Application of ADAM10 in occlusive cardiovascular and cerebrovascular diseases

Abstract

The present invention relates to the field of medicine, and in particular to the diagnosis and treatment of the establishment of collateral circulation in occlusive cardiovascular and cerebrovascular diseases. More specifically, the present invention relates to ADAM10 and its use as a biomarker and therapeutic target in the diagnosis of the establishment of collateral circulation in patients with vascular occlusion. The present invention provides the use of ADAM10 as a biological marker for the establishment of collateral circulation in patients with occlusive cardiovascular and cerebrovascular diseases, which can effectively predict the establishment of collateral circulation in patients with occlusive cardiovascular and cerebrovascular diseases and prevent the development of adverse cardiovascular events, which is of great value for clinical cardiovascular events, can improve the survival rate of patients with occlusive cardiovascular and cerebrovascular diseases and can improve long-term prognosis.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the diagnosis and treatment of collateral circulation establishment. More specifically, the present invention relates to the use of ADAM10 as a biomarker and therapeutic target in the diagnosis of collateral circulation establishment in patients with vascular occlusion. Background Art

[0002] Occlusive cardiovascular and cerebrovascular diseases have a common pathological basis. When the main blood supply vessels of patients are occluded, both nerve cells and myocardial cells will suffer irreversible damage, resulting in high mortality and disability rates. Many studies have shown that if a good collateral circulation can be formed after occlusion, it can not only effectively reduce the infarct area, maintain myocardial and cerebral blood perfusion, but also reduce the overall incidence of adverse cardiovascular events, while improving the patient's survival rate, quality of life, and prolonging the patient's survival time. Therefore, based on the concept of collateral circulation, "therapeutic angiogenesis" has emerged as a treatment method. Therefore, it is particularly important to find new therapeutic targets and biomarkers that can give exogenous pro-angiogenic factors to promote the formation of new blood vessels or the maturation of pre-existing blood vessels to bypass occluded blood vessels and restore organ blood supply.

[0003] A disintegrin and metalloproteinase 10 (ADAM10) is a member of the disintegrin metalloproteinase family. It is an important α-secretase that can trigger continuous transmembrane protein hydrolysis, regulate the transmission of intracellular signals, and regulate the activity of growth factors, adhesion molecules, receptors, and cytokines. It is expressed in endothelial cells, vascular smooth muscle cells, and leukocytes. Many substrates related to ADAM10, such as tumor necrosis factor α and chemokine CX3CL1, are related to the process of angiogenesis, development, inflammation, and regeneration. At present, the research on ADAM10 mainly focuses on neurological diseases such as Alzheimer's disease and lacunar cerebral infarction, as well as malignant tumors, and plays different roles in different diseases. For example, ADAM10 is highly expressed in oral squamous cell carcinoma tissues and is significantly associated with tumor metastasis. It is speculated that ADAM10 may increase angiogenesis and promote tumor metastasis by shearing vascular endothelial cadherin (VE-cadherin). In addition, in breast cancer, it was found that ADAM10 expression increased, which could promote cancer cell growth by cleaving human epidermal growth factor receptor 2. After applying the ADAM10 inhibitor GI254023X, it was found that cell proliferation in the inhibitor group decreased, angiogenesis decreased, apoptosis increased, and invasion and migration ability weakened. Other studies have shown that ADAM10 can reduce the release of inflammatory mediators by blocking the RAGE / NFκB signaling pathway, thereby reducing endothelial cell damage and improving the symptoms of chronic occlusive vasculitis. Other studies have found that after ADAM10 is silenced, VEGFR-2 protein expression in endothelial cells is reduced, inhibiting angiogenesis in pancreatic cancer; after porcine arterial endothelial cells are stimulated by VEGF, ADAM10 expression is upregulated to promote increased angiogenesis. Summary of the invention

[0004] The present invention provides the application of ADAM10 protein as a biomarker and therapeutic target for the formation of collateral circulation in occlusive cardiovascular and cerebrovascular diseases.

[0005] The technical solution of the present invention is: the application of ADAM10 as a biomarker for promoting collateral circulation in patients with occlusive cardiovascular and cerebrovascular diseases. In patients with chronic total occlusion (chronic total occlusion), ADAM10 protein is significantly upregulated in patients with well-established collateral circulation, which provides the application of ADAM10 protein as a collateral circulation biomarker; and the expression level of ADAM10 protein is correlated with the prognosis of patients with occlusive vascular diseases, and it is found that patients with high ADAM10 protein expression have better prognosis than patients with low expression.

[0006] This indicates that ADAM10 can not only be used as a biomarker for early prediction of the establishment of collateral circulation, but also as a biomarker for predicting the long-term prognosis of patients with occlusive vascular disease.

[0007] The second technical solution provided by the present invention is: the application of ADAM10 as a therapeutic target for promoting the establishment of collateral circulation. First, we found that when exogenous ADAM10 protein was given to mice with lower limb vascular occlusion, it was found that it could promote collateral circulation and blood supply in the lower limbs of mice.

[0008] At the same time, the present invention is based on in vitro research that adeno-associated virus overexpression can significantly promote endothelial cell proliferation, migration and angiogenesis, which can indicate that increased expression of ADAM10 protein can significantly promote angiogenesis.

[0009] ADAM10 promoted an increase in the density of microvessels (CD31-MDV) at the histological level, while the number of new blood vessels did not increase significantly. It is speculated that ADAM10 may increase blood flow by increasing shear force to open collateral circulation. Based on this, it may provide a new target for new drugs to promote the opening of collateral circulation in patients with vascular occlusion.

[0010] The beneficial technical effect of the present invention is: the present invention provides the application of ADAM10 as a biological marker for the establishment of collateral circulation in patients with occlusive vascular disease, which can effectively predict the establishment of collateral circulation in patients with occlusive vascular disease and prevent the development of adverse cardiovascular events, which is of great value for clinical cardiovascular events, can improve the survival rate of patients with occlusive vascular disease and can improve long-term prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] 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 will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0012] Figure 1 ADAM10 can be used as a biomarker to predict the establishment of collateral circulation and patient prognosis in patients with occlusive vascular disease. ADAM10 expression is higher in patients with good collateral circulation.

[0013] Figure 2 ADAM10 can be used as a biomarker to predict the establishment of collateral circulation;

[0014] Figure 3 Patients with high ADAM10 expression had a better prognosis;

[0015] Figure 4In the mouse lower limb femoral artery occlusion model, injection of ADAM10 recombinant protein can promote the formation of collateral circulation and blood perfusion in the lower limbs. (Number of mice N = 8)

[0016] Figure 5 At the tissue level, after ADAM10 injection, the number of new blood vessels increased, the vascular density increased, and the collateral circulation was more open;

[0017] Figure 6 Overexpression of ADAM10 in human umbilical vein endothelial cells promotes endothelial cell proliferation. DETAILED DESCRIPTION

[0018] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Example 1

[0020] 1. In the embodiment, patients who were diagnosed with occlusive vascular disease by coronary angiography were selected and divided into a good collateral circulation group and a poor collateral circulation group during intraoperative diagnosis. Serum samples were retained before opening, and the expression of ADAM10 was measured by double antibody sandwich ELISA method. The results showed that the expression of ADAM10 in the good collateral circulation group was significantly higher than that in the poor collateral circulation group ( Figure 1 ).

[0021] 2. In addition, the receiver operating curve showed that ADAM10 can well predict collateral circulation ( Figure 2 ), indicating that this protein can enrich the study of the pathological mechanism of collateral circulation generation in occlusive patients and can be used for early diagnosis without intervention.

[0022] 3. Finally, we followed up the patients and found that after at least 12 months of follow-up, the incidence of adverse cardiovascular events in the group with elevated ADAM10 protein was significantly lower than that in the group with low ADAM10 protein expression ( Figure 3 ). In summary, ADAM10 protein can be used as a therapeutic target to promote collateral circulation, providing a theoretical basis for clinical treatment and drug development for patients with vascular occlusion.

[0023] Example 2

[0024] 1. This example uses male 8-12 week old C57BL / 6J mice. After 1.25% isoflurane induced anesthesia, the mice were fixed on the operating table. The bilateral lower limbs were depilated with a depilatory, the residual depilatory was washed with water, iodine was disinfected, alcohol was deiodinated, and a sterile surgical sheet was laid. A longitudinal incision of about 2-4 mm long was made from the midpoint of the inguinal ligament to the inner side of the knee, and the subcutaneous tissue was bluntly separated to expose the femoral nerve vascular sheath and its branches. The external iliac artery and femoral artery were separated with a glass needle under a dissecting microscope. The upper end of the external iliac artery and the femoral artery were ligated with No. 6 silk thread, and the skin was sutured with No. 4 surgical sutures. The right femoral artery was sham-operated, and only the femoral artery and vein were separated without ligation, and the skin was sutured with No. 4 surgical sutures. A mouse lower limb unilateral limb ischemia model was established. The experimental group and the control group were matched in age. 0.5 ml of 5 mg / ml antibiotics were injected intraperitoneally, and the mice were then placed in a 37°C incubator until they woke up.

[0025] Laser Doppler blood perfusion was measured after surgery. The criteria for judgment were: the blood velocity after surgery was reduced to less than 10% of the preoperative value, and the blood flow spectrum changed significantly, which was considered a successful model. After the model was successfully established, the experimental group mice were given intraperitoneal injection of ADAM10 recombinant protein intervention model: ADAM10 was 6ug / kg, once a day, for 14 days.

[0026] The results showed that the blood perfusion of the affected limbs of the experimental group of mice was significantly higher than that of the control group starting from the 7th day. The blood perfusion increased further on the 7th, 14th, and 21st days, and was still higher than that of the control group. Figure 4 )

[0027] 2. After 21 days, the mice were deeply anesthetized and then killed by cervical dislocation. The adductor muscles and gastrocnemius muscles were removed (the gastrocnemius and intimal muscles are the main muscles of the mouse lower limbs, which are mainly supplied by the deep femoral artery. When the femoral artery and its branches are ligated, these two muscles will be ischemic). The formation of collateral circulation was evaluated by pathology ( Figure 5 ).

[0028] (1) HE staining;

[0029] (2) Immunohistochemistry: CD31 to calculate microvessel density;

[0030] (3) Immunofluorescence: CD31 / Ki67 double staining to calculate the density of new blood vessels;

[0031] The results showed that: first, in HE staining, the experimental group had more blood vessels than the control group; in immunohistochemistry, the microvascular density (CD31 positive) in the experimental group was significantly higher than that in the control group. In double-stained immunofluorescence, the Ki67 positive in the experimental group was higher than that in the control group.

[0032] Example 3

[0033] 1. First, an adeno-associated virus-based overexpression plasmid of ADAM10 was constructed through genetic engineering, and the vector was transfected into human umbilical vein endothelial cells (HUVEC) to promote the overexpression of ADAM10 protein level, so as to study the mechanism by which ADAM10 protein regulates endothelial cell angiogenesis in vitro.

[0034] 2. After the cell model is successfully constructed, the cell proliferation, migration and angiogenesis phenotypes are tested respectively;

[0035] The results showed that compared with the control group, overexpression of ADAM10 promoted endothelial cell proliferation, migration and angiogenesis ( Figure 6 ).

[0036] In summary, the above combined with the accompanying drawings has made a detailed and multi-dimensional description of the embodiments of the present invention: ADAM10 protein can promote the formation of collateral circulation, is a potential therapeutic target for angiogenesis, and provides a theoretical basis for the clinical treatment and drug development of patients with vascular occlusion.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. Application of ADAM10 protein in the preparation of biomarkers for the establishment of collateral circulation in patients with occlusive cardiovascular and cerebrovascular diseases.

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