WO2020118911A1 - Application of transthyretin to angiogenesis inhibition - Google Patents

Abstract

The present invention relates to the field of biochemistry, molecular biology and medicine. Disclosed is an application of transthyretin (TTR) to angiogenesis inhibition. In the present invention, it is found out that after the TTR is given to mammals, in STZ induced diabetes rat and mouse models, the ocular neovascularization thereof are obviously reduced. It is verified that the TTR has the effect of inhibiting the angiogenesis by means of tumor neovascularization, diabetes neovascularization, ocular neovascularization and cerebral neovascularization in-vitro experiment models; the result shows that the TTR can act on an in-vitro cell model in a brain anaerobic environment and can continuously and efficiently inhibit the generation of neovascularization. When the TTR acts on umbilical vein endothelial cells, the formation of 90% of vessels can be obviously inhibited; when the TTR acts on cerebral microvascular endothelial cell, the formation of 95% of vessels can be effectively inhibited; when the TTR acts on ocular microvascular endothelial cells, the formation of 90% of vessels can be effectively inhibited; when the TTR acts on rat and mouse models, as high as 90% of the generation of neovascularization can be effectively inhibited.

Description

Application of transthyretin in inhibiting angiogenesis Technical field

The invention relates to the application of transthyretin protein in inhibiting angiogenesis, and belongs to the fields of biochemistry, molecular biology and medicine.

Background technique

Angiogenesis refers to the development of new blood vessels from existing capillaries or retrocapillary veins, mainly including: degradation of vascular basement membrane during activation; activation, proliferation, migration of vascular endothelial cells; reconstruction and formation of new blood vessels And the vascular network is a complex process involving multiple molecules of multiple cells. Angiogenesis is a complex process that promotes the coordination of angiogenic factors and inhibitors. Under normal circumstances, the two are in a balanced state. Once this balance is broken, the vascular system will be activated, causing excessive angiogenesis or inhibiting the vascular system to degrade the blood vessels. The mechanism of angiogenesis is complex, and there are many factors involved in and promoting angiogenesis. The number of macrophages in EMT peritoneal fluid is significantly increased. The secreted TNF-α and IL-8 can promote the proliferation of vascular endothelial cells and transform growth factor-β ( TGF-β), platelet-derived endothelial growth factor (PD-ECGF), heparanase, angiopoietin (angs), osteopoietin (OPN), cyclooxygenase (COX-2), hypoxia-inducible factor -1, laminin (LN), placental growth factor (PLGF), Survivin, erythropoietin (Epo) are involved in the EMT angiogenesis process (De Palma M. Nat. Rev. Cancer 201708; 17(8)457 -474).

Pathological neovascularization-related diseases include tumor neovascularization (Li X. Science 2018 Mar 23; 359 (6382) 1335-1336), cerebral ischemia neovascularization (Chang J. Nat. Med. 2017; 23 (4) 450- 460), renal neovascularization (Amin EM. Cancer Cell 2011; 20 (6) 768-780), diabetic neovascularization (Hu J. Nature 2017 12 12; 552 (7684)), etc. The generation of new blood vessels is a complex biological process that involves multiple growth factors and their signaling receptors, and a single molecule targeted in the signaling pathway may not be uncontrollable in diseases such as cancer Of angiogenesis provides effective clinical treatment. Therefore, the need to develop innovative therapies that can synergistically combine several key angiogenesis factors to effectively inhibit angiogenesis and disease progression will continue to increase. At present, the treatment of angiogenesis mainly uses targeted drug anti-VEGF therapy, but there is no effective way to overcome the clinical insensitivity and drug resistance of targeted drugs.

Summary of the invention

The first object of the present invention is to provide a method for inhibiting angiogenesis using transthyretin.

In one embodiment of the present invention, the Genbank accession number of the transthyretin protein is CAG33189.1.

In one embodiment of the present invention, the transthyretin protein is at least 99%, or 98%, or 95%, or 90% homologous to the protein whose amino acid sequence is Genbank accession number CAG33189.1, and A protein with angiogenesis inhibiting function.

In one embodiment of the present invention, the effective dose of transthyretin is ≥4 mmol/L.

In one embodiment of the invention, the method includes inhibiting tumor angiogenesis, diabetic angiogenesis, ocular angiogenesis or brain angiogenesis.

In one embodiment of the invention, the method is to inhibit the tumor angiogenesis factor VEGF with transthyretin.

In one embodiment of the present invention, the tumor angiogenesis includes liver cancer, lung cancer, bladder cancer, breast cancer, rectal cancer, osteosarcoma, gastric cancer, pancreatic cancer, leukemia, lymphoma, myeloma, blood vessel angiogenesis, Hemangiomas, hemangiomas complicated with tissue angiogenesis, multiple hemangiomas, hemangioblastomas, benign vascular hyperplasia.

In one embodiment of the invention, the method is to treat angiogenic ophthalmopathy.

In one embodiment of the present invention, the angiogenic eye disease includes iris angiogenic eye disease, choroidal angiogenic eye disease, retinal angiogenic eye disease or corneal angiogenic eye disease.

In one embodiment of the present invention, the corneal angiogenesis eye disease includes corneal angiogenesis disease caused by contact lens, alkali and other substance burns, corneal surgery, bacterial infection, chlamydia infection, viral infection or protozoan infection Corneal angiogenesis eye disease.

In one embodiment of the present invention, the iris angiogenesis ophthalmopathy includes angiogenesis glaucoma, diabetic retinopathy or central retinal vein embolism.

In one embodiment of the present invention, the choroidal angiogenesis eye disease includes age-related macular degeneration, central exudative retinal choroiditis, ocular histoplasmosis syndrome, or glucocorticoid choriopathies.

In one embodiment of the present invention, the retinal angiogenesis eye diseases include diabetes, tumor, retinal detachment, central retinal vein occlusion, periretinal vein inflammation, and systemic lupus erythematosus.

In one embodiment of the present invention, the method is to use transthyretin to improve the proliferation, migration and tube forming ability of ocular vascular endothelial cells in a high glucose environment.

In one embodiment of the present invention, the method further includes treating diabetic retinopathy, diabetic macular edema, age-related macular degeneration (AMD), retinal vein occlusion, and polypoidal choroidal vasculopathy.

The second object of the present invention is to provide a pharmaceutical composition containing the transthyretin protein and a pharmaceutically acceptable carrier.

In one embodiment of the present invention, the Genbank accession number of the transthyretin protein is CAG33189.1.

In one embodiment of the present invention, the dosage form of the pharmaceutical composition is an injection preparation.

In one embodiment of the present invention, the dosage form of the pharmaceutical composition is eye drops.

Advantages and effects of the present invention: The present invention found that administration of TTR to mammals significantly reduced ocular neovascularization in STZ-induced diabetic rat and mouse models. The in vitro experimental model of tumor neovascularization, diabetic neovascularization, ocular neovascularization and brain neovascularization was used to verify the effect of TTR on inhibiting angiogenesis. The results show that the in vitro cell model of TTR acting on the hypoxic environment of the brain is sustainable and Effectively inhibit the generation of new blood vessels. TTR acting on umbilical vein endothelial cells can significantly inhibit 90% of blood vessel formation; acting on brain microvascular endothelial cells can effectively inhibit 95% of blood vessel formation; acting on ocular microvascular endothelial cells can effectively inhibit 90% blood vessel formation; acting on rats In the mouse model, it effectively inhibits neovascularization by up to 90%.

BRIEF DESCRIPTION

Figure 1 is the chemical structure of TTR (A); and cell activity at different TTR concentrations (B);

Figure 2 is the migration diagram of umbilical vein endothelial cells; where, A is normal umbilical vein endothelial cells cultured with high-glucose DMEM for 48 hours, observed under the microscope and photographed, and randomly selected 5 fields for cell counting to calculate the cell migration rate; B is exogenous addition Observe the photograph under the microscope after TTR, take 5 fields randomly to count the cells and calculate the cell migration rate;

Figure 3 is a migration diagram of cerebral vascular endothelial cells; where A is the migration rate of cerebral vascular endothelial cells after 48 hours, and B is the migration rate of cells after exogenous addition of TTR;

Figure 4 is a diagram of the migration of ocular retinal endothelial cells, where A is the migration rate of the retinal endothelial cells after 48 hours, and B is the migration rate of the retinal endothelial cells after 48 hours of exogenously added TTR;

Figure 5 is a diagram of tube formation of ocular retinal endothelial cells, where L is normal low glucose (5mmol/L) cultured retinal endothelial cell vascularization, H is high glucose (30mmol/l); T is exogenously added TTR blood vessels newborn;

Fig. 6 is a tube formation experiment of umbilical vein endothelial cells, where L is normal umbilical vein endothelial cell angiogenesis, H is angiogenesis after exogenous addition of TTR; T is angiogenesis after exogenous addition of TTR;

Figure 7 is the retinal vascular leakage in rats under different treatment conditions; where, A is the retinal vascular leakage after intravitreal injection of TTR protein (4mmol/L, 5ul per eye) in SD rats; B is STZ-induced Leakage of retinal vessels in rat model, C is the ratio of leakage fluid;

Figure 8 is the retinal vascular leakage in mice under different treatment conditions; among them, A is the retinal vascular leakage after injection of TTR protein (4mmol/L, 2ul per eye) in C57 mouse eye; B is STZ induced Leakage of retinal vessels in a mouse model, C is the ratio of leakage fluid;

Figure 9 is the fundus optical coherence tomography image (A) and VEGF level (B) in the eye of C57 mice; where L is low sugar; H is high sugar; T is high sugar + TTR;

Fig. 10 is the fundus pictures of rats under different treatment conditions; among them, A is the fundus picture of normal rats, B is the fundus picture of SD diabetic rats, and C is the fundus picture after TTR protein injection.

detailed description

Example 1

As shown in FIG. 1A, the structure of TTR is center-symmetric and axis-symmetric; the monomer includes 147 amino acids, and the Gene ID encoding TTR is 7276. TTR corresponds to the amino acid sequence of Genbank accession number CAG33189.1.

Under the high glucose DMEM culture conditions, the MTT method was used to detect the cell viability of different concentrations of TTR on human microvascular endothelial cells (3000/well). The proliferation of cells in each group after 48h showed that the A value in the 0μmol/L group was (0.40±0.03), and the A value in the 4μmol/L group was (0.10±0.02). The inhibitory effect of the 4μmol/L group on proliferation was higher than that of the 0μmol/L group, and the difference was statistically significant (t=15.47, P=0.001) (Figure 1B). The cell proliferation in the 4μmol/L group was reduced by 75.4% compared with the 0μmol/L group. It can be seen that TTR has an inhibitory effect on the proliferation of human microvascular endothelial cells.

Example 2 Umbilical vein endothelial cell migration experiment

Add 600μl of high-sugar DMEM medium containing 0μM and 4μM TTR to each well of the 24-well plate, and put into a transwell chamber with a pore size of 8.0μm. Add 200μl of FBS-free 1×10 ⁴ human umbilical vein endothelial cells to the chamber Culture medium. After 48h, aspirate the medium in the upper and lower chambers, wipe the cells in the upper chamber with a cotton swab, add 0.4g/L paraformaldehyde 600μl to the 24-well plate and fix at room temperature for 30min, aspirate the paraformaldehyde, rinse the back of the chamber with PBS Two times, 600 μl of crystal violet was added to the 24-well plate, and stained at room temperature for 20 min. After aspiration, the back of the chamber was washed twice with PBS, observed under the microscope and photographed, and 5 fields were randomly selected for cell counting.

The results of the 48h cell migration experiment showed that the number of human umbilical vein endothelial cells migrating out of the 0μmol/L group was (227±14), and the number of human umbilical vein endothelium migrating out of the 4μmol/L group was (140±7). Comparing the number of cells migrating outward between the two groups, 4μmol/L was 87±7 lower than that in the 0μmol/L group, and the difference was statistically significant (t=6.75, P=0.0005) (Figure 2). It can be seen that TTR has an inhibitory effect on the migration of human umbilical vein endothelial cells.

Example 3 Migration experiment of cerebral vascular endothelial cells

Add 600 μl of high-sugar DMEM medium containing 0 μM and 4 μM TTR to each well of a 24-well plate, and place in a transwell chamber with a pore size of 8.0 μm. Add 200 μl of FBS-free cells containing 1×10 ⁴ cerebrovascular endothelial cells Culture medium. After 48h, aspirate the upper and lower chamber media, wipe the upper chamber cells with a cotton swab, add 0.4g/L paraformaldehyde 600μl to the 24-well plate and fix at room temperature for 30min, aspirate the paraformaldehyde, rinse the back of the chamber twice with PBS, 24 Add 600μl of crystal violet to the well plate, stain at room temperature for 20min, wash the back of the chamber twice with PBS after aspiration, observe and photograph under the microscope, and randomly take 5 fields for cell counting.

The results are shown in Figure 3. The 48h cell migration experiment results showed that the number of outward migration of cerebral vascular endothelial cells in the 0μmol/L group was (300±25), and the number of outward migration of cerebral vascular endothelium in the 4μmol/L group was (150±2 ). Comparing the number of cells migrating outward between the two groups, the 4μmol/L group was lower than the 0μmol/L group, and the difference was statistically significant (t = 7.85, P = 0.0003) (Figure 3). It can be seen that TTR has an inhibitory effect on the migration of cerebral vascular endothelial cells.

Example 4 Eye retinal endothelial cell migration experiment

Add 600 μl of high-sugar DMEM medium containing 0 μM and 4 μM TTR to each well of the 24-well plate, and simultaneously put into a transwell chamber with a pore size of 8.0 μm. Add 200 μl of FBS-free cells containing 1×10 ⁴ eye retinal endothelial cells Culture medium. After 48h, aspirate the upper and lower chamber media, wipe the upper chamber cells with a cotton swab, add 0.4g/L paraformaldehyde 600μl to the 24-well plate, fix at room temperature for 30min, aspirate the paraformaldehyde, rinse the back of the chamber twice with PBS, 600μl of crystal violet was added to the 24-well plate, and stained at room temperature for 20min. After aspiration, the back of the chamber was rinsed twice with PBS.

The results are shown in Figure 4. The 48h cell migration experiment showed that the number of outward migration of retinal vascular endothelial cells in the 0μmol/L group was (350±30), and the number of outward migration of the retinal endothelium in the 4μmol/L group was (110± 17). Comparing the number of cells migrating outward between the two groups, the 4μmol/L group was lower than the 0μmol/L group, and the difference was statistically significant (t=8.59, P=0.0002). It can be seen that TTR has an inhibitory effect on the migration of cerebral vascular endothelial cells.

Example 5 Tube formation experiment of eye retinal endothelial cells

Place Matrigel Matrigel stored at -20°C in a refrigerator at 4°C overnight, and use it only when it has completely melted into a red gel-like liquid. Using a pre-chilled pipette tip, add Matrigel glue to the pre-chilled 48-well plate at 150 μl per well. Gently shake the culture plate on ice to make Matrigel glue flat on the bottom of the plate. The 48-well plate was then placed in a 37°C incubator for 30 min to allow Matrigel gel to solidify. When the cells grow to 80%-90% confluence, replace with FBS-free medium and starve for 24h. Wash the cells 3 times with PBS, add 0.25% trypsin to digest the cells, the volume is limited to cover the bottom of the bottle, gently shake the bottle to make the trypsin fully contact with the cells, and incubate at 37°C for several minutes. Observe the cell morphology under an inverted microscope. When the cytoplasm is found to shrink and the cell gap increases, but the cells have not floated, immediately add complete medium to stop the digestion. Blow the bottom of the flask repeatedly with the culture solution to make the cells completely fall off, collect all the liquid into the centrifuge tube, and count them on the cell counting plate. Centrifuge at room temperature, 1000rpm, 5min. Discard the supernatant, add new culture solution, pipet into cell suspension, aliquot into four centrifuge tubes, centrifuge again, 1000rpm, 5min. Discard the supernatant in each tube and add an equal volume of each group of conditioned medium to make a cell suspension. Inoculate the gel-coated culture plates at a density of 3×10 ⁴ cells/well, and set 2 double wells for each group, and incubate at 37°C in a 5% CO ₂ incubator for 8 hours. Observe the cells using an inverted microscope and collect images with a camera system. Use ImagePro-Plus software to analyze the image and calculate the number of vascular cavities.

The results are shown in Figure 5. L is the normal glucose DMEM medium, H is the high glucose DMEM medium, and T is the high glucose DMEM medium + TTR. The retinal endothelial cells in normal eyes show a lumen state, and TTR is added exogenously. After angiogenesis is inhibited, the application of Matrigel can induce angiogenesis of cultured retinal endothelial cells in vitro and form a lumen structure. Endothelial cells begin to form luminal structures after 4h of culture. Figure 5 shows that 8h can establish a stable in vitro model of vascularization. The number of lumens in the high-glucose+TTR group (50±2) was significantly lower than that in the high-glucose group (500±12) (P=0.025, <0.05), and the differences were statistically significant.

Example 6 Umbilical vein endothelial cell tube formation experiment

Place Matrigel Matrigel stored at -20°C in a refrigerator at 4°C overnight, and use it only when it has completely melted into a red gel-like liquid. Using a pre-chilled pipette tip, add Matrigel glue to the pre-chilled 48-well plate at 150 μl per well. Gently shake the culture plate on ice to make Matrigel glue flat on the bottom of the plate. The 48-well plate was then placed in a 37°C incubator for 30 min to allow Matrigel gel to solidify. When the cells grow to 80%-90% confluence, replace with FBS-free medium and starve for 24h. Wash the cells 3 times with PBS, add 0.25% trypsin to digest the cells, the volume is limited to cover the bottom of the bottle, gently shake the bottle to make the trypsin fully contact with the cells, and incubate at 37°C for several minutes. Observe the cell morphology under an inverted microscope. When the cytoplasm is found to shrink and the cell gap increases, but the cells have not floated, immediately add complete medium to stop the digestion. Blow the bottom of the flask repeatedly with the culture solution to make the cells completely fall off, collect all the liquid into the centrifuge tube, and count them on the cell counting plate. Centrifuge at room temperature, 1000rpm, 5min. Discard the supernatant, add new culture solution, pipet into cell suspension, aliquot into four centrifuge tubes, centrifuge again, 1000rpm, 5min. Discard the supernatant in each tube and add an equal volume of each group of conditioned medium to make a cell suspension. Inoculate the gel-coated culture plates at a density of 3×10 ⁴ cells/well, and set 2 double wells for each group, and incubate at 37°C in a 5% CO ₂ incubator for 8 hours. Observe the cells using an inverted microscope and collect images with a camera system. Use ImagePro-Plus software to analyze the image and calculate the number of vascular cavities.

The results are shown in Fig. 6, L is normal sugar concentration DMEM medium, H is high sugar DMEM medium, T is high sugar DMEM medium + TTR, normal umbilical vein endothelial cells have a lumen state, and TTR is added exogenously. After angiogenesis is inhibited, the application of Matrigel Matrigel can induce angiogenesis of cultured umbilical vein endothelial cells in vitro, forming a lumen structure. Endothelial cells begin to form luminal structures 4 hours after culture, and a stable in vitro model of vascularization can be established at 8 hours. The number of lumens in the high-glucose+TTR group (50±1) was significantly lower than that in the high-glucose group (500±20) (P = 0.013, <0.05), and the differences were statistically significant.

Example 7

Preparation of STZ (Destruction of Islet Ureazocin) Reagents:

Citric acid buffer: citric acid solution A: weigh 2.1g citric acid in 100ml ddH2O; trisodium citrate solution B: weigh 2.9g trisodium citrate in 100ml ddH2O; solution A: solution B = 1.06: 1; 1.5 %STZ: 1.5g is added to 100ml mixed solution;

Establishment of animal models:

Rats: at a dose of 60mg/kg=6mg/100g, each rat is injected with 0.4ml/100g of 1.5% STZ; C57 mice: at a dose of 150mg/kg=1.5mg/10g, each mouse is injected with 0.1 1.5% STZ in ml/10g. Measure the blood glucose of tail vein after 1W,

Blood sugar ≥16.7, diabetes modeling success.

The experiment was carried out according to the following steps: first, anesthetize the rats with 3.5ml/kg 10% chloral hydrate solution, compound tropicamide eye drops to dilate the pupils, the surface of Nobile eyeballs was anesthetized, antibiotic gel was dropped on the surface of the cornea and covered A coverslip with a diameter of about 1cm (can directly look at the fundus), use a micro syringe to aspirate 5μL of adenovirus under the operating microscope, avoid the lens and retinal blood vessels, pierce the vitreous cavity 1mm behind the corneal sclera, and inject Apply antibiotic gel to the needle and pull out the needle. Put it on the insulation board until it wakes up. Observe whether there is postoperative fundus hemorrhage or net detachment. Chloramphenicol eye drops are applied once a day to prevent infection.

The results are shown in Figure 7. After the injection of TTR protein (4mmol/L, 5ul) into the vitreous cavity of SD rats, the retinal vascular leakage of each eye was 10%; the retinal vascular leakage of the rat model induced by STZ It is 90%, and the leakage range after intraocular injection of TTR is reduced from 90% to 10%. It can be seen that the injection of TTR will significantly inhibit the formation of new blood vessels due to diabetes.

Example 8

An animal model was established in the manner of Example 7, and the experiment was as follows

The procedure is carried out: diabetic mice and control groups (normal C57 mice) are anesthetized with ketamine (80 mg/kg) and dimethylprazine (4 mg/kg). The right jugular vein and right iliac artery were cannulated, and then perfused with heparinized saline. Evans blue (45mg/kg) was injected through the jugular vein for 10 seconds. After two hours, approximately 0.2 ml of blood was obtained from anesthetized mice. Animals were perfused with PBS through the left ventricle, followed by 1% paraformaldehyde. The cornea, lens and vitreous were removed. The remaining retina and sclera were fixed with 4% paraformaldehyde in phosphate buffered saline at room temperature for 30 minutes. The retina was treated with dimethylformamide (SigmaAldrich, St. Louis, MO) at 78°C overnight, then centrifuged at 12000g for 15 minutes, and the supernatant was subjected to spectrophotometric detection at 620nm (blue) and 740nm (background) . According to the standard curve of Evans blue in formamide, the concentration of the dye in the plasma was calculated to detect the degree of blood vessel leakage.

C57 mice were treated with vitreous injection of mice, and the results are shown in Figure 8. After injection of TTR protein (4mmol/L, 2ul per eye) into the vitreous cavity of C57 mice, retinal vascular leakage was 90%; STZ was used to induce The retinal vascular leakage of the rat model was 90%, and the range of serum leakage was reduced from 90% to 10%. It can be seen that the injection of TTR will significantly inhibit the formation of new blood vessels due to diabetes.

The optical coherence tomography of C57 mice before and after the treatment was scanned. The results are shown in Figure 9 (VEGF is vascular growth factor and GAPDH is the internal reference gene). From left to right, it can be seen that the deep retinal exudation is obvious after the addition of TTR cut back. B. The level of VEGF in the eye decreased after the addition of TTR (t=7.89, p<0.05).

Example 10

The mice were treated according to the treatment method of Example 9, diabetic mice were anesthetized with ketamine/dimethylperazine, and compound tropicamide (Alcon, Fort Worth, TX, USA) was dilated. Under the guidance of bright-field live fundus images, the Micron IV image-guided OCT system (Phoenix Research Laboratories, Pleasanton, CA, USA) was used for spectral domain optical coherence tomography (OCT). As a result, as shown in FIG. 10, the tortuous phenomenon caused by diabetes due to the addition of TTR is significantly improved.

Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. The protection scope of the present invention shall be defined by the claims.

Claims (17)

1. The application of transthyretin with Genbank accession number CAG33189.1 in the preparation of drugs that inhibit angiogenesis.
2. The use according to claim 1, characterized in that the effective dose of transthyretin is ≥4mmol/L or ≥4mmoL/g.
3. The use according to claim 1 or 2, wherein the inhibition of angiogenesis includes inhibition of tumor angiogenesis, diabetic angiogenesis, ocular angiogenesis or brain angiogenesis.
4. A method for treating angiogenesis, characterized in that transthyretin is used to inhibit angiogenesis; the transthyretin protein is (a) or (b):

   (a) The protein with Genbank accession number CAG33189.1;

   (b) A protein having at least 99%, or 98%, or 95%, or 90% homology with the amino acid sequence of (a) and having a function of inhibiting angiogenesis.
5. The method according to claim 4, comprising inhibiting tumor angiogenesis, diabetic angiogenesis, ocular angiogenesis or brain angiogenesis.
6. The method according to claim 5, wherein the tumor angiogenesis factor VEGF is inhibited by transthyretin.
7. The method according to claim 4 or 5, wherein the tumor angiogenesis includes liver cancer, lung cancer, bladder cancer, breast cancer, rectal cancer, osteosarcoma, gastric cancer, pancreatic cancer, leukemia, lymphoma, myeloma blood Cancer angiogenesis, hemangioma, hemangioma complicated tissue angiogenesis, multiple hemangioma, hemangioblastoma, benign vascular hyperplasia.
8. The method according to claim 4, wherein the method is to treat angiogenic ophthalmopathy.
9. The method according to claim 8, wherein the angiogenic ophthalmopathy comprises iris angiogenesis ophthalmopathy, choroidal angiogenesis ophthalmopathy, retinal angiogenesis ophthalmopathy, or corneal angiogenesis ophthalmopathy.
10. The method according to claim 9, wherein the corneal angiogenesis eye disease includes corneal angiogenesis disease caused by contact lens, alkali and other substance burns, corneal surgery, bacterial infection, chlamydia infection, viral infection or Corneal angiogenesis eye disease caused by protozoan infection.
11. The method according to claim 9, wherein the iris angiogenesis ophthalmopathy includes angiogenesis glaucoma, diabetic retinopathy or central retinal vein embolism.
12. The method according to claim/9, wherein the choroidal angiogenesis eye disease includes age-related macular degeneration, central exudative retinal choroiditis, ocular histoplasmosis syndrome or glucocorticoid choroid Lesions.
13. The method according to claim/9, wherein the retinal angiogenesis eye diseases include diabetes, tumor, retinal detachment, central retinal vein occlusion, periretinal vein inflammation, and systemic lupus erythematosus.
14. The method according to claim/8, characterized in that the method is to use transthyretin to improve the proliferation, migration and tube forming ability of ocular vascular endothelial cells under high glucose environment.
15. A pharmaceutical composition, characterized in that it contains transthyretin and a pharmaceutically acceptable carrier; the transthyretin is (a) or (b):

    (a) The protein with Genbank accession number CAG33189.1;

    (b) A protein having at least 99%, or 98%, or 95%, or 90% homology with the amino acid sequence of (a) and having a function of inhibiting angiogenesis.
16. The composition according to claim 15, wherein the dosage form is an injection preparation.
17. The composition according to claim 15, wherein the dosage form is eye drops.

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