CN109125731B - Application of Sema4D/PlexinB1 inhibitor in the preparation of drugs for the treatment and prevention of fundus vascular diseases - Google Patents

Abstract

The invention belongs to the field of biological medicine, and particularly discloses an application of a Sema4D/PlexinB1 inhibitor in preparation of a medicine for treating and preventing fundus vascular diseases, wherein an applicant finds that Sema4D is increased in aqueous humor of a diabetic retinopathy patient, the Sema4D level is negatively related to the response of the patient to anti-VEGF, and a Sema4D/PlexinB1 signal channel can act on endothelial cells to promote migration and lumen formation of the endothelial cells, so that the Sema4D/PlexinB1 signal channel is inhibited by promoting migration of pericytes to aggravate leakage, the neogenesis and leakage of fundus vessels can be effectively inhibited, and a new thought is provided for fundus vascular hyperplasia and leakage diseases such as diabetic retinopathy and the like.

Description

Sema4D/PlexinB1 inhibitors in the preparation of drugs for the treatment and prevention of fundus vascular diseases application in things

technical field

The invention belongs to the field of biomedicine, and specifically relates to the application of Sema4D/PlexinB1 inhibitors in the preparation of medicines for treating and preventing fundus vascular diseases.

Background technique

Diabetic retinopathy (DR), as the first cause of blindness in adults aged 20-75, has a high prevalence in patients with type 1 and type 2 diabetes. Globally, the prevalence of DR in diabetic population is 35.4%. The prevalence of proliferative diabetic retinopathy (PDR) in the most severe stage is 7.5% (2017 Diabetes Care IF=13.397). In China: the prevalence of DR in different stages of diabetes is also as high as 24.7%-37.5 (2014 guidelines), The World Health Organization (WHO) estimates that by 2025, the number of diabetic patients worldwide will exceed 30 billion (2009EYE), which shows that the number of DR patients will further increase.

The treatment of DR has been mainly three types of treatment since the study of diabetic retinopathy vitrectomy (DRVS study) was carried out in 1990: ① laser photocoagulation therapy ② vitrectomy ③ application of anti-angiogenic drugs (mainly a variety of anti- VEGF treatment, 2004-present), laser photocoagulation therapy is the treatment method of abandoning the car to keep handsome, sacrificing the peripheral visual field to retain the central visual field, but only 30% of the patients receiving anti-VEGF treatment are effective, it can be seen that anti-VEGF treatment resistance , repeated administration of infection, neuronal toxicity, and heavy economic burden all bring inconvenience and risk to the treatment, which urgently requires us to develop new treatment strategies.

The pathological process of DR is different from tumor angiogenesis, and its treatment has certain specificity. The initiating factor of DR is the slowing down of fundus blood vessel flow velocity, leading to local metabolic changes, activation of inflammation and related signaling pathways, resulting in vascular leakage, blood-retinal Barrier damage, parietal cell detachment, and cell matrix damage lead to loss of function of blood vessels, local formation of hypoxic areas, resulting in compensatory abnormal vascular proliferation, aggravation of leakage and even incomplete abnormal new blood vessels, resulting in fundus hemorrhage and retinal detachment (CurrDiab Rep 2011; Diabetes. 2006). In tumors, angiogenesis starts from the induction of pro-angiogenic factors and hypoxia, and after the activation of endothelial cells, the extension of pseudopodia, the attachment of surrounding parietal cells and the fusion with adjacent neovascular lumens, form a vascular network. The purpose of angiogenesis in tumors is to provide support for tumor growth. Therefore, the treatment for angiogenesis in tumors focuses on simply inhibiting angiogenesis, but it is different in the fundus. The purpose of anti-angiogenesis treatment is to inhibit abnormal blood vessels while restoring normal blood vessels. It can provide blood supply to the area and reduce leakage. It can be seen that although the two diseases both inhibit angiogenesis, the treatment results can be quite different.

The Semaphorin protein family is a protein that plays an important role in the development of the nervous system. It is currently considered to play an important role in the mechanism of angiogenesis and vascular development. Among them, the single transmembrane protein Sema4D (Class 4 semaph orins) is hotly discussed in angiogenesis The active extracellular free Sema4D fragments are mainly derived from the cleavage of MMP-MT1 in various cells or the cleavage of ADAM17 in platelets. It is involved in angiogenesis in tumor, bone, and trauma models, and its effects are specific in different tissues and cells , in tumors, Sema4D promotes the phosphorylation of Met and Ron by binding to PlexinB receptors to promote cell migration and angiogenesis leading to tumor cell metastasis (2009Valente). Inhibiting cell migration (Swiercz 2008), it can be seen that Sema4D plays different roles in different tissues and diseases.

In the present invention, the applicant has clarified that inhibiting the Sema4D/PlexinB1 signaling pathway can effectively inhibit fundus angiogenesis and reduce fundus vascular leakage, and more importantly, early inhibition of the Sema4D/PlexinB1 signaling pathway can effectively reduce the loss of pericytes and contribute to Protecting and restoring the function of fundus blood vessels can prolong the therapeutic effect to a certain extent, reduce the frequency of administration, and provide a new strategy for the treatment of fundus angiogenesis and leakage.

Contents of the invention

The object of the present invention is to provide the application of Sema4D/PlexinB1 inhibitors in the preparation of drugs for the treatment and prevention of fundus vascular diseases, which include but not limited to fundus angiogenesis or fundus vascular leakage or loss of pericytes.

In order to achieve the above object, the present invention takes the following technical measures:

In view of the limited treatment methods for fundus angiogenesis and leakage, the applicant collected aqueous humor from patients with diabetic retinopathy, screened members closely related to angiogenesis, and found that Sema4D/PlexinB1 signaling pathway inhibitors can effectively inhibit fundus angiogenesis and leakage. leak.

The application of Sema4D/PlexinB1 signaling pathway inhibitors in the preparation of drugs for the treatment and prevention of fundus vascular diseases, said inhibitors include but are not limited to Sema4D/PlexinB1 signaling pathway inhibitory viruses, or Sema4D/PlexinB1 signaling pathway neutralizing antibodies, or Compounds that inhibit the function of the Sema4D/PlexinB1 signaling pathway.

In the above-mentioned applications, preferably, the fundus angiogenesis and leakage are caused by diabetes.

A method for treating fundus vascular disease, the method is to inject an inhibitor of Sema4D protein into the vitreous of the eye;

A method for treating fundus vascular disease, the method is to inject Sema4D protein inhibitor and anti-VEGF into eye vitreous.

The fundus vascular disease includes but not limited to fundus angiogenesis or fundus vascular leakage or loss of pericytes.

In the above method, preferably, the inhibitor is a Sema4D protein neutralizing antibody or a Sema4D/PlexinB1 signaling pathway inhibitor.

Compared with the prior art, the present invention has the following advantages:

In view of the current limited treatment methods for fundus angiogenesis and leakage, the existing main treatment methods are ① laser photocoagulation therapy ② vitrectomy ③ application of anti-angiogenic drugs, among which anti-angiogenic drugs are mainly anti-VEGF fundus Injection, but only 30% of patients receiving anti-VEGF therapy are effective. In view of the limitations of current treatment, the applicant took aqueous humor from patients with diabetic retinopathy, screened Sema family members closely related to angiogenesis, and found Sema4D/PlexinB1 signaling pathway Inhibitors can effectively inhibit fundus angiogenesis and leakage, and proved the effect of inhibiting Sema4D/PlexinB1 signaling pathway on fundus pathological angiogenesis and leakage in vivo, in vitro and in gene knockout mice, and clarified the related mechanism.

Description of drawings

Figure 1 is a schematic diagram of the close correlation between Sema4D and DR;

Among them, A: schematic diagram of optical coherence tomography (OCT);

B-C. The expression of Sema4D in the aqueous humor of DR patients was significantly increased (Western Blot)

D. The expression of Sema4D in the aqueous humor of DR patients was significantly increased (ELISA detection)

Free Sema4D in the aqueous humor of E-F.DR patients was negatively correlated with central macular thickness (CST) and retinal macular volume (MV) after 3 months of anti-VEGF treatment (statistics obtained from the ophthalmology database of Wuhan Union Medical College Hospital)

Figure 2 is a schematic diagram of the increased expression of Sema4D in the mouse OIR and STZ models;

Among them, A: mRNA expression of Sema4D in OIR model mouse retina increased (q-PCR);

B: Western Blot results show that the expression of Sema4D protein in the retina of OIR model mice is increased;

C-D: Western Blot shows that the expression of Sema4D protein in the retina of STZ model mice increased at 3 and 6 months.

Figure 3 is a schematic diagram of Sema4D gene knockout inhibiting fundus angiogenesis;

Among them, A-C: Gene knockout mouse verification;

D-H: Immunofluorescence staining, Evan's blue leakage test, and HE staining results showed that Sema4D gene knockout can significantly inhibit pathological fundus angiogenesis and vascular leakage.

Figure 4 is a schematic diagram showing that the Sema4D/PlexinB1 signaling pathway can promote endothelial cell lumen formation and leakage;

Among them, A-B: Scratch test confirmed that Sema4D can promote endothelial cell migration;

C-D: The lumen formation experiment confirmed that Sema4D can promote the lumen formation of endothelial cells;

E: TEER experiment confirmed that Sema4D can promote endothelial cell leakage;

F: Fluorescein leakage experiment confirmed that Sema4D can promote endothelial cell leakage;

G: PlexinB1 down-regulation verification;

H-I: Scratch test confirmed that blocking the silencing of PlexinB1 receptors can block the role of Sema4D in promoting endothelial cell migration;

J-K: Lumen formation experiments confirmed that silencing PlexinB1 receptors can block the role of Sema4D in promoting endothelial cell lumen formation;

L-M: TEER and fluorescein leakage assays confirmed that silencing PlexinB1 receptors can block the effect of Sema4D on promoting endothelial cell leakage;

Figure 5 is a schematic diagram of the Sema4D/PlexinB1 signaling pathway increasing the leakage of endothelial and pericyte co-culture models;

Among them, A-B: TEER experiments of co-cultured pericytes and endothelial cells and fluorescein leakage experiments confirmed that Sema4D signal promotes leakage;

C-D: In the co-cultured pericyte and endothelial cell model, TEER assay and fluorescein leakage assay confirmed that silencing PlexinB1 receptor blocked Sema4D-induced leakage;

Figure 6 is a schematic diagram of anti-Sema4D inhibiting fundus angiogenesis in OIR model mice and fundus vascular leakage in STZ model mice; where, A: anti-Sema4D treatment can inhibit fundus angiogenesis in OIR model mice in a concentration-dependent manner;

B: Evan's blue test confirmed that anti-Sema4D treatment can inhibit fundus vascular leakage in OIR model mice in a concentration-dependent manner C-D: Anti-Sema4D antibody specificity verification;

E-I: In the OIR model, anti-Sema4D treatment can inhibit angiogenesis and the effect of combined anti-VEGF treatment is significantly better than anti-Sema4D alone or anti-VEGF treatment alone;

J-K: Single administration, anti-Sema4D treatment in STZ model fundus can inhibit fundus vascular leakage, and combined with anti-VEGF treatment is significantly better than anti-Sema4D alone or anti-VEGF treatment alone.

Figure 7 is a schematic diagram of the effect of multiple anti-Sema4D treatments;

Among them, A-B: multiple administrations, observation after 1 week, STZ model fundus anti-Sema4D treatment can inhibit fundus vascular leakage, and the combined effect of anti-VEGF treatment is significantly better than anti-Sema4D alone or anti-VEGF treatment alone

C-F: STZ model fundus anti-Sema4D treatment can inhibit pericyte loss and improve pericyte coverage, and combined with anti-VEGF treatment is significantly better than anti-Sema4D alone or anti-VEGF treatment alone, more importantly, anti-Sema4D Inhibition of pericyte loss and improvement of pericyte coverage was superior to anti-VEGF treatment (P<0.05)

G-H: STZ model fundus anti-Sema4D treatment can increase the continuity of VE-cadherin, and combined with anti-VEGF treatment is significantly better than anti-Sema4D alone or anti-VEGF treatment alone

I-J: STZ model fundus anti-Sema4D treatment can increase the coverage ratio of N-cadherin, and combined with anti-VEGF treatment is significantly better than anti-VEGF treatment alone

K-L: Multiple administrations, observed after 2 weeks, the STZ model fundus anti-Sema4D treatment can inhibit fundus vascular leakage, and the combined anti-VEGF treatment effect is significantly better than anti-Sema4D alone or anti-VEGF treatment alone, more importantly Multiple anti-Sema4D treatments for fundus vascular leakage were better than multiple anti-VEGF treatments (P<0.05).

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. The technical solutions described in the present invention are conventional solutions in the art unless otherwise specified. The reagents or materials, unless otherwise specified, were obtained from commercial sources.

Example 1:

Findings that Sema4D is closely related to DR:

1) retinal tomography

Comparing the OCT tomographic images of diabetic retinopathy patients with normal OCT tomographic images, it was found that the thickness of the macular fovea was thickened in patients with diabetic retinopathy (A in Figure 1)

2) Detection of the expression level of Sema4D in aqueous humor

The experimental group was the aqueous humor of patients with diabetic retinopathy, and the control group was the aqueous humor of cataract patients, which were extracted with a sterile syringe.

The expression of Sema4D protein in the aqueous humor was detected by Western Blot, and the results showed that the expression of Sema4D in the aqueous humor of DR patients was significantly increased (B-C in Figure 1).

ELISA kit (MyBioSource) was used to detect the expression of Sema4D protein in aqueous humor, and the results showed that the expression of Sema4D in aqueous humor of DR patients was significantly increased, and n represents the sample size.

3) Retinal tomography and detection of Sema4D protein in aqueous humor of DR patients after treatment

Figures E-F show that free Sema4D in aqueous humor of DR patients is negatively correlated with foveal thickness (CST) and macular volume change (MV) after 3 months of anti-VEGF treatment, and n represents the sample size.

Example 2:

Increased expression of Sema4D in mouse OIR and STZ models:

1) Establish mouse OIR model

Experimental group (OIR): 3-4 months old C57BL/6 mother mice were put into an oxygen chamber containing 75% oxygen together with the pups on the 7th day of birth, and the pups were separated from the mother mice on the 12th day of birth. Take it out and leave it in normal air for 5 days. The young mice treated above were anesthetized on the 12th, 14th, and 17th day after birth, and after cardiac perfusion with saline, the eyeballs were removed, the retina was removed, and the RNA was extracted and reverse-transcribed into cDNA or protein.

Control group (Normal): young mice after birth, grown under normal oxygen conditions, anesthetized mice on the 12th, 14th, and 17th day of life, and harvested eyeballs after heart perfusion with normal saline, peeled off the retina, extracted RNA and reverse transcribed into cDNA or extracted protein.

2) q-PCR detection of Sema4D mRNA expression

Using primers (CCTGGTGGTAGTGTTGAGAAC and GCAAGGCCGAGTAGTTAAAGAT), the cDNA in step 1) was used as a template to detect the expression level of Sema4D. The results showed that in the OIR group, the mRNA expression level of Sema4D was significantly increased (A in Figure 2). (P12 in Fig. 2 refers to the mouse sample on the 12th day of birth, and so on, the same below)

3) Western Blot detection of Sema4D protein in the experimental group (OIR) and the control group

The protein extracted in step 1) was used as the antigen, and sheep anti-SEMA4Dantibody (source: R&DSystems) was used as the antibody for Western Blot detection, with β-action as the control. The results showed that the expression of Sema4D protein in the retina of OIR model mice was significantly increased (B in Figure 2)

4) Establish mouse STZ model

Experimental group (STZ): 8-week-old C57BL/6 mice were starved for 4 hours and injected STZ (streptozotocin, 50 mg/kg) intraperitoneally for 5 consecutive days, once a day, and blood glucose was detected on the 7th day after the injection. Blood glucose greater than 15mmol/L was considered as successful modeling. At the 3rd and 6th month after modeling, the mice were anesthetized, and the heart was perfused with normal saline, then the eyeballs were removed, the retina was peeled off, and the protein was extracted.

Control group (Vehicle): The streptozotocin in the experimental group was replaced with normal saline, and the rest of the operations were the same.

5) Western Blot detection of Sema4D protein in the experimental group (STZ) and the control group (Vehicle)

The protein extracted in step 4) was used as the antigen, and sheep anti-SEMA4D antibody (R&D Systems) was used as the antibody to perform Western Blot detection, and β-action was used as the control. The results showed that the expression of Sema4D protein in the retina of STZ model mice was significantly increased at 3 and 6 months after modeling (C and D in Figure 2).

Example 3:

Knockout of Sema4D gene can significantly inhibit fundus angiogenesis and leakage in mice

The Sema4D knockout mice used in this example were purchased from Huazhong Agricultural University, and were obtained by designing sgRNA to the Sema4d exon region of the mice to knock out the expression of Sema4D.

1) Verification of successful knockout of the target gene (sema4D) in knockout mice:

Extract DNA from the mouse tail to verify the successful knockout of the mouse target gene (sema4D). Take the wild-type mouse as the control: take a 3-5cm mouse tail and put it in the DNA lysate at 56°C for 10 hours. After the precipitation of isopropanol, 95% After washing with alcohol, configure the following system to amplify, and electrophoresis in agarose gel.

Sema4d-GT-F1 TCTGGGGCTCTAAGAGGTCCTT Sema4d-GT-R1 AGCCACTGAGGTCACATACACC

A in Figure 3 is a schematic diagram of the sgRNA targeting region, and B in Figure 3 shows that the sema4D gene of the knockout group is truncated (Sema4D-KO), so the molecular weight is lower than that of the wild group, indicating that the knockout is successful.

Use mouse retinal protein to do Western Blot to detect Sema4D protein, take mouse retinal tissue, the extraction procedure is the same as the general tissue protein extraction procedure, 6 mice per group, with β-action as the control; Sema4D protein antibody is SEM A4D antibody (R&D Systems).

C in Figure 3 shows that homozygous mice for sema4D knockout have no expression of sema4D protein, and the expression of sema4D protein in knockout heterozygous mice is less than that of wild type. Zygote, knockout heterozygote.

2) IB4 immunofluorescence staining showed that Sema4D knockout mice can significantly inhibit fundus angiogenesis in OIR model

The OIR model was established using Sema4D gene knockout mice. The mice were anesthetized on the 17th day after birth, and after heart perfusion with saline, the eyeballs were fixed in 4% paraformaldehyde at 4°C overnight. After the retina was peeled off, Isolectin B4 was stained overnight at 4°C, and the slices were photographed with Isolectin B4 (1:100, Vector Laboratories). The OIR model established by wild-type mice was used as a control, with 6 mice in each group.

D in Figure 3 shows the reduction of abnormal new blood vessels after OIR modeling in sema4D-knockout mice, and E in Figure 3 is a statistical schematic diagram of the proportion of abnormal blood vessels to the total blood vessel area using ImageJ.

3) Evan's blue extraction test confirmed that Sema4D gene knockout mice can significantly inhibit fundus vascular leakage in the OIR model. Experiments were divided into wild-type OIR model mice and sema4D knockout OIR model mice, with 6 mice in each group, as follows:

OIR model mice were intraperitoneally injected with Evans blue dye (200 mg/kg) on the 17th day of birth, anesthetized the mice after circulating for 4 hours, and after cardiac perfusion with normal saline, their retinas were placed in formamide (70°C water bath) overnight, and used A microplate reader (wavelength 620-740mm) was used to measure the absorbance of the extracted liquid. The concentration is calculated according to the standard curve (with different concentrations of Evans blue dye as the abscissa, and the liquid absorbance measured by the microplate reader at a wavelength of 620-740mm as the vertical axis to make the standard curve), and the weight of the retina and the level of the retina in the blood are converted Post statistics.

F in Figure 3 shows that the Sema4D knockout mice can significantly inhibit the fundus vascular leakage in the OIR model.

4) In the OIR model, Sema4D knockout mice can significantly inhibit the number of preretinal neovascularization cells (HE staining)

The experiment was divided into four groups, namely: wild-type mouse group, sema4D knockout mouse group, wild-type OIR model mice and sema4D knockout OIR model mice, with 6 mice in each group. The specific steps are as follows:

All the above four groups of mice were anesthetized on the 17th day after birth, and after cardiac perfusion with normal saline, their eyeballs were fixed in 4% paraformaldehyde, paraffin-embedded and sectioned for HE staining, observed under an optical microscope and counted to break through the inner boundary of the retina The number of vascular endothelial cells in the membrane.

G in Figure 3 shows the number of neovascularization cells in the preretinal retina stained by HE staining. There was no neovascularization in the wild-type mouse group and the sema4D knockout mouse group. The wild-type OIR model mice and the sema4D knockout OIR model mice All have neovascularization, but the number of new blood vessels in the sema4D knockout OIR model mice is significantly smaller than that in the wild type OIR model mice. The arrows indicate the preretinal neovascularization cells. H in Figure 3 shows that in the OIR model, the Sema4D gene knockout mice can significantly Inhibits the number of preretinal neovascularization cells.

Example 4:

The enhancement or inhibition of Sema4D/PlexinB1 signaling pathway affects the lumen formation and leakage of endothelial cells:

The cells or endothelial cells involved in this embodiment all refer to mouse brain microvascular endothelial cells.

1) Sema4D can promote endothelial cell migration (scratch test)

The culture wells were filled with mouse brain microvascular endothelial cells, starved with 0.5% FBS overnight, and vertical vertical lines were drawn on the cells with a 200ul pipette tip. After washing the marked cells, they were added to DMEM respectively The sema4D protein with the final concentration of 400, 800 and 1600ng/ml was added to the culture wells and incubated for 24 hours, and the micrograph was used to take pictures, and image J was used to count the cell migration.

The results showed that: Sema4D can promote the migration of endothelial cells (A in FIG. 4 ), and the migration ability of endothelial cells increases with the concentration of sema4D (B in FIG. 4 ).

2) Sema4D can promote the formation of endothelial cell lumen (tube formation experiment)

Add 150ul pre-cooled Matrigel/well (BD Biosciences) to a 48-well plate, place in a cell culture incubator at 37°C for 30min, plant ² ×104 mouse brain microvascular endothelial cells per well, and culture for 3 days until the cells are full Afterwards, sema4D with a final concentration of 400, 800, and 1600 ng/ml was added to DMEM, and incubated for 24 hours after adding to the culture wells. After 24 hours, images were taken with a microscope for statistics, and DMEM without sema4D was used as a control.

The results showed that Sema4D could promote the lumen formation of endothelial cells, and it increased as the concentration of sema4D increased (C-D in Figure 4).

3) Sema4D can promote endothelial cell leakage (TEER experiment)

First coat a layer of fibronectin on the upper layer of a 24-well transwell chamber (0.4um), and incubate at room temperature for 1 h; after coating, ⁵ ×104 mouse brain microvascular endothelial cells are planted, and cultured for 3 days until the cells are full. The final concentration of 400, 800, and 1600 ng/ml sema4D protein was added to the medium respectively, and the DMEM without sema4D was used as a control. After incubation for 24 hours, the resistance value was measured with a cell transmembrane resistance measuring instrument.

E in Figure 4 shows that the TEER of endothelial cells gradually decreased after adding 400, 800, and 1600ng/ml sema4D, indicating that Sema4D can promote the leakage of endothelial cells, and the leakage increased with the increase of concentration.

4) Sema4D can promote endothelial cell leakage (fluorescence leakage experiment)

First coat a layer of fibronectin on the upper layer of a 24-well transwell chamber (0.4um), incubate at room temperature for 1 hour, and seed mouse brain microvascular endothelial cells after coating. After culturing for 3 days, add The final concentration of sema4D is 400, 800, 1600ng/ml, and the DMEM without sema4D is used as the control. After 24 hours, fluorescently labeled dextran with a final concentration of 300μg/ml is added to the DMEM in the upper layer of the chamber, and 30ul of the lower medium is extracted in 1h. A microplate reader measures the absorbance at a wavelength of 625 nm.

F in Figure 4 shows that the dextran fluorescence value of endothelial cell leakage increases after adding sema4D at a final concentration of 400, 800, and 1600 ng/ml, indicating that Sema4D can aggravate endothelial cell leakage, and the leakage increases with the increase of concentration.

5) PlexinB1 down-regulation in endothelial cells caused by lentivirus transfection, protein level verification (Western Blot)

The lentiviruses involved in the embodiments of the present invention were all purchased from Jikai Company, wherein CRISPR-wt is an empty lentivirus with GFP, and CRISPR-plB1 is a PlexinB1 knockout CRISPR/Cas9 lentivirus, which is used to down-regulate PlexinB1; experiment Divided into 3 groups: 1. Normal group (Normal), that is, the endothelial cell group without any substance; 2. Transfected with empty lentivirus group with GFP (CRISPR-wt); 3. Transfected with CRISPR-plB1 Each group has 5 repetitions, and the MOI value of the virus group is 50.

48 hours after lentivirus transfection of mouse brain microvascular endothelial cells, protein was extracted and Western Blot was used to detect the expression effect of PlexinB1. β-action was used as the control, and the antibody used in the WB process was PlexinB1 antibody (1:500, Abcam).

The results showed that: CRISPR-plB1 can significantly reduce the expression of PlexinB1 (G in Figure 4, the left picture is the WB picture, and the right picture is the WB statistical picture).

6) The endothelial cell migration mediated by sema4D was weakened after PlexinB1 was down-regulated (scratch test)

Experimental steps: mouse brain microvascular endothelial cells were transfected with lentivirus (CRISPR-wt or CRISPR-plB1) for 48 hours, digested and planted in cell plates, filled with mouse brain microvascular endothelial cells in culture wells, starved with 0.5% FBS After overnight, draw a vertical line on the cells with a 200ul pipette tip, wash away the drawn cells, and incubate for 24h directly or add sema4D protein with a final concentration of 1600ng/ml and then incubate for 24h, with 6 replicates in each group, Using microscopic photography, image J counted cell migration.

The results showed that: sema4D protein promotes the increase of the migration number of endothelial cells (H-I in Fig. 4), and the down-regulation of PlexinB1 can inhibit this process.

7) After down-regulating PlexinB1, the sema4D-mediated lumen formation of endothelial cells is weakened (lumen formation experiment)

Add 150ul pre-cooled Matrigel/well to a 48-well plate, place in a cell culture incubator at 37°C for 30min, and plant ² ×104 mouse brain microvessels transfected with lentivirus (CRISPR-wt or CRISPR-plB1) per well Endothelial cells: cultured for 3 days and incubated for 24 hours after the cells were congested, or incubated for 24 hours after adding sema4D protein at a final concentration of 1600ng/ml, with 6 replicates in each group.

J-K in Figure 4 shows that the sema4D-mediated endothelial cell lumen formation is weakened after down-regulation of PlexinB1.

8) The sema4D-mediated leakage of endothelial cells was reduced after down-regulation of PlexinB1 (TEER experiment)

The upper layer of the 24-well transwell chamber (0.4um) was first coated with a layer of adhesion factor (incubated at room temperature for 1 h), and then seeded into the mouse brain microvascular endothelium transfected with lentivirus (CRISPR-wt or CRISPR-plB1) Cells were cultured for 3 days and incubated for 24 hours directly after the cells were confluent or added with sema4D protein at a final concentration of 1600ng/ml and then incubated for 24 hours, with 6 replicates in each group. After incubation for 24 h, the resistance value was measured with a cell transmembrane resistance measuring instrument.

L in Figure 4 shows that sema4D promotes increased leakage of endothelial cells, and downregulation of PlexinB1 can inhibit this process.

9) The sema4D-mediated leakage of endothelial cells was reduced after down-regulation of PlexinB1 (fluorescence leakage experiment)

Coat the upper layer of a 24-well transwell chamber (0.4um) with a layer of adhesion factor, incubate at room temperature for 1 hour, and seed into mouse brain microvascular endothelial cells transfected with lentivirus (CRISPR-wt or CRISPR-plB1) ( ⁵ ×10 cells), cultivated for 3 days and waited until the cells were full, directly incubated for 24 hours or added sema4D protein with a final concentration of 1600ng/ml and then incubated for another 24 hours, and then added 300 μg/ml fluorescently labeled dextran to the DMEM on the upper layer of the small chamber, After 1 h, 30 ul of the lower culture medium was extracted, and the absorbance at a wavelength of 625 nm was measured with a microplate reader.

M in Figure 4 shows that fluorescence leakage experiments show that sema4D promotes the leakage of endothelial cells, and the downregulation of PlexinB1 can inhibit this process.

Example 5:

Sema4D/PlexinB1 signaling pathway can promote leakage in endothelial-pericyte mixed culture model;

The endothelial cells used in this example are mouse brain microvascular endothelial cells, and the pericytes are primary mouse brain microvascular pericytes.

1) Sema4D can promote the leakage of the co-culture system of endothelial cells and pericytes (TEER experiment).

A 24-well transwell chamber (0.4um) was first coated with a layer of fibronectin, and incubated at room temperature for 1 hour; mouse brain microvascular endothelial cells (5×10 ⁴ ) were seeded in the lower layer of the chamber, and after 12 hours, seeded in the upper layer of the chamber Mouse primary brain microvascular pericytes (2.5×10 ⁴ ), cultured for 3 days, replaced the medium, and added sema4D protein at the final concentration of 400, 800, and 1600 ng/ml to the upper layer of DMEM respectively. The DMEM without sema4D was used as the As for the control, the resistance value was measured with a cell transmembrane resistance measuring instrument after incubation for 24 hours. A in Figure 5 shows that Sema4D can reduce the TEER value of the endothelial-pericyte co-culture system, and the reduction value increases with the increase of the Sema4D protein concentration.

2) Sema4D can promote the leakage of the co-culture system of endothelial cells and pericytes (fluorescence leakage experiment).

A 24-well transwell chamber (0.4um) was first coated with a layer of fibronectin, incubated at room temperature for 1 hour, and seeded with brain microvascular endothelial cells in the lower layer of the chamber. After 12 hours, seeded primary mouse brain microvascular pericytes in the upper layer of the chamber. After culturing for 3 days, replace the medium, add sema4D protein with a final concentration of 400, 800, and 1600 ng/ml to the upper layer of DMEM respectively, and use DMEM without sema4D as a control, add 300 μg/ml fluorescent label to the upper layer of the chamber after 24 hours dextran, and the cell sample without sema4D was used as a control, and 30ul of the lower culture medium was extracted after 1h, and the absorbance at a wavelength of 625nm was measured with a microplate reader.

B in Figure 5 shows that Sema4D can promote the leakage of endothelial and pericyte co-culture system in a concentration-dependent manner.

3) In the co-culture model, silencing the PlexinB1 protein receptor of pericytes alone can reverse the effect of Sema4D to a greater extent than silencing the PlexinB1 protein receptor of endothelial cells alone

A 24-well transwell chamber (0.4um) was first coated with a layer of fibronectin, incubated at room temperature for 1 hour, and treated mouse brain microvascular endothelial cells (5×10 ⁴ ) were seeded into the lower layer, and after 12 hours, seeded into the upper layer of the chamber Treated mouse primary brain microvascular pericytes (2.5×10 ⁴ ); after 3 days of culture, replace the medium, add Sema4D protein at a final concentration of 1600ng/ml to the upper layer of DMEM, and use no Sema4D protein as a control; then follow the steps 1) and 2) in the method, carry out TEER experiment and fluorescence leakage experiment respectively;

The specific groups are as follows:

(1), CRISPR-wt group: the lower layer was planted with endothelial cells transfected with empty lentivirus, and the upper layer of the small chamber was planted with pericytes transfected with empty lentivirus;

(2), PC-CRISPR-plB1 group: the lower layer was seeded with endothelial cells transfected with empty lentivirus, and the upper layer of the chamber was seeded with pericytes transfected with CRISPR-plB1;

(3) EC-CRISPR-plB1 group: endothelial cells transfected with CRISPR-plB1 were inoculated in the lower layer, and pericytes transfected with empty lentivirus were inoculated in the upper layer of the chamber.

The results showed that in the co-cultured pericyte and endothelial cell model, TEER assay and fluorescein leakage assay showed that pericyte PlexinB1 receptor silencing could more significantly block Sema4D-induced leakage than endothelial cell PlexinB1 receptor silencing (Figure 5 Middle C-D)

Embodiment 6:

A single injection of anti-Sema4D can inhibit fundus angiogenesis and fundus vascular leakage in OIR model mice and STZ model mice

1) IB4 immunofluorescence staining experiment (single injection):

After the 3-4 month old C57BL/6 mother mouse gave birth, put the pups together with the pups on the 7th day of life in an oxygen chamber with 75% oxygen, and after the 12th day after birth, the pups and the mother mice were taken out, and then the pups were anesthetized Different doses of Sema-4D neutralizing antibody (BMA-12) (0.5ug, 1ug, 2ug) were injected into the vitreous, 2ug of IgG was injected as a control, an equal volume of PBS was used as a control, and normal air feeding was continued for 5 days; The pups were anesthetized on the 17th day of birth, and after cardiac perfusion with saline, the eyeballs were separated and fixed, and the retina was stripped for IB4 staining. The results showed that anti-Sema4D concentration-dependently reduced the percentage of abnormal blood vessels in the total retinal area (A in Figure 6), It shows that anti-Sema4D treatment can inhibit fundus angiogenesis in OIR model mice in a concentration-dependent manner.

2) Evans blue fluorescence experiment (single injection):

After the 3-4 month old C57BL/6 mother mouse gave birth, put the pups together with the pups on the 7th day of life in an oxygen chamber with 75% oxygen, and after the 12th day after birth, the pups and the mother mice were taken out, and then the pups were anesthetized Inject different doses of sema4d neutralizing antibodies (0.5ug, 1ug, 2ug) into the vitreous, (injecting 2ug of IgG as a control, and an equal volume of PBS as a control, continue to feed in normal air for 5 days; pups were born at 17 days Inject Evans blue dye (200 mg/kg) intraperitoneally, and anesthetize the young mice 4 hours later. After the heart is perfused with normal saline, take the retina and place it in formamide (70°C water bath) overnight, and use a microplate reader (wavelength 620-740mm) Determination of the extraction liquid. Calculate the concentration according to the standard curve, and use the weight of the retina and the retinal Evans blue level in the blood to calibrate. The results show that anti-Sema4D treatment can inhibit the leakage of fundus blood vessels in OIR model mice, and it is concentration-dependent (Figure 6 Middle B).

3) IB4 immunofluorescence staining experiment (wild-type mice and knockout mice, single injection):

wt group: C57BL/6 mother mice of 3-4 months were put into an oxygen chamber with 75% oxygen together with the pups on the 7th day of birth after giving birth, and the pups and mother mice were taken out after the 12th day of birth, and then the pups were After anesthesia, the mice were injected with Sema4D neutralizing antibody (2ug) or IgG (2ug) in the vitreous and continued to be fed in normal air for 5 days; then the IB4 immunofluorescence staining experiment was performed;

Sema4D-KO group: C57BL/6 mother mice with knockout Sema4D gene at 3-4 months were put into an oxygen chamber with 75% oxygen together with pups on the 7th day after birth, and the pups were placed in an oxygen chamber with 75% oxygen after 12 days after birth. The mice and mother mice were taken out, and then the pups were injected with Sema4D neutralizing antibody (2ug) or IgG (2ug) in the vitreous after anesthesia, and continued to be fed in normal air for 5 days; then IB4 immunofluorescence staining experiments were performed;

The results showed that both anti-Sema4D treatment and Sema4D gene knockout could reduce fundus angiogenesis (IB4 immunofluorescence staining) caused by the OIR model, and vitreous anti-Sema4D injection could not further inhibit abnormal angiogenesis in Sema4D knockout mice (Figure 6 Middle C).

4) Evans blue fluorescence experiment (wild-type mice and knockout mice, single injection):

wt group: C57BL/6 mother mice of 3-4 months were put into an oxygen chamber with 75% oxygen together with the pups on the 7th day of birth after giving birth, and the pups and mother mice were taken out after the 12th day of birth, and then the pups were After the mice were anesthetized, inject Sema4D neutralizing antibody (2ug) or IgG (2ug) into the vitreous and continue feeding in normal air for 5 days; follow the method in step 2), and then perform the Evans blue fluorescence experiment;

Sema4D-KO group: C57BL/6 mother mice with knockout Sema4D gene at 3-4 months were put into an oxygen chamber with 75% oxygen together with pups on the 7th day after birth, and the pups were placed in an oxygen chamber with 75% oxygen after 12 days after birth. The mice and mother mice were taken out, and then the pups were injected with Sema4D neutralizing antibody (2ug) and IgG (2ug) in the vitreous body after anesthesia and continued to be fed in normal air for 5 days; follow the method in step 2), and then perform the Evans blue fluorescence experiment ;

The results showed that both anti-Sema4D treatment and Sema4D gene knockout could reduce the fundus vascular leakage caused by the OIR model (Evan blue fluorescence test), and the injection of anti-Sema4D in the vitreous body could not further inhibit the vascular leakage of Sema4D gene knockout mice ( Figure 6D).

5) IB4 immunofluorescence staining experiment (anti-Sema4D combined with anti-VEGF treatment, single injection)

After the 3-4 month old C57BL/6 mother mouse gave birth, put the pups together with the pups on the 7th day of life in an oxygen chamber with 75% oxygen, and after the 12th day after birth, the pups and the mother mice were taken out, and then the pups were anesthetized Simultaneous injection of sema4d neutralizing antibody (2ug/eye) and VEGF neutralizing antibody (2ug/eye) in the vitreous of the mouse eye; or separate injection of anti-Sema4D (2ug), anti-VEGF (2ug), IgG (2ug), PBS (2ug); then perform IB4 immunofluorescence staining experiment according to the method in step 1).

The results showed that anti-Sema4D combined with anti-VEGF treatment inhibited fundus angiogenesis in OIR model mice, and the effect was significantly better than anti-Sema4D alone or anti-VEGF treatment alone (Fig. 6 E-F).

6) Evan's blue experiment (anti-Sema4D combined with anti-VEGF treatment, single injection)

After the 3-4 month old C57BL/6 mother mouse gave birth, put the pups together with the pups on the 7th day of birth in an oxygen chamber with 75% oxygen, and after the 12th day after birth, the pups and the mother mice were taken out, and then the pups were anesthetized Simultaneous injection of sema4d neutralizing antibody (2ug/eye) and VEGF neutralizing antibody (2ug/eye) in the vitreous of the mouse eye; or separate injection of anti-Sema4D (2ug), anti-VEGF (2ug), IgG (2ug), PBS (1ul); then perform Evans blue fluorescence experiment according to the method in step 2).

The results showed that anti-Sema4D combined with anti-VEGF treatment inhibited fundus vascular leakage in OIR model mice, and the effect was significantly better than anti-Sema4D alone or anti-VEGF treatment alone (Evan's blue experiment) (G in Figure 6)

7) HE staining experiment (anti-Sema4D combined with anti-VEGF treatment, single injection)

After the 3-4 month old C57BL/6 mother mouse gave birth, put the pups together with the pups on the 7th day of life in an oxygen chamber with 75% oxygen, and after the 12th day after birth, the pups and the mother mice were taken out, and then the pups were anesthetized Simultaneous injection of sema4d neutralizing antibody (2ug/eye) and VEGF neutralizing antibody (2ug/eye) in the vitreous of the mouse eye; or separate injection of anti-Sema4D (2ug), anti-VEGF (2ug), IgG (2ug), PBS (1ul); the mice were anesthetized on the 17th day of birth, and after cardiac perfusion with normal saline, their eyeballs were fixed in 4% paraformaldehyde, paraffin-embedded sections were stained with HE, observed under an optical microscope and counted the number of breakthroughs in the retina The number of vascular endothelial cells in the limiting membrane.

The results showed that anti-Sema4D combined with anti-VEGF treatment inhibited the number of preretinal neovascularization cells in OIR model mice, and the effect was significantly better than that of anti-Sema4D or anti-VEGF treatment alone (HE staining) (Figure 6H-I).

8) Evans blue fluorescence experiment on STZ model mice (anti-Sema4D combined with anti-VEGF treatment, single injection)

The STZ model mice were anesthetized 5 months after the model was established, and sema4d neutralizing antibody (2ug/eye) and VEGF neutralizing antibody (2ug/eye) were simultaneously injected into the vitreous of the mouse eye, or anti-Sema4D (2ug) was injected alone , anti-VEGF (2ug), IgG (2ug), PBS (1ul); one week after the injection, inject Evan's blue dye (45mg/kg) into the tail vein, anesthetize the mice after 2 hours, and perfuse the heart with normal saline, whichever The retina was stored in formamide (70°C water bath) overnight, and measured using a microplate reader (wavelength 620-740mm). The concentration is calculated according to the standard curve, and the retinal weight and retinal level in the blood are used for statistics after conversion. At the same time, a part of the eyeball was fixed in 4% PFA at room temperature for 2 hours, and then the retina was sliced and photographed under fluorescence.

The results showed that a single combined anti-Sema4D and anti-VEGF treatment inhibited fundus vascular leakage in STZ model mice, and the effect was significantly better than that of anti-Sema4D or anti-VEGF treatment alone.

Embodiment 7:

Repeated anti-Sema4D treatment can inhibit fundus vascular leakage in STZ model mice and is superior to anti-VEGF in inhibiting fundus vascular pericyte loss

1) Evans blue fluorescence experiment (STZ model mice were treated multiple times)

STZ model mice were simultaneously injected with sema4d neutralizing antibody (2ug/eye) and VEGF neutralizing antibody (2ug/eye) in the vitreous of the eye 4 months after the model was established, or injected with anti-Sema4D (2ug), anti-VEGF (2ug ), IgG (2ug), PBS (1ul) with injection of anti-Sema4D (2ug) or anti-VEGF (2ug) or IgG (2ug) or PBS (2ug) as a control, injected once a week, one week after the fifth injection Inject the Evans blue dye (45mg/kg) into the tail vein, anesthetize the mouse after circulating for 2 hours, and after the heart is perfused with normal saline, take the retina and place it in formamide (70°C water bath) overnight, and use a microplate reader (wavelength 620-740mm) Determination. The concentration is calculated according to the standard curve, and the retinal weight and retinal level in the blood are used for statistics after conversion. At the same time, a part of the eyeball was fixed in 4% PFA at room temperature for 2 hours, the retina was separated, and the slices were taken for fluorescence photography.

The results showed that multiple anti-Sema4D treatments were injected once a week, and one week after the fifth injection, it was found that it could inhibit the fundus vascular leakage of the STZ model. Multiple treatments with anti-Sema4D alone or anti-VEGF alone (A and B in Figure 7). (NO DM in the figure is the normal control group, the model group refers to the intraperitoneal injection of STZ group, and the normal control group refers to the intraperitoneal injection of citrate group.

2) Glycogen staining experiment (STZ model mice were treated multiple times)

STZ model mice were simultaneously injected with sema4d neutralizing antibody (2ug/eye) and VEGF neutralizing antibody (2ug/eye) in the vitreous of the eye 4 months after the model was established, or injected with anti-Sema4D (2ug), anti-VEGF (2ug ), IgG (2ug), PBS (1ul), injected once a week, a total of 5 injections, anesthetized after the fifth injection, and after heart perfusion with normal saline, fresh eyeballs were fixed in 4% PFA for 24 hours , and then the retina was isolated and digested with 3% trypsin (dissolved in tris-hcl, pH.7.8) at 37 degrees Celsius. When the retina began to disintegrate, the retina was then shaken until the vascular network was completely separated. The vascular network was transferred to a clean glass slide, dried and stained with glycogen, and photographed under a microscope to count the number of vascular non-irrigated areas. (NO DM in the figure is the normal control group)

The results showed that multiple treatments of anti-Sema4D could inhibit the formation of fundus vascular avascular zone in the STZ model, and the therapeutic effect of anti-Sema4D combined with anti-VEGF was significantly better than multiple treatments of anti-Sema4D or anti-VEGF alone (Figure 7 C and D).

3) Desmin immunofluorescence staining (STZ model mice treated multiple times)

STZ model mice were simultaneously injected with sema4d neutralizing antibody (2ug/eye) and VEGF neutralizing antibody (2ug/eye) in the vitreous of the eye 4 months after the model was established, or injected with anti-Sema4D (2ug), anti-VEGF (2ug ), IgG (2ug), PBS (1ul), injected once a week, a total of 5 injections, anesthetized after the fifth injection, and after heart perfusion with normal saline, fresh eyeballs were fixed in 4% PFA for 24 hours , and then the retina was isolated and digested with 3% trypsin (dissolved in tris-hcl, pH.7.8) at 37 degrees Celsius. When the retina began to disintegrate, the retina was then shaken until the vascular network was completely separated. Transfer the vascular network to a clean glass slide for Desmin immunofluorescent staining, and use a fluorescent microscope to take pictures for statistics. Desmin (1:100, Abcam).

The results showed that multiple times of anti-Sema4D treatment could inhibit the loss of pericytes on the fundus blood vessel surface of STZ model, and it could improve pericyte coverage better than that of anti-VEGF treatment, and the effect of multiple anti-Sema4D combined with anti-VEGF treatment was significantly better than that of multiple treatments. Anti-Sema4D or anti-VEGF treatment alone (E and F in Fig. 7).

4) Immunofluorescence staining to investigate the continuity of VE-cadherin in fundus vessels (STZ model mice were treated multiple times)

STZ model mice were simultaneously injected with sema4d neutralizing antibody (2ug/eye) and VEGF neutralizing antibody (2ug/eye) in the vitreous of the eye 4 months after the model was established, or injected with anti-Sema4D (2ug), anti-VEGF (2ug ), IgG (2ug), PBS (1ul), injected once a week, a total of 5 injections, anesthetized after the 5th injection, after heart perfusion with normal saline, fresh eyeballs were fixed in anhydrous methanol at -20°C for 2 hours, and then stripped The retina was removed, placed in blocking solution (1% BSA, 0.5% Triton X-100, PBS) overnight at 4°C, incubated with the primary antibody (VE-cadherin/Collagen IV) for 5 days, incubated with the secondary antibody at room temperature for 3 hours, and then photographed. Antibody: VE-cadherin (1:50, BD Biosciences), Collagen IV (1:100, Southern Biotech).

The results show that multiple anti-Sema4D treatments can increase the continuity of VE-cadherin in the fundus of the STZ model, and the effect of multiple treatments of anti-Sema4D combined with anti-VEGF is significantly better than multiple anti-Sema4D alone or multiple anti-Sema4D alone. - VEGF treatment (G-H in Fig. 7).

5) Immunofluorescence staining to examine the coverage of fundus vascular N-cadherin

STZ model mice were simultaneously injected with sema4d neutralizing antibody (2ug/eye) and VEGF neutralizing antibody (2ug/eye) in the vitreous of the eye 4 months after the model was established, or injected with anti-Sema4D (2ug), anti-VEGF (2ug ), IgG (2ug), PBS (1ul), injected once a week, a total of 5 injections, anesthetized after the 5th injection, after cardiac perfusion with normal saline, fresh eyeballs were fixed in 4% PFA at 4°C for 2h, peeled off Remove the retina, place it in blocking solution (1% BSA 0.5% Triton X-100, PBS), incubate the primary antibody (N-cadherin/Collagen IV) for 5 days after overnight at 4°C, incubate the secondary antibody at room temperature for 3 hours, and then take pictures . Antibodies: N-cadherin (1:50, Life Technologies), Collagen IV (1:100, Southern Biotech).

The results show that multiple anti-Sema4D treatments can increase the coverage of fundus vascular N-cadherin in the STZ model, and the effect is better than that of anti-VEGF alone, and the effect of anti-Sema4D combined with anti-VEGF is significantly better than that of anti-Sema4D alone Or anti-VEGF treatment alone. (I-J in Figure 7)

6) Evans blue fluorescence experiment

According to the experimental scheme in step 1), carry out the Evan's blue fluorescence experiment and prolong the observation time.

The results showed that repeated anti-Sema4D treatment and prolonging the observation time proved that it could inhibit the fundus vascular leakage of the STZ model after two weeks, and the effect was better than multiple anti-VEGF treatments. Treatment with anti-Sema4D or anti-VEGF alone (K-L in Figure 7).

Claims (3)

1.Sema4D inhibitor treats or prevents the application in optical fundus blood vessel leakage or pericyte depigmentation drug in preparation, described Sema4D inhibitor is the Sema4D neutralizing antibody that clone number is BMA-12.

2. application according to claim 1, the optical fundus blood vessel leakage or pericyte depigmentation are as caused by diabetes.

3. application according to claim 1 or 2, the drug contains the Sema4D inhibitor and anti-VEGF.