Molecular diagnosis and intervention method for early warning of vascular rupture

Abstract

The present invention provides fibrin and / or fibrinogen as a biomarker of vascular rupture. Specifically, the invention provides application of detection reagents and / or visual reagents of fibrin and / or fibrinogen, and all subtypes, degradation products, compounds and functional fragments thereof in early warning of vascular rupture. Also provided are uses of down-regulating agents of fibrin and / or fibrinogen in prevention of vascular rupture and uses of intervention agents or instruments thereof in treatment of vascular rupture related diseases. The invention also provides a kit and a method for indicating and preventing the risk of vascular rupture before the occurrence of vascular rupture.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a molecular diagnostic and intervention reagent for early warning of blood vessel rupture, as well as methods for preventing and intervening in blood vessel rupture. Background Technology

[0002] Ruptured blood vessels are a major manifestation of hemorrhagic diseases. Ruptured cerebral blood vessels are a manifestation of hemorrhagic stroke. Ruptured aorta is a manifestation of abdominal aortic aneurysm and aortic dissection. All organs of the body may bleed due to ruptured blood vessels, which may even endanger life.

[0003] Current technologies provide diagnosis after a blood vessel ruptures, but cannot provide early warning monitoring. However, blood vessel rupture is characterized by rapid onset and rapid progression, and existing methods such as angiography often cannot provide early warning when there is a risk of blood vessel rupture.

[0004] Furthermore, ruptured blood vessels can endanger a patient's life and lead to subsequent disability, significantly reducing their quality of life. Effective and timely intervention for patients with ruptured blood vessels is therefore of great importance.

[0005] Therefore, there is an urgent need to provide a biomarker for vascular rupture so that the risk of vascular rupture can be detected during routine physical examinations and other procedures, and timely and effective intervention can be carried out. Summary of the Invention

[0006] The purpose of this invention is to provide a biomarker for vascular rupture, and based on this biomarker, to provide preventive drugs for vascular rupture, and based on this biomarker, to provide ideas and methods for the diagnosis and treatment of diseases related to vascular rupture.

[0007] In a first aspect of the invention, there is provided the use of a detection reagent for fibrin and / or fibrinogen for preparing a composition or formulation for assessing the risk of vascular rupture in a subject.

[0008] In another preferred embodiment, the fibrin and / or fibrinogen refers to fibrin and / or fibrinogen distributed on the blood vessel wall.

[0009] In another preferred embodiment, the fibrin and / or fibrinogen refers to fibrin and / or fibrinogen distributed in the blood vessel wall.

[0010] In another preferred embodiment, the blood vessels include: cardiovascular vessels, cerebral vessels, peripheral vessels, or combinations thereof.

[0011] In another preferred embodiment, the blood vessels include coronary arteries, cerebral arteries, aortic vessels, or combinations thereof.

[0012] In another preferred embodiment, the blood vessel is the aorta.

[0013] In another preferred embodiment, the blood vessel is an ascending aorta.

[0014] In another preferred embodiment, the blood vessel is the descending aorta.

[0015] In another preferred embodiment, the ruptured blood vessel is an acute ruptured blood vessel.

[0016] In another preferred embodiment, the ruptured blood vessel is a chronic ruptured blood vessel.

[0017] In another preferred embodiment, the fibrin and / or fibrinogen comprises: different fibrin and / or fibrinogen subtypes, degradation products of fibrin and / or fibrinogen, functional fragments of fibrin and / or fibrinogen, or combinations thereof.

[0018] In a second aspect of the invention, there is provided the use of a reagent for visualizing fibrin and / or fibrinogen for preparing a composition or formulation for assessing the risk of vascular rupture in a subject.

[0019] In another preferred embodiment, the fibrin and / or fibrinogen refers to fibrin and / or fibrinogen distributed on the blood vessel wall.

[0020] In another preferred embodiment, the fibrin and / or fibrinogen refers to fibrin and / or fibrinogen distributed in the blood vessel wall.

[0021] In another preferred embodiment, the blood vessels include: cardiovascular vessels, cerebral vessels, peripheral vessels, or combinations thereof.

[0022] In another preferred embodiment, the blood vessels include coronary arteries, aortas, cerebral arteries, or combinations thereof.

[0023] In another preferred embodiment, the blood vessel is the aorta.

[0024] In another preferred embodiment, the blood vessel is an ascending aorta.

[0025] In another preferred embodiment, the blood vessel is the descending aorta.

[0026] In a third aspect of the invention, there is provided the use of a therapeutic agent of fibrin and / or fibrinogen for preparing a medicament or combination of medicaments for preventing vascular rupture in a subject.

[0027] In another preferred embodiment, the blood vessels include: cardiovascular vessels, cerebral vessels, peripheral vessels, or combinations thereof.

[0028] In another preferred embodiment, the blood vessels include coronary arteries, aortas, cerebral arteries, or combinations thereof.

[0029] In another preferred embodiment, the blood vessel is the aorta.

[0030] In another preferred embodiment, the blood vessel is an ascending aorta.

[0031] In another preferred embodiment, the blood vessel is the descending aorta.

[0032] In another preferred embodiment, the therapeutic agent for fibrin and / or fibrinogen includes: an agent that downregulates the expression level of fibrin and / or fibrinogen, an agent that degrades fibrin and / or fibrinogen, an agent that interferes with fibrin and / or fibrinogen, an agent that blocks the activity of fibrin and / or fibrinogen, or a combination thereof.

[0033] In another preferred embodiment, the reagent that intervenes in fibrin and / or fibrinogen is a reagent that negatively intervenes in the activity of fibrin and / or fibrinogen.

[0034] In another preferred embodiment, the negative intervention refers to downregulating the activity of fibrin and / or fibrinogen, or binding fibrin and / or fibrinogen to inhibit their function.

[0035] In another preferred embodiment, the reagent includes: a biological drug, a compound drug, a herbal drug, or a combination thereof.

[0036] In another preferred embodiment, the reagent is selected from the group consisting of batroxobin, snake venom drugs, fibrinolytic enzymes for injection, gene editing reagents, sodium valproate, antibodies, or combinations thereof.

[0037] In another preferred embodiment, the snake venom drug is snake venom hemagglutinin.

[0038] In another preferred embodiment, the reagent is batroxobin.

[0039] In another preferred embodiment, the prevention includes: preventing blood vessel rupture, delaying blood vessel rupture, or a combination thereof.

[0040] In another preferred embodiment, the ruptured blood vessel is an acute ruptured blood vessel.

[0041] In another preferred embodiment, the ruptured blood vessel is a chronic ruptured blood vessel.

[0042] In another preferred embodiment, the therapeutic agent is an agent that significantly reduces the expression levels of fibrin and / or fibrinogen γ chains.

[0043] In another preferred embodiment, "significantly reduced" means that the ratio M1 / M0 of the expression level M1 of fibrin and / or fibrinogen γ chain in the test subject after administration to the expression level M0 of fibrin and / or fibrinogen γ chain in the test subject before administration is ≤0.5, preferably ≤0.25, and most preferably ≤0.1.

[0044] In a fourth aspect of the invention, a pharmaceutical combination for preventing and / or treating ruptured blood vessels is provided, the pharmaceutical combination comprising:

[0045] (i) a therapeutic component comprising a therapeutic agent of fibrin and / or fibrinogen, and

[0046] (ii) a detection component comprising a detection reagent and / or a visualization reagent for fibrin and / or fibrinogen.

[0047] In another preferred embodiment, the drug combination also includes pharmaceutically acceptable excipients.

[0048] In another preferred embodiment, the blood vessels include: cardiovascular vessels, cerebral vessels, peripheral vessels, or combinations thereof.

[0049] In another preferred embodiment, the blood vessels include coronary arteries, aortas, cerebral arteries, or combinations thereof.

[0050] In another preferred embodiment, the blood vessel is the aorta.

[0051] In another preferred embodiment, the blood vessel is an ascending aorta.

[0052] In another preferred embodiment, the blood vessel is the descending aorta.

[0053] In another preferred embodiment, the therapeutic agent for fibrin and / or fibrinogen includes: an agent that downregulates the expression level of fibrin and / or fibrinogen, an agent that degrades fibrin and / or fibrinogen, an agent that interferes with fibrin and / or fibrinogen, an agent that blocks the activity of fibrin and / or fibrinogen, or a combination thereof.

[0054] In another preferred embodiment, the reagent that intervenes in fibrin and / or fibrinogen is a reagent that negatively intervenes in the activity of fibrin and / or fibrinogen.

[0055] In another preferred embodiment, the negative intervention refers to downregulating the activity of fibrin and / or fibrinogen, or binding fibrin and / or fibrinogen to inhibit their function.

[0056] In another preferred embodiment, the therapeutic agent includes: a biological drug, a compound drug, a herbal drug, or a combination thereof.

[0057] In another preferred embodiment, the therapeutic agent is selected from the group consisting of batroxobin, snake venom drugs, fibrinolytic enzymes for injection, gene editing reagents, sodium valproate, antibodies, or combinations thereof.

[0058] In another preferred embodiment, the snake venom drug is snake venom hemagglutinin.

[0059] In another preferred embodiment, the therapeutic agent is batroxobin.

[0060] In another preferred embodiment, the therapeutic agent is an agent that significantly reduces the expression levels of fibrin and / or fibrinogen γ chains.

[0061] In a fifth aspect of the invention, the use of the pharmaceutical combination described in the fourth aspect of the invention is provided, the pharmaceutical combination being used to prepare a kit for treating diseases related to ruptured blood vessels.

[0062] In another preferred embodiment, the relevant diseases include: aortic dissection, aortic aneurysm, Marfan syndrome, or a combination thereof.

[0063] In another preferred embodiment, the aortic aneurysm includes: thoracic aortic aneurysm and abdominal aortic aneurysm.

[0064] In another preferred embodiment, the aortic aneurysm is an abdominal aortic aneurysm.

[0065] In another preferred embodiment, the treatment refers to alleviating the symptoms of the related disease, slowing the deterioration of the symptoms of the related disease, and / or restoring the physiological indicators of the related disease to normal levels.

[0066] In a sixth aspect of the invention, a kit is provided comprising the drug combination described in the fourth aspect of the invention.

[0067] In another preferred embodiment, the kit comprises the following components:

[0068] (a) Reagents for visualizing fibrin and / or fibrinogen; and / or

[0069] (b) Therapeutic agents for fibrin and / or fibrinogen.

[0070] In another preferred embodiment, the kit includes: (c) reagents capable of reflecting vascular mechanics and / or hemodynamic parameters that reflect fibrin and / or fibrinogen performance.

[0071] In another preferred embodiment, the kit further includes: (d) instructions for use of the kit in warning and / or preventing vascular rupture in test subjects.

[0072] In another preferred embodiment, the warning refers to indicating to the subject that there is a risk of blood vessel rupture before the blood vessel ruptures.

[0073] In another preferred embodiment, the prevention includes: preventing blood vessel rupture, delaying blood vessel rupture, or a combination thereof.

[0074] In another preferred embodiment, the blood vessels include: cardiovascular vessels, cerebral vessels, peripheral vessels, or combinations thereof.

[0075] In another preferred embodiment, the blood vessels include coronary arteries, aortas, cerebral arteries, or combinations thereof.

[0076] In another preferred embodiment, the blood vessel is the aorta.

[0077] In another preferred embodiment, the therapeutic agent for fibrin and / or fibrinogen includes: an agent that downregulates the expression level of fibrin and / or fibrinogen, an agent that degrades fibrin and / or fibrinogen, an agent that interferes with fibrin and / or fibrinogen, an agent that blocks the activity of fibrin and / or fibrinogen, or a combination thereof.

[0078] In another preferred embodiment, the reagent that intervenes in fibrin and / or fibrinogen is a reagent that negatively intervenes in the activity of fibrin and / or fibrinogen.

[0079] In another preferred embodiment, the negative intervention refers to downregulating the activity of fibrin and / or fibrinogen, or binding fibrin and / or fibrinogen to inhibit their function.

[0080] In another preferred embodiment, the therapeutic agent includes: a biological drug, a compound drug, a herbal drug, or a combination thereof.

[0081] In another preferred embodiment, the therapeutic agent is selected from the group consisting of batroxobin, snake venom drugs, fibrinolytic enzymes for injection, gene editing reagents, sodium valproate, antibodies, or combinations thereof.

[0082] In another preferred embodiment, the snake venom drug is snake venom hemagglutinin.

[0083] In another preferred embodiment, the therapeutic agent is batroxobin.

[0084] In another preferred embodiment, the therapeutic agent is an agent that significantly reduces the expression levels of fibrin and / or fibrinogen γ chains.

[0085] In another preferred embodiment, the kit is used to assess the risk of vascular rupture in subjects and / or to prevent vascular rupture.

[0086] In a seventh aspect of the invention, a system for assessing the risk of blood vessel rupture is provided, the system comprising the following modules:

[0087] (Z1) Data acquisition module, the data acquisition module is configured to: quantify the relative amount of fibrin and / or fibrinogen in the blood vessel wall of the test subject, defined as Y1, and quantify the relative amount of fibrin and / or fibrinogen in the blood vessel wall of the normal control, defined as Y0.

[0088] (Z2) Blood vessel rupture risk analysis module, wherein the blood vessel rupture risk analysis module is configured to: compare the relative amount Y1 of fibrin and / or fibrinogen in the blood vessel wall of the test subject with the relative amount Y0 of fibrin and / or fibrinogen in the blood vessel wall of the normal control, thereby indicating whether the test subject has a risk of blood vessel rupture.

[0089] (Z3) Output module, which is configured to output whether the subject has a risk of blood vessel rupture.

[0090] In another preferred embodiment, the vascular rupture risk analysis module is configured to indicate that the subject has a risk of vascular rupture if the relative amount Y1 of fibrin and / or fibrinogen in the vascular wall of the test subject is significantly increased compared with the relative amount Y0 of fibrin and / or fibrinogen in the vascular wall of the normal control.

[0091] In another preferred embodiment, the term "significant increase" refers to an increase that is statistically significant.

[0092] In another preferred embodiment, “significant increase” means that Y1 / Y0 ≥ 2, preferably ≥ 10, and most preferably ≥ 20.

[0093] In another preferred embodiment, the quantification includes: imaging and quantification.

[0094] In an eighth aspect of the invention, the use of fibrin and / or fibrinogen and their promoters is provided for the preparation of experimental reagents for generating non-human animal models of blood vessel rupture.

[0095] In another preferred embodiment, the non-human animal model of ruptured blood vessels is used for:

[0096] (Z1) Screening or identifying drugs that can prevent, alleviate or treat ruptured blood vessels;

[0097] (Z2) Screening or identifying vaccines that can prevent blood vessel rupture; and / or

[0098] (Z3) Analyze the pathogenesis of blood vessel rupture.

[0099] In another preferred embodiment, the non-human animal is a non-human mammal.

[0100] In another preferred embodiment, the non-human mammal is a rat or a mouse.

[0101] In another preferred embodiment, the non-human mammal is a mouse.

[0102] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0103] Figure 1 The diagram shows (A) the establishment of a mouse model of acute vascular rupture and (B).

[0104] Figure 2 The diagram shows (A) construction and (B) schematic of the mouse model of chronic vascular rupture.

[0105] Figure 3 The results showed that proteomics analysis confirmed elevated fibrin(ogen) levels associated with ruptured blood vessels.

[0106] Among them, (A) shows the experimental procedure; (B) shows the comparison of the number of differentially expressed proteins between the CON and AR groups; (C) shows the biological process that changes most significantly with blood vessel rupture; and (D) shows the top 20 upregulated proteins.

[0107] Figure 4 It shows that as vascular rupture progresses, fibrin(ogen) is upregulated and deposited in the adventitia of the aorta.

[0108] Among them, (A) shows the immunohistochemical staining results of the vascular rupture process; (B) shows the immunofluorescence staining results of the vascular rupture process; and (C) shows the protein immunoblotting results of the vascular rupture process.

[0109] Figure 5 This study demonstrates the deposition of fibrinogen prior to vascular rupture during the development of chronic vascular injury.

[0110] Among them, (A) shows the immunohistochemical staining results of fibrin(ogen) deposition in chronic vascular injury; (B) shows the immunofluorescence staining results of fibrin(ogen) deposition in chronic vascular injury; and (C) shows the protein immunoblotting results of fibrin(ogen) deposition in chronic vascular injury.

[0111] Figure 6 This demonstrates that fibrin(ogen) is more specific as a marker of vascular rupture than albumin.

[0112] (A) shows the immunohistochemical results of albumin, the main component of plasma during the development of vascular injury, and (B) shows its statistical analysis.

[0113] Figure 7 This showed that batroxobin significantly downregulated fibrinogen.

[0114] Figure 8 The study showed that batroxobin inhibition of fibrin(ogen) significantly reduced aortic rupture.

[0115] Among them, (A) shows the experimental procedure; (B) shows a schematic diagram of the reduction of blood vessel wall rupture rate under the action of batroxobin and (C) its statistical analysis; (D) shows representative echocardiograms of each group and (E) statistical analysis of blood vessel wall pulse wave velocity (PWV) of each group.

[0116] Figure 9 The study showed that knocking down the expression level of fibrinogen γ chain (Fgg) in mice significantly reduced aortic rupture.

[0117] (A) shows the experimental procedure; (B) shows a representative aorta after Fgg knockdown; and (C) shows a statistical analysis of the incidence of vascular rupture.

[0118] Figure 10 The study showed that knocking down the fibrinogen γ chain (Fgg) in mice with abdominal aortic aneurysms reduced the rate of vascular rupture and mortality.

[0119] Among them, (A) shows the experimental procedure; (B) shows the effect of Fgg knockdown on the diameter of the abdominal aortic aneurysm; and (C) shows the quantitative analysis of the aneurysm diameter and survival rate of the abdominal aortic aneurysm model mouse after Fgg knockdown. Detailed Implementation

[0120] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that both acute and chronic vascular rupture processes are accompanied by elevated fibrin(ogen) levels in the vascular adventitia. Furthermore, inhibiting fibrin(ogen) activity or downregulating fibrin(ogen) levels can significantly reduce vascular wall hardening and the incidence of aortic rupture. Therefore, on the one hand, fibrin(ogen) can serve as a biomarker for vascular rupture; on the other hand, the regulation of fibrin(ogen) plays a crucial role in the prediagnosis and prevention of vascular rupture. This invention is based on these findings.

[0121] As used herein, the terms "fibrin(ogen)" and "fibrin and / or fibrinogen" are used interchangeably and refer to fibrin, fibrinogen, or combinations thereof. In specific embodiments, the fibrin and / or fibrinogen comprises: degradation products of fibrin and / or fibrinogen, complexes containing fibrin and / or fibrinogen, functional fragments of fibrin and / or fibrinogen, fibrin and / or fibrinogen subtypes, or combinations thereof. In specific embodiments, fibrinogen subtypes include, but are not limited to: unmodified, phosphorylated, and glycosylated fibrinogen. In specific embodiments, fibrin subtypes include, but are not limited to: soluble fibrin monomers and cross-linked fibrin polymers. In specific embodiments, in the complex containing fibrin and / or fibrinogen, fibrin and / or fibrinogen are the main active ingredients, i.e., the mass percentage of fibrin and / or fibrinogen in the complex exceeds 50%, preferably exceeds 60%, more preferably exceeds 70%, and most preferably exceeds 80%. Those skilled in the art should understand that the detection reagents for fibrin and / or fibrinogen of the present invention refer to reagents that can obtain the relative or absolute levels of fibrin and / or fibrinogen through direct or indirect means, or through qualitative and / or quantitative detection.

[0122] As used in this article, "warning before blood vessel rupture," "early warning of blood vessel rupture," and "assessing the risk of blood vessel rupture" can be used interchangeably. They all refer to assessing the risk of blood vessel rupture before it occurs, so as to promptly indicate the presence of the risk.

[0123] As used in this article, “agents for downregulating fibrin and / or fibrinogen,” “inhibitors of fibrin and / or fibrinogen,” and “therapeutic agents for fibrin and / or fibrinogen” are used interchangeably and all refer to downregulating the expression level of fibrin and / or fibrinogen, downregulating the activity of fibrin and / or fibrinogen, blocking the activity of fibrin and / or fibrinogen, blocking the function of fibrin and / or fibrinogen, inhibiting the function of fibrin and / or fibrinogen, downregulating the deposition of fibrin and / or fibrinogen, destroying fibrin and / or fibrinogen, restoring the content and / or activity of fibrin and / or fibrinogen to a level that is not statistically significantly different from that of normal subjects, or combinations thereof.

[0124] As used herein, the “visualization” refers to imaging fibrin and / or fibrinogen to obtain the location, content, or combination thereof of fibrin and / or fibrinogen.

[0125] As used herein, “treatment” includes stopping the rupture process of a chronically ruptured blood vessel, delaying the rupture process of a chronically ruptured blood vessel, restoring a chronically ruptured blood vessel to the state of a healthy control vessel, stopping an acute rupture of a blood vessel, restoring an acutely ruptured blood vessel to the state of a healthy control vessel, or a combination thereof.

[0126] As used in this article, the term "subject" refers to the person who undergoes the experiment.

[0127] In a specific implementation, the test subjects are humans or non-human mammals.

[0128] In a specific implementation, the test subject refers to an individual at risk of blood vessel rupture.

[0129] In a specific implementation, the test subject refers to an object with ruptured blood vessels.

[0130] In a specific implementation, the patient is a patient with cardiovascular or cerebrovascular risks.

[0131] In a specific implementation, the patient with cardiovascular or cerebrovascular risks refers to a patient with a family history of cardiovascular or cerebrovascular diseases.

[0132] In a specific implementation, the test subjects refer to those with a family history of blood vessel rupture.

[0133] In a specific implementation, the subjects refer to those with a family history of aortic dissection.

[0134] In a specific implementation, the subjects refer to those with a family history of aortic aneurysm.

[0135] In a specific implementation, the test subjects refer to those with a family history of cerebral aneurysms.

[0136] In a specific implementation, the test subject refers to a Marfan patient.

[0137] In a specific implementation, the test subjects refer to those with a Marfan family history.

[0138] In a specific implementation, the test subjects refer to males aged 55 and above who smoke.

[0139] In a specific implementation, the test subjects include those undergoing physical examinations.

[0140] In a specific implementation, the non-human mammals include (but are not limited to): dogs, cats, rabbits, rats, mice, guinea pigs, and quails.

[0141] In a specific implementation, the non-human mammal is a mouse.

[0142] ruptured blood vessel

[0143] The blood vessel wall, composed of endothelial cells and a muscular layer, maintains the stability of blood flow. Blood vessels can become fragile and prone to rupture due to abnormal protein deposition in the vessel wall (such as in amyloidosis, cryoglobulinemia, and hypergammaglobulinemia-related purpura) or inflammation (such as autoimmune vasculitis).

[0144] When blood vessels are subjected to external impact or abnormal internal changes, the vessel walls are damaged, which in turn triggers a series of inflammatory and coagulation reactions.

[0145] Based on the type of blood vessel, ruptured blood vessels are generally classified as capillary rupture, arterial rupture, and venous rupture. Based on the location of the blood vessel, they are also classified as cardiovascular, cerebrovascular, etc. The symptoms of different types of ruptured blood vessels vary. Mild cases may present with massive bleeding at the site of injury, pulsatile hematoma, significant swelling of the limb, and disappearance of distal arterial pulses. Severe cases can lead to death in a short period of time (such as in diseases like aortic dissection).

[0146] Therefore, early detection of the risk of vascular rupture before it occurs is beneficial for timely intervention, thereby preventing or delaying vascular rupture and avoiding inflammation and coagulation reactions caused by vascular damage.

[0147] Physical signs of ruptured blood vessels typically include asymmetry in pulse or blood pressure bilaterally, decreased blood flow to the lower extremities, and a systolic murmur in the precordial area. Diagnosis can be inferred based on the characteristics of the physical signs, the mechanism of injury, and / or chest X-ray results, and confirmed by CT, ultrasound, or aortic angiography.

[0148] While these diagnostic mechanisms are effective, the time from diagnosis to rupture of the blood vessel is short, leaving little time for human intervention.

[0149] Therefore, this invention, through extensive screening, provides a biomarker for vascular rupture—fibrin(ogen), and all its subtypes, degradation products, complexes, and functional fragments. In this invention, if a subject's fibrin(ogen) level differs statistically from that of a normal healthy control, the subject is at risk of vascular rupture.

[0150] Fibrinogen

[0151] Fibrin(ogen) is a pleiotropic blood protein that regulates coagulation, inflammation, and tissue repair. Fibrin(ogen) comprises two components: fibrin and fibrinogen. Specifically, fibrinogen is a glycoprotein synthesized and secreted by hepatocytes, composed of three pairs of different polypeptide chains: α (Fga), β (Fgb), and γ (Fgg). Fibrinogen plays a role in thrombin, fibrin stabilizing factor (FXⅢa), and calcium... 2+Under the action of clotting factors, fibrin monomers are formed, and the fibrin monomers covalently bind to each other to form fibrin polymers and a stable fibrin network.

[0152] In this invention, fibrin(ogen) can serve as a biomarker for vascular rupture. Early detection and intervention of excessive fibrin(ogen) deposits on the vessel wall in the early stages of vascular rupture risk, before rupture occurs, can achieve early warning and prevention of vascular rupture.

[0153] The method of the present invention

[0154] The method of this invention refers to a method for preventing and / or warning of blood vessel rupture, specifically, a method for indicating the risk of blood vessel rupture and taking preventive measures before rupture. The method includes the following steps:

[0155] (N1) The relative amount of fibrin(ogen) in the blood vessel wall of the test subject was imaged and quantified, and was defined as Y1. The relative amount of fibrin(ogen) in the blood vessel wall of the normal control was imaged and quantified, and was defined as Y0.

[0156] (N2) The relative amount of fibrin(ogen) in the blood vessel wall of the test subject, Y1, is compared with the relative amount of fibrin(ogen) in the blood vessel wall of the normal control, Y0. If the relative amount of fibrin(ogen) in the blood vessel wall of the test subject, Y1, is significantly increased compared with the relative amount of fibrin(ogen) in the blood vessel wall of the normal control, it indicates that the test subject has a risk of blood vessel rupture.

[0157] In another preferred embodiment, the method further includes: (N3) administering a reagent or kit to downregulate fibrin(ogen) to subjects who are indicated to have a risk of vascular rupture.

[0158] In another preferred embodiment, the reagent refers to batroxobin reagent.

[0159] In another preferred embodiment, the kit is a kit that knocks down the expression level of mouse fibrinogen γ chain using gene editing technology.

[0160] In another preferred embodiment, “significant increase” means that Y1 / Y0 ≥ 2, preferably ≥ 10, and most preferably ≥ 20.

[0161] In another preferred embodiment, the method is a method for scientific research purposes.

[0162] The main advantages of this invention include:

[0163] (a) The present invention provides a method for providing early warning before a blood vessel ruptures, namely, detecting or imaging the fibrin(ogen) deposited on the blood vessel wall to identify the risk of blood vessel rupture as early as possible.

[0164] (b) The present invention provides a method for preventing and / or improving vascular rupture by downregulating fibrin(ogen) in subjects who are judged to be at risk of vascular rupture, thereby preventing or delaying vascular rupture.

[0165] (c) This invention provides reagents for preventing blood vessel rupture. Specifically, batroxobin reagent reduced the incidence of blood vessel rupture by 45%; reagents that knock down the expression level of fibrinogen γ chain (Fgg) in mice reduced the incidence of blood vessel rupture by approximately 54%.

[0166] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0167] Example 1: Fibrin is a type of protein that is upregulated in the event of blood vessel rupture.

[0168] 1.1 Laboratory Animals

[0169] C57BL / 6 mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. All experimental animals were housed in an SPF-grade environment at the experimental animal center, with a room temperature of 25℃±2℃ and a 12-hour circadian rhythm maintained during the experiments, and free access to food and water was ensured.

[0170] 1.2 Construction of the mouse model

[0171] Mouse model of vascular rupture: C57BL / 6 mice were fed a diet containing β-aminopropionitrile (BAPN, TCI, Japan) for 15 consecutive days to establish a vascular rupture model. On day 15, mice were subcutaneously injected with 1.44 mg / kg angiotensin II (Ang II, Calbiochem, USA), and tissue samples were collected 24 hours later to observe the occurrence of vascular rupture. To assess the progression of acute vascular rupture, mice fed BAPN plus Ang II were subdivided into the NAR group (non-aortic rupture, mice before vascular rupture) and the AR group (aortic rupture, mice with vascular rupture), while the CON group (normal control group) was fed a normal diet and injected with the same volume of physiological saline (NAR). Figure 1 A and Figure 1 B).

[0172] A mouse model of chronic vascular rupture: C57BL / 6 mice were fed diets containing different doses of BAPN for 15 consecutive days. The mice were then divided into three groups based on the BAPN content in the diet: 0%, 0.125%, and 1%, corresponding to normal diet, 0.125% BAPN diet, and 1% BAPN diet, respectively. On day 15 of modeling, mice were subcutaneously injected with 1.44 mg / kg angiotensin II, and tissue samples were collected 24 hours later to observe the occurrence of vascular rupture. Figure 2 A and Figure 2 B), in which mice that did not rupture were selected as the research subjects.

[0173] 1.3 Proteomics experiments.

[0174] An appropriate amount of protein lysis buffer was added to a lysis tube containing fresh mouse aortic samples, and protein extraction was performed using a high-throughput tissue homogenizer. After determining the protein concentration, 100 μg of protein was quantitatively aspirated and lysed into peptides using trypsin, followed by probe labeling with iTRAQ reagent. The peptides were then separated using high-pH liquid chromatography, and detected using liquid chromatography-tandem mass spectrometry (Easy-nLC 1200 combined with Q Exactive HF-X mass spectrometer). The mass spectrometry results were analyzed using Proteome Discoverer™ Software 2.4, and the protein types were determined by comparison with a database. Analysis was performed using the Meiji Cloud platform (cloud.majorbio.com). The p-value for statistical significance between samples was calculated using the t.test function in R, and the fold change (FC) between groups was also calculated, with a significance test p < 0.05. GO (gene ontology, http: / / geneontology.org / ) was used to perform GO annotation functional cluster analysis on all differentially expressed proteins from three aspects: biological process, cellular component, and molecular function.

[0175] 1.4 Protein immunoblotting analysis.

[0176] Fresh mouse aortas were minced on ice, lysed with an appropriate amount of Ripa lysis buffer, and the resulting protein extract was obtained using a high-throughput tissue homogenizer. After denaturation, the protein extract was separated by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane via electrotransfer. The membrane was incubated overnight with antibodies such as GAPDH (Boster, BA2913) and fibrin(ogen) (Abcam, ab189490), then incubated with a horseradish peroxidase-conjugated (HRP) secondary antibody and detected using an enhanced chemiluminescence (ECL) kit (absin, abs921).

[0177] 1.5 Immunohistochemistry and immunofluorescence staining.

[0178] Aortic tissue sections were incubated overnight at 4°C with antibodies such as antifibrin(ogen), followed by incubation at 37°C for 1 hour with HRP-conjugated or fluorescently labeled secondary antibodies. The signal was detected using a DAB detection kit or by direct fluorescence detection. Images were acquired using an Olympus BX51 microscope or a confocal microscope (Zeiss LSM 900, Japan). For quantification, five fields of view of the stained tissue were randomly selected, and images were quantified using ImageJ by personnel not involved in this project.

[0179] 1.6 Batroxobin Intervention Experiment.

[0180] C57BL / 6 mice were fed a diet containing β-aminopropionitrile (BAPN, TCI, Japan) for 15 consecutive days to induce a mouse model of vascular rupture. On day 15 of modeling, mice were subcutaneously injected with 1.44 mg / kg angiotensin II (Ang II, Calbiochem, USA), and tissue samples were collected 24 hours later to observe the occurrence of vascular rupture. To assess the effect of reducing fibrin(ogen) on vascular rupture, mice in the batroxobin group were intraperitoneally injected with batroxobin on days 11, 13, and 15, while the control and model groups were injected with the same volume of physiological saline.

[0181] 1.7 Ultrasonic testing.

[0182] Mice were anesthetized with isoflurane, and their limbs were attached to four electrocardiogram (ECG) electrodes on an ultrasonically heated platform for ECG and respiratory rate measurements. Body temperature was monitored via a rectal probe and maintained at 36-38°C. Ultrasonic images of the aortic root to the beginning of the descending aorta were obtained using an MX400 ultrasound probe (20-46MHz, center emission: 30MHz, axial resolution: 50μm). Pulsed Doppler signals were recorded at the aortic root and the beginning of the descending aorta in PW mode. Doppler waveforms were measured based on the corresponding Doppler images. The time to blood flow to the aortic root (t1) and the beginning of the descending aorta (t2) was measured, starting from the peak value of the ECG R wave (t1 and t2 values ​​are the average of 10 cardiac cycles). The aortic arch length (d) is the propagation distance between the two points where the Doppler signal was recorded. Finally, the aortic arch length (d) is divided by the aortic arch passage time (t2-t1) to calculate the pulse wave velocity (PWV), i.e., PWV = d / (t2-t1).

[0183] 1.8HE staining

[0184] Paraffin tissue sections with a thickness of 4 μm were baked on a slide baking machine (65℃, 60 min), then dewaxed to an aqueous phase (xylene 15 min → anhydrous ethanol 5 min → 95% ethanol 5 min → 75% ethanol 5 min → rinsed with running water for 5 min); stained with hematoxylin staining solution for 15 min, then rinsed with running water for 5 min; differentiated with 1% hydrochloric acid ethanol for 3 s (differentiation time adjusted according to the time of placement of the differentiation solution), then blued with running water for 5 min; stained with eosin staining solution for 5 min, then placed in water, dehydrated, and then permeated with xylene (75% ethanol 5 min → 95% ethanol 5 min → anhydrous ethanol 5 min → xylene 15 min), and mounted with neutral resin.

[0185] Example 2: Blood vessel rupture accompanied by elevated fibrin(ogen)

[0186] Proteomics analysis was used to analyze normal control mice (CON) and AR group mice (with ruptured blood vessels) to identify differentially expressed proteins. The research procedure is as follows: Figure 3 As shown in Figure A.

[0187] The results are as follows Figure 3 As shown in B. Compared with CON mice, 183 differentially expressed proteins were identified in AR mice, of which 146 were upregulated and 37 were downregulated.

[0188] Figure 3 Gene set enrichment analysis of C differential proteome revealed the most significant changes in biological processes following blood vessel rupture, caused by... Figure 3 As shown in D, among the first 20 upregulated proteins in the vascular rupture process, there are 3 fibrin(ogen) subtypes, indicating that fibrin(ogen) increases with the vascular rupture process.

[0189] Subsequently, immunohistochemical staining, immunofluorescence staining, and Western blotting were used to further determine that the vascular rupture process was accompanied by an increase in fibrin(ogen), and the results were as follows: Figure 4 As shown.

[0190] Figure 4 Immunohistochemical staining results for A showed that, compared with CON mice, the proportion of fibrinogen in the adventitia of mice with ruptured blood vessels increased from 0.21% ± 0.04% to 40.46% ± 1.22%, indicating significant fibrin(ogen) deposition. In NAR mice before blood vessel rupture, the proportion of fibrinogen in the adventitia also increased to 4.25% ± 1.92%, 20 times that of CON mice, indicating a correlation between fibrinogen percentage and the progression of blood vessel rupture, and suggesting that it can provide an indication before blood vessel rupture.

[0191] Figure 4Immunofluorescence staining results of B showed that before vascular rupture, fibrin(ogen) was significantly deposited in the adventitia; after vascular rupture, fibrin(ogen) was significantly deposited in the adventitia.

[0192] Figure 4 The results of Western blotting of C showed that the proportion of fibrinogen in the aorta of NAR mice before vascular rupture was 0.087±0.018, which was 3.78 times that of CON mice; while compared with CON mice, the proportion of fibrinogen in the aorta of mice with vascular rupture increased from 0.023±0.018 to 0.379±0.008.

[0193] All of the above results confirm that aortic fibrin(ogen) gradually increases as vascular rupture progresses, and that fibrin(ogen) in the aorta can serve as a biological indicator of vascular rupture, providing a warning before vascular rupture occurs.

[0194] Example 3: Fibrin(ogen) deposition increases with the duration of chronic vascular injury.

[0195] To eliminate the influence of acute vascular rupture and bleeding, this invention induced and established an animal model of vascular rupture induced by chronic vascular injury, and observed the deposition of fibrin(ogen) as the duration of chronic vascular injury increased.

[0196] Experimental results are as follows Figure 5 As shown in A-5C.

[0197] Figure 5 Immunohistochemical staining results for A showed that, compared with CON mice, the proportion of fibrinogen in the adventitia of 1% BAPN mice increased from 0.06% ± 0.05% to 4.33% ± 1.70%, exhibiting significant fibrin(ogen) deposition. In 0.125% BAPN mice, the proportion of fibrinogen in the adventitia also increased to 0.57% ± 0.22%, which was 9.5 times that of CON mice, indicating a correlation between fibrinogen percentage and the progression of vascular rupture, and suggesting that it may provide an indication before vascular rupture.

[0198] Figure 5 Immunofluorescence staining results of B showed that fibrin(ogen) was significantly deposited in the adventitia before vascular rupture; and fibrin(ogen) was significantly deposited in the adventitia as the rupture progressed.

[0199] Figure 5The results of Western blotting in C showed that the proportion of fibrinogen in the aorta of 0.125% BAPN mice was 0.655±0.136, which was 3.38 times that of CON mice; while compared with CON mice, the proportion of fibrinogen in the aorta of 1% BAPN mice increased from 0.193±0.107 to 1.084±0.220.

[0200] Example 4: Fibrin(ogen) deposition exhibits specificity

[0201] This invention also detected the leakage of blood constituent albumin (66kD) into the blood vessel wall during the development of chronic vascular injury.

[0202] The results are as follows Figure 6 A and Figure 6 As shown in B, immunohistochemistry revealed that, compared with CON mice, no further albumin deposition was found during the development of chronic vascular injury. In blood vessels that had undergone chronic injury but not rupture, the amount of albumin deposition was comparable to that in the control group, indicating that fibrinogen (340kD) is specific in the deposition of fibrinogen in the blood vessel wall as a biological indicator of vascular rupture.

[0203] Example 5: Inhibition of fibrin(ogen) can significantly reduce aortic rupture.

[0204] Batroxobin, also known as fibrinolytic enzyme, is a clinically used drug that reduces fibrinogen by breaking down fibrinogen. Figure 7 As shown, immunohistochemical detection results clearly indicate that batroxobin significantly downregulates fibrin(ogen) deposition in the blood vessel wall.

[0205] In this invention, such as Figure 8 As shown in Figure A, on days 11, 13, and 15, mice in the batroxobin group were injected intraperitoneally with batroxobin, while the control and model groups were injected with the same volume of physiological saline to investigate the relationship between fibrin(ogen) and blood vessel rupture.

[0206] The results are as follows Figure 8 B and Figure 8 As shown in Figure C, under the action of batroxobin, the deposition of fibrin(ogen) in the blood vessel wall is reduced, and the incidence of blood vessel rupture is significantly reduced from 75% to 30%.

[0207] Figure 8 D and Figure 8 Echocardiographic results from E showed that significant vascular wall sclerosis was observed in the mouse model of ruptured blood vessels. Under the action of batroxobin, fibrin(ogen) deposition in the blood vessel wall decreased, and vascular wall sclerosis was inhibited.

[0208] like Figure 9As shown in Figure A, the expression level of fibrinogen γ chain (Fgg) in mice was knocked down using gene editing technology, and the incidence of blood vessel rupture in mice was tested after induction with 1% BAPN for 16 days.

[0209] The results are as follows Figure 9 B and Figure 9 As shown in C, knockdown of Fgg significantly reduced the incidence of vascular rupture from 70% to 16.67%.

[0210] The results of this embodiment show that inhibiting fibrin(ogen) can significantly reduce the incidence of aortic rupture and improve vascular wall sclerosis.

[0211] Example 6: Blocking fibrin(ogen) function can significantly reduce aortic rupture.

[0212] The effect of the small peptide Fibγ377-395 on the occurrence of blood vessel rupture was investigated using the following experimental methods:

[0213] (I) Preparation of animal models and intervention with Fibγ377-395 small peptide: AR animal models were prepared using BAPN+AngII. Fibγ377-395 small peptide (KETTMKIIPFNRLSI) was administered to mice via intraperitoneal injection every other day. Control mice were given the same dose of Scramble small peptide (KMMISYTFPIERTGLISNK).

[0214] (II) Evaluation of the degree of damage to vascular structure: Hematoxylin-eosin staining is used to evaluate the entire vascular structure, lichen red staining is used to evaluate whether the elastic layer of the blood vessel has been degraded, Sirius red staining is used to evaluate whether the adventitia of the blood vessel is intact, and transmission electron microscopy is used to evaluate the vascular structure.

[0215] In addition, the effect of ancrolein on the occurrence of blood vessel rupture was investigated using the following experimental methods:

[0216] (I) Preparation of animal models and intervention with Angolase: AR animal models were prepared using BAPN+AngII, and Angolase was administered at a stable and uniform dose of 2.4U / day to achieve defibrinogenization in mice.

[0217] (II) Evaluation of the degree of damage to vascular structure: Hematoxylin-eosin staining is used to evaluate the entire vascular structure, lichen red staining is used to evaluate whether the elastic layer of the blood vessel has been degraded, Sirius red staining is used to evaluate whether the adventitia of the blood vessel is intact, and transmission electron microscopy is used to evaluate the vascular structure.

[0218] The results of this embodiment show that blocking fibrin(ogen) function can significantly reduce aortic rupture.

[0219] Example 7: Effect of fibrinogen γ chain on rupture of abdominal aortic aneurysm in mice

[0220] Fibrinogen γ chain knockout mice were purchased from Jicui Pharmaceutical Co., Ltd. Due to the fibrinogen γ chain knockout mice (Fgg... - / - In homozygous strains, fibrinogen levels are undetectable in circulation, and prenatal death or spontaneous neonatal hemorrhage may occur. Therefore, heterozygous Fgg strains were selected. + / - Mice used in this study had low fibrinogen levels in their blood, but no delay in clotting time. Male mice were used in this study. All experimental animals were housed in a specific pathogen-free environment (SPF) with a 12-hour light / dark cycle.

[0221] In the experimental grouping and construction of the abdominal aortic aneurysm model, this experiment was divided into two groups: wild-type mice (Fgg) + / + Fibrinogen γ chain knockout mice are marked as Fgg + / - Construction of the abdominal aortic aneurysm model: Mice were anesthetized with a mixture of Zoltil@50 and xylazine (injection dose: Zoltil@50 50 mg / kg, xylazine 10 mg / kg), with an injection volume of 0.1 ml / 10 g. After the mice were anesthetized, their abdominal hair was shaved with a razor, and further hair removal was performed using depilatory cream. The abdomen was cleaned and the mice were fixed on the operating table, with a heating pad used to maintain body temperature. The abdominal skin was wiped with iodine and then deiodized with 75% ethanol. A 1.5 cm incision was made in the midline of the abdomen, and the organs were carefully dissected with forceps. The intestines were then separated to the left and right sides using gauze moistened with 3% penicillin-streptomycin. The connective tissue and muscle tissue on the surface of the aorta were carefully separated with forceps to fully expose the abdominal aorta. The exposed length of the abdominal aorta was approximately 0.5 cm, proximally not exceeding the bilateral renal arteries and distally not exceeding the femoral artery.

[0222] Sterile absorbent paper (0.3cm × 0.3cm) was thoroughly soaked in porcine pancreatic elastase (PPE) and then applied to the exposed abdominal aorta for 50 minutes. A gauze pad moistened with 3% penicillin-streptomycin saline was placed above the abdominal incision to prevent excessive loss of abdominal fluid. After 50 minutes, the absorbent paper was carefully removed from the aorta, and the abdominal cavity was flushed with saline containing 3% penicillin-streptomycin. The peritoneum and skin were sutured with 3 / 8 suture needles and 5 / 0 sutures. After suturing, the wound was wiped with iodine, and the iodine was removed with 75% ethanol after 1 minute. Mice were placed in clean cages, fed a diet containing 1% β-aminopropionitrile (BAPN), and given free access to water. The diet was changed every other day. After 15 days of rearing, the rate of vascular rupture was evaluated. All animal deaths were due to vascular rupture.

[0223] Figure 10 A shows the experimental flowchart for constructing an AAA mouse model through co-induction of PPE and BAPN. (Fgg) + / - and Fgg + / +Mice were used to establish a model by feeding them PPE combined with 1% BAPN, and then tissue samples were collected to observe the effect of Fibrinogen γ chain knockdown on aortic diameter. Figure 10 B); From Figure 10 From the C survival rate and diameter quantification, it can be seen that Fgg + / - The diameter of the aneurysm in the group (1139±62.16μm) mice was compared with that in Fgg. + / + The expression level of Fbrinogenγ chain in the group (1962±193.30μm) showed a significant decrease. Furthermore, regarding survival, the group with higher Fbrinogenγ chain expression levels experienced abdominal aortic rupture and death on day 13, while no such deaths occurred after the Fbrinogenγ chain level decreased.

[0224] Example 8: Construction of a blood vessel rupture model

[0225] By constructing conditionally overexpressing fibrin(ogen) mice through transgenic means to increase fibrin(ogen) expression, or by increasing fibrin(ogen) content in mice through tail vein injection, an animal model of chronic vascular injury leading to vascular rupture can be induced and established.

[0226] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of a reagent for detecting fibrin and / or fibrinogen, characterized in that, This is used to prepare a composition or formulation for assessing the risk of vascular rupture in a test subject.

2. The use of a reagent for visualizing fibrin and / or fibrinogen, characterized in that, This is used to prepare a composition or formulation for assessing the risk of vascular rupture in a test subject.

3. The use of a therapeutic agent for fibrin and / or fibrinogen, characterized in that, This is used to prepare a drug or combination of drugs for preventing vascular rupture in a test subject.

4. The use as described in claim 3, characterized in that, The therapeutic agent is an agent that significantly reduces the expression levels of fibrin and / or fibrinogen γ chains.

5. A drug combination, characterized in that, The drug combination includes: (i) a therapeutic component comprising a therapeutic agent of fibrin and / or fibrinogen, and (ii) a detection component comprising a detection reagent and / or a visualization reagent for fibrin and / or fibrinogen.

6. The use of the pharmaceutical composition according to claim 5, characterized in that, The drug combination is used to prepare a kit for treating diseases related to ruptured blood vessels.

7. A reagent kit, characterized in that, The kit comprises the drug combination as described in claim 5.

8. A system for assessing the risk of blood vessel rupture, characterized in that, The system includes the following modules: (Z1) Data acquisition module, the data acquisition module is configured to: image and quantify the relative amount of fibrin and / or fibrinogen in the blood vessel wall of the test subject, defined as Y1, and image and quantify the relative amount of fibrin and / or fibrinogen in the blood vessel wall of the normal control, defined as Y0. (Z2) Blood vessel rupture risk analysis module, wherein the blood vessel rupture risk analysis module is configured to: compare the relative amount Y1 of fibrin and / or fibrinogen in the blood vessel wall of the test subject with the relative amount Y0 of fibrin and / or fibrinogen in the blood vessel wall of the normal control, thereby indicating whether the test subject has a risk of blood vessel rupture. (Z3) Output module, which is configured to output whether the subject has a risk of blood vessel rupture.

9. The judgment system as described in claim 8, characterized in that, The blood vessel rupture risk analysis module is configured to indicate that the subject has a risk of blood vessel rupture if the relative amount Y1 of fibrin and / or fibrinogen in the blood vessel wall of the test subject is significantly increased compared with the relative amount Y0 of fibrin and / or fibrinogen in the blood vessel wall of the normal control.

10. Use of fibrin and / or fibrinogen and their promoters, characterized in that, Experimental reagents used to prepare non-human animal models of blood vessel rupture.

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