WO2021029436A1 - Method for screening vascular endothelial cell activation substances - Google Patents

Abstract

The present invention relates to a method which screens substances for promoting or inhibiting vascular endothelial activation and uses, as an index, the uptake quantity of VEGF into vascular endothelial cells. More specifically, the present invention relates to a method for screening substances for promoting or inhibiting vascular endothelial activation, the method comprising the following steps of: 1) culturing vascular endothelial cells along with test substances; 2) determining the uptake quantity of VEGF into the vascular endothelial cells; and 3) evaluating the vascular endothelial cell activation effect of the test substances on the basis of the uptake quantity of VEGF into the vascular endothelial cells.

Description

Screening method for vascular endothelial cell activator

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

Transplantation of bone marrow mononuclear cells (Bone Marlow-Monocular Cell: BM-MNC) is known to activate angiogenesis in ischemic tissue. BM-MNC contains a relatively high proportion of hematopoietic stem cells and can be easily prepared from autologous tissue, making it a suitable source of cell therapy, and experiments on ischemic diseases such as limb ischemia, myocardial infarction, and cerebral infarction. Although frequently used in therapeutic treatment, it is not known how BM-MNC activates angiogenesis.

Initially, the differentiation of hematopoietic stem cells contained in BM-MNC into endothelial cells was proposed as a basic mechanism, but in subsequent studies, very few transplanted cells differentiate into endothelial cells, which is fundamental. It turned out that it was not a typical mechanism. Secretion of multiple growth factors and exosomes has also been proposed, but no clear evidence has been shown. In addition, cell survival after transplantation is known to be relatively short, and growth factor and exosome secretion is fundamental to the potent angiogenesis-activating effects observed long-term after BM-MNC transplantation. It is not considered a mechanism.

Vascular Endothelial Growth Factor (VEGF) is one of the most important angiogenesis-promoting factors. By binding to VEGF receptors on the cell surface, VEGF activates tyrosine kinase, transmits signals inside the cell, and changes the function and structure of the cell. Using a method for screening endothelial cells with high angiogenic potential using the expression ratio of VEGF receptors VGFR1 and VGFR2 as an index (Patent Document 1), or using a cultured vascular endothelial cell line expressing VEGFR1 on the cell surface, VEGFR1 A method for screening a substance having an angiogenesis-promoting action using the phosphorylation of the cell and the migration of the cell as an index has been reported (Patent Document 2). In addition, a method for evaluating angiogenic ability by measuring blood concentrations of VEGF and endostatin has been reported (Patent Document 3). As described above, conventionally, the angiogenesis-promoting ability has been measured based on the concentration of VEGF, the dynamics of VEGF receptors, etc., but as in the present invention, the amount of uptake of vascular endothelial cells of VEGF is a candidate. The idea of directly measuring the angiogenesis-promoting capacity of a substance was completely lacking.

Japanese Unexamined Patent Publication No. 2005-0565697 JP-A-2007-244273 WO 2002/031497

The subject of the present invention is to apply the mechanism of angiogenesis by BM-MNC transplantation, establish a method for screening low molecular weight substances that substitute for its function, and ischemic including cerebral infarction, myocardial infarction, and limb ischemia. The purpose is to provide new therapeutic drug candidates for diseases or diseases in which angiogenesis such as cancer, inflammatory disease, and rheumatism is involved in the progression of the pathological condition.

Conventionally, BM-MNC has been considered to promote angiogenesis by secreting various growth factors related to promotion of angiogenesis. The present inventors do not promote angiogenesis by secreting various growth factors related to angiogenesis promotion, but BM-MNC acts on vascular endothelial cells to cause angiogenesis. We have found that promoting the uptake of promoting factors in vascular endothelial cells results in the promotion of angiogenesis. Then, by screening for low molecular weight compounds having the same action as BM-MNC using the change in the amount of uptake of VEGF or other angiogenesis-promoting factors in vascular endothelial cells as an index, a new candidate for a therapeutic drug for ischemic disease I found that it is possible to search for.

That is, the present invention relates to the following (1) to (13).
(1) Screening method for substances that promote or suppress vascular endothelial cell activation, including the following steps:
1) A step of culturing vascular endothelial cells together with a test substance in the presence of labeled VEGF,
2) Step of determining the amount of VEGF uptake into vascular endothelial cells,
3) A step of evaluating the vascular endothelial cell activating effect of the test substance based on the amount of VEGF taken up by the vascular endothelial cells.
Examples of the label include a fluorescent label, an enzyme label, and a radioactive label, and a fluorescent label is preferable.
(2) The method according to (1), wherein the amount of VEGF uptake into the vascular endothelial cells is determined by the change in the labeled VEGF-derived signal in the vascular endothelial cells before and after culturing with the test substance.
(3) The method according to (1), wherein the amount of labeled VEGF uptake into vascular endothelial cells is determined by a change in the labeled VEGF-derived signal in the culture supernatant before and after culturing with the test substance.
(4) Any of (1) to (3), further comprising a step of comparing the change in the amount of VEGF uptake into the vascular endothelial cell by the test substance with the change in the amount of VEGF uptake into the vascular endothelial cell by the standard substance. The method described in Crab.
(5) In step 3), when the amount of VEGF uptake into the vascular endothelial cells is increased, the test substance is evaluated to be useful as a vascular endothelial cell activation promoting substance, and the uptake of VEGF into the vascular endothelial cells is evaluated. The method according to any one of (1) to (4), wherein the test substance is evaluated to be useful as a vascular endothelial cell inhibitor when the amount is reduced.
(6) Further, it comprises a step of determining the concentration in the culture supernatant of one or more cytokines selected from the group consisting of angiopoetin 2, endoglin, IGFBP (insulin-like growth factor binding protein), TNFα, and TGFα. , (1) to (5).
(7) The method according to (6), wherein the cytokine contains at least angiopoietin 2.
(8) By culturing with the test substance, when the cytokine concentration in the culture supernatant decreases in addition to the increase in the amount of VEGF uptake into the vascular endothelial cells, the test substance is used as a vascular endothelial cell activation promoting substance. When the cytokine concentration in the culture supernatant increases in addition to the decrease in the amount of VEGF uptake into vascular endothelial cells, the test substance is evaluated as useful as a vascular endothelial cell activation inhibitor. The method according to (6) or (7).
(9) Described in any of (1) to (8), wherein the vascular endothelial cell activation promoting substance is a candidate for a therapeutic agent for ischemic disease, and the vascular endothelial cell activation inhibitor is a candidate for an angiogenesis inhibitor. the method of.
In other words, the methods (1) to (8) can be a screening method for an ischemic disease therapeutic agent or an angiogenesis inhibitor. Examples of the ischemic disease include limb ischemia, ischemic heart disease including myocardial infarction / angina / heart failure, and ischemic cerebrovascular disorder such as cerebral infarction. Angiogenesis inhibitors include cancers such as solid tumors and hemangiomas, rheumatoid arthritis, Kron's disease, Bechet's disease, psoriasis, porphyritic arteriosclerosis, diabetic retinopathy, angiogenic glaucoma, retinopathy of prematurity, and aging. It is useful for the treatment of diseases (angiogenic diseases) in which angiogenesis is involved in the progression of pathological conditions, such as retinopathy of prematurity.
(10) Screening method for angiogenesis inhibitor including the following steps:
1) A step of culturing vascular endothelial cells together with a test substance in the presence of labeled VEGF,
2) Step of determining the amount of VEGF uptake into vascular endothelial cells,
3) A step of selecting the test substance as a candidate for a therapeutic agent for ischemic disease when the amount of VEGF taken up by the vascular endothelial cells increases.
(11) Screening method for angiogenesis inhibitor including the following steps:
1) A step of culturing vascular endothelial cells together with a test substance in the presence of labeled VEGF,
2) Step of determining the amount of VEGF uptake into vascular endothelial cells,
3) A step of selecting the test substance as an angiogenesis inhibitor when the amount of VEGF taken up by the vascular endothelial cells decreases.
(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.

Until now, a method for evaluating angiogenesis-activating action using the expression of VEGF receptor and the amount of VEGF secretion as an index has been known (above), but the promotion of the uptake amount of VEGF is used as an index to evaluate the vascular endothelial cells. No method is known to evaluate the activating effect.

According to the present invention, a substance having an action of activating vascular endothelial cells can be screened from known low molecular weight compounds by a mechanism similar to that of BM-MNC, and the function of BM-MNC in vivo can be reduced to low molecular weight. It can be replaced with a compound. A screening system using the intracellular uptake of labeled VEGF as an index enables high-throughput screening of substances that promote or suppress vascular endothelial cell activation. The identified test substance is also advantageous from the viewpoint of drug delivery because the target is vascular endothelial cells.

FIG. 1 shows Vascular Endothelial Growth Factor (VEGF) uptake into human Umbilical Vein Endothelial Cell (HUVEC) by co-culture with BM-MNC.
(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 with cell culture inserts did not reduce VEGF levels. (B) HUVEC and BM-MNC could be clearly distinguished by anti-CD31 antibody and anti-CD45 antibody. No uptake of (CG) streptavidin-Allophycocyanin (APC) into HUVEC was observed (C). It was observed that the uptake of VEGF into HUVEC was increased after BM-MNC co-culture as compared to HUVEC culture alone (D) (E). Blocking BM-MNC gap junctions with 1-octanol (F) or carbenoxolone (G) negated the co-culture effect. No binding of (HJ) streptavidin-APC to BM-MNC was observed (H). When BM-MNC was cultured alone (I) or co-cultured with HUVEC (J), uptake of VEGF into BM-MNC was hardly observed. * P <0.01 versus medium, ** p <0.01 versus medium, n = 3 (A) in each group.
FIG. 2 shows the BCECF (2', 7'-Bis (carboxyethyl) -4 or 5-carboxyfluorescein) transition from BM-MNC to HUVEC via gap junctions in vitro.
(AD) FACS analysis of HUVEC (AC) co-cultured with naive (A) and BCECF-labeled BM-MNC (B). BCECF-positive HUVEC was observed after co-culture with BCECF-labeled BM-MNC. BCECF migration was blocked when co-cultured with cell culture inserts (C) or when the gap junction decoupling agent 1-octanol was applied (D). (E, F) HUVEC was stained with Alexa Fluor® 488 fluorescently labeled antibody (hereafter Alexa488) alone (E) or anti-Cx37 (connexin 37) antibody and Alexa488 (F). In HUVEC, almost no expression of Cx37 was observed. HUVEC stained with (G, H) isotype control antibody (G) or anti-Cx43 (connexin 43) antibody (H). Expression of Cx43 was observed on HUVEC. (I, J) BM-MNC was stained with Alexa488 alone (I) or anti-Cx37 antibody and Alexa488 (J). Expression of Cx37 was observed in BM-MNC. (K, L) BM-MNC stained with isotype control antibody (K) or anti-Cx43 antibody (L). Expression of Cx43 was observed on HUVEC.
FIG. 3 shows the transfer of BCECF from BM-MNC to endothelial cells via gap junctions in vivo.
(AD) BCECF-positive BM-MNC transplanted 5 minutes after intravenous injection into the brain after cerebral infarction. BCECF-positive cells (A) were observed in the cerebral microvascular system (B). Overlaying the images demonstrates the transfer of BCECF from BM-MNC to endothelial cells through the CD31-positive endothelial cell membrane (C, D). (E, F) Connexin 37 (Cx37) was not observed in the contralateral healthy cerebral cortex (E), but was clearly expressed 48 hours after the induction of cerebral infarction on the ipsilateral side (F). Expression of (G, H) connexin 43 (Cx43) was rarely observed in healthy cortex (G), but increased expression was observed after cerebral infarction (H). Accumulation (J) of Cx37 with (IL) BCECF infiltration (J) was observed in endothelial cells (K, L). Accumulation (M) of Cx43 with (MP) BCECF infiltration (N) was observed in endothelial cells (O, P). Scale bar, 20 μm (A), 5 μm (D, I), 40 μm (E)
FIG. 4 shows that changes in endothelial cell microstructure suggest a reduction in autophagy in BM-MNC transplanted animals.
(A) Normal capillaries consisting of endothelial cells, basal layer, pericytes and astrocyte foot processes in non-cerebral infarction mice. (B, C) Representative photographs of capillaries in lesions. A significant number of autophagosome-like vacuoles were observed in the endothelial cells of PBS-treated mice (B). In contrast, despite the disappearance of astrocyte foot processes, several autophagosomes with attached pericytes were found in the endothelial cells of BM-MNC-treated mice 72 hours after cerebral infarction induction. Astrocytes were observed (C). (D, E) A significant number of autophagosome-like vacuoles were observed in the endothelial cells of PBS-treated mice (D). In contrast, few autophagosome-like vacuoles were observed in the endothelial cells of BM-MNC-treated mice (E). (F, G) Photographs of capillaries in the intact 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 the 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 region. Scale bar, 2 μm (A), p <0.01 versus PBS, n = 5 (H) in each group.
FIG. 5 shows the expression of the 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 peri-lesion region, LC3-positive endothelial cells were rarely observed in BM-MNC-treated mice, but most endothelial cells were LC3-positive in PBS-treated mice. (C) In the undamaged contralateral cortex, some LC3-positive endothelial cells were observed in mice treated with PBS, but no LC3-positive endothelial cells were observed in mice treated with BM-MNC. It was. Scale bar, 20 μm (A)
FIG. 6 shows Hif-1α expression after BM-MNC transplantation.
(AC) At 3 hours (B) or 48 hours (C) after the induction of brain injury, almost no expression of Hif-1α was observed in the undamaged brain (A) and the post-cerebral infarction region. .. (D) However, one hour after BM-MNC transplantation, increased Hif-1α expression was observed in the capillary-like structure. (EH) BCECF signal (E) was co-localized with Hif-1α expression in CD31-positive endothelial cells (G, H). Scale bar, 40 μm (A), 10 μm (E)
FIG. 7 shows changes in the concentrations of various cytokines in the HUVEC culture supernatant.
HUVEC single culture (PBS), co-culture with BM-MNC (BM-MNC). The graph shows the amount of change (pM) in the cytokine concentration in the supernatant after 6 hours of culturing. (A) Angiopoietin-2, (B) Endoglin, (C) TGFα, (D) IGFBP-1, (E) TNFα.
FIG. 8 compares the VEGF uptake promoting action of HUVEC (human umbilical vein vascular endothelial cells) by a low molecular weight compound with a standard substance.
(A) Fluorescent background, (B) Standard substance / no test substance, (C) Standard substance (Cafeic acid), (D) Test substance 1 (4-0-Cafeoylequic acid), (E) Test substance 2 (Caffeic acid) acid).

The present invention screens vascular endothelial cell activation-promoting or inhibitory substances using VEGF uptake in vascular endothelial cells as an index, particularly substances having an action of controlling vascular endothelial cell activation by a mechanism similar to BM-MNC. Regarding the method.

1. 1. Step 1) “Vascular endothelial cells” in which vascular endothelial cells are cultured together with a test substance in the presence of labeled VEGF.
The "vascular endothelial cell" used in the present invention is not particularly limited as long as it is a culturable vascular endothelial cell expressing a receptor for VEGF, but a mammalian-derived cell is preferable, and a human-derived cell is more preferable. preferable. Examples of human vascular endothelial cells include human umbilical venous endothelial cells (HUVEC), human brain microvascular endothelial cells (HBMEC), human umbilical artery endothelial cells (HUAEC), human aortic endothelial cells (HAoEC), and human coronary endothelial cells. (HCAEC), Human Pulmonary Arterial Endothelial Cell (HPAEC), Human Saphenous Vinegar Endothelial Cell (HSaVEC), Human Cutaneous Vascular Endothelial Cell (HDBEC), Human Skin Microvascular Endothelial Cell (HDMEC), Human Uterine Microvascular Endothelial Cell (HUtMEC) ), Human lung microvascular endothelial cells (HPMEC), human heart microvascular endothelial cells (HCMEC), human skin microlymphocyte endothelial cells (HDLEC), etc., and commercially available cultured cell lines can be used for all of them. .. 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 particularly preferable. Human-derived VEGF is more preferred. There are seven families of VEGF, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, PIGF-1, and PIGF-2. Generally, VEGF-A may be referred to as VEGF, and even in the present specification, when simply referred to as VEGF, it means VEGF-A.

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

Examples of the enzyme used for enzyme labeling include HRP (Horseradish peroxidase) and AP (Alkaline phosphatase). HRP uses a coloring substrate such as DAB or TAB, and AP uses a coloring substrate such as BCIP / NBT or pPNPP. To develop color.
The radioisotope used in radiolabeled, can be used like ^{125 I,} 3 ^H, using protein iodine labeling reagent is incorporated into the protein to be detected by enzymatic or chemical oxidation.

Examples of the fluorescent substance used for the fluorescent label include fluorescent proteins such as PE (Phycoerythrin), APC (Allophycocyanin), GFP, CFP, RFP, etc., FITC (Fluorescein Isothiocyanate), Coumarin, and TRITC (Tetramethyllo). ), Cy (registered trademark), Texas Red (registered trademark), BODIPY (registered trademark) FL, Super Bright, and other low-molecular-weight fluorescent dyes can be preferably used, but are not limited thereto. The signal of the fluorescent label can be detected and quantified by a detection method such as an optical microscope, a high-sensitivity CCD camera, a confocal laser microscope, or flow cytometry, and an image processing technique.

For detection, the fluorescence signal may be amplified by using a biotin-avidin system or a biotin-streptavidin system in order to increase the sensitivity.

"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 diseases described later, low molecular weight compounds that are easy to synthesize and handle are preferable. The low molecular weight compound means a compound having a molecular weight of about 10,000 or less.

"Culture of vascular endothelial cells"
Vascular endothelial cells are cultured with the test substance in the presence of labeled VEGF. For culturing, a commercially available medium for endothelial cells may be used, or various nutrient sources necessary for maintenance and proliferation of cells and various components necessary for inducing differentiation may be added to the basal medium. As the basal medium, DMEM, Keratinocite-SFM, BME medium, BGJb medium, CMRL 1066 medium, Glassgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, α MEM, ham medium, RPMIs Any medium that can be used for culturing mammalian cells, such as a medium, McCoy's medium, Williams E medium, and a mixed medium thereof, can be used.

Nutrient sources include carbon sources such as glycerol, glucose, fructose, sucrose, lactose, honey, starch and dextrin, hydrocarbons such as fatty acids, fats and oils, lecithin and alcohols, ammonium sulfate, ammonium nitrate, ammonium chloride and urea. , Nitrogen sources such as sodium nitrate, salt, potassium salt, phosphate, magnesium salt, calcium salt, iron salt, manganese salt and other inorganic salts, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, sulfuric acid It can contain ferrous iron, sodium molybdate, sodium tungstate and manganese sulfate, various vitamins, amino acids and the like.

In the medium, if necessary, amino acid reducing agents such as retinoic acid, pyruvate, β-mercaptoethanol, transferrin, fatty acids, insulin, collagen precursors, trace elements, 3'thiolglycerol, growth factors (epidermal growth factor; EGF, basic fibrlast growth factor; bFGF, Insulin-like growth factor; IGF, etc.), serum or serum substitute may be added. Examples of serum substitutes include albumin (for example, lipid-rich albumin), commercially available Knockout Serum Supplement (KSR), bovine pituitary extract (BPE), Chemically-defined Lipid coordinated (Gibco), and Gibco. ), B27 supplement (manufactured by Gibco), Y-27632 (Wako Junyaku).

The pH of the medium obtained by blending 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.

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

2. 2. Step 2) Determining the amount of VEGF uptake into vascular endothelial cells "Determining the amount of VEGF uptake into vascular endothelial cells"
The amount of VEGF uptake into vascular endothelial cells can be directly determined by measuring the change in signal intensity derived from labeled VEGF in vascular endothelial cells before and after culturing with the test substance. Alternatively, the amount of VEGF uptake into vascular endothelial cells may be indirectly determined by measuring the change in signal intensity derived from the labeled VEGF remaining in the culture solution before and after culturing with the test substance.

When measuring changes in the labeled VEGF-derived signal within vascular endothelial cells, the vascular endothelial cells and the test substance are subjected to at least 10 minutes or more, 20 minutes or more, 30 minutes or more, more preferably in the presence of the labeled VEGF. It is recommended to measure after culturing with the test substance for 30 minutes to 4 hours, more preferably about 1 to 3 hours, but it may be appropriately changed depending on the culture conditions and the cells to be used.

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

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

3. 3. Step 3) Evaluating the vascular endothelial cell activating effect of the test substance The vascular endothelial cell activating effect of the test substance is evaluated based on the amount of VEGF taken up by the test substance into the vascular endothelial cells. The evaluation may be carried out by the test substance alone, or a compound known to have a VEGF uptake promoting action on vascular endothelial cells may be used as a standard substance and evaluated in comparison with the standard substance.

When evaluating the test substance alone, the simplest case is that when the amount of VEGF uptake into vascular endothelial cells is significantly increased by culturing with the test substance, the test substance is used as a vascular endothelial cell activating substance. Identify as a candidate. Alternatively, if a certain reference value can be set, the test substance is identified as a candidate for a vascular endothelial cell activating substance when an uptake amount of VEGF exceeding the reference value is observed.

When evaluating the test substance alone, the simplest case is that when the amount of VEGF uptake into vascular endothelial cells is significantly reduced by culturing with the test substance, the test substance is a candidate for a vascular endothelial cell inhibitor. Identify as. Alternatively, if a certain reference value can be set, the test substance is identified as a candidate for a vascular endothelial cell inhibitor when an uptake amount of VEGF exceeding the reference value is observed.

"Standard substance"
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 is compared to vascular endothelial cells. Evaluate the activation effect. The standard substance is not particularly limited as long as it has been confirmed to have a VEGF uptake promoting effect on vascular endothelial cells. In the examples described later, the method of the present invention is used to obtain VEGF uptake of other low molecular weight compounds into vascular endothelial cells by using Caffeic acid, for which the inventors have confirmed the effect of promoting VEGF uptake on vascular endothelial cells. Although evaluated, the reference material is not limited to this.

"Vascular endothelial cell activation promoter"
The "vascular endothelial cell activation promoting substance" screened by the method of the present invention is a substance having an action of promoting the activation of vascular endothelial cells by the same mechanism as BM-MNC.

"Vascular endothelial cell activation inhibitor"
The "vascular endothelial cell activation inhibitor" screened by the method of the present invention is a substance that exerts an action opposite to that of BM-MNC and has an action of suppressing the activation of vascular endothelial cells.

4. Evaluation of Cytokine Concentration in Culture Supernatants If necessary, the above method further comprises one or more selected from the group consisting of angiopoetin 2, endoglin, IGFBP (insulin-like growth factor binding protein), TNFα, and TGFα. It may include a step of determining the concentration of the cytokine in the culture supernatant. Cytokine concentration can be measured in a similar manner to VEGF, but multiple cytokines may be measured simultaneously using a commercially available multiplex system. For example, when the cytokine concentration in the culture supernatant is decreased in addition to the increase in the amount of VEGF uptake into the vascular endothelial cells by culturing with the test substance, the test substance is used as a substance for promoting vascular endothelial cell activation. Identify as a candidate. On the contrary, when the cytokine concentration in the culture supernatant is increased in addition to the decrease in the amount of VEGF uptake into the vascular endothelial cells by culturing with the test substance, the test substance is used as the inhibitor of vascular endothelial cell activation. Identify as a candidate. As cytokines used for evaluation, angiopoietin 2 and TGFα are particularly preferable, and angiopoietin 2 is more preferable.

5. Screening of therapeutic agents for ischemic diseases "Therapeutic agents for ischemic diseases"
The vascular endothelial cell activation-promoting substance identified by the screening method of the present invention can activate angiogenesis by vascular endothelial cells by a mechanism of action similar to that of BM-MNC. Therefore, bed slip / skin ulcer, surgical scar, wound including refractory digestive ulcer, inflammatory disease including chronic inflammatory bowel disease such as ulcerative colitis and Crohn's disease, severe limb ischemia, myocardial infarction / angina・ It is useful as a therapeutic agent for ischemic heart disease including heart failure, ischemic cerebrovascular disorder such as cerebral infarction, diabetic neuropathy, and ischemic disease such as cancer with severe ischemia (ischemic disease). .. In other words, the screening method of the present invention can be used for screening candidate substances for therapeutic agents for ischemic diseases.

6. Screening for 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 by a mechanism that antagonizes the action of BM-MNC. Therefore, as angiogenesis inhibitors, cancer, chronic rheumatoid arthritis, which is closely related to the progression of pathological conditions and angiogenesis, Clone's disease, Bechet's disease, psoriasis, porphyritic arteriosclerosis, diabetic retinopathy, angiogenic glaucoma, It is useful as a therapeutic agent for angiogenic diseases such as retinopathy of prematurity and age-related yellow spot degeneration. In other words, the screening method of the present invention can be used for screening the above-mentioned candidate substances for angiogenesis inhibitors.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

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

-Preparation of endothelial cells and co-culture with BM-MNC Human umbilical vein endothelial cells (HUVEC, Kurabo) were cultured with medium (HuMedia-EB2, Kurabo), serum and growth additives according to the manufacturer's protocol. HUVEC 6-well plates (1000 .mu.l, 1.5 × 10 ⁵ cells / well) were seeded in, were incubated for 72 hours. A supernatant (200 μl) was collected from the sample. Next, for co-culture experiments, only HuMedia-EB2 or a BM-MNC suspension suspended in HuMedia-EB2 (1 × 10 ⁶ cells / well) was added to HUVEC. After co-culturing for 6 hours, supernatant (200 μl) was collected from the wells.

To assess the association of gap junction-mediated cell-cell interactions between HUVEC and BM-MNC, BM-MNC was used as a gap junction inhibitor 1-octanol (final concentration 1 mM) or a gap junction inhibitor. It was incubated with a carbenoxolone (CDX: final concentration 100 μM, Sigma) and washed twice with HuMedia-EB 2 before co-culturing with HUVEC.

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

-Introduction of low-molecular-weight fluorescent molecules into the cytosol of BM-MNC 1 μM BCECF-AM (2', 7'-bis- (2-carboxyethyl) -5- (and-6) -carboxyfluorescein, acetoxyformater) , Thermo Fisher) at room temperature for 30 minutes. Prior to use, BCECF labeled BM-MNC was washed twice with PBS.

Incorporation of VEGF by HUVEC Biotin-bound VEGF (R & D Systems) was incubated with streptavidin-bound APC (Thermo Fisher, molar ratio 4: 1) for 10 minutes at room temperature. HUVEC was suspended in PBS. 2 × 10 ⁵ BM-MNCs and APC-labeled VEGF (final concentration 10 nM) were added to 1 × 10 ⁵ HUVECs and incubated at 37 ° C. for 3 hours. After co-incubation, the cell mixture was washed twice with PBS and Phycoerythrin (PE) -labeled anti-human CD31 antibody (BD Bioscience), FITC (Fluorescein isothiociate) -labeled anti-mouse CD45 antibody (BD Bioscience) and 7-AAD (BD Bioscience). Stained with. Levels of APC in HUVEC (CD31 positive, CD45 negative and 7AAD negative) were evaluated on a FACS Calibur fluorescent cell sorter (BD Bioscience). BM-MNC was incubated with 1-octanol to assess the role of gap junctions in VEGF uptake.

-Induction of local 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 Cerembral Issue Machine in CB-17". J Exp Stroke Transl Med. 2010; 3 (1): 28-33.). That is, a permanent local cerebral deficiency was obtained by ligating and cutting the distal part of the left middle cerebral artery (MCA) of a 7-week-old male CB-17 mouse (Oriental yeast) using bipolar forceps under 3% halothane inhalation anesthesia. Induced blood. During the operation, rectal temperature was monitored and controlled to 37.0 ± 0.2 ° C. with a feedback-adjustable heating pad. Cerebral blood flow (CBF) in the MCA region was also monitored. Mice with a CBF reduction of 75% or more immediately after MCA occlusion were used in the experiment (success rate 100%). Preoperative animals weighed 20-25 grams. Forty-eight hours after the induction of cerebral infarction, 5 × 10 ⁵ BM-MNC or PBS were injected through the tail vein.

-Immunohistochemistry To analyze the infarct cortex, mice were deeply anesthetized with pentobarbital sodium and perfused with saline containing paraformaldehyde at a final concentration of 4%. The brain was carefully removed and a coronary section (20 μm) was prepared using a Vibratome (Leica). Sections of non-cerebral infarcted CB-17 mice were also prepared to assess the expression of connexins 37 and 43 in the cerebral cortex. Sections were CD31 (BD Harmingen, dilution 1:50), LC3 (Medical & Biological Laboratories, 1: 500), HIF-1α (Hif-1α R & D, 1:50), Connexin 37 (Cloud-Clone, 1:50), Immunostaining was performed with a primary antibody against Connexin 43 (Proteintech, 1: 200) or DAPI (BD Bioscience, 1: 1000). The anti-Hif-1α antibody was visualized by the 3,3'-diaminobenzidine (DAB) method and counterstained with Mayer's hematoxylin solution (Wako). Alexa 555 binding antibody (Novus Biologicals) was used as the secondary antibody to visualize CD31, connexin 37 and connexin 43. Alexa647, Alexa488 or Alexa633 (all Novus Biologicals) -binding antibodies were used as secondary antibodies to detect Hif-1α, LC3, Connexin 37 or Connexin 43 antibodies, respectively.

-Electron Microscopic Analysis Animals were perfused with a fixative containing PBS containing 4% paraformaldehyde and 2% glutaraldehyde (pH 7.4) to analyze hyperfine structure. The brain was carefully removed and immersed in paraformaldehyde at 4 ° C. for 24 hours. The coronary brain section was sliced with a microslicer to a thickness of 100 μm. Sections were fixed at 1% OsO ₄ . It was dehydrated for 1 hour and flatly embedded on a siliconized slide glass in epoxy resin. Ultrathin sections of the cerebral cortex containing ischemic lesions, peri-cerebral infarction regions or corresponding contralateral cortical regions were excised and placed on a Formvar-coated single slot (2x1 mm) grid and stained with 2% uranyl acetate and lead citrate. Electron micrographs were taken at 80 kV using JEM-1400EX + (JDEL). The number of autophagosome-like vacuoles in ischemic lesions, peri-cerebral infarction and corresponding contralateral cortical areas was 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. When instructed, individual comparisons were made using Student's t-test. Shown as mean ± SD in all experiments.

2. 2. Results (1) BM-MNC promotes uptake of VEGF into HUVEC through gap junction-mediated interactions VEGF is one of the most prominent angiogenesis-promoting factors. First, the effect of BM-MNC on VEGF levels in HUVEC medium was evaluated. Mouse BM-MNC was co-cultured with HUVEC and the level of hVEGF in the medium was evaluated before and 6 hours after co-culture. hVEGF levels decreased significantly from baseline after co-culture with BM-MNC, but increased in controls (FIG. 1A). Co-culture with BM-MNC isolated by cell culture inserts did not reduce hVEGF levels. This finding supports the hypothesis that BM-MNC may promote the uptake of hVEGF into HUVEC through cell-cell interactions.

To confirm the hypothesis, APC-labeled VEGF was added to HUVEC 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 co-culture with BM-MNC (Fig. 1C-E). Gap junctions are known to play an important role in cell-cell interactions, including interactions between bone marrow hematopoietic stem cells (HSCs) and endothelial cells. The essential contribution of cell-cell interactions through gap junctions to VEGF uptake was investigated. Incorporation of APC-VEGF into HUVEC was not improved when BM-MNC was pretreated with a gap junction inhibitor (1-octanol or carbenoxolone) prior to co-culture (FIGS. 1F, G). It should be noted that little VEGF uptake was observed in BM-MNC when co-cultured in the presence or absence of HUVEC (Fig. 1HJ).

(2) In vitro transport of low molecular weight material from BM-MNC to endothelial cells via gap junction Low to confirm the assumed gap junction-mediated interaction between BM-MNC and HUVEC, low molecular weight fluorescence A molecular fluorescent substance (BCECF) was introduced into the cytoplasm of BM-MNC. BCECF has a molecular weight of about 520 and is known to cross gap junctions. The transition of BCECF from BM-MNC to HUVEC was evaluated by FACS (FIGS. 2A, B). No migration was observed when BM-MNC and HUVEC were separated and co-cultured with a cell culture insert (Fig. 2C), and no migration was observed when the gap junction decoupling agent 1-octanol was applied (Fig. 2D). ). Expression of connexins 37 and 43 was examined to confirm the gap junction-mediated interaction between BM-MNC and HUVEC. Expression of connexin 37 was not observed (FIGS. 2E, F), but expression of connexin 43 was observed on the cell surface of HUVEC (FIGS. 2G, H). In contrast, expression of both connexin 37 (FIGS. 2I, J) and connexin 43 (FIG. 2K) was observed in BM-MNC.

(3) In vivo transfer of low molecular weight substance from BM-MNC to endothelial cells via gap junction BCECF-labeled BM-MNC was intravenously transplanted into mice 48 hours after the induction of cerebral infarction. Mice were euthanized 5 minutes after BM-MNC injection and the localization of BCECF was evaluated by fluorescence microscopy. BCECF-positive cells were observed in the microvasculature of the cerebral infarct region, with evidence of BCECF transfer from BM-MNC to endothelial cells (FIGS. 3A-D). To investigate gap junction-mediated cell-cell interactions between BM-MNC and endothelial cells, we investigated the expression of connexin 37 in the brain after cerebral infarction. Almost no expression of connexin 37 was observed in non-cerebral infarction mice (Fig. 3E), and increased expression of connexin 37 was observed in the lesion area 48 hours after the induction of cerebral infarction (Fig. 3F). Similarly, no expression of connexin 43 was observed in non-cerebral infarcted mice (Fig. 3G). However, connexin 43 expression increased in the lesion area 48 hours after the induction of cerebral infarction (Fig. 3H). To confirm gap junction-mediated cell-cell interactions between BM-MNC and endothelial cells in vivo, BCECF-labeled BM-MNC was intravenously implanted in mice 48 hours after cerebral infarction induction. BCECF positive signals were observed in endothelial cells with accumulation of connexin 37 (FIG. 3IL) and connexin 43 (FIG. 3MP).

(4) BM-MNC transplantation reduces autophagy in endothelial cells after cerebral infarction In order to investigate hyperfine structure changes in endothelial cells after BM-MNC transplantation, test pieces for electron microscopy were prepared. FIG. 4A shows intact capillaries with a normal blood-brain barrier (BBB) of the lesion-free brain composed of endothelial cells, basement membranes, pericytes, and astrocyte foot processes.

As expected, neuronal and glial cell degeneration and necrosis were significant in the focal center 72 hours after cerebral infarction induction (ie, 24 hours after PBS and BM-MNC administration, respectively), but vascular structure was still treated with PBS. It was maintained relatively well in both mice (FIG. 4B) and BM-MNC-treated mice (FIG. 4C). In PBS-treated mice (Fig. 4B), a large number of autophagosome-like vacuoles were formed in the endothelial cells, sometimes containing an amorphous substance or having a layered structure. Denatured mitochondria with obscure cristae were also observed in the EC cytoplasm, and nuclear heterochromatin was also reduced. In contrast, little change was seen in BM-MNC-treated mice (Fig. 4C).

In the region around the cerebral infarction, vacuoles of various sizes were formed in the cytoplasm of mouse endothelial cells to which PBS was administered (Fig. 4D). In contrast, BM-MNC-treated mice showed little autophagosome-like vacuolization in endothelial cells (Fig. 4E). Some vacuoles in endothelial cells were observed in the lesion-free contralateral cortex of mice treated with PBS (Fig. 4F), whereas such vacuoles were observed in mice treated with BM-MNC. There was no (Fig. 4G). When the number of autophagosome-like vacuoles in different regions was measured, it was confirmed that BM-MNC transplantation reduced the formation of autophagosome-like vacuoles in all related regions (Fig. 4H).

To examine autophagosome-like vacuoles in endothelial cells, brain sections 72 hours after cerebral infarction induction (ie, 24 hours after injection of PBS or BM-MNC) were stained with the autophagy marker LC3. LC3-positive vascular endothelial cells were observed in both PBS-treated and BM-MNC-treated mice in the area of cerebral infarction. In the region around cerebral infarction, LC3-positive vascular endothelial cells were observed in PBS-administered mice, but hardly observed in BM-MNC-treated mice (Fig. 5B). On the opposite side of the cerebral infarction, LC3-positive vascular endothelial cells were observed in PBS-administered mice, but not in BM-MNC-treated mice. (Fig. 5C). These data demonstrate that BM-MNC transplantation suppresses autophagy in endothelial cells after cerebral ischemia.

(5) Increased Hif-1α expression in the microvascular system after cerebral infarction after BM-MNC transplantation Hif-1α in the brain after cerebral infarction in order to finally investigate upstream regulators of VEGF uptake in endothelial cells. The expression of was investigated. Similar to previous reports, little Hif-1α expression was observed in the undamaged brain 3 hours (FIG. 6B) or 48 hours (FIG. 6C) after cerebral infarction induction (FIG. 6A). BM-MNC was intravenously administered 48 hours after the induction of cerebral infarction, and expression of Hif-1α in vascular system-like cells of the brain after cerebral infarction was observed 1 hour after cell administration. The increased likelihood of Hif-1α activation after transplantation of BCECF-labeled BM-MNC was investigated. BCECF-labeled BM-MNC was injected intravenously 48 hours after the induction of cerebral infarction, and the localization of BCECF and Hif-1α in endothelial cells was examined 30 minutes after the cell injection. As a result, co-localization of BCECF and Hif-1α was observed in endothelial cells (Fig. 6E-H). These data showed that the transfer of low molecular weight material from BM-MNC could potentially induce Hif-1α expression in cerebral endothelial cells after cerebral infarction.

3. 3. Discussion This result demonstrates that BM-MNC upregulates VEGF uptake and supports endothelial cell activation by increasing Hif-1α expression through gap junction-mediated cell-cell exchange of small molecules. It was.

Endothelial cells are known to communicate with the bone marrow cell population in the bone marrow vascular niche, which is mediated by gap junctions. In this experiment, we demonstrated that the expression of connexins 37 and 43 in cerebral endothelial cells is up-regulated after cerebral infarction. The low molecular weight water-soluble substance migrated from the transplanted BM-MNC to the endothelial cells, and this migration increased the Hif-1α content. Activation of Hif-1α is known to induce up-regulation of VEGF uptake into endothelial cells (Gledle 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 by cell therapy in cerebral infarction. If a small molecule can replace the complicated process leading to VEGF uptake by BM-MNC, it will 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 of HUVEC by BM-MNC 1. Materials and Methods BM-MNC was prepared by the method described in Example 1. According to Example 1, HUVEC was co-cultured with BM-MNC, and the amount of change in various cytokine concentrations in the culture supernatant after 6 hours of culture was examined. The concentration of various cytokines (Angiopoietin, Endoglin, IGFBP-1 (Insulin-like growth factor binding protein-1), TNF-α, TGF-α (all derived from humans)) is determined by Bio-Plex Pro human Cancer Simultaneous measurements were made using Plex panel # 171AC600M (BioRad). For comparison, the concentrations of various cytokines when the same amount of medium was added instead of BM-MNC and HUVEC was cultured in the same manner were also measured.

2. 2. Results The results are shown in FIG. The concentration of Angiopoietin-2 in the culture supernatant increased to 7081 pM when HUVEC alone was used, but when co-cultured with BM-MNC, the concentration in the culture supernatant decreased by 1618 pM, resulting in a decrease in the concentration of BM-MNC. Decreases Angiopoietin-2 in culture supernatant by 8699 pM. Similarly, the concentrations of Endoglin, IGFBP-1, TNFα, and TGFα in the culture supernatant decreased significantly by co-culturing with BM-MNC. In this experimental system, changes in the concentration in the culture supernatant were observed before co-culturing with BM-MNC and after 6 hours of co-culturing. If the value is positive, the concentration in the culture supernatant is observed. Was increased, a negative value indicates a decrease in the concentration in the culture supernatant, and a value of 0 indicates that there was no change in the concentration in the culture supernatant.

3. 3. Discussion The decrease in the concentration of cytokines such as Angiopoietin-2, Endoglin, IGFBP-1, TNFα, and TGFα in the culture supernatant is caused by the uptake of these cytokines into cells in addition to VEGF by co-culture with BM-MNC. Suggests an increase. These cytokines are also molecules that are deeply involved in angiogenesis, and it is considered that co-culture with BM-MNC increased their uptake in HUVEC as well as VEGF. Therefore, by reducing the concentration of these cytokines in the culture supernatant or measuring the amount taken into HUVEC, it is possible to screen for substances that promote or suppress vascular endothelial cell activation.

Example 3: Standardization of a method for quantitative evaluation of a vascular activator using a standard substance The inventors of the present invention used a 100 μg / ml Caffeic acid (3,4-dihydroxycinnamic acid) according to the experimental system (screening system) of the present invention. It was confirmed that CAS registration number 331-39-5) has a VEGF uptake effect of HUVEC. In fact, caffeic acid is a polyphenol belonging to chlorogenic acids, and the polyphenol belonging to chlorogenic acids is known to have an effect of improving vascular function (Am J Hypertension. 2007 May; 20 (5): 508-13. Functional acid restaurants -Dependent vasodilation in aortas of spontaneously hypertension.). From this, it was judged that this experimental system is useful for screening the vascular endothelial cell activating action.
In this example, it is shown that the effect of other low molecular weight compounds on promoting VEGF uptake on vascular endothelial cells can be quantitatively evaluated using this caffeic acid (100 μg / ml) as a standard substance.

1. 1. Materials and Methods Biotin-bound VEGF (R & D Systems) was incubated with streptavidin-bound APC (Thermo Fisher, molar ratio 4: 1) for 10 minutes at room temperature to create APC-bound VEGF. HUVEC was recovered by the same method as described above and suspended in PBS.

The following 1-6 were prepared as experimental samples.
1. 1. Sample without APC-bound VEGF added to HUVEC (fluorescent background)
2. 2. APC-bound VEGF added to HUVEC, PBS added, and then cultured for 3 hours (standard substance / no test substance)
3. 3. APC-bound VEGF added to HUVEC, 100 μg / ml caffeic acid added, and then cultured for 3 hours (standard substance).
4. APC-binding VEGF was added to HUVEC, and 4-0-Cafeoylequic acid (CAS Registry Number 905-99-7), whose VEGF uptake promoting action was unknown, was added to 100 μg / ml and then cultured for 3 hours (subject). Test substance 1)
5. APC-binding VEGF was added to HUVEC, and Chicoric acid (CAS Registry Number 6537-80-0), whose VEGF uptake promoting action was unknown, was added to 100 μg / ml and then cultured for 3 hours (test substance 2).

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 (Fig. 8A). The sample without standard / test material (with PBS added) showed a fluorescence intensity of 4.92 (Fig. 8B). The sample to which the standard substance (100 μg / ml Caffeic acid) was added showed a fluorescence intensity of 6.78. Samples supplemented with Test Substances 1 and 2 (100 μg / ml each of 4-0-Cafeoylequic acid, Chicoric acid) showed fluorescence intensities of 6.56 and 3.90, respectively (FIGS. 8D and E).

The amount of VEGF uptake without the standard substance / test substance is 3.38 (value obtained by subtracting 1.54 of background fluorescence from the fluorescence intensity of 4.92), and the amount of VEGF uptake of the sample to which the standard substance (caffeic acid) is added is It was 5.24. These results indicate that the addition of the standard substance increased VEGF uptake in HUVEC by 55% ((VEGF uptake with standard substance addition [5.24] -VEGF uptake without standard substance / test substance). Amount [3.38]) / Standard substance / VEGF uptake without test substance [3.38]).

In test substance 1 (4-0-Cafeoylquinic acid), HUVEC has been shown to have a 49% increase in VEGF uptake, resulting in 88% of its uptake promoting capacity compared to the standard substance. It turned out ([49%] / [55%]).

In test substance 2 (Choric acid), HUVEC was shown to have a 30% reduction in VEGF uptake, and as a result, its uptake promoting capacity was found to be -55% compared to the standard substance. ([-30%] / [55%]).

3. 3. Discussion As described above, by using this measurement system, it is possible to determine the degree of vascular endothelial cell activating effect as compared with 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 be changed as appropriate.

It should be noted that the coffee extract acid acid is known to have an vascular endothelium activating effect (Int J Food Sci Nutr. 2019 May; 70 (3): 267-284. TNF-α-induced oxidative stress and endothelial 9th endothelial cells is presented by mate and green coffee extends, 5-caffeoylquinic acid and it's microbial metabolite, dihydrocaffic acid. Wang S, et. In addition, Hydroxycinnamic acid is Hydroxycinnamic acid, but it is known that they have angiogenesis-inhibiting action (2011 KAKEN expense performance report (research subject / area number: 21390033), for angiogenesis-inhibiting therapy. Bioprobe molecular design and chemical genetics, principal investigator: Hideko Nagasawa), consistent with the results of this measurement system.

According to the present invention, a substance having an action of activating vascular endothelial cells by a mechanism similar to that of BM-MNC can be easily screened in vitro. The substance identified by the method of the present invention replaces the function of BM-MNC in vivo and is a candidate substance for a therapeutic agent for ischemic diseases such as cerebral infarction and limb ischemia, or angiogenic diseases such as cancer. It is useful.

All publications, patents and patent applications cited in this specification shall be incorporated herein by reference as is.

Claims (13)

1. Screening method for substances that promote or suppress vascular endothelial cell activation, including the following steps:
   1) A step of culturing vascular endothelial cells together with a test substance in the presence of labeled VEGF,
   2) Step of determining the amount of VEGF uptake into vascular endothelial cells,
   3) A step of evaluating the vascular endothelial cell activating effect of the test substance based on the amount of VEGF taken up by the vascular endothelial cells.
2. The method according to claim 1, wherein the amount of VEGF uptake into the vascular endothelial cells is determined by the change in the labeled VEGF-derived signal in the vascular endothelial cells before and after culturing with the test substance.
3. The method according to claim 1, wherein the amount of labeled VEGF uptake into vascular endothelial cells is determined by a change in the labeled VEGF-derived signal in the culture supernatant before and after culturing with the test substance.
4. The invention according to any one of claims 1 to 3, further comprising a step of comparing a change in the amount of VEGF uptake into vascular endothelial cells by a test substance with a change in the amount of VEGF uptake into vascular endothelial cells by a standard substance. the method of.
5. In step 3), when the amount of VEGF uptake into the vascular endothelial cells increased, the test substance was evaluated as useful as a vascular endothelial cell activation promoting substance, and the amount of VEGF uptake into the vascular endothelial cells decreased. The method according to any one of claims 1 to 4, wherein the test substance is evaluated to be useful as a vascular endothelial cell inhibitor.
6. The claim further comprises the step of determining the concentration in the culture supernatant of one or more cytokines selected from the group consisting of angiopoetin 2, endoglin, IGFBP (insulin-like growth factor binding protein), TNFα, and TGFα. The method according to any one of 1 to 5.
7. The method according to claim 6, wherein the cytokine contains at least angiopoietin 2.
8. When the cytokine concentration in the culture supernatant decreases in addition to the increase in VEGF uptake into vascular endothelial cells, the test substance is evaluated as useful as a candidate for vascular endothelial cell activation promoting substance, and vascular endothelial cells The method according to claim 6 or 7, wherein the test substance is evaluated as useful as a vascular endothelial cell activation inhibitor when the cytokine concentration in the culture supernatant increases in addition to the decrease in the amount of VEGF uptake into the cell. ..
9. The method according to any one of claims 1 to 8, wherein the vascular endothelial cell activation promoting substance is a candidate for a therapeutic agent for ischemic disease, and the vascular endothelial cell activation inhibitor is a candidate for an angiogenesis inhibitor.
10. Screening method for angiogenesis inhibitors, including the following steps:
    1) A step of culturing vascular endothelial cells together with a test substance in the presence of labeled VEGF,
    2) Step of determining the amount of VEGF uptake into vascular endothelial cells,
    3) A step of selecting the test substance as a candidate for a therapeutic agent for ischemic disease when the amount of VEGF taken up by the vascular endothelial cells increases.
11. Screening method for angiogenesis inhibitors, including the following steps:
    1) A step of culturing vascular endothelial cells together with a test substance in the presence of labeled VEGF,
    2) Step of determining the amount of VEGF uptake into vascular endothelial cells,
    3) A step of selecting the test substance as an angiogenesis inhibitor when the amount of VEGF taken up by the vascular endothelial cells decreases.
12. The method according to any one of claims 1 to 11, wherein the label is a fluorescent label.
13. The method according to any one of claims 1 to 12, wherein the vascular endothelial cells are human vascular endothelial cells.

Images