Serum biomarker ficolin-2 for assessing carotid atherosclerotic plaque and application thereof

Abstract

This invention relates to ficolin-2, a serum biomarker for assessing carotid atherosclerotic plaques, and its applications, belonging to the field of biomedical technology. To address the urgent need for readily available and repeatable circulating biomarkers that reflect the unstable state of atherosclerotic plaques, particularly their hemorrhagic characteristics, this invention provides the application of ficolin-2 as a serum biomarker in the preparation of products for assessing the stability of atherosclerotic plaques and the prognosis of sonodynamic therapy for atherosclerosis. This invention is the first to use serum ficolin-2 protein as an efficacy monitoring indicator for SDT treatment of hemorrhagic carotid plaques, monitoring the level of complement activation-related inflammation within the plaque, enabling dynamic management of patient treatment, filling the gap in the quantification of efficacy in hemorrhagic plaque treatment, and providing a key tool for the clinical translation of emerging therapies such as sonodynamic therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to ficolin-2, a serum biomarker for evaluating carotid atherosclerotic plaques, and its application. Specifically, it relates to the application of ficolin-2 as a serum biomarker in the preparation of products for evaluating the stability of atherosclerotic plaques and the prognosis of sonodynamic therapy for atherosclerosis. Background Technology

[0002] Activation of the complement system has both promoting and inhibiting effects on the progression of atherosclerosis. When the central component of complement, C3, cleaves in a pro-inflammatory direction, it forms a membrane attack complex, which promotes the progression of atherosclerosis and increases plaque instability. When the central component of complement, C3, cleaves in an anti-inflammatory direction, opsonization is enhanced, which can promote the clearance of dead cells. This clearance is achieved through ficolin-2 (MG Kiss, CJ Binder. (2022). The multifaceted impact of complement on atherosclerosis. Atherosclerosis. 351, 29-40. http: / / doi.org / 10.1016 / j.atherosclerosis.2022.03.014.). Studies have found that patients with smaller carotid infarct hemorrhage (IPH) have higher plasma ficolin-2 levels compared to those with larger carotid infarct hemorrhage (S. Fumagalli, C. Perego, R. Zangari, D. De Blasio, M. Oggioni, F. DeNigris, F. Snider, P. Garred, AM Ferrante, MG De Simoni. (2017). LectinPathway of Complement Activation Is Associated with Vulnerability of Atherosclerotic Plaques. Front Immunol. 8, 288. http: / / doi.org / 10.3389 / fimmu.2017.00288.).Immunohistochemical results of carotid artery pathological sections from patients with atherosclerosis showed that ficolin-2 in cholesterol-rich plaque regions was associated with macrophage recruitment (F. Carbone, A. Valente, C. Perego, M. Bertolotto, B. Pane, G. Spinella, D. Palombo, MG De Simoni, F. Montecco, S. Fumagalli. (2021). Ficolin-2 serum levels predict the occurrence of acute coronary syndrome in patients with severe carotid arterystenosis. Pharmacol Res. 166, 105462. http: / / doi.org / 10.1016 / j.phrs.2021.105462.). In high-risk plaques, macrophages undergo programmed death, resulting in impaired necrolysis (GRY De Meyer, M. Zurek, P. Puylaert, W. Martinet. (2024). Programmed death of macrophages in atherosclerosis: mechanisms and therapeutic targets. Nat RevCardiol. 21, 312-325. http: / / doi.org / 10.1038 / s41569-023-00957-0.). Therefore, ficolin-2 may enhance the clearance of dead cells within plaques by promoting macrophage recruitment. However, no studies have yet correlated serum ficolin-2 levels with carotid atherosclerotic plaques, particularly hemorrhagic plaques with high-risk characteristics. Whether ficolin-2 can serve as a convenient serological marker for non-invasively indicating the risk of carotid intraplaque hemorrhage, assessing plaque instability, and even predicting future cerebrovascular events remains a technological gap, lacking relevant systematic research and feasible implementation plans. Summary of the Invention

[0003] To address the urgent need for readily available and repeatable circulating biomarkers that reflect the unstable state of atherosclerotic plaques, especially hemorrhagic characteristics, this invention provides the application of ficolin-2 as a serum biomarker in the preparation of products for assessing the stability of atherosclerotic plaques and the prognosis of sonodynamic therapy for atherosclerosis. Furthermore, it provides the application of ficolin-2 as a target for developing drugs to prevent and / or treat the vulnerability of atherosclerotic plaques.

[0004] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first object of the present invention is to provide an application of a serum biomarker for non-disease diagnostic purposes, the application being used to assess the stability of atherosclerotic plaques, wherein the serum biomarker is ficolin-2, the serum biomarker being derived from the serum of a subject.

[0005] In one embodiment of the present invention, the decrease in ficolin-2 content indicates that the subject's atherosclerotic plaque is a vulnerable plaque with a risk of instability.

[0006] In one embodiment of the present invention, the serum biomarker can be combined with other serum biomarkers as biomarkers to assess the stability of atherosclerotic plaques, and the other serum biomarkers include C3, C5, C1q, and MASP1.

[0007] A second object of the present invention is to provide the use of reagents for detecting the above-mentioned serum biomarkers in the preparation of products for assessing the stability of atherosclerotic plaques.

[0008] A third object of the present invention is to provide a product for assessing the stability of atherosclerotic plaques, the product comprising reagents for detecting the aforementioned serum biomarkers.

[0009] A fourth object of the present invention is to provide the use of ficolin-2 as a target in the development of medicaments for the prevention and / or treatment of vulnerability to atherosclerotic plaques, said medicaments comprising inhibitors of serum ficolin-2.

[0010] In one embodiment of the present invention, the drug comprises an antagonist or antibody targeting the ficolin-2 signaling pathway.

[0011] A fifth object of the present invention is to provide an application of a serum biomarker for non-disease diagnostic purposes, the application being used to assess the prognosis of sonodynamic therapy for atherosclerosis, wherein the serum biomarker is ficolin-2, and the serum biomarker is derived from the serum of a subject.

[0012] A sixth object of the present invention is to provide the use of reagents for detecting the above-mentioned serum biomarkers in the preparation of products for evaluating the prognosis of sonodynamic therapy for atherosclerosis.

[0013] A seventh object of the present invention is to provide a product for evaluating the prognosis of sonodynamic therapy for atherosclerosis, the product comprising reagents for detecting the aforementioned serum biomarkers.

[0014] The beneficial effects of this invention are: This invention is the first to use serum ficolin-2 protein concentration as an indicator for monitoring the efficacy of slow-dose therapy (SDT) for carotid artery hemorrhage plaques. It allows for dynamic monitoring of complement activation-related inflammation levels within the plaque, enabling dynamic management of patient treatment. This application fills a gap in the quantification of efficacy for hemorrhage plaque treatment and provides a key tool for the clinical translation of emerging therapies such as sonodynamic therapy.

[0015] By detecting changes in serum ficolin-2 and C3 levels, complement activation-related proteins, before and after treatment, the impact of SDT on complement activation-related inflammation in high-risk plaques can be dynamically assessed, thus serving as an alternative or supplementary indicator for monitoring the efficacy of carotid MRI in quantifying infarct-associated hemorrhage (IPH). Specifically, this invention aims to replace carotid MRI in predicting patient efficacy by detecting dynamic changes in serum ficolin-2. Compared to existing technologies, serum ficolin-2 and C3 complement activation-related proteins can highly sensitively indicate the level of complement-related inflammation within plaques, and the negative correlation between ficolin-2 and IPH volume facilitates accurate monitoring of the efficacy of carotid artery hemorrhage plaque treatment by healthcare professionals.

[0016] This invention combines serum ficolin-2 concentration with other complement activation-related proteins as an indicator for assessing carotid plaque vulnerability and treatment efficacy. By detecting the levels of these proteins, plaque vulnerability and the risk of cardiovascular and cerebrovascular events can be evaluated. Simultaneously, by increasing serum ficolin-2 and decreasing serum C3, C1q, and MASP1 levels, it is possible to reduce IPH, inhibit plaque progression, and enhance plaque stability, thereby preventing cardiovascular events such as plaque rupture.

[0017] Furthermore, this invention can be combined with the detection of other serum markers or complement C3, C5, C1q, MASP1, etc., to construct a multi-marker prediction model, thereby improving the accuracy of subtyping or efficacy prediction. Based on the above mechanism, agonists targeting the ficolin-2 signaling pathway to enhance opsonization can also be developed to inhibit complement-related inflammation and prevent plaque rupture. Attached Figure Description

[0018] Figure 1This image shows the results of MRI assessment of the maximum wall thickness of carotid atherosclerotic plaques at different time points before and after SDT treatment; among them, Figure 1 Figure A in the image shows the results of MRI assessment of the maximum wall thickness of all carotid atherosclerotic plaques at different time points before and after SDT treatment. Figure 1 Figure B in the figure shows the results of MRI assessment of the maximum wall thickness of the hemorrhagic plaque (IPH) at different time points before and after SDT treatment; *P<0.05; Figure 2 The images show the results and carotid plaque characteristics of the well-responsive (GR) and poorly-responsive (PR) groups using MRI; among them, Figure 2 In the figure, A represents the results of MRI assessment of the good response group (GRs) and the poor response group (PRs) to sonodynamic therapy. The horizontal axis represents the percentage decrease in the maximum IPH volume of the carotid atherosclerotic hemorrhage plaque (IPH). Blue represents the GRs group (patients with IPH volume reduction >10%, n=7), and red represents the PRs group (patients with IPH volume reduction ≤10% or increase, n=3). Figure 2 B in the image represents a representative MRI image of the plaque on an MPRAGE sequence; the arrow indicates the IPH region (high signal intensity area). Figure 3 This figure shows the differences in protein levels between the GRs and PRs groups at baseline and at the point of maximum IPH volume reduction after SDT intervention, as assessed by proteomics. Figure 3 Figure A shows the results of a heatmap illustrating the differences in serum protein levels between the GRs and PRs groups before and after SDT treatment, at the point of maximum reduction in IPH volume. Figure 3 B in the figure shows the results of using volcano plots to show the difference in serum protein levels between the GRs and PRs groups when the IPH volume decreased most before and after SDT treatment; Figure 4 This is a graph showing the results of using ELISA to detect serum ficolin-2 levels in all subjects before and after SDT treatment; Figure 5 The figure shows the results of serum ficolin-2 levels in the GRs group before and after SDT treatment using ELISA. Figure 6 The figure shows the results of serum ficolin-2 levels in the PRs group before and after SDT treatment using ELISA. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the objectives of the invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention. In the art, embodiments obtained by other those skilled in the art without creative effort are all protected by this invention.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents and instruments used are conventional materials, reagents and instruments in the art, which can be obtained by those skilled in the art through commercial channels.

[0021] 1. The research object of this invention: A total of 12 patients with carotid atherosclerosis were included in the study, 10 of whom had intraplaque hemorrhage (IPH), with a total of 15 IPH plaques.

[0022] 2. The intervention method of the present invention: All participants received a single SDT treatment.

[0023] The target lesion for SDT intervention is an atherosclerotic plaque in the carotid artery with a diameter stenosis rate of 30-70% and a plaque thickness >2.5 mm, as detected by Doppler ultrasound. Record the location of the target lesion: common carotid artery (CCA), carotid bulb, internal carotid artery (ICA), and external carotid artery (ECA). Mark the corresponding skin areas with dye, record and preserve the corresponding ultrasound images.

[0024] SDT treatment was administered to enrolled patients who signed informed consent forms and completed baseline data collection. The treatment steps and precautions are as follows: (1) Timing of treatment: Intravenous injection of prepared DVDMS solution, followed by ultrasound irradiation treatment 4 hours later; (2) Treatment position: The patient lies supine with the head slightly tilted to the opposite side to fully expose the marked skin area; (3) Treatment parameters: Ultrasound intensity: 1.6 W / cm 2 Frequency: 1.0 MHz, duty cycle: 30%, irradiation time: 15 minutes.

[0025] Precautions: 1) During the treatment, the ultrasound probe and the marked area should be tightly fitted with coupling gel, and the subject should avoid moving; 2) Avoid ultraviolet radiation for 10 days after the treatment ends. When going out, wear sunglasses, masks, long-sleeved clothing and pants to cover your skin and avoid photosensitivity reactions caused by direct exposure to strong light.

[0026] 3. Sample collection and processing methods: Collect 3-5 mL of peripheral venous blood from the subject, place it in a coagulation tube, let it stand at room temperature for 30 min, centrifuge at 3000 rpm for 10 min, separate the serum, aliquot and store for later use.

[0027] Test reagents and instruments: Serum ficolin-2 concentration was detected using an enzyme-linked immunosorbent assay (ELISA) with an ELISA reader (wavelength 450 nm).

[0028] Ficolin-2 can also be quantitatively detected using chemiluminescence immunoassay (CLIA), electrochemiluminescence immunoassay (ECLIA), or protein chips.

[0029] Assessment process: Changes in maximum wall thickness (MaxWT), IPH volume, and plaque volume of carotid atherosclerotic plaques were measured by magnetic resonance imaging (MRI) at baseline and at 1, 3, and 6 months after treatment. Serological indicators were analyzed by proteomics and metabolomics.

[0030] 4. The statistical methods involved in this invention: The Shapiro-Wilk test was used to assess the data distribution. A two-tailed t-test was used for normally distributed data, and the Wilcoxon signed-rank test was used for non-normally distributed data. P < 0.05 was considered statistically significant.

[0031] Example 1: Application of serum ficolin-2 protein as a biomarker for classifying and evaluating the efficacy of sonodynamic therapy in patients with carotid artery hemorrhage plaques. (1) MRI assessment of changes in the maximum wall thickness (MaxWT) of carotid atherosclerotic plaques MaxWT of carotid atherosclerotic plaques was assessed using MRI before SDT treatment (baseline) and at 1, 3, and 6 months after treatment. Results are as follows: Figure 1As shown, the MaxWT of all plaques decreased significantly 1 month after SDT treatment ( P =0.0011), the MaxWT of plaques with IPH was significantly reduced 1 month after SDT treatment ( P =0.0094); IPH volume showed a decreasing trend 3 months after SDT treatment ( Figure 1 ).

[0032] (2) Subgroup analysis of the efficacy of SDT intervention in IPH plaques Thirty days after sonodynamic therapy, patients were divided into groups based on MRI assessment of IPH volume changes: Good response group (GRs): IPH volume decreased by more than 10% after intervention (n=7); Poor Response Group (PRs): Participants whose IPH decreased by less than or equal to 10% or whose IPH increased after intervention were classified as poor responders (PRs) (n=3) (see [link to relevant documentation]). Figure 2 ).

[0033] (3) Serological analysis of IPH plaques Serum proteomics analysis showed that serum ficolin-2 levels were significantly elevated in all IPH subjects after SDT treatment. Serum ficolin-2 levels were significantly elevated in the GRs group, while serum C3, C1q, and MASP1 levels were significantly decreased. The change in ficolin-2 was the most significant, increasing 20-fold. Figure 3 Therefore, serum ficolin-2 protein can serve as a biomarker for evaluating the efficacy of sonodynamic therapy in patients with carotid artery hemorrhage plaques.

[0034] The complement system inhibits the progression of atherosclerosis by cleaving the central component C3 of complement towards anti-inflammatory effects, enhancing opsonization and promoting the clearance of dead cells. This clearance is achieved through ficolin-2.

[0035] Enzyme-linked immunosorbent assay (ELISA) of peripheral blood serum further validated the results of peripheral blood proteomics: serum ficolin-2 levels in all IPH subjects were compared before and after treatment, and the results showed that the mean level increased significantly after treatment. Figure 4 Further, all patients were divided into GRs and PRs groups. Results showed that serum ficolin-2 levels were significantly elevated in the GRs group after treatment. Figure 5 ); while the ficolin-2 level in the PRs group showed no significant change ( Figure 6 ).

[0036] Based on the above experimental results, the following evaluation indicators are derived: Therapeutic efficacy monitoring indicators: Serum ficolin-2 levels were measured before SDT treatment and at 1, 3, and 6 months after treatment. If the serum ficolin-2 level increased by ≥10% after treatment, SDT treatment was considered effective; if the level increased by <10% or decreased, the treatment was considered ineffective and the treatment strategy needed to be adjusted.

[0037] Based on the above experimental results, Figure 2 The results showed that magnetic resonance imaging revealed changes in plaque structure after treatment, with a reduction in intraplaque hemorrhage components. This reduction in plaque components indicates improved plaque stability. Simultaneously, serum proteomics (…) Figure 3 ) and ELISA results ( Figures 4-6 The results showed that serum ficolin-2 levels increased after treatment. Therefore, we inferred that ficolin-2 levels are related to plaque structure and composition. Thus, detecting ficolin-2 levels in peripheral blood can indicate plaque composition and stability, and play the role of a diagnostic biomarker.

[0038] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An application of a serum biomarker for non-disease diagnostic purposes, characterized in that, The application is used to assess the stability of atherosclerotic plaques, and the serum biomarker is ficolin-2, which is derived from the serum of the subject.

2. The application according to claim 1, characterized in that, The decreased ficolin-2 level indicates that the subject's atherosclerotic plaques are vulnerable plaques with a risk of instability.

3. The application according to claim 1, characterized in that, The serum biomarkers can be combined with other serum biomarkers to assess the stability of atherosclerotic plaques. The other serum biomarkers include C3, C5, C1q, and MASP1.

4. The use of the reagent for detecting the serum biomarker of claim 1 in the preparation of a product for assessing the stability of atherosclerotic plaques.

5. A product for evaluating the stability of atherosclerotic plaques, characterized in that, The product includes reagents for detecting the serum biomarkers of claim 1.

6. The use of ficolin-2 as a target in the development of drugs for the prevention and / or treatment of vulnerability to atherosclerotic plaques.

7. The application according to claim 6, characterized in that, The drug includes an agonist that targets the ficolin-2 signaling pathway.

8. An application of a serum biomarker for non-disease diagnostic purposes, characterized in that, The application is used to evaluate the prognosis of sonodynamic therapy for atherosclerosis, and the serum biomarker is ficolin-2, which is derived from the serum of the subject.

9. The use of the reagent for detecting the serum biomarker of claim 8 in the preparation of a product for evaluating the prognosis of sonodynamic therapy for atherosclerosis.

10. A product for evaluating the prognosis of sonodynamic therapy for atherosclerosis, characterized in that, The product includes reagents for detecting the serum biomarkers of claim 8.

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