Serum biomarker VE-cadherin for assessing carotid atherosclerotic plaque and application thereof

Abstract

The application relates to a serum biomarker VE-cadherin for evaluating carotid atherosclerotic plaques and an application thereof, and belongs to the biomedical technology field. In order to solve the technical problems such as the urgent need to find a circulating biomarker which can reflect the unstable state of atherosclerotic plaques, especially the hemorrhagic characteristics, is easy to obtain, and can be repeatedly detected, the application provides an application of VE-Cadherin as a serum biomarker in preparation of products for evaluating the stability of atherosclerotic plaques and the prognosis of sonodynamic treatment of atherosclerosis. The serum VE-cadherin concentration reflects the damage degree of the endothelial adhesion connection of the neovessels, realizes accurate typing and dynamic management of the patients, fills the blank of the prior art in the fields of pre-treatment evaluation and curative effect quantification, and provides a key tool for clinical transformation of emerging therapies such as sonodynamic technology.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a serum biomarker for assessing carotid atherosclerotic plaques, especially unstable or hemorrhagic plaques, and its application, particularly the use of vascular endothelial cadherin (VE-Cadherin) as a serum biomarker in the preparation of products for diagnosing or assessing the risk of carotid hemorrhagic plaques. Background Technology

[0002] Atherosclerosis is the primary pathological basis for cardiovascular and cerebrovascular events (such as cerebral infarction and myocardial infarction). Among these, the nature of carotid atherosclerotic plaques, especially their stability, is a key factor determining stroke risk. Unstable plaques, particularly those with intraplaque hemorrhage, are characterized by a thin fibrous cap, inflammatory cell infiltration, and abundant and fragile neovascularization, making them highly susceptible to rupture, secondary thrombosis, and acute vascular events. Therefore, early, non-invasive, and accurate identification and assessment of carotid hemorrhage plaques is of significant clinical importance for risk stratification and intervention strategy development in high-risk populations.

[0003] Currently, the main methods for assessing carotid artery plaques rely on imaging examinations such as ultrasound, computed tomography angiography, and magnetic resonance imaging (MRI). Among these, high-resolution magnetic resonance imaging (MRI) can accurately identify high-risk features such as intraplaque hemorrhage and lipid necrosis cores, and is considered the "gold standard" for assessing plaque stability. However, MRI examinations are expensive, time-consuming, and require highly skilled equipment and interpreters, making them difficult to popularize as a routine screening or dynamic monitoring method in primary healthcare institutions and large populations. Therefore, there is an urgent need to find easily obtainable and repeatable circulating biomarkers that can reflect plaque instability, especially hemorrhagic characteristics.

[0004] Vascular endothelial cadherin (VE-Cadherin) is an important member of the cadherin family. It is mainly expressed at the cell adhesion junctions of vascular endothelial cells and is a core molecule for maintaining the integrity of the endothelial barrier and regulating vascular permeability (Lampugnani MG, Dejana E, Giampietro C. Vascular Endothelial (VE)-Cadherin, Endothelial Adherens Junctions, and Vascular Disease. Cold Spring Harb Perspect Biol. 2018 Oct 1;10(10):a029322.). Studies have shown that VE-cadherin not only acts as a structural protein but also as a signaling hub, playing a crucial role in angiogenesis, inflammatory response, and maintenance of vascular homeostasis (Delgado-Bellido D, Serrano-Saenz S, Fernández-Cortés M, Oliver FJ. Vasculogenic mimicry signaling revisited: focus on non-vascular VE-cadherin. Mol Cancer. 2017 Mar 21;16(1):65.).

[0005] Existing research has preliminarily explored the role of VE-cadherin in atherosclerosis. For example, in the histopathological sections of carotid plaque tissue from patients with atherosclerosis, immunohistochemical results showed that the VE-cadherin level in the unstable plaque group was higher than that in the stable plaque group, and VE-cadherin was positively correlated with intimal thickness and plaque area (Huang Ying, Hu Cunkun. Relationship between serum ANGPTL4, VE-cadherin levels and vulnerable carotid plaques in patients with acute cerebral infarction [J]. Central South Medical Science Journal, 2022, 50(5):762-764.). However, this study focused on protein expression at the tissue level and failed to provide information on this indicator in circulating blood. To date, no study has conducted an association analysis between the level of VE-cadherin in circulating blood (serum or plasma) and carotid atherosclerotic plaques, especially hemorrhagic plaques with high-risk characteristics. Whether VE-Cadherin can serve as a convenient serological marker for non-invasively indicating the risk of intraplaque hemorrhage in the carotid artery, 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

[0006] To address the urgent need for readily available and repeatable circulating biomarkers that reflect the unstable state of atherosclerotic plaques, especially their hemorrhagic characteristics, this invention provides the application of VE-Cadherin 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 VE-Cadherin as a target for developing drugs to prevent and / or treat the vulnerability of atherosclerotic plaques.

[0007] 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, characterized in that the application is used to assess the stability of atherosclerotic plaques, wherein the serum biomarker is VE-Cadherin, and the serum biomarker is derived from the serum of a subject.

[0008] In one embodiment of the present invention, the elevated VE-Cadherin content in the peripheral blood serum indicates that more VE-Cadherin has detached and entered the bloodstream, resulting in more severe vascular damage. The atherosclerotic plaques of the subject are vulnerable plaques and are at risk of instability.

[0009] 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 heme-binding protein and MASP1.

[0010] In one embodiment of the present invention, VE-Cadherin can be combined with other serum indicators to construct a multi-indicator prediction model and improve the accuracy of subtyping; for example, it can be combined with the detection of heme-binding protein (Hemopexin), MASP1, etc. to construct a multi-indicator prediction model and improve the accuracy of efficacy prediction.

[0011] 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.

[0012] 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.

[0013] A fourth object of the present invention is to provide the use of VE-Cadherin as a target for the development of medicaments for the prevention and / or treatment of vulnerability to atherosclerotic plaques, said medicaments comprising inhibitors of serum VE-Cadherin.

[0014] In one embodiment of the present invention, the drug comprises an antagonist or antibody targeting the VE-Cadherin signaling pathway.

[0015] In one embodiment of the present invention, antagonists or neutralizing antibodies targeting the VE-Cadherin signaling pathway are developed to stabilize plaque endothelial structure and prevent plaque rupture.

[0016] 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 VE-Cadherin, and the serum biomarker is derived from the serum of a subject.

[0017] 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.

[0018] 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.

[0019] The beneficial effects of this invention are: This invention is the first to use serum VE-cadherin concentration as a biomarker to assess the stability of atherosclerotic plaques (the degree of disruption of neovascular endothelial adhesion junctions within carotid artery plaques). Specifically, elevated VE-cadherin levels indicate that the subject's atherosclerotic plaques are vulnerable plaques with a risk of instability.

[0020] Furthermore, this invention has found that serum VE-cadherin concentration can highly sensitively indicate the degree of neovascular endothelial damage, thereby enabling accurate screening of patients with carotid artery hemorrhage plaques before SDT treatment and avoiding ineffective treatment. Serum VE-cadherin concentration can also serve as a predictive indicator of the responsiveness of patients with carotid artery hemorrhage plaques to sonodynamic therapy (SDT). By detecting serum VE-cadherin concentration, good responders (GRs) and poor responders (PRs) can be differentiated in advance to guide appropriate intervention timing and further serve as an indicator for monitoring and evaluating treatment efficacy. Serum VE-cadherin concentration can be used alone or in combination with other indicators to complete patient classification and treatment efficacy evaluation, which is beneficial for medical staff to accurately conduct pre-treatment assessment and post-treatment management of carotid artery hemorrhage plaques.

[0021] Serum VE-cadherin concentration serves as a predictive indicator of the responsiveness to SDT treatment for carotid artery hemorrhage plaques and a key indicator for monitoring efficacy. By reflecting the degree of disruption of neovascular endothelial adhesion junctions, it enables precise patient classification and dynamic treatment management, filling the gap in existing technologies for pre-treatment assessment and efficacy quantification. It also provides a crucial tool for the clinical translation of emerging therapies such as sonodynamic technology. Attached Figure Description

[0022] Figure 1 This 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 different time points after SDT intervention, when the IPH volume decreased most significantly. 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 display the differences in serum protein levels between the GRs and PRs groups when the IPH volume decreased most before and after SDT treatment; Figure 4 Figure showing the results of serum VE-Cadherin levels in the GRs and PRs groups at baseline using ELISA; *P<0.05. Detailed Implementation

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 2. The intervention method of the present invention: All participants received a single SDT treatment.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] Test reagents and instruments: Serum VE-Cadherin concentration was detected using an enzyme-linked immunosorbent assay (ELISA) with an ELISA reader (wavelength 450 nm).

[0032] VE-Cadherin can also be quantitatively detected using chemiluminescence immunoassay (CLIA), electrochemiluminescence immunoassay (ECLIA), or protein chips.

[0033] Assessment process: Changes in maximum wall thickness (MaxWT) were measured by magnetic resonance imaging (MRI) at baseline and at 1, 3 and 6 months after treatment, and serological indicators were analyzed by proteomics and metabolomics.

[0034] 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 a Wilcoxon signed-rank test was used for non-normally distributed data. P < 0.05 was considered statistically significant.

[0035] Example 1: Application of serum VE-cadherin 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<0.05), and the MaxWT of plaques with IPH decreased significantly 1 month after SDT treatment (P<0.05); the volume of IPH showed a decreasing trend 3 months after SDT treatment.

[0036] (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 ).

[0037] (3) Serological analysis of IPH plaques Serological analysis showed that at baseline, serum VE-Cadherin levels in the PRs group were significantly higher than those in the GRs group. After SDT treatment, serum VE-Cadherin levels were significantly reduced in all IPH participants. Figure 3 ).

[0038] At baseline, proteomics analysis showed that serum VE-Cadherin levels in the PRs group were significantly higher than those in the GRs group. Figure 3 VE-Cadherin is a key component of adhesion junctions and plays a central role in maintaining and regulating vascular endothelial barrier function. During leukocyte migration, its extracellular domains detach, leading to increased capillary permeability and erythrocyte extravasation.

[0039] Enzyme-linked immunosorbent assay (ELISA) of peripheral blood serum further validated the results of peripheral blood proteomics: serum VE-Cadherin levels in the PRs group were significantly higher than those in the GRs group. Figure 4 ).

[0040] Based on the above experimental results, the following evaluation indicators are derived: Pre-treatment screening indicators for SDT: The cutoff value for serum VE-Cadherin was set at ≥200 ng / mL (based on...). Figure 4 (Baseline differences between GRs and PRs are shown). If a patient's serum VE-Cadherin ≥200 ng / mL is considered a poor SDT response (PR), it is recommended to adjust the treatment plan or combine it with other interventions.

[0041] Therapeutic efficacy monitoring indicators: Serum VE-Cadherin levels were measured before SDT treatment and at 1, 3, and 6 months after treatment. Peripheral blood proteomics results showed that a significant decrease in serum VE-Cadherin levels after treatment was considered an effective treatment. If the decrease in VE-Cadherin levels was not significant (<10%) or even increased after treatment, the treatment was considered ineffective, and the treatment strategy needed to be adjusted.

[0042] 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 VE-Cadherin, which is derived from the serum of the subject.

2. The application according to claim 1, characterized in that, The elevated VE-Cadherin levels indicated that the subject's atherosclerotic plaques were vulnerable and at 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 heme-binding protein 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 VE-Cadherin as a target in the development of drugs for the prevention and / or treatment of vulnerability to atherosclerotic plaques, characterized in that, The drug includes an inhibitor of serum VE-Cadherin.

7. The application according to claim 6, characterized in that, The drug includes antagonists or antibodies that target the VE-Cadherin 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 VE-Cadherin, 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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