WO2021029436A1 - Procédé de criblage de substances d'activation de cellules endothéliales vasculaires - Google Patents

Procédé de criblage de substances d'activation de cellules endothéliales vasculaires Download PDF

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WO2021029436A1
WO2021029436A1 PCT/JP2020/030921 JP2020030921W WO2021029436A1 WO 2021029436 A1 WO2021029436 A1 WO 2021029436A1 JP 2020030921 W JP2020030921 W JP 2020030921W WO 2021029436 A1 WO2021029436 A1 WO 2021029436A1
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vascular endothelial
endothelial cells
vegf
uptake
test substance
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明彦 田口
映恵 田浦
優子 小川
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公益財団法人神戸医療産業都市推進機構
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    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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  • 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.
  • BM-MNC bone marrow mononuclear cells
  • 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.
  • VEGF Vascular Endothelial Growth Factor
  • 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).
  • 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.
  • 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.
  • 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.
  • promoting the uptake of promoting factors in vascular endothelial cells results in the promotion of angiogenesis.
  • a new candidate for a therapeutic drug for ischemic disease I found that it is possible to search for.
  • the present invention relates to the following (1) to (13).
  • 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.
  • the label include a fluorescent label, an enzyme label, and a radioactive label, and a fluorescent label is preferable.
  • 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.
  • cytokine contains at least angiopoietin 2.
  • the test substance 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.
  • the test substance is evaluated as useful as a vascular endothelial cell activation inhibitor.
  • the methods (1) to (8) can be a screening method for an ischemic disease therapeutic agent or an angiogenesis inhibitor.
  • ischemic disease examples 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.
  • 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.
  • 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.
  • 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.
  • VEGF Vascular Endothelial Growth Factor
  • 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).
  • CG streptavidin-Allophycocyanin
  • BCECF 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).
  • 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.
  • BM-MNC was stained with Alexa488 alone (I) or anti-Cx37 antibody and Alexa488 (J). Expression of Cx37 was observed in BM-MNC.
  • 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).
  • 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).
  • 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).
  • 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.
  • FIG. 6 shows Hif-1 ⁇ expression after BM-MNC transplantation.
  • FIG. 7 shows changes in the concentrations of various cytokines in the HUVEC culture supernatant.
  • 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.
  • 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.
  • human vascular endothelial cells examples 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.
  • HBMEC human brain microvascular endothelial cells
  • HAAEC human umbilical artery endothelial cells
  • HAoEC human aortic endothelial cells
  • coronary endothelial cells examples include human coronary endothelial cells.
  • HCAEC Human Pulmonary Arterial Endothelial Cell
  • HPAEC Human Pulmonary Arterial Endothelial Cell
  • HaVEC Human Saphenous Vinegar Endothelial Cell
  • HBEC Human Cutaneous Vascular Endothelial Cell
  • HDMEC Human Skin Microvascular Endothelial Cell
  • HMMEC Human Uterine Microvascular Endothelial Cell
  • HPMEC Human lung microvascular endothelial cells
  • HCMEC human heart microvascular endothelial cells
  • HDLEC human skin microlymphocyte endothelial cells
  • VEGF Vascular Endothelial Growth Factor
  • 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.
  • VEGF-A vascular endothelial growth factor
  • VEGF-B vascular endothelial growth factor
  • VEGF-C vascular endothelial growth factor
  • VEGF-D vascular endothelial growth factor
  • VEGF-E PIGF-1
  • PIGF-2 PIGF-1
  • PIGF-2 PIGF-2.
  • VEGF-A may be referred to as VEGF, and even in the present specification, when simply referred to as VEGF, it means VEGF-A.
  • 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
  • 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.
  • fluorescent substance used for the fluorescent label examples 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.
  • 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.
  • Vascular endothelial cells are cultured with the test substance in the presence of labeled VEGF.
  • 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.
  • DMEM 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, ⁇ 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.
  • amino acid reducing agents such as retinoic acid, pyruvate, ⁇ -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.
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the test 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.
  • the test substance 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.
  • 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.
  • 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.
  • the above method further comprises one or more selected from the group consisting of angiopoetin 2, endoglin, IGFBP (insulin-like growth factor binding protein), TNF ⁇ , and TGF ⁇ . 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.
  • the test substance 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.
  • the test substance 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.
  • cytokines used for evaluation angiopoietin 2 and TGF ⁇ are particularly preferable, and angiopoietin 2 is more preferable.
  • 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.
  • 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). ..
  • the screening method of the present invention can be used for screening candidate substances for therapeutic agents for ischemic diseases.
  • 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.
  • 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).
  • HUVEC 6-well plates 1000 .mu.l, 1.5 ⁇ 10 5 cells / well were seeded in, were incubated for 72 hours. A supernatant (200 ⁇ l) was collected from the sample.
  • 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 ⁇ l) was collected from the wells.
  • 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 ⁇ M, Sigma) and washed twice with HuMedia-EB 2 before co-culturing with HUVEC.
  • CDX carbenoxolone
  • hVEGF human vascular endothelial growth factor
  • BCECF-AM (2', 7'-bis- (2-carboxyethyl) -5- (and-6) -carboxyfluorescein, acetoxyformater) , Thermo Fisher) at room temperature for 30 minutes.
  • BCECF labeled BM-MNC was washed twice with PBS.
  • BM-MNC was incubated with 1-octanol to assess the role of gap junctions in VEGF uptake.
  • CBF Cerebral blood flow
  • 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 ⁇ m) 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-1 ⁇ (Hif-1 ⁇ 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-1 ⁇ antibody was visualized by the 3,3'-diaminobenzidine (DAB) method and counterstained with Mayer's hematoxylin solution (Wako).
  • DAB 3,3'-diaminobenzidine
  • 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-1 ⁇ , LC3, Connexin 37 or Connexin 43 antibodies, respectively.
  • BM-MNC promotes uptake of VEGF into HUVEC through gap junction-mediated interactions VEGF is one of the most prominent angiogenesis-promoting factors.
  • BM-MNC promotes uptake of VEGF into HUVEC through gap junction-mediated interactions VEGF is one of the most prominent angiogenesis-promoting factors.
  • 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.
  • BCECF 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.
  • FACS FACS
  • 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).
  • connexin 37 was expressed 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).
  • 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).
  • 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.
  • BBB blood-brain barrier
  • vacuoles of various sizes were formed in the cytoplasm of mouse endothelial cells to which PBS was administered (Fig. 4D).
  • 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).
  • BM-MNC transplantation reduced the formation of autophagosome-like vacuoles in all related regions (Fig. 4H).
  • 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).
  • 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.
  • BCECF-labeled BM-MNC was injected intravenously 48 hours after the induction of cerebral infarction, and the localization of BCECF and Hif-1 ⁇ in endothelial cells was examined 30 minutes after the cell injection. As a result, co-localization of BCECF and Hif-1 ⁇ 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-1 ⁇ expression in cerebral endothelial cells after cerebral infarction.
  • Endothelial cells are known to communicate with the bone marrow cell population in the bone marrow vascular niche, which is mediated by gap junctions.
  • 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-1 ⁇ content.
  • Activation of Hif-1 ⁇ is known to induce up-regulation of VEGF uptake into endothelial cells (Gledle JM and Ratcliffe PJ., (1997) Blood.15; 89 (2): pp503-9. ).
  • BM-MNC-mediated activation of Hif-1 ⁇ 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- ⁇ , TGF- ⁇ (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.
  • cytokines such as Angiopoietin-2, Endoglin, IGFBP-1, TNF ⁇ , and TGF ⁇ in the culture supernatant
  • 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 ⁇ g / 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.
  • 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.).
  • 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 ⁇ g / ml and then cultured for 3 hours (test substance 2).
  • 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]).
  • 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%]).
  • test substance 2 Choleic 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%]).
  • 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- ⁇ -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.
  • 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.
  • 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.

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Abstract

La présente invention concerne un procédé criblant des substances pour favoriser ou inhiber l'activation endothéliale vasculaire et utilisant, en tant qu'indice, la quantité d'absorption de VEGF dans des cellules endothéliales vasculaires. Plus particulièrement, la présente invention concerne un procédé de criblage de substances pour favoriser ou inhiber l'activation endothéliale vasculaire, le procédé comprenant les étapes suivantes : 1) culture de cellules endothéliales vasculaires conjointement avec des substances de test ; 2) détermination de la quantité d'absorption de VEGF dans les cellules endothéliales vasculaires ; et 3) évaluation de l'effet d'activation des cellules endothéliales vasculaires des substances de test sur la base de la quantité d'absorption de VEGF dans les cellules endothéliales vasculaires.
PCT/JP2020/030921 2019-08-09 2020-08-04 Procédé de criblage de substances d'activation de cellules endothéliales vasculaires WO2021029436A1 (fr)

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JP2012502114A (ja) * 2008-09-12 2012-01-26 クライオプラキス クライオバイオロヒア エリテーデーアー． 虚血組織の細胞療法

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KIKUCHI-TAURA AKIE, OKINAKA YUKA, TAKEUCHI YUKIKO, OGAWA YUKO, MAEDA MITSUYO, KATAOKA YOSKY, YASUI TERUHITO, KIMURA TAKAFUMI, GUL : "Bone Marrow Mononuclear Cells Activate Angiogenesis via Gap Junction- Mediated Cell - Cell Interaction", STROKE, vol. 51, no. 4, 19 February 2020 (2020-02-19), pages 1279 - 1289, XP055793488 *
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