Application of CXC chemokine ligand 13 as vascular calcification diagnostic marker

Abstract

The invention provides application of a CXC chemokine ligand 13 as a vascular calcification diagnosis marker, and belongs to the technical field of medicines. The high expression of the CXCL13 prompts the high risk of vascular calcification, and the reagent for detecting the expression level of the CXC chemotactic factor ligand 13 can be used for preparing a vascular calcification early-warning reagent or early-warning kit. The invention studies the effect and molecular mechanism of CXCL13 in macrophages and vascular smooth muscle cells for regulating and controlling vascular calcification, provides an early intervention target for vascular calcification, and has great significance for early diagnosis and prevention of vascular calcification.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of CXC chemokine ligand 13 as a diagnostic marker for vascular calcification. Background Technology

[0002] Vascular calcification (VC) is one of the most common pathological changes in cardiovascular disease. VC mainly includes three types: atherosclerotic plaque calcification, Münckeberg medial calcification, and initial calcification. Each type of VC has its own characteristics but also shares commonalities. The commonality is that the occurrence of VC is similar to the process of bone development, and it is a regulated pathological process. Vascular smooth muscle cells (VSMCs) play a crucial role in the pathological process of VC. However, the pathological mechanisms involved in vascular calcification are complex, and the core molecular regulatory network and key nodes in the pathogenesis process remain unclear, requiring urgent basic research to resolve. Therefore, in-depth research into the pathogenesis of vascular calcification will be of great significance for the prevention and treatment of cardiovascular and cerebrovascular diseases.

[0003] CXC chemokine ligand 13 (CXCL13) is a 10.7 kDa chemokine containing 87 amino acids. It is primarily recognized on B lymphocytes (BLCs) and B-cell-attracting 1 (BCA-1) cells, belonging to the CXC chemokine superfamily. This homologous chemokine attracts B lymphocytes and also functions on macrophages and T cells. Nearly 50% of CXCL13 chemokines are distributed in the liver, with the remainder found in serum, lymph nodes, and the stomach. It regulates B lymphocytes through its unique membrane protein receptor, CXCR5. Studies have shown that extra-lymphoid organs, CXCL13-CXCR5 plays a decisive role in regulating the selective recruitment of B lymphocytes to sites of inflammatory damage, such as atherosclerotic plaques. CXCL13-CXCR5 also plays an important role in regulating tumor microenvironment formation and tumor growth, progression, and metastasis, as well as liver injury, macrophage recruitment, induction of perioperative neurocognitive impairment, adipocyte inflammation, and lipid metabolism disorders. Furthermore, reduced CXCL13 expression is associated with decreased osteoclast differentiation. However, the relationship between CXCL13 and vascular calcification is still unclear. Summary of the Invention

[0004] The purpose of this invention is to provide the application of CXC chemokine ligand 13 as a diagnostic marker for vascular calcification, where high CXCL13 expression indicates a high risk of vascular calcification.

[0005] To achieve the above objectives, the present invention provides the application of CXC chemokine ligand 13 as a diagnostic biomarker for vascular calcification.

[0006] This invention also provides the application of reagents for detecting the expression level of CXC chemokine ligand 13 in the preparation of vascular calcification early warning reagents or early warning kits.

[0007] As a preferred embodiment, the warning reagent comprises a reagent for detecting the content of CXC chemokine ligand 13 in biological samples.

[0008] As a preferred embodiment, the early warning kit contains reagents for detecting the content of CXC chemokine ligand 13 in biological samples.

[0009] In a preferred embodiment of the present invention, an enzyme-linked immunosorbent assay (ELISA) is used to detect the CXCL13 content in the patient's serum. Alternatively, a kit for detecting CXCL13 content can be used to detect the CXCL13 content in the serum.

[0010] As a preferred embodiment, the biological sample is obtained from the subject's blood.

[0011] The present invention also provides the use of CXC chemokine ligand 13 in the preparation of a drug for treating vascular calcification, wherein the drug blocks the expression of the CXC chemokine ligand 13 specific receptor CXCR5.

[0012] In a preferred embodiment of the present invention, a CXCR5 receptor blocker is used to inhibit CXC chemokine ligand 13-induced smooth muscle cell calcification.

[0013] In a preferred embodiment of the present invention, transfection with ADV-CXCR5-siRNA knocks down the expression of the CXCL13-specific receptor CXCR5 in smooth muscle cells, reduces cellular calcification deposition, and improves macrophage-induced smooth muscle cell calcification.

[0014] The advantage of this invention is that it studies the role and molecular mechanism of CXCL13 in regulating vascular calcification in macrophages and vascular smooth muscle cells, providing early intervention targets for vascular calcification, which is of great significance for early diagnosis and prevention of vascular calcification. Attached Figure Description

[0015] Figure 1 ac: ApoE- / - mice were fed a high-phosphorus, high-calcium, and high-fat diet for 12 weeks to induce aortic calcification. Aortic tissue was taken for single-cell sequencing (scRNA-seq) and 7 cell subpopulations were obtained.

[0016] Figure 2 ac: Macrophages were re-clustered into 25 subgroups.

[0017] Figure 3 Clusters 4, 8, and 12 highly expressed the resident macrophage markers Lyve, Timd4, and Folr2, and were defined as resident macrophages.

[0018] Figure 4 Gene enrichment analysis and KEGG and GO analysis were performed on the resident macrophage subsets of each group.

[0019] Figure 5 ab experimental group mice had aortic macrophage subsets (Lyve + Timd4 + Folr2 + The expression level of CXCL13 was significantly increased in ) and VSMCs.

[0020] Figure 6 ab used flow cytometry to sort resident macrophages and then performed Western blotting. CXCL13 expression was increased in resident macrophages induced by OX-LDL.

[0021] Figure 7 ac: Calcification was induced in mouse vascular smooth muscle cells using calcified culture medium. VSMCs were intervened with recombinant CXCL13 protein. The CXCL13-specific receptor CXCR5 was knocked down by adenovirus. Alizarin Red staining showed that calcification deposition was significantly increased in the CXCL13 recombinant protein intervention compared with the control group, while calcification deposition was reduced after CXCR5 knockdown. Figure 7 dg: Western blot analysis of calcification markers RUNX2 and BMP2 revealed that the CXCL13 recombinant protein intervention significantly increased calcification marker expression compared to the control group, while CXCR5 knockdown reduced calcification marker expression.

[0022] Figure 8 There were no significant differences in baseline data among the groups, but the plasma CXCL13 concentration in patients with calcification was significantly higher than that in the control group (P < 0.001).

[0023] Figure 9 a: Plasma CXCL13 concentration was detected by ELISA. Compared with patients without calcification (calcification score of 0), the plasma CXCL13 concentration was significantly higher in patients with mild to moderate calcification (calcification score of 1-3) to severe calcification (calcification score of 4). Figure 9 b: Correlation analysis was performed on the absolute value of the patient's calcium score (CACS) and plasma CXCL13 concentration. CACS and CXCL13 concentration were linearly correlated. Figure 9 c: ROC curve analysis results showed that the AUC value for judging vascular calcification based on plasma CXCL13 level was 0.778, the optimal cutoff value was >6.849 pg / mL, the sensitivity was 79.55%, and the specificity was 66.67%. Detailed Implementation

[0024] The present invention will now be described in detail with reference to specific embodiments. It should be understood that the following specific embodiments are only for the purpose of helping those skilled in the art to understand the present invention, and are not intended to limit the present invention.

[0025] Example 1. CXCL13 regulates vascular calcification in macrophages and vascular smooth muscle cells.

[0026] To investigate the mechanisms of vascular calcification and identify key targets in its development, we induced vascular calcification in 8-week-old C57BL / 6-derived ApoE- / - mice. After one week of acclimatization under standard conditions, the control group was fed a normal diet, while the calcified group was fed a high-phosphorus, high-fat diet for 16 weeks. Single-cell sequencing was performed on the aorta and surrounding tissues of the mice.

[0027] Our research found that ApoE animals fed a high-phosphorus, high-calcium, and high-fat diet... - / - Single-cell sequencing (scRNA-seq) of mouse aortic tissue was performed. Figure 1 a) , obtained 7 cell subsets ( Figure 1 b, c), the macrophages were further clustered into 25 subgroups (b, c), Figure 2 Clusters 4, 8, and 12 highly expressed the resident macrophage markers Lyve, Timd4, and Folr2, and were defined as resident macrophages. Figure 3 Gene enrichment analysis and KEGG / GO analysis were performed on the resident macrophage subsets in each group. We found that compared with the control group mice, the experimental group mice had aortic resident macrophage subset (Lyve... + Timd4 + Folr2 + The expression level of CXCL13 on VSMCs was significantly increased (FC = 17.48). Figure 4 , Figure 5 Western blot analysis of resident macrophages by flow cytometry revealed increased CXCL13 expression in OX-LDL-induced resident macrophages. Figure 6 ).

[0028] Example 2. CXCL13 recombinant protein intervention in VSMCs

[0029] To verify the role of CXCL13 in vascular calcification, we conducted in vitro studies. Mouse vascular smooth muscle cells (VSMCs) were resuspended and seeded into culture dishes, cultured in an incubator, and the medium was changed every 2 days. Cells were passaged until 90%–100% confluence, and cells from passages 3–5 were used. Calcification was induced using a calcification medium containing 10 mM β-glycerophosphate sodium, 50 μg / mL ascorbic acid, and 100 nM dexamethasone. VSMCs (0.5 μL / mL medium) were treated with recombinant CXCL13 protein (recombinant mouse CXCL13 protein (ab50165), abcam, Shanghai). The expression of the CXCL13-specific receptor CXCR5 in smooth muscle cells was knocked down by transfection with ADV-CXCR5-siRNA (Hanbio, Shanghai). After removing the culture medium, the cells were fixed with 4% paraformaldehyde for 10 min, rinsed with 95% ethanol, and stained with 1% alizarin red for 10 min. The staining solution was removed, and the cells were rinsed with 95% ethanol before observation and photography under a microscope. Orange-red nodules were identified as calcified nodules. Alizarin red staining and calcification marker detection revealed that calcification deposition was significantly increased in the CXCL13 recombinant protein intervention group compared to the control group. However, knockout of the CXCL13-specific receptor CXCR5 reduced cellular calcification deposition and decreased expression of calcification markers. Figure 7 (ag). This indicates that CXCL13 can exacerbate the transformation of VSMCs to a calcified phenotype through its specific receptor CXCR5.

[0030] Example 3. Early warning effect of CXCL13 on vascular calcification in clinical patients

[0031] To verify the predictive effect of CXCL13 on vascular calcification in clinical patients, we established a clinical cohort with coronary artery calcification and a control group without calcification and collected clinical characteristics: A cohort of patients with coronary heart disease was established at Xinhua Hospital affiliated with Shanghai Jiao Tong University School of Medicine. Informed consent was obtained from patients, and clinical characteristics were collected, including age, sex, weight, BMI, blood pressure, serum calcium, serum phosphorus, calcium-phosphorus product, ALP, blood glucose, and blood lipids. Coronary CT was used to detect the calcification index, and plasma CXCL13 concentration was measured by ELISA. Clinical correlation analysis was performed. Results showed no significant differences in baseline data between the two groups, but the plasma CXCL13 concentration in patients with calcification was significantly higher than that in the control group. Figure 8 Compared to patients without calcification (calcification score of 0), patients with mild to moderate calcification (calcification score of 1-3) to severe calcification (calcification score of 4) showed significantly higher plasma CXCL13 concentrations. Figure 9 a). Pearson correlation analysis of the absolute value of the patient's calcium score (CACS) and plasma CXCL13 concentration showed a linear correlation between CACS and CXCL13 concentration. Figure 9b, r = 0.3568, P < 0.01). ROC curve analysis showed that the AUC value for judging vascular calcification based on plasma CXCL13 levels was 0.778, the optimal cutoff value was >6.849 pg / mL, the sensitivity was 79.55%, and the specificity was 66.67%. Figure 9 c). We believe that plasma CXCL13 concentration can serve as a warning indicator of vascular calcification in clinical patients; vascular calcification is present when plasma CXCL13 concentration > 6.849 pg / mL. The above studies indicate that CXCL13 plays an important role in vascular calcification and can serve as a potential warning indicator of vascular calcification in clinical practice.

[0032] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications 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. Application of CXC chemokine ligand 13 as a diagnostic biomarker for vascular calcification.

2. Application of reagents for detecting the expression level of CXC chemokine ligand 13 in the preparation of vascular calcification early warning reagents or early warning kits.

3. The application of the reagent for detecting the expression level of CXC chemokine ligand 13 according to claim 2 in the preparation of vascular calcification early warning reagents or early warning kits, characterized in that, The warning reagent includes a reagent for detecting the content of CXC chemokine ligand 13 in biological samples.

4. The application of the reagent for detecting the expression level of CXC chemokine ligand 13 according to claim 2 in the preparation of vascular calcification early warning reagents or early warning kits, characterized in that, The early warning kit contains reagents for detecting the content of CXC chemokine ligand 13 in biological samples.

5. The application of the reagent for detecting the expression level of CXC chemokine ligand 13 according to claim 3 or 4 in the preparation of vascular calcification early warning reagents or early warning kits, characterized in that, The biological sample was obtained from the subject's blood.

6. The application of CXC chemokine ligand 13 in the preparation of drugs for treating vascular calcification, characterized in that, The drug blocks the expression of CXC chemokine ligand 13-specific receptor CXCR5.

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