Application of CCN2 as therapeutic target in preparation of medicine for preventing and / or treating anti-phospholipid syndrome

Abstract

The invention relates to the technical field of biomedicine, in particular to application of CCN2 as a therapeutic target to preparation of a medicine for preventing and / or treating anti-phospholipid syndrome. The invention provides an application of CCN2 as a therapeutic target in preparation of drugs for preventing and / or treating antiphospholipid syndromes and an establishment method of an APS-related vasculopathy model. It is found for the first time that the CCN2 target spot in the APS blood vessel plays a crucial role in promoting communication between vascular endothelial cells and vascular smooth muscle cells, a new view angle is provided for knowing cell and molecular mechanisms of APS vasculopathy, and it is determined that the CCN2 is a potential target spot for clinical anti-proliferation treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of CCN2 as a therapeutic target in the preparation of a drug for preventing and / or treating antiphospholipid syndrome. Background Art

[0002] Antiphospholipid syndrome (APS) is an acquired autoimmune disease with thrombosis and late pregnancy loss as its main manifestations, with an incidence of approximately 1:2000. Although APS is usually associated with acute thrombotic events such as stroke, deep vein thrombosis or large vessel pulmonary embolism, some APS patients also suffer from chronic organ damage due to occlusive neointimal formation in small vessels (i.e., APS vasculopathy). Chronic vasculopathy is a serious vascular event of antiphospholipid syndrome, manifested as arterial stenosis and occlusion leading to multiple organ dysfunction. Its core pathological changes are the proliferation of endothelial cells and vascular smooth muscle cells, leading to hyperplasia, stenosis and occlusion of the arterial intima. Vascular lesions can affect multiple important organs throughout the body, such as the heart, brain, and kidneys, leading to multiple organ dysfunction. There is still a large knowledge gap about the molecular pathways of proliferative lesions in APS vasculopathy. In terms of treatment, lifelong anticoagulation is the main treatment for APS, which can prevent the occurrence of large vessel events to a certain extent. However, its efficacy in preventing small vessel lesions and long-term organ deterioration is still limited. There is currently a lack of effective treatment for APS vascular lesions, especially for chronic occlusive vascular lesions of the kidneys, skin, and nervous system and the corresponding organ dysfunction. Only in-depth mechanism research can propose more effective treatments.

[0003] In addition, in the study of APS, the existing animal models mainly study APS-related thrombosis, and lack a special animal model of APS-related vascular disease. Therefore, the establishment of an animal model of APS-related vascular disease is crucial for subsequent in-depth mechanism research. Summary of the invention

[0004] In view of this, the present invention provides the use of CCN2 as a therapeutic target in the preparation of a drug for preventing and / or treating antiphospholipid syndrome. The present invention first discovered that the CCN2 target in APS blood vessels plays a vital role in promoting the communication between vascular endothelial cells and vascular smooth muscle cells, providing a new perspective for understanding the cellular and molecular mechanisms of APS vascular lesions, and identifying CCN2 as a potential target for clinical antiproliferative therapy.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides the use of CCN2 as a therapeutic target in the preparation of a drug for preventing and / or treating antiphospholipid syndrome.

[0007] In some embodiments of the invention, the prevention and / or treatment comprises inhibiting intimal thickening.

[0008] In some specific embodiments of the invention, the inhibiting of intimal thickening comprises reducing the average cross-sectional thickness and / or cross-sectional area of ​​the femoral artery.

[0009] The present invention also provides the use of the CCN2 inhibitor in preparing a drug for preventing and / or treating antiphospholipid syndrome.

[0010] In some specific embodiments of the present invention, the CCN2 inhibitor comprises an anti-CCN2 monoclonal antibody.

[0011] In some specific embodiments of the present invention, the heavy chain of the anti-CCN2 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:1; and the light chain of the anti-CCN2 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:2.

[0012] The present invention also provides a drug for preventing and / or treating antiphospholipid syndrome, comprising a CCN2 inhibitor.

[0013] In some specific embodiments of the present invention, the CCN2 inhibitor includes any of the following:

[0014] (I) substances that inhibit CCN2 activity; and / or

[0015] (II) a substance that knocks out or knocks down CCN2; and / or

[0016] (III) A substance that increases the level or activity of a CCN2 inhibitor.

[0017] The present invention also provides a method for establishing an antiphospholipid syndrome-related vascular disease model, comprising intraperitoneal injection of APS IgG into an animal to obtain the antiphospholipid syndrome-related vascular disease model.

[0018] In some specific embodiments of the present invention, before the step of intraperitoneal injection of APS IgG into the animal, the step further includes making an arteriotomy at the branch point of the deep femoral artery and the superficial femoral artery, dilating the arteriotomy and inserting a 0.015-inch guide wire, and then pushing it forward 5-~10 mm toward the iliac artery, staying for 3 minutes, removing the guide wire, suturing the blood vessel wall and restoring blood flow.

[0019] In some specific embodiments of the present invention, the injection amount of APS IgG comprises 20 mg / kg.

[0020] In some specific embodiments of the invention, the animal comprises a C57BL6 / J mouse.

[0021] In some specific embodiments of the present invention, the frequency of intraperitoneal injection includes once every 3 days.

[0022] In some specific embodiments of the present invention, the APS-related vascular disease model is obtained after 2 weeks.

[0023] The present invention also provides an APS-related vascular disease model, which is obtained by using the establishment method.

[0024] The present invention includes but is not limited to providing the following beneficial effects:

[0025] The present invention provides the use of CCN2 as a therapeutic target in the preparation of drugs for preventing and / or treating antiphospholipid syndrome and a method for establishing an APS-related vascular lesion model. The present invention first discovered that the CCN2 target in APS blood vessels plays a vital role in promoting the communication between vascular endothelial cells and vascular smooth muscle cells, providing a new perspective for understanding the cellular and molecular mechanisms of APS vascular lesions and identifying CCN2 as a potential target for clinical antiproliferative therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0027] Figure 1 The APS-related mouse vascular proliferation model provided in the examples of the present application is shown. The mice were subjected to femoral artery guidewire injury at 10 weeks of age. APS-IgG was injected starting from the first day after surgery, once every three days, and blood vessels were collected for analysis 2 or 4 weeks after injury.

[0028] Figure 2 The figure shows the vascular proliferation of mice 2 weeks or 4 weeks after guidewire injury; A is a representative HE staining picture of blood vessels in each group of mice; B is a statistical chart of vascular proliferation in each group of mice, using one-way ANOVA, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, **** represents P < 0.0001;

[0029] Figure 3 Schematic diagram of the experimental scheme for treating APS mice with femoral artery injury with anti-CCN2 monoclonal antibodies; mice underwent femoral artery guidewire injury at 10 weeks of age, and APS-IgG was injected on the first day after surgery. Anti-CCN2 monoclonal antibodies or an equal dose of PBS were injected intraperitoneally every three days, and blood vessels were collected for analysis 2 weeks after injury;

[0030] Figure 4Figure 4 shows the vascular proliferation of APS mice with femoral artery injury treated with anti-CCN2 monoclonal antibody; A is a representative HE staining image of blood vessels in each group of mice; B is a statistical graph of vascular proliferation in each group of mice, using one-way ANOVA, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, **** represents P < 0.0001. DETAILED DESCRIPTION

[0031] The present invention discloses the use of CCN2 as a therapeutic target in the preparation of a drug for preventing and / or treating antiphospholipid syndrome. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0032] The present invention establishes an APS-related vascular lesion model based on a conventional femoral artery guidewire injury model, which replicates the CCN2-mediated vascular proliferative lesions after administration of APS IgG. At the same time, we also confirmed that in this new model, the thickening of the APS vascular endothelium can be inhibited by using a monoclonal antibody targeting CCN2.

[0033] The monoclonal antibody sequence information is as follows:

[0034] >CLNHv (SEQ ID NO:1)

[0035] QLVQSGGGLVHPGGSLRLSCAGSGFTFSSYGMHWVRQAPGKGLEWVSSGIGTGGGTYSTDSVKGRFTISRDNAKNSLYLQMNSLRAEDMAVYYCARGDYYGSGSFFDCWGQGTLVTVSS

[0036] >CLNLv (SEQ ID NO:2)

[0037] DIQMTQSPSSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTFGQGTKLEIK

[0038] In summary, the present invention discovered for the first time that the CCN2 target in APS vessels plays a vital role in promoting the communication between vascular endothelial cells and vascular smooth muscle cells, providing a new perspective for understanding the cellular and molecular mechanisms of APS vascular lesions and identifying CCN2 as a potential target for clinical anti-proliferative therapy.

[0039] Unless otherwise specified, the raw materials and reagents used in the application of CCN2 provided by the present invention as a therapeutic target in the preparation of a drug for preventing and / or treating antiphospholipid syndrome can be purchased from the market.

[0040] The present invention will be further described below in conjunction with embodiments:

[0041] Example 1 Construction of a femoral artery guidewire injury vascular proliferation model in mice

[0042] This example provides the construction of a vascular proliferation model in mice with femoral artery guidewire injury after administration of APS IgG.

[0043] Twenty male C57BL6 / J mice aged more than 10 weeks and weighing more than 25 g were selected. After one week of adaptive feeding, femoral artery guidewire injury was performed. Mice were anesthetized with 2% isoflurane gas and placed on a heating pad to maintain core temperature. Under a microscope, veins and connective tissues around the artery were carefully removed with microsurgical forceps to isolate the femoral artery. An arterial incision was made with microscissors at the branch point of the deep femoral artery and the superficial femoral artery. The incision was expanded and a 0.015-inch guidewire was inserted, which was then pushed forward 5-10 mm toward the iliac artery and stayed for 3 minutes. After the guidewire was removed, the blood flow was restored after suturing the vascular wall with 11-0 nylon thread. APS IgG or control IgG purified from the plasma of APS patients or healthy people (2.5 mg / ml, 20 mg / kg) was injected intraperitoneally every 3 days starting on the first day after surgery. Ten mice were given the drug for 2 consecutive weeks, and the other 10 mice were given the drug for 4 consecutive weeks. After 2 or 4 weeks, the mice were euthanized and immediately perfused with 4% paraformaldehyde (PFA) through the left ventricle for 10 minutes, and then the injured left femoral artery was collected for analysis, and the uninjured femoral artery was used as a control group. Figure 1 The mean cross-sectional thickness and cross-sectional area of ​​the mouse femoral artery were quantified using the analysis module of Image Pro Plus 6.0 software.

[0044] The results of observation and statistical analysis are as follows Figure 2 As shown. A is a representative HE staining picture of blood vessels in each group of mice; B is a statistical chart of vascular proliferation in each group of mice, using one-way ANOVA, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, **** represents P < 0.0001. Figure 2It can be seen that after the guidewire injured the femoral artery, causing endothelial injury and activation in mice, the mice were injected with control IgG or APS IgG. Compared with the injured blood vessels of mice receiving control IgG, the injured blood vessels of mice receiving APS IgG showed more obvious vascular proliferation at 2 weeks and 4 weeks. The average cross-sectional thickness and cross-sectional area of ​​the blood vessels of APS IgG mice were significantly greater than those of control IgG group mice (P< 0.01).

[0045] Example 2 Pamrevlumab in the treatment of femoral artery guidewire injury mouse vascular proliferation model

[0046] This example provides the construction of a vascular proliferation model in mice with femoral artery guidewire injury after administration of APS IgG.

[0047] Fifteen male C57BL6 / J mice aged more than 10 weeks and weighing more than 25 g were selected. After one week of adaptive feeding, the femoral artery guidewire injury was performed. Mice were anesthetized with 2% isoflurane gas and placed on a heating pad to maintain core temperature. Under a microscope, the veins and connective tissues around the artery were carefully removed with microsurgical forceps to isolate the femoral artery. An arterial incision was made with microscissors at the branch point of the deep femoral artery and the superficial femoral artery. The incision was expanded and a 0.015-inch guidewire was inserted, which was then pushed forward 5-10 mm toward the iliac artery and stayed for 3 minutes. After the guidewire was removed, the blood flow was restored after suturing the vascular wall with 11-0 nylon thread. APS IgG or control IgG purified from the plasma of APS patients or healthy people (2.5 mg / ml, 20 mg / kg) was injected intraperitoneally every 3 days starting on the first day after surgery. The mice treated with APS IgG were injected intraperitoneally with anti-CCN2 monoclonal antibody (Beijing Kono Xincheng Technology Co., Ltd., US20190127456A1) (10 mg / kg) or an equal volume of PBS once every 3 days. After 2 weeks of continuous administration, the mice were euthanized and immediately perfused with 4% paraformaldehyde (PFA) through the left ventricle for 10 minutes, and then the injured left femoral artery was collected for analysis. The implementation plan is as follows Figure 3 The mean cross-sectional thickness and cross-sectional area of ​​the mouse femoral artery were quantified using the analysis module of ImagePro Plus 6.0 software.

[0048] The results of observation and statistical analysis are as follows Figure 4 As shown. A is a representative HE staining picture of blood vessels in each group of mice; B is a statistical chart of vascular proliferation in each group of mice, using one-way ANOVA, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, **** represents P < 0.0001. Figure 4It can be seen that compared with APS IgG-treated mice receiving PBS, the average cross-sectional thickness and cross-sectional area of ​​blood vessels in APS IgG-treated mice treated with anti-CCN2 monoclonal antibody were significantly smaller than those in the PBS group mice (P < 0.01).

[0049] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of CCN2 as a therapeutic target in the preparation of drugs for preventing and / or treating antiphospholipid syndrome.

2. The use according to claim 1, characterized in that The prevention and / or treatment includes inhibiting vascular intimal thickening.

3. The use according to claim 1 or 2, characterized in that: The inhibiting of intimal thickening includes reducing the average cross-sectional thickness and / or cross-sectional area of ​​the femoral artery.

4. Use of CCN2 inhibitors in the preparation of drugs for preventing and / or treating antiphospholipid syndrome.

5. The use according to claim 4, characterized in that The CCN2 inhibitor includes anti-CCN2 monoclonal antibodies.

6. A drug for preventing and / or treating antiphospholipid syndrome, characterized in that: Including CCN2 inhibitors.

7. The drug according to claim 6, characterized in that The CCN2 inhibitor includes any of the following: (I) substances that inhibit CCN2 activity; and / or (II) a substance that knocks out or knocks down CCN2; and / or (III) A substance that increases the level or activity of a CCN2 inhibitor.

8. A method for establishing a model of antiphospholipid syndrome-related vascular disease, characterized in that: The method comprises injecting APSIgG into the animal intraperitoneally to obtain the antiphospholipid syndrome-related vascular disease model.

9. The method of establishing as claimed in claim 8, characterized in that: Prior to the step of intraperitoneal injection of APS IgG into the animal, an arterial incision is made at the branch point of the deep femoral artery and the superficial femoral artery, the arterial incision is dilated and a 0.015-inch guide wire is inserted, which is then pushed forward 5 to 10 mm toward the iliac artery and left there for 3 minutes. After the guide wire is removed, the blood vessel wall is sutured and blood flow is restored.

10. The establishment method according to claim 8 or 9, characterized in that: The injection amount of the APS IgG included 20 mg / kg.

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