Use of angiopoietin receptor Tie-2 as a biomarker for the diagnosis of moyamoya disease
By detecting the expression of the angiopoietin receptor Tie-2, the differential diagnosis problem of smoke disease and intracranial atherosclerotic diseases is solved, and the accurate diagnosis and prognosis prediction of smoke disease is achieved, providing a non-invasive and reliable means to improve the early diagnosis and treatment effect.
Patent Information
- Application Number
- CN202211589865.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-12-12
- Publication Date
- 2025-06-27
- Estimated Expiration
- 2042-12-12
AI Technical Summary
The prior art is difficult to accurately diagnose smoke disease and intracranial atherosclerotic diseases in adults, especially in the early stages. The lack of non-invasive diagnostic methods affects the timely identification and treatment of the disease.
The diagnosis and prognosis prediction of fume disease are achieved by using the angiopoietin receptor Tie-2 as a biomarker by detecting its expression. Specific methods include detecting the levels of soluble Tie-2 protein in the patient's venous blood sample and determining the diagnostic and predicted cutoff values through ROC curve analysis.
It achieves accurate diagnosis and prognosis prediction of smoke disease, overcomes the subjectivity and traumaticity of traditional diagnostic methods, and provides a non-invasive and reliable means to help improve the early diagnosis and treatment effect of smoke disease.
Smart Images
Figure CN115792220B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of medicine and relates to the diagnosis of Moyamoya disease. Specifically, the present invention relates to the use of the angiopoietin receptor Tie-2 as a biomarker for the diagnosis of Moyamoya disease. Background Art
[0002] Moyamoya disease is a rare cerebrovascular disease characterized by the progressive stenosis or occlusion of the internal carotid artery (ICA) and its proximal branches within the circle of Willis, resulting in the formation of an abnormal vascular network at the base of the skull. According to epidemiological studies, in China, the incidence of Moyamoya disease is high in Henan, Anhui, and Hubei provinces (standardized incidence > 1 - 5 / 100,000 inhabitants / year). Currently, the results of cerebral angiography (DSA) are the gold standard for the diagnosis of Moyamoya disease. However, before diagnosing Moyamoya disease, cerebrovascular diseases with intracranial arteriosclerosis must be excluded. In the early stage of adult Moyamoya disease, the development of the moyamoya vessels is incomplete, and typical angiographic features may not be observed. Therefore, the differential diagnosis between adult Moyamoya disease and intracranial arteriosclerotic diseases is not straightforward, and new non-invasive methods are needed to assist in the differential diagnosis of the disease.
[0003] In short, Moyamoya disease has two major categories of clinical manifestations. One category is caused by cerebral ischemia, including transient ischemic attack (TIA), cerebral infarction, reversible ischemic neurological deficit (RIND), and seizure; the other category is the harmful consequences caused by the compensatory mechanism in response to ischemia, including the rupture and bleeding of fragile collateral vessels and microaneurysms, and headache caused by transdural vasodilation. If not treated promptly, these symptoms will lead to a high disability rate and mortality in patients. Unfortunately, the disease does not respond to any drug treatment. Surgical vascular reconstruction by direct or indirect bypass techniques is the preferred treatment method. The long-term effect of the surgery highly depends on the patient's own angiogenesis ability. Therefore, how to judge the patient's vascular reconstruction ability before surgery is of great significance for the treatment of Moyamoya disease.
[0004] Tie-2 is a member of the receptor tyrosine kinase family, expressed in vascular endothelial cells, and is the receptor of angiopoietin (Ang). 【1-3】 The Ang-Tie-2 signaling pathway is involved in vascular development and maturation, the balance and stability of the vascular microenvironment, and angiogenesis, etc. 【4】 Ang-1 binds to the Tie-2 receptor, phosphorylates it, and plays an activating role; Ang-2 can competitively bind to the Tie-2 receptor with Ang-1, inhibit the phosphorylation of Tie-2, and inactivate it. 【4】 In recent years, studies have shown that the content of sTie-2 is a marker for the malignancy of tumors and an evaluation index for tumor prognosis and treatment effect. 【5-7】; There are also studies showing that the level of sTie-2 in peripheral blood can also predict the prognosis of patients with acute coronary syndrome and congestive heart failure. 【8-9】 . SUMMARY OF THE INVENTION
[0005] The object of the present invention is to provide a new method for diagnosing moyamoya disease, which uses the angiopoietin receptor Tie-2 as a biomarker to achieve the clinical diagnosis and prognosis prediction of moyamoya disease by detecting its expression level.
[0006] To achieve the above object, the applicant recruited 36 patients diagnosed with moyamoya disease (MMD) and 10 patients with cerebrovascular disease (ACVD) caused by intracranial arteriosclerosis through routine examinations. Before surgery, venous blood was collected and the expression level of soluble Tie-2 was detected and analyzed. The results showed that there was low expression of Tie-2 protein in the serum of moyamoya disease patients, which was significantly different from that of the ACVD control group (P < 0.01). The ROC curve further indicated that Tie-2 had considerable diagnostic ability for moyamoya disease. The above test results showed that Tie-2 protein could be used as a biomarker for the diagnosis of moyamoya disease.
[0007] Furthermore, the applicant performed prognostic grading on 29 moyamoya disease patients during the 6-month follow-up after surgery and found that there was low expression of Tie-2 protein in the serum of moyamoya disease patients with good revascularization, which was significantly different from that of moyamoya disease patients with poor revascularization (P < 0.01). Through univariate and multivariate regression analyses, it was found that the Tie-2 level was a key factor affecting postoperative revascularization. The ROC curve further confirmed that Tie-2 had considerable predictive ability for the prognosis of moyamoya disease. The above test results showed that Tie-2 protein could be used as a biomarker for the prognosis prediction of moyamoya disease.
[0008] Therefore, the angiopoietin receptor Tie-2 can be used as a biomarker for the clinical diagnosis and prognosis prediction of moyamoya disease. On this basis, the applicant completed the following invention, that is, to provide the use of a reagent for detecting the angiopoietin receptor Tie-2 in the preparation of a diagnostic device for moyamoya disease.
[0009] The reagents for detecting the angiopoietin receptor Tie-2 include but are not limited to antibodies and probes. Among them, antibodies include monoclonal antibodies and polyclonal antibodies, and can detect antigens in the test sample through biological immune reactions; while probes detect target genes from the genome through the principle of molecular hybridization. Detecting known proteins or genes is a conventional technical means in the art, and there is no technical obstacle for those skilled in the art to design corresponding antibodies or probes for known sequences of antigens or genes. The object of the present invention is not to provide antibodies or probes for detecting Tie-2, and there are already records of detection antibodies or probes for Tie-2 in the prior art.
[0010] The instruments for diagnosing moyamoya disease include, but are not limited to, gene chips and test kits. The former contains probes for detecting target genes through molecular hybridization, and the latter contains antibodies for detecting target proteins through biological immune reactions. On the basis of obtaining the probes and antibodies, there is no technical obstacle for those skilled in the art to assemble the gene chips or test kits.
[0011] The present invention can diagnose moyamoya disease by detecting soluble Tie-2 in human peripheral blood, which causes less trauma to the human body and has high compliance.
[0012] The present invention can judge the diagnostic result through the cut-off value of the ROC curve. When the test result is lower than the cut-off value (2730 pg / ml), it is diagnosed as moyamoya disease and distinguished from cerebrovascular diseases with intracranial arteriosclerosis. The diagnostic result is accurate and reliable, overcoming the subjectivity of disease diagnosis.
[0013] The present invention identifies and screens differentially expressed factors between moyamoya disease and cerebrovascular diseases with intracranial arteriosclerosis through transcriptomics methods, realizing the rapid diagnosis of moyamoya disease. At the same time, it can effectively predict the revascularization effect after surgery for moyamoya disease patients, which has important value and clinical significance for reducing the occurrence of cerebrovascular events and improving the prognosis.
[0014] For more detailed technical solutions, please refer to the specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 are the protein contents of Ang-1, Ang-2, sTie-2, and VEGF in the sera of moyamoya disease patients in the embodiments.
[0016] Figure 2 is the ROC curve graph of diagnosing moyamoya disease with sTie-2 protein in the embodiments.
[0017] Figure 3 are the protein contents of Ang-1, Ang-2, sTie-2, and VEGF in the sera of patients with different revascularization effects after surgery in the embodiments.
[0018] Figure 4 is the ROC curve graph of using sTie-2 protein to judge the revascularization effect after surgery for moyamoya disease patients in the embodiments. DETAILED DESCRIPTION OF THE INVENTION
[0019] To make the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the scope of protection of the present invention. For the experimental methods without specific conditions noted in the embodiments, they are carried out according to the conditions recommended by the kit manufacturing company or according to conventional experimental conditions. Among them, ELISA technology uses the kits of Jingmei Company, product numbers: Ang-1 JM-03156H1, Ang-2 JM-03183H1, sTie-2 JM-5571H1, VEGF JM-03270H1.
[0020] Example 1
[0021] I. Test method
[0022] 1. Case inclusion
[0023] From March 2021 to November 2021, 36 inpatients with moyamoya disease (MMD) were recruited from the Department of Neurosurgery of Zhongnan Hospital of Wuhan University. During the same period, 10 patients with cerebrovascular diseases of intracranial arteriosclerosis (ACVD) were recruited from Zhongnan Hospital of Wuhan University as the control group.
[0024] 2. Exclusion criteria and diagnostic methods
[0025] Patients with a history of infectious, inflammatory, hematological, or cancerous diseases, or patients diagnosed with other cerebrovascular diseases with pathological angiogenesis, such as cerebral arteriovenous malformations, cavernous hemangiomas, or aneurysms, are not within the scope of this study. All MMD patients and ACVD control group received a comprehensive diagnostic work before surgery, including neurological examination, computed tomography (CT) or magnetic resonance imaging (MRI) scans, digital subtraction angiography (DSA), and functional regional cerebral blood flow measurement. Venous blood of the patients was collected, and the blood collection tube was gently inverted and mixed 5-10 times immediately after blood collection to ensure that the anticoagulant took effect. As much as possible, the blood sample was centrifuged at 1000 rpm at 4°C for 10 minutes after blood collection, the serum was separated and placed in a cryopreservation tube, and frozen at -80°C until ELISA analysis.
[0026] 3. Prognosis grading method
[0027] According to the DSA results of moyamoya disease patients 6 months after surgery, based on the Matsushima grading
[10] The patients were divided into a group with good revascularization (GOOD) and a group with poor revascularization (POOR). Among them, those with Matsushima grade A were classified into the group with good revascularization, and those with Matsushima grades B and C were classified into the group with poor revascularization.
[0028] 4. Detection method
[0029] ELISA detection: Commercialized kits for Ang-1, Ang-2, sTie-2, and VEGF were purchased and used for detection according to the instructions. Three replicates were set for each sample for quality control.
[0030] II. Test results
[0031] 1. The levels of Ang-1, Ang-2, sTie-2, and VEGF in serum were evaluated by ELISA. The results confirmed that there was low expression of sTie-2 protein in the serum of Moyamoya disease patients, which was significantly different from that in the ACVD control group (P < 0.01). However, there were no obvious differences in the other three markers, Ang-1, Ang-2, and VEGF, between the MMD and ACVD groups (P > 0.05)( Figure 1 ), suggesting that sTie-2 protein can be used as a new biomarker for the diagnosis of Moyamoya disease.
[0032] 2. The ROC curve was used to analyze the value of sTie-2 in diagnosing Moyamoya disease. The diagnostic ability of sTie-2 for Moyamoya disease (AUC 0.84, 95% CI 0.49 - 0.96, sensitivity 75.8%, specificity 80.0%, cut-off value 2730 pg / mL) was considerable( Figure 2 ), suggesting that sTie-2 can be used as a new diagnostic tool for Moyamoya disease.
[0033] 3. DSA data of 29 Moyamoya disease patients during the 6-month follow-up after surgery were collected. Among them, 16 had good revascularization effects and 13 had poor revascularization effects. The levels of Ang-1, Ang-2, sTie-2, and VEGF in serum before the test were evaluated. The results showed that there was low expression of sTie-2 protein in the serum of Moyamoya disease patients with good revascularization, which was significantly different from that in the POOR group with poor revascularization (P < 0.01)( Figure 3 ), suggesting that sTie-2 protein can be used as a biomarker for predicting the prognosis of Moyamoya disease.
[0034] 4. Through univariate analysis, it was found that age, Suzuki grading, and the level of peripheral blood sTie-2 were related to the revascularization effect (Table 1). Then, multivariate regression analysis was performed on these factors, and it was found that the factors significantly related to good revascularization were Suzuki grading and the level of peripheral blood sTie-2 (Table 2). The ROC curve was used to analyze the value of sTie-2 in predicting the prognosis of Moyamoya disease. The prognostic ability of sTie-2 for Moyamoya disease (AUC 0.83, 95% CI 0.49 - 0.96, sensitivity 93.8%, specificity 69.2%, cut-off value < 2512 pg / mL) was considerable (P < 0.01)(Figure 4 ), suggesting that sTie-2 can be used to predict the effect of postoperative revascularization.
[0035] Table 1. Comparison of baseline characteristics between the group with good postoperative revascularization and the group with poor revascularization
[0036]
[0037] Table 2. Multivariate logistic regression analysis of good postoperative revascularization
[0038]
[0039]
[0040] Explanation of related terms:
[0041] Moyamoya disease: Moyamoya disease is a cerebrovascular disease with unknown etiology, characterized by chronic progressive stenosis or occlusion at the terminal ends of bilateral internal carotid arteries and the origins of the anterior cerebral artery and middle cerebral artery, and secondary formation of abnormal vascular networks at the base of the skull. Since this abnormal vascular network at the base of the skull resembles "smoke" in cerebral angiography images, it is called "Moyamoya disease".
[0042] Tie-2: It is a tyrosine kinase receptor mainly expressed in vascular endothelial cells, and its ligand is angiopoietin. The amino acid sequence of the Tie-2 protein can be found in NP_000450. sTie-2 (soluble Tie-2) refers to the soluble protein in human peripheral blood.
[0043] Ang: Angiopoietins, a family of vascular growth factors, binds to the Tie receptor. This family mainly consists of four factors: Ang-1, Ang-2, Ang-3, and Ang-4. The Ang / Tie pathway plays an important role in regulating vascular stability, angiogenesis under physiological and pathological conditions, and inflammation.
[0044] Ang-1: It is a homohexamer composed of 498 amino acids. Ang-1 binds to Tie-2 to form a dimer, phosphorylating and activating the latter.
[0045] Ang-2: It consists of 496 amino acids and has 60% homology with Ang-1. Binding of Ang-2 to Tie-2 can inhibit the activity of the latter.
[0046] VEGF: Vascular endothelial growth factor, which has the functions of promoting increased vascular permeability, extracellular matrix degeneration, migration and proliferation of vascular endothelial cells, and blood vessel formation.
[0047] ELISA: Enzyme-linked immunosorbent assay, which is a qualitative and quantitative detection method that binds soluble antigens or antibodies to a solid-phase carrier and uses the specific binding of antigens and antibodies for immune reactions.
[0048] Revascularization: A medical term referring to the restoration of blood perfusion to narrowed or occluded arterial blood vessels through pharmacological or surgical means to restore blood supply to the corresponding ischemic organs for therapeutic purposes.
[0049] Related literature:
[0050] 1. Sato, T.N. et al. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature 376, 70 - 74, doi:10.1038 / 376070a0 (1995).
[0051] 2. Davis, S. et al. Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning. Cell 87, 1161 - 1169, doi:10.1016 / s0092 - 8674(00)81812 - 7 (1996).
[0052] 3. Maisonpierre, P.C. et al. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science (New York, N.Y.) 277, 55 - 60, doi:10.1126 / science.277.5322.55 (1997).
[0053] 4. Yancopoulos, G.D. et al. Vascular-specific growth factors and blood vessel formation. Nature 407, 242 - 248, doi:10.1038 / 35025215 (2000).
[0054] 5. Jayson, G. C. et al. Plasma Tie2 is a tumor vascular response biomarker for VEGF inhibitors in metastatic colorectal cancer. Nature communications 9, 4672, doi:10.1038 / s41467-018-07174-1 (2018).
[0055] 6. Zhou, C. et al. Systematic analysis of circulating soluble angiogenesis-associated proteins in ICON7 identifies Tie2 as a biomarker of vascular progression on bevacizumab. British journal of cancer 115, 228 - 235, doi:10.1038 / bjc.2016.194 (2016).
[0056] 7. Sopo, M. et al. High expression of Tie-2 predicts poor prognosis in primary high grade serous ovarian cancer. PloS one 15, e0241484, doi:10.1371 / journal.pone.0241484 (2020).
[0057] 8. Lee, K. W., Lip, G. Y. & Blann, A. D. Plasma angiopoietin-1, angiopoietin-2, angiopoietin receptor tie-2, and vascular endothelial growth factor levels in acute coronary syndromes. Circulation 110, 2355 - 2360, doi:10.1161 / 01.Cir.0000138112.90641.7f (2004).
[0058] 9. Chong, A.Y., Caine, G.J., Freestone, B., Blann, A.D. & Lip, G.Y. Plasma angiopoietin-1, angiopoietin-2, and angiopoietin receptor tie-2 levels in congestive heart failure. Journal of the American College of Cardiology 43, 423 - 428, doi:10.1016 / j.jacc.2003.08.042 (2004).
[0059] 10. Matsushima, T. et al. Surgical treatment of moyamoya disease in pediatric patients--comparison between the results of indirect and direct revascularization procedures. Neurosurgery 31, 401 - 405, doi:10.1227 / 00006123-199209000-00003 (1992).
[0060] The preferred embodiments of the present invention have been specifically described above. However, the present invention is not limited to the described embodiments. Those skilled in the art can also make various equivalent variations or substitutions without departing from the spirit of the present invention. These equivalent variations or substitutions are all included within the scope defined by the claims of this application.
Claims
1. Use of a reagent for detecting angiopoietin receptor Tie-2 in the preparation of a diagnostic instrument for differentiating moyamoya disease from intracranial arteriosclerotic cerebrovascular disease, characterized in that: Detecting the expression level of soluble angiopoietin receptor Tie-2 in human peripheral blood using the said instrument can effectively predict the revascularization effect after the operation for patients with moyamoya disease.
2. The application according to claim 1, wherein: The said reagent is an antibody against angiopoietin receptor Tie-2, and the said instrument is an ELISA kit.