. Particularly preferred are HUVEC and HBMEC.

"Vascular endothelial growth factor (VEGF)"
The vascular endothelial growth factor (VEGF) used in the present invention is not particularly limited as long as it can bind to the VEGF receptor expressed in the vascular endothelial cells used, but mammalian-derived VEGF is preferred, and human-derived VEGF is more preferred. There are seven VEGF families, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, PIGF-1, and PIGF-2. VEGF-A is sometimes generally referred to as VEGF, and in this specification, when simply referring to VEGF, it means VEGF-A.

In the present invention, labeled VEGF can be used. The labeling method is not particularly limited, and any known method such as enzyme labeling, radioactive labeling, or fluorescent labeling can be appropriately selected and used. In the present invention, fluorescent labeling is preferred.

Enzymes used for enzyme labeling include horseradish peroxidase (HRP) and alkaline phosphatase (AP). HRP is colored using a chromogenic substrate such as DAB or TAB, while AP is colored using a chromogenic substrate such as BCIP/NBT or pPNPP.
Radioisotopes such as ¹²⁵ I and ³ H can be used for radiolabeling, and are incorporated into the protein to be detected by enzymatic or chemical oxidation using a protein iodine labeling reagent.

Fluorescent substances used for fluorescent labeling include, but are not limited to, fluorescent proteins such as PE (Phycoerythrin), APC (Allophycocyanin), GFP, CFP, RFP, etc., and low molecular weight fluorescent dyes such as FITC (Fluorescein Isothiocyanate), Coumarin, TRITC (Tetramethylrhodamine), Alexa Fluor (registered trademark), Cy (registered trademark), Texas Red (registered trademark), BODIPY (registered trademark) FL, and Super Bright. Fluorescent labeling signals can be detected and quantified by detection methods such as optical microscopes, high-sensitivity CCD cameras, confocal laser microscopes, and flow cytometry, as well as image processing techniques.

To increase sensitivity, detection may involve amplifying the fluorescent signal using the biotin-avidin or biotin-streptavidin system.

"Test substance"
The type of test substance to be screened in the method of the present invention is not particularly limited. Considering the use as a therapeutic agent for ischemic disease described later, a low molecular weight compound that is easy to synthesize and handle is preferable. A low molecular weight compound means a compound with a molecular weight of about 10,000 or less.

"Culture of vascular endothelial cells"
Vascular endothelial cells are cultured with a test substance in the presence of labeled VEGF. For the culture, a commercially available medium for endothelial cells may be used, or a basal medium may be prepared by adding various nutrients necessary for the maintenance and proliferation of cells and various components necessary for differentiation induction. As the basal medium, any medium that can be used for the culture of mammalian cells, such as DMEM, Keratinocyte-SFM, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, α MEM, Ham's medium, RPMI 1640 medium, Fischer's medium, McCoy's medium, Williams E medium, and a mixture of these, may be used.

Nutrient sources may include carbon sources such as glycerol, glucose, fructose, sucrose, lactose, honey, starch, dextrin, etc., hydrocarbons such as fatty acids, oils and fats, lecithin, alcohols, etc., nitrogen sources such as ammonium sulfate, ammonium nitrate, ammonium chloride, urea, sodium nitrate, etc., inorganic salts such as table salt, potassium salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, sodium molybdate, sodium tungstate, manganese sulfate, various vitamins, amino acids, etc.

If necessary, the medium may contain additional ingredients such as retinoic acid, pyruvic acid, amino acid reducing agents such as β-mercaptoethanol, transferrin, fatty acids, insulin, collagen precursors, trace elements, 3'-thiolglycerol, growth factors (epidermal growth factor; EGF, basic fibroblast growth factor; bFGF, insulin-like growth factor; IGF, etc.), serum or serum substitutes, etc. Examples of serum substitutes include albumin (e.g., lipid-rich albumin), commercially available Knockout Serum Replacement (KSR), bovine pituitary extract (BPE), chemically-defined lipid concentrated (Gibco), Glutamax (Gibco), B27 supplement (Gibco), and Y-27632 (Wako Pure Chemical Industries, Ltd.).

The pH of the medium obtained by mixing these components is in the range of 5.5 to 9.0, preferably 6.0 to 8.0, and more preferably 6.5 to 7.5.

The culture is carried out at 36° C. to 38° C., preferably 36.5° C. to 37.5° C., under conditions of 1% to 25% O ₂ and 1% to 15% CO ₂ .

2. Step 2) Determining the amount of VEGF taken up into vascular endothelial cells "Determining the amount of VEGF taken up into vascular endothelial cells"
The amount of VEGF taken up into vascular endothelial cells can be determined directly by measuring the change in the signal intensity derived from labeled VEGF in vascular endothelial cells before and after incubation with a test substance, or the amount of VEGF taken up into vascular endothelial cells can be determined indirectly by measuring the change in the signal intensity derived from labeled VEGF remaining in the culture medium before and after incubation with a test substance.

When measuring changes in signals derived from labeled VEGF in vascular endothelial cells, it is recommended to culture the vascular endothelial cells and the test substance in the presence of labeled VEGF for at least 10 minutes, 20 minutes, or 30 minutes, more preferably 30 minutes to 4 hours, and even more preferably 1 to 3 hours, before performing the measurement; however, this may be modified as appropriate depending on the culture conditions and the cells used.

When measuring changes in signals derived from labeled VEGF in the culture supernatant, it is recommended to measure after culturing the vascular endothelial cells and the test substance in the presence of labeled VEGF for at least 30 minutes, 1 hour, 2 hours, or more, 3 hours, more preferably 4 hours or more, and particularly preferably about 6 hours, but this may be changed as appropriate depending on the culture conditions and the cells used.

The amount of labeled VEGF in the cells can be easily measured, for example, by using FACS. The amount of labeled VEGF remaining in the culture supernatant can be measured, for example, by using a spectrofluorometer, a microplate reader, or a Bio-Plex reader.

3. Step 3) Evaluating the vascular endothelial cell activation effect of the test substance The vascular endothelial cell activation effect of the test substance is evaluated based on the amount of VEGF uptake into vascular endothelial cells by the test substance. The evaluation may be performed using the test substance alone, or using a compound known to promote VEGF uptake into vascular endothelial cells as a standard substance and evaluating the test substance in comparison with the standard substance.

When evaluating a test substance alone, the simplest case is when the amount of VEGF taken up by vascular endothelial cells is significantly increased by culturing the test substance with the test substance, the test substance is identified as a candidate for a vascular endothelial cell activator. Alternatively, if a certain standard value can be set, the test substance is identified as a candidate for a vascular endothelial cell activator when an amount of VEGF taken up that exceeds the standard value is observed.

When evaluating a test substance alone, the simplest approach is to identify a test substance as a candidate for a vascular endothelial cell inhibitor if the amount of VEGF taken up by vascular endothelial cells is significantly reduced by culturing the test substance with the test substance. Alternatively, if a certain standard value can be set, the test substance is identified as a candidate for a vascular endothelial cell inhibitor if the amount of VEGF taken up by vascular endothelial cells exceeds the standard value.

"Standard materials"
When evaluating in comparison with a standard substance, the amount of VEGF uptake into vascular endothelial cells when the standard substance is added and when the test substance is added under the same conditions are compared to evaluate the vascular endothelial cell activation effect. The standard substance is not particularly limited as long as it is a substance whose VEGF uptake promoting effect on vascular endothelial cells has been confirmed. In the examples described below, the VEGF uptake of other low molecular weight compounds into vascular endothelial cells was evaluated by using caffeic acid, which the inventors have confirmed to have a VEGF uptake promoting effect on vascular endothelial cells, by the method of the present invention, but the standard substance is not limited thereto.

"Substances that promote vascular endothelial cell activation"
The "vascular endothelial cell activation-promoting substance" screened for by the method of the present invention is a substance that has the effect of promoting the activation of vascular endothelial cells through a mechanism similar to that of BM-MNC.

"Substance that inhibits vascular endothelial cell activation"
The "substances that inhibit vascular endothelial cell activation" screened for by the method of the present invention are substances that exert an action opposite to that of BM-MNC and have the effect of inhibiting the activation of vascular endothelial cells.

4. Evaluation of cytokine concentration in culture supernatant If necessary, the above method may further include a step of determining the concentration of one or more cytokines selected from the group consisting of angiopoietin 2, endoglin, IGFBP (insulin-like growth factor binding protein), TNFα, and TGFα in the culture supernatant. The cytokine concentration can be measured in the same manner as VEGF, but a commercially available multiplex system may be used to simultaneously measure multiple cytokines. For example, when the amount of VEGF uptake by vascular endothelial cells is increased and the cytokine concentration in the culture supernatant is decreased by culturing with the test substance, the test substance is identified as a candidate for a substance that promotes vascular endothelial cell activation. Conversely, when the amount of VEGF uptake by vascular endothelial cells is decreased and the cytokine concentration in the culture supernatant is increased by culturing with the test substance, the test substance is identified as a candidate for a substance that inhibits vascular endothelial cell activation. As cytokines to be used for evaluation, angiopoietin 2 and TGFα are particularly preferable, and angiopoietin 2 is more preferable.

5. Screening of drugs for treating ischemic diseases "Drugs for treating ischemic diseases"
The vascular endothelial cell activation promoter identified by the screening method of the present invention can activate angiogenesis by vascular endothelial cells through the same mechanism of action as BM-MNC. Therefore, it is useful as a therapeutic agent for diseases caused by ischemia (ischemic diseases), such as wounds including bedsores/skin ulcers, surgical scars, and intractable digestive ulcers, inflammatory diseases including chronic inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, severe limb ischemia, ischemic heart diseases including myocardial infarction, angina pectoris, and heart failure, ischemic cerebrovascular disorders such as cerebral infarction, diabetic neuropathy, and cancer accompanied by severe ischemia. In other words, the screening method of the present invention can be used to screen candidate substances for therapeutic agents for ischemic diseases.

6. Screening of angiogenesis inhibitors "Angiogenesis inhibitors"
The vascular endothelial cell activation inhibitor identified by the screening method of the present invention can suppress angiogenesis activation by vascular endothelial cells through a mechanism that antagonizes the action of BM-MNC. Therefore, as an angiogenesis inhibitor, it is useful as a therapeutic agent for angiogenic diseases in which the progression of pathology and angiogenesis are closely related, such as cancer, chronic rheumatoid arthritis, Crohn's disease, Behcet's disease, psoriasis, atherosclerosis, diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, and age-related macular degeneration. In other words, the screening method of the present invention can be used to screen candidate substances for the above-mentioned angiogenesis inhibitors.

The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1: Promotion of VEGF uptake by HUVECs by BM-MNCs 1. Materials and Methods - Preparation of BM-MNCs Bone marrow was obtained from 6-week-old male C57BL/6 mice (Japan SLC). The femurs and tibias were dissected, and bone marrow was extracted from the bones using PBS. The bone marrow was mechanically separated, and BM-MNCs were isolated by density gradient centrifugation using Ficoll-Paque Premium (GE Healthcare).

Preparation of endothelial cells and co-culture with BM-MNCs Human umbilical vein endothelial cells (HUVECs, Kurabo) were cultured with medium (HuMedia-EB2, Kurabo), serum and growth additives according to the manufacturer's protocol. HUVECs were seeded in 6-well plates (1000 μl, 1.5× ¹⁰⁵ cells/well) and incubated for 72 hours. Supernatants (200 μl) were collected from the samples. Then, for co-culture experiments, HuMedia-EB2 alone or BM-MNC suspension suspended in HuMedia-EB2 (1× ¹⁰⁶ cells/well) were added to HUVECs. After 6 hours of co-culture, supernatants (200 μl) were collected from the wells.

To evaluate the relevance of gap junction-mediated cell-cell interactions between HUVECs and BM-MNCs, BM-MNCs were incubated with the gap junction inhibitor 1-octanol (final concentration 1 mM) or the gap junction inhibitor carbenoxolone (CDX: final concentration 100 μM, Sigma) and washed twice with HuMedia-EB 2 before co-culture with HUVECs.

- VEGF measurement Human vascular endothelial growth factor (hVEGF) concentration was measured using Bio-Plex Multiplex System (Bio-Rad) according to the manufacturer's protocol. As expected, the hVEGF level in the BM-MNC suspension was below the detection limit. To evaluate the effect of cell-cell interactions between HUVECs and BM-MNCs, cell culture inserts (1 μm pore size: Thermo Fisher) were used to prevent direct cell-cell interactions.

Introduction of small fluorescent molecules into the cytosol of BM-MNC BM-MNC were incubated with 1 μM BCECF-AM (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethylester, Thermo Fisher) at room temperature for 30 minutes. Before use, BCECF-labeled BM-MNC were washed twice with PBS.

- VEGF uptake by HUVEC Biotin-conjugated VEGF (R&D Systems) was incubated with streptavidin-conjugated APC (Thermo Fisher, molar ratio 4:1) for 10 min at room temperature. HUVEC were suspended in PBS. ^2x105 BM-MNC and APC-labeled VEGF (final concentration 10 nM) were added to ^1x105 HUVEC and incubated at 37°C for 3 h. After co-incubation, the cell mixture was washed twice with PBS and stained with Phycoerythrin (PE)-conjugated anti-human CD31 antibody (BD Bioscience), FITC (Fluorescein isothiocyanate)-conjugated anti-mouse CD45 antibody (BD Bioscience), and 7-AAD (BD Bioscience). The levels of APCs (CD31 positive, CD45 negative, and 7AAD negative) in HUVECs were assessed using a FACS Calibur fluorescent cell sorter (BD Bioscience). To assess the role of gap junctions on VEGF uptake, BM-MNCs were incubated with 1-octanol.

Induction of focal cerebral ischemia and administration of BM-MNC A mouse cerebral infarction model with excellent reproducibility was adopted (Taguchi A et al., "A Reproducible and Simple Model of Permanent Cerebral Ischemia in CB-17 and SCID Mice." J Exp Stroke Transl Med. 2010; 3(1): 28-33.). That is, the distal part of the left middle cerebral artery (MCA) of 7-week-old male CB-17 mice (Oriental Yeast) was ligated and cut using a bipolar forceps under 3% halothane inhalation anesthesia to induce permanent focal cerebral ischemia. During the operation, rectal temperature was monitored and controlled at 37.0 ± 0.2 ° C. with a feedback-regulated heating pad. Cerebral blood flow (CBF) in the MCA territory was also monitored. Mice with a 75% or greater decrease in CBF immediately after MCA occlusion were used in the experiment (success rate 100%). The animals weighed 20-25 grams before surgery. 48 hours after induction of cerebral infarction, 5×10 ⁵ BM-MNCs or PBS were injected via the tail vein.

Immunohistochemistry: To analyze the infarcted cortex histochemically, mice were deeply anesthetized with sodium pentobarbital and perfused with saline containing 4% final concentration of paraformaldehyde. Brains were carefully removed and coronal sections (20 μm) were made using a vibratome (Leica). Sections from non-infarcted CB-17 mice were also made to evaluate the expression of connexins 37 and 43 in the cerebral cortex. Sections were immunostained with primary antibodies against CD31 (BD Pharmingen, dilution 1:50), LC3 (Medical & Biological Laboratories, 1:500), HIF-1α (Hif-1α R&D, 1:50), connexin 37 (Cloud-Clone, 1:50), connexin 43 (Proteintech, 1:200) or DAPI (BD Bioscience, 1:1000). Anti-Hif-1α antibodies were visualized by the 3,3′-diaminobenzidine (DAB) method and counterstained with Mayer's hematoxylin solution (Wako). Alexa 555-conjugated antibodies (Novus Biologicals) were used as secondary antibodies to visualize CD31, connexin 37 and connexin 43. Alexa 647, Alexa 488 or Alexa 633 (all Novus Biologicals)-conjugated antibodies were used as secondary antibodies to detect Hif-1α, LC3, connexin 37 or connexin 43 antibodies, respectively.

Electron microscopy analysis. To analyze the ultrastructure, animals were perfused with a fixative solution containing 4% paraformaldehyde and 2% glutaraldehyde in PBS (pH 7.4). Brains were carefully removed and immersed in paraformaldehyde at 4°C for 24 h. Coronal brain slices were cut with a microslicer to a thickness of 100 μm. Sections were fixed in 1% _OsO4 . They were dehydrated for 1 h and flat embedded on siliconized glass slides in epoxy resin. Ultrathin sections of the cerebral cortex, including the ischemic lesion, peri-infarct area or corresponding contralateral cortical area, were cut, mounted on Formvar-coated single-slot (2x1 mm) grids, and stained with 2% uranyl acetate and lead citrate. Electron micrographs were taken at 80 kV using a JEM-1400EX+ (JDEL). The numbers of autophagosome-like vacuoles in the ischemic lesion, peri-infarct area and corresponding contralateral cortical area were counted.

Data analysis: Statistical comparisons between groups were determined using one-way analysis of variance (ANOVA) followed by post-hoc analysis using Dunnett's test. Where indicated, individual comparisons were performed using Student's t-test. Data are presented as mean ± SD in all experiments.

2. Results (1) BM-MNC promotes VEGF uptake into HUVECs through gap junction-mediated interactions VEGF is one of the most prominent angiogenesis-promoting factors. We first evaluated the effect of BM-MNC on VEGF levels in HUVEC culture medium. Mouse BM-MNCs were cocultured with HUVECs, and the levels of hVEGF in the culture medium were evaluated before and 6 hours after coculture. hVEGF levels were significantly decreased from baseline after coculture with BM-MNCs, but increased in the control (Figure 1A). Coculture with BM-MNCs separated by cell culture inserts did not decrease hVEGF levels. This finding supports the hypothesis that BM-MNCs can promote hVEGF uptake into HUVECs through cell-cell interactions.

To confirm the hypothesis, APC-labeled VEGF was added to the HUVEC culture medium, and the uptake of APC-labeled VEGF into HUVEC was evaluated by FACS. HUVEC and BM-MNC were distinguished by antibodies against CD31 and CD45 (Fig. 1B). Increased uptake of VEGF into HUVEC was observed after coculture with BM-MNC (Fig. 1C-E). Gap junctions are known to play an important role in cell-cell interactions, including those between hematopoietic stem cells (HSCs) and endothelial cells in the bone marrow. The essential contribution of gap junction-mediated cell-cell interactions to VEGF uptake was examined. When BM-MNCs were pretreated with gap junction inhibitors (1-octanol or carbenoxolone) before coculture, APC-VEGF uptake into HUVEC was not improved (Fig. 1F, G). It should be noted that little VEGF uptake was observed in BM-MNCs when cocultured with or without HUVECs ( Figures 1H-J ).

(2) In vitro transport of low molecular weight substances from BM-MNC to endothelial cells via gap junctions To confirm the hypothesized gap junction-mediated interaction between BM-MNC, HUVEC, and low molecular weight fluorescent substance, a low molecular weight fluorescent substance (BCECF) was introduced into the cytoplasm of BM-MNC. The molecular weight of BCECF is approximately 520, and it is known to pass through gap junctions. The transfer of BCECF from BM-MNC to HUVEC was evaluated by FACS (Figures 2A and 2B). No transfer was observed when BM-MNC and HUVEC were separated and co-cultured on a cell culture insert (Figure 2C), and no transfer was observed when the gap junction uncoupler 1-octanol was applied (Figure 2D). To confirm the gap junction-mediated interaction between BM-MNC and HUVEC, the expression of connexins 37 and 43 was examined. Connexin 37 expression was not observed (Fig. 2E, F), whereas connexin 43 expression was observed on the cell surface of HUVECs (Fig. 2G, H). In contrast, expression of both connexin 37 (Fig. 2I, J) and connexin 43 (Fig. 2K) was observed in BM-MNCs.

(3) In vivo transfer of low molecular weight substances from BM-MNC to endothelial cells via gap junctions BCECF-labeled BM-MNCs were intravenously transplanted into mice 48 hours after the induction of cerebral infarction. Five minutes after BM-MNC injection, mice were euthanized and the localization of BCECF was evaluated by fluorescence microscopy. BCECF-positive cells were observed in the microvasculature of the cerebral infarcted area, along with evidence of BCECF transfer from BM-MNC to endothelial cells (Figures 3A-D). To investigate gap junction-mediated cell-cell interactions between BM-MNC and endothelial cells, the expression of connexin 37 in the brain after cerebral infarction was investigated. Little expression of connexin 37 was observed in non-cerebral infarcted mice (Figure 3E), whereas increased expression of connexin 37 was observed in the lesion area 48 hours after the induction of cerebral infarction (Figure 3F). Similarly, no expression of connexin 43 was observed in non-cerebral infarcted mice (Figure 3G). However, connexin 43 expression was increased in the lesion area 48 hours after the induction of cerebral infarction (Fig. 3H). To confirm the gap junction-mediated cell-cell interaction between BM-MNCs and endothelial cells in vivo, BCECF-labeled BM-MNCs were intravenously transplanted into mice 48 hours after the induction of cerebral infarction. BCECF-positive signals were observed in endothelial cells accompanied by the accumulation of connexin 37 (Fig. 3I-L) and connexin 43 (Fig. 3M-P).

(4) BM-MNC transplantation reduces autophagy in endothelial cells after cerebral infarction To examine the ultrastructural changes in endothelial cells after BM-MNC transplantation, specimens were prepared for electron microscopy. Figure 4A shows an intact capillary with a normal blood-brain barrier (BBB) in a lesion-free brain, which is composed of endothelial cells, basement membrane, pericytes, and astrocytes (astrocyte foot processes).

As expected, degeneration and necrosis of neurons and glial cells were significant in the lesion center 72 hours after the induction of cerebral infarction (i.e., 24 hours after administration of PBS and BM-MNC, respectively), but the vascular structure was still relatively well maintained in both PBS-treated (Fig. 4B) and BM-MNC-treated (Fig. 4C) mice. In PBS-treated mice (Fig. 4B), numerous autophagosome-like vacuoles were formed in endothelial cells, sometimes containing amorphous material or having a lamellar structure. Degenerated mitochondria with unclear cristae were also observed in the EC cytoplasm, and nuclear heterochromatin was also reduced. In contrast, little change was observed in BM-MNC-treated mice (Fig. 4C).

In the peri-infarct area, various sizes of vacuoles were formed in the cytoplasm of endothelial cells of mice treated with PBS (Fig. 4D). In contrast, mice treated with BM-MNC showed little autophagosome-like vacuole formation in endothelial cells (Fig. 4E). Some vacuoles in endothelial cells were observed in the non-lesioned contralateral cortex of mice treated with PBS (Fig. 4F), but no such vacuoles were observed in mice treated with BM-MNC (Fig. 4G). Counting the number of autophagosome-like vacuoles in different areas confirmed that BM-MNC transplantation reduced the formation of autophagosome-like vacuoles in all involved areas (Fig. 4H).

To examine autophagosome-like vacuoles in endothelial cells, brain sections 72 hours after induction of cerebral infarction (i.e., 24 hours after PBS or BM-MNC injection) were stained with LC3, an autophagy marker. LC3-positive vascular endothelial cells were observed in both PBS-treated and BM-MNC-treated mice in the cerebral infarct region. In the peri-infarct region, LC3-positive vascular endothelial cells were observed in PBS-treated mice, but were hardly observed in BM-MNC-treated mice (Figure 5B). On the contralateral side of the cerebral infarct, LC3-positive vascular endothelial cells were observed in PBS-treated mice, but were not observed at all in BM-MNC-treated mice (Figure 5C). These data demonstrate that BM-MNC transplantation suppresses autophagy in endothelial cells after cerebral ischemia.

(5) Increase in Hif-1α expression in microvasculature after cerebral infarction after BM-MNC transplantation To finally investigate the upstream regulators of VEGF uptake in endothelial cells, the expression of Hif-1α in the brain after cerebral infarction was investigated. Similar to previous reports, almost no expression of Hif-1α was observed in uninjured brains 3 hours (Fig. 6B) or 48 hours (Fig. 6C) after the induction of cerebral infarction (Fig. 6A). BM-MNCs were intravenously administered 48 hours after the induction of cerebral infarction, and expression of Hif-1α in vasculature-like cells in the brain after cerebral infarction was observed 1 hour after cell administration. The possibility of increased Hif-1α activation after transplantation of BCECF-labeled BM-MNCs was examined. BCECF-labeled BM-MNCs were intravenously injected 48 hours after the induction of cerebral infarction, and the localization of BCECF and Hif-1α in endothelial cells was examined 30 minutes after cell injection. As a result, colocalization of BCECF and Hif-1α was observed in endothelial cells (Figure 6E-H). These data indicated that the transfer of low molecular weight substances from BM-MNCs could potentially induce the expression of Hif-1α in endothelial cells in the brain after cerebral infarction.

3. Discussion These results demonstrated that BM-MNCs increase Hif-1α expression through gap junction-mediated intercellular exchange of small molecular substances, thereby upregulating VEGF uptake and supporting endothelial cell activation.

Endothelial cells are known to communicate with bone marrow cell populations in the bone marrow vascular niche, and this is mediated by gap junctions. In this experiment, we demonstrated that the expression of connexins 37 and 43 in brain endothelial cells was upregulated after cerebral infarction. Small molecular weight water-soluble substances were transferred from transplanted BM-MNCs to endothelial cells, and this transfer increased Hif-1α content. Activation of Hif-1α is known to induce upregulation of VEGF uptake into endothelial cells (Gleadle JM and Ratcliffe PJ., (1997) Blood. 15; 89(2): pp503-9.). These findings indicate that BM-MNC-mediated activation of Hif-1α in endothelial cells, followed by upregulation of VEGF uptake, is an important mechanism of angiogenesis induced by cell therapy in cerebral infarction. If it were possible to substitute a small molecule for the complex process leading to VEGF uptake by BM-MNCs, this would be useful as an alternative treatment to cell therapy for diseases involving angiogenesis, such as cerebral infarction.

Example 2: Promotion of uptake of various cytokines by HUVECs by BM-MNCs 1. Materials and methods BM-MNCs were prepared by the method described in Example 1. HUVECs were co-cultured with BM-MNCs according to Example 1, and the changes in the concentrations of various cytokines in the culture supernatants after 6 hours of culture were examined. The concentrations of various cytokines (angiopoietin, endogliin, IGFBP-1 (insulin-like growth factor binding protein-1), TNF-α, and TGF-α (all human-derived)) were simultaneously measured using Bio-Plex Pro human Cancer Biomarker 2 18-Plex panel #171AC600M (BioRad). For comparison, the concentrations of various cytokines were also measured when HUVECs were similarly cultured with the addition of the same volume of medium instead of BM-MNCs.

2. Results The results are shown in FIG. 7. The concentration of Angiopoetin-2 in the culture supernatant increased to 7081 pM in the case of HUVEC alone, but the concentration in the culture supernatant decreased by 1618 pM when co-cultured with BM-MNC, and as a result, BM-MNC reduces Angiopoetin-2 in the culture supernatant by 8699 pM. Similarly, the concentrations of Edoglin, IGFBP-1, TNFα, and TGFα in the culture supernatant decreased with a significant difference due to co-culture with BM-MNC. In this experimental system, the change in the concentration in the culture supernatant before and 6 hours after co-culture with BM-MNC was observed, and a positive value indicates that the concentration in the culture supernatant increased, a negative value indicates that the concentration in the culture supernatant decreased, and a value of 0 indicates that there was no change in the concentration in the culture supernatant.

3. Discussion The decrease in the concentrations of cytokines such as Angiopoetin-2, Edoglin, IGFBP-1, TNFα, and TGFα in the culture supernatant suggests that the uptake of these cytokines into cells, in addition to VEGF, is increased by co-culture with BM-MNC. These cytokines are also molecules deeply involved in angiogenesis, and it is considered that the uptake of these cytokines into HUVECs, like VEGF, was increased by co-culture with BM-MNC. Therefore, by measuring the decrease in the concentration of these cytokines in the culture supernatant or the uptake amount into HUVECs, it is possible to screen for substances that promote or inhibit vascular endothelial cell activation.

Example 3: Standardization of quantitative evaluation method of vasoactivators using standard substances Using the experimental system (screening system) of the present invention, the inventors confirmed that 100 μg/ml caffeic acid (3,4-dihydroxycinnamic acid, CAS registration number 331-39-5) has the effect of activating VEGF uptake by HUVECs. In fact, caffeic acid is a polyphenol belonging to the chlorogenic acid group, and polyphenols belonging to the chlorogenic acid group are known to have an effect of improving vascular function (Am J Hypertens. 2007 May; 20(5):508-13. Ferulic acid restores endothelium-dependent vasodilator activity in aortas of spontaneously hypertensiverats.). For this reason, this experimental system was determined to be useful for screening of vascular endothelial cell activation activity.
In this example, it is shown that the VEGF uptake promoting effect of other low molecular weight compounds into vascular endothelial cells can be quantitatively evaluated using caffeic acid (100 μg/ml) as a standard substance.

1. Materials and Methods Biotin-conjugated VEGF (R&D Systems) was incubated with streptavidin-conjugated APC (Thermo Fisher, molar ratio 4:1) at room temperature for 10 minutes to prepare APC-conjugated VEGF. HUVECs were harvested in the same manner as above and suspended in PBS.

The following 1-6 were prepared as experimental samples.
1. Sample in which APC-bound VEGF was not added to HUVEC (fluorescence background)
2. APC-bound VEGF was added to HUVEC, PBS was added, and the cells were cultured for 3 hours (standard substance/no test substance)
3. APC-bound VEGF was added to HUVEC, 100 μg/ml of caffeic acid was added, and the cells were cultured for 3 hours (standard material).
4. APC-bound VEGF was added to HUVEC, and 4-0-Caffeoylquinic acid (CAS registration number 905-99-7), the function of which for promoting VEGF uptake is unknown, was added at 100 μg/ml, followed by incubation for 3 hours (test substance 1).
5. APC-bound VEGF was added to HUVECs, and chicoric acid (CAS registration number 6537-80-0), the function of which for promoting VEGF uptake is unknown, was added at 100 μg/ml, followed by incubation for 3 hours (test substance 2).

2. Results The background fluorescence of this measurement system (fluorescence intensity of the sample to which APC-bound VEGF was not added) was 1.54 (Figure 8A). The sample without the standard substance/test substance (with PBS added) showed a fluorescence intensity of 4.92 (Figure 8B). The sample to which the standard substance (100 μg/ml Caffeic acid) was added showed a fluorescence intensity of 6.78. The samples to which test substances 1 and 2 (100 μg/ml each of 4-0-Caffeoylquinic acid and Chicoric acid) were added showed fluorescence intensities of 6.56 and 3.90, respectively (Figures 8D and E).

The amount of VEGF uptake in the sample without the standard/test substance was 3.38 (fluorescence intensity 4.92 minus background fluorescence 1.54), and the amount of VEGF uptake in the sample with added standard substance (caffeic acid) was 5.24. These results indicate that the addition of the standard substance increased VEGF uptake in HUVECs by 55% ((VEGF uptake when standard substance was added [5.24] - VEGF uptake in the sample without the standard/test substance [3.38]) / VEGF uptake in the sample without the standard/test substance [3.38]).

Test substance 1 (4-0-Caffeoylquinic acid) was shown to increase VEGF uptake by HUVECs by 49%, and as a result, its uptake-promoting ability was found to be 88% compared to the standard substance ([49%]/[55%]).

Test substance 2 (chicoric acid) was shown to reduce VEGF uptake by HUVECs by 30%, and as a result, its ability to promote uptake was found to be -55% compared to the standard substance ([-30%]/[55%]).

3. Discussion As described above, by using this measurement system, it is possible to determine the degree of vascular endothelial cell activation activity compared to the standard substance caffeic acid. Furthermore, by using this measurement system, it is possible to quantitatively determine not only substances that promote vascular activation, but also substances that suppress vascular activation. The standard substance can also be changed as appropriate.

Caffeoylquinic acid is known to activate vascular endothelium (Int J Food Sci Nutr. 2019 May; 70(3):267-284. TNF-α-induced oxidative stress and endothelial dysfunction in EA. hy926 cells is prevented by mate and green coffee extracts, 5-caffeoylquinic acid and its microbial metabolism, dihydrocaffeic acid. Wang S, et al.). In addition, chicoric acid is a hydroxycinnamic acid, which is known to have angiogenesis inhibitory effects (2011 KAKEN Fund Results Report (Research Project/Area Number: 21390033), Bioprobe Molecular Design and Chemical Genetics for Antiangiogenic Therapy, Research Leader: Hideko Nagasawa), which is consistent with the results of this measurement system.

The present invention makes it possible to easily screen in vitro for substances that have the effect of activating vascular endothelial cells through a mechanism similar to that of BM-MNC. Substances identified by the method of the present invention can replace the function of BM-MNC in the body, and are useful as candidate substances for therapeutic agents for ischemic diseases such as cerebral infarction and limb ischemia, or angiogenic diseases such as cancer.

All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims (13)

A method for screening for a substance that promotes or inhibits vascular endothelial cell activation, comprising the steps of:
1) culturing vascular endothelial cells with a test substance in the presence of labeled VEGF;
2) determining the amount of VEGF taken up into vascular endothelial cells;
3) A step of evaluating the vascular endothelial cell activation effect of the test substance based on the amount of VEGF taken up into the vascular endothelial cells. The method according to claim 1, wherein the amount of VEGF taken up into vascular endothelial cells is determined by a change in a signal derived from labeled VEGF in the vascular endothelial cells before and after incubation with a test substance. The method according to claim 1, wherein the amount of labeled VEGF taken up by vascular endothelial cells is determined by a change in the signal derived from the labeled VEGF in the culture supernatant before and after incubation with the test substance. The method according to any one of claims 1 to 3, further comprising a step of comparing the change in the amount of VEGF uptake into vascular endothelial cells caused by the test substance with the change in the amount of VEGF uptake into vascular endothelial cells caused by a standard substance. The method according to any one of claims 1 to 4, wherein in step 3, if the amount of VEGF uptake into the vascular endothelial cells is increased, the test substance is evaluated as being useful as a substance that promotes vascular endothelial cell activation, and if the amount of VEGF uptake into the vascular endothelial cells is decreased, the test substance is evaluated as being useful as a substance that inhibits vascular endothelial cells. The method according to any one of claims 1 to 5, further comprising a step of determining the concentration in the culture supernatant of one or more cytokines selected from the group consisting of angiopoietin 2, endoglin, IGFBP (insulin-like growth factor binding protein), TNFα, and TGFα. The method of claim 6, wherein the cytokine comprises at least angiopoietin 2. The method according to claim 6 or 7, wherein if the amount of VEGF taken up by vascular endothelial cells is increased and the concentration of cytokines in the culture supernatant is decreased, the test substance is evaluated as being useful as a candidate for a substance that promotes vascular endothelial cell activation, and if the amount of VEGF taken up by vascular endothelial cells is decreased and the concentration of cytokines in the culture supernatant is increased, the test substance is evaluated as being useful as a substance that inhibits vascular endothelial cell activation. The method according to any one of claims 1 to 8, wherein the vascular endothelial cell activation promoter is a candidate for a therapeutic drug for ischemic disease, and the vascular endothelial cell activation inhibitor is a candidate for an angiogenesis inhibitor. A method for screening a therapeutic agent for ischemic disease , comprising the steps of:
1) culturing vascular endothelial cells with a test substance in the presence of labeled VEGF;
2) determining the amount of VEGF taken up into vascular endothelial cells;
3) selecting the test substance as a candidate for a therapeutic agent for ischemic disease when the amount of VEGF uptake into the vascular endothelial cells is increased. A method for screening an angiogenesis inhibitor, comprising the steps of:
1) culturing vascular endothelial cells with a test substance in the presence of labeled VEGF;
2) determining the amount of VEGF taken up into vascular endothelial cells;
3) selecting the test substance as an angiogenesis inhibitor when the amount of VEGF taken up by the vascular endothelial cells is reduced. The method according to any one of claims 1 to 11, wherein the label is a fluorescent label. The method according to any one of claims 1 to 12, wherein the vascular endothelial cells are human vascular endothelial cells.

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