Atherosclerotic plaque instability and its application in diagnosis of acute coronary syndrome

Abstract

The application belongs to the technical field of biological medicine, and discloses an auxiliary marker for diagnosing atherosclerotic plaque instability and acute coronary syndrome and application thereof. The auxiliary marker is selected from at least one of the following: a) Proteus mirabilis; b) extracellular vesicles derived from Proteus mirabilis; and c) HpmA in the extracellular vesicles. The auxiliary marker is used for preparing a product for diagnosing, screening or evaluating atherosclerotic plaque instability and / or acute coronary syndrome. The application proves that Proteus mirabilis is significantly enriched in ACS patients, extracellular vesicles carrying HpmA cross the intestinal barrier and are enriched in atherosclerotic plaques. These vesicles inhibit the FAK / SRC signaling pathway through HpmA, induce VSMC to undergo phenotypic transformation, promote plaque instability, and provide an action target and a screening model for drug development. The application is easy to popularize because it is detected through non-invasive samples such as feces and blood.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an auxiliary biomarker for the diagnosis of atherosclerotic plaque instability and acute coronary syndrome, and its application in the preparation of products for the diagnosis, screening, or evaluation of atherosclerotic plaque instability or acute coronary syndrome (ACS). Background Technology

[0002] Atherosclerotic cardiovascular disease is one of the leading causes of disease prevalence and mortality worldwide. Acute coronary syndrome (ACS) is one of the most serious cardiovascular diseases, its pathogenesis being the instability of atherosclerotic plaques in the coronary arteries. Epidemiological studies show that up to 85% of fatal acute coronary events are caused by the rupture or erosion of unstable plaques. Therefore, accurate prediction and effective intervention of unstable plaques are crucial for the prevention of ACS.

[0003] However, current diagnostic and assessment methods have limitations. While circulating biomarkers, such as high-sensitivity C-reactive protein (hs-CRP) and cardiac troponin (cTn), can provide prognostic information, their correlation with actual plaque stability and vulnerability is poor. Although advanced imaging techniques such as intravascular optical coherence tomography (OCT) can identify unstable plaques, their invasiveness and high cost limit their widespread use in routine clinical practice. Therefore, there is an urgent need to find non-invasive biomarkers that can accurately reflect plaque vulnerability.

[0004] One of the core pathophysiological mechanisms of plaque instability is the phenotypic transformation of vascular smooth muscle cells (VSMCs). In vulnerable lesions, VSMCs transform from a stable contractile type to a pro-inflammatory type, leading to thinning of the fibrous cap and expansion of the necrotic core, ultimately causing plaque rupture.

[0005] Finding a marker that influences VSMC phenotype and plaque stability, as well as an auxiliary marker for the diagnosis, screening, or assessment of atherosclerotic plaque instability or acute coronary syndrome (ACS), remains an urgent question to be explored. Summary of the Invention

[0006] To overcome the shortcomings and deficiencies of existing technologies, the present invention aims to provide an auxiliary biomarker for the diagnosis of atherosclerotic plaque instability and acute coronary syndrome, and its application. The present invention utilizes *Proteus mirabilis*, a Gram-negative bacterium found in the intestine. Proteus mirabilis Or extracellular vesicles of *Proteus mirabilis* (Pm-EVs), or the HPMA protein or its encoding gene in Pm-EVs, can be used as biomarkers for the diagnosis of atherosclerotic plaque instability and acute coronary syndrome. *Proteus mirabilis* ( Proteus mirabilisIt was significantly enriched in ACS patients and positively correlated with myocardial injury markers. In addition, Proteus mirabilis delivers its virulence factor HPMA to the circulatory system and targets atherosclerotic plaques by releasing extracellular vesicles (Pm-EVs) as a carrier, thereby causing atherosclerotic plaque instability.

[0007] The objective of this invention is achieved through the following technical solution: An auxiliary biomarker for the diagnosis of atherosclerotic plaque instability and acute coronary syndrome, selected from at least one of the following: a) Proteus mirabilis; b) Extracellular vesicles (Pm-EVs) derived from Proteus mirabilis; c) HPMA hemolysin A present in the extracellular vesicles of Proteus mirabilis.

[0008] The extracellular vesicles of the *Proteus mirabilis* were prepared by the following method: The cultured Proteus mirabilis was subjected to differential centrifugation at the following conditions: 200 g–400 g for 10–20 minutes, 2000–4000 g for 15–25 minutes, and 11,000–13,000 g for 25–35 minutes. The supernatant was collected, and the precipitate was collected by ultracentrifugation. The precipitate was resuspended in sterile PBS, and ultracentrifuged again to remove the supernatant and obtain extracellular vesicles. The extracellular vesicles were resuspended in PBS for later use.

[0009] The ultracentrifugation is performed at 134,000~136,000g for 65~75 minutes. The second ultracentrifugation is performed at 119,000~121,000 g for 30~40 min.

[0010] The temperature for the ultracentrifugation is 3~5℃. The temperature for the second ultracentrifugation is 3~5℃.

[0011] The temperature of the differential centrifuge is 3~5℃.

[0012] The auxiliary markers can also be referred to as auxiliary substances.

[0013] The aforementioned auxiliary biomarkers for the diagnosis of atherosclerotic plaque instability and acute coronary syndrome are used in products for the diagnosis, screening, or evaluation of atherosclerotic plaque instability and / or acute coronary syndrome (ACS).

[0014] The use of an inhibitor in the preparation of a medicament for the prevention or treatment of atherosclerotic plaque instability or acute coronary syndrome (ACS), said inhibitor being selected from at least one of the following: a) Inhibitors that suppress the growth or activity of Proteus mirabilis; b) Substances that inhibit the production or release of extracellular vesicles (Pm-EVs) by Proteus mirabilis; c) Inhibitors that suppress HPMA protein activity or expression; d) Substances that block the interaction between HPMA (hemolysin A) protein and focal adhesion kinase (FAK).

[0015] A method for screening drugs for the prevention or treatment of atherosclerotic plaque instability or acute coronary syndrome (ACS) includes the following steps: a) Establish a cell model by co-culturing vascular smooth muscle cells (VSMCs) with Proteus mirabilis extracellular vesicles (Pm-EVs) or HPMA protein; b) Contact the candidate drug with the cell model; c) Examine the effects of candidate drugs on VSMC phenotypic transformation or FAK / SRC signaling pathway activity. Candidate drugs that can inhibit VSMC transformation to a pro-inflammatory phenotype or restore FAK / SRC signaling pathway activity are potential therapeutic drugs.

[0016] A biomarker for diagnosing atherosclerotic plaque instability includes Proteus mirabilis; extracellular vesicles (Pm-EVs) derived from Proteus mirabilis; and one or more of HPMA hemolysin A present in the extracellular vesicles of Proteus mirabilis.

[0017] An auxiliary biomarker for the diagnosis of acute coronary syndrome, selected from at least one of the following: a) Proteus mirabilis; b) Extracellular vesicles (Pm-EVs) derived from Proteus mirabilis; c) HPMA hemolysin A present in the extracellular vesicles of Proteus mirabilis.

[0018] This invention has discovered that Proteus mirabilis and its secreted extracellular vesicles (Pm-EVs) and the HPMA protein enriched within the vesicles are key pathogenic factors leading to atherosclerotic plaque instability.

[0019] In this invention, the HPMA protein carried by Pm-EVs can directly bind to focal adhesion kinase (FAK) in vascular smooth muscle cells (VSMCs). By inhibiting phosphorylation of the FAK / SRC signaling pathway, it drives VSMCs to transform from a stable contractile phenotype to a pro-inflammatory, unstable phenotype. This phenotypic transformation leads to decreased collagen synthesis, increased secretion of inflammatory factors, and exacerbated intracellular lipid accumulation, ultimately promoting thinning of the fibrous cap and expansion of the necrotic core, increasing the risk of plaque rupture.

[0020] This invention utilizes Proteus mirabilis, Pm-EVs, or HPMA proteins as biomarkers for diagnosing or assessing the risk of ACS and plaque instability. The levels of these biomarkers in samples such as feces, blood, or serum can be detected, providing a basis for the early, non-invasive diagnosis of ACS.

[0021] This invention aims to stabilize plaques and prevent the occurrence of acute coronary syndrome (ACS) by developing drugs that target and inhibit HPMA protein, block HPMA-FAK interaction, or regulate gut microbiota to reduce Proteus mirabilis levels.

[0022] Compared with the prior art, the present invention has the following beneficial effects and advantages: 1) This invention is the first to directly link the intestinal bacteria Proteus mirabilis and its product HPMA to the core mechanism of plaque instability (VSMC phenotypic transformation). The discovered biomarkers have higher pathological mechanism specificity than traditional inflammatory markers (such as CRP).

[0023] 2) The HPMA of this invention works by inhibiting the FAK / SRC signaling pathway, providing a clear target and screening model for drug development.

[0024] 3) The biomarkers proposed in this invention can be detected using non-invasive samples such as feces or blood, making them easy to clinically apply and suitable for early screening and risk stratification of ACS patients. Furthermore, the therapeutic targets proposed based on well-defined mechanisms open up entirely new avenues for developing drugs to stabilize plaques. Attached Figure Description

[0025] Figure 1 A diagram showing the association between Gram-negative gut bacteria and vascular plaque formation and instability; Figure 2 Figure: Proteus mirabilis promotes plaque progression and instability in atherosclerotic mice; Figure 3 Figure showing the role of extracellular vesicles (Pm-EVs) derived from Proteus mirabilis in systemic translocation and atherosclerotic plaque instability. Figure 4 Pm-EVs induce phenotypic transformation and lipid accumulation in vascular smooth muscle cells (VSMCs); Figure 5 Transcriptomic analysis revealed that FAK / SRC pathway inhibition is a key mechanism by which Pm-EVs induce VSMC phenotypic transformation. Figure 6 The virulence factor HpmA mediates Pm-EVs by inhibiting FAK-induced VSMC phenotypic transformation; Figure 7 EV-mediated delivery enhances the pro-atherosclerotic effect of HPMA in vivo. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to the embodiments, but the implementation and protection of the present invention are not limited thereto.

[0027] Example 1: The association between Proteus mirabilis and ACS Twenty-nine patients with acute coronary syndrome (ACS) and 23 patients with stable angina (SAP) were recruited. Fecal samples were collected from all subjects, and the gut microbiota composition was analyzed using 16S rRNA amplicon sequencing. Results showed that the species composition of the gut microbiota was significantly altered in the ACS group compared to the SAP group. Figure 1 A. At the phylum level, Proteobacteria (… Proteobacteria The abundance of ) was significantly increased in the ACS group ( Figure 1 E). At the genus level, *Proteus* is the most significantly enriched genus in the ACS group. Figure 1 F). Furthermore, mass spectrometry was used to detect multiple proteins derived from the genus *Proteus* in unstable atherosclerotic plaques in humans. Figure 1 H).

[0028] For Proteus mirabilis ( P. mirabilis Spearman correlation analysis was performed on the abundance of ) and clinical myocardial injury indicators, and the results showed that P. mirabilis The abundance of NT-proBNP, high-sensitivity troponin T (hsTNT), creatine kinase isoenzyme (CK-MB), C-reactive protein (CRP), and white blood cell count (WBC) were all significantly positively correlated. Figure 1 I).

[0029] These results indicate that the enrichment of *Proteus mirabilis* in the gut is closely associated with the occurrence and severity of acute coronary syndrome (ACS).

[0030] Figure 1Gram-negative gut bacteria are associated with vascular plaque formation and instability. A: Beta-diversity principal coordinate analysis based on the unweighted unifrac matrix shows that, at the species level, the microbial composition of patients with acute coronary syndrome (ACS) (n=29) differs significantly from that of patients with stable angina (SAP) (n=23). BD: Comparison of α-diversity indices (Shannon, Simpson, and Chao1 indices) of gut microbiota in ACS and SAP patients. E: Stacked bar charts show the top 5 phyla of relative abundance in ACS and SAP patients. F: Volcano plots show the enrichment of gut bacteria at the genus level in ACS patients compared to SAP patients (-log10(P)>1, |log2 (fold change)| ≥2). G: Schematic diagram of the association between atherosclerotic plaques and gut microbiota. H: Representative charts show the protein composition of Proteus mirabilis in human atherosclerotic plaques through mass spectrometry data analysis. I: Spearman correlation between the relative abundance of specific bacteria (Escherichia coli and Proteus mirabilis) and clinical parameters. Red indicates a positive correlation, and blue indicates a negative correlation. Results are expressed as mean ± standard error (Figure BD). Data were analyzed using t-tests. *** P < 0.001; ** P < 0.01; * P < 0.05. ACS, Acute Coronary Syndrome; SAP, Stable Angina.

[0031] Figure 1 The study revealed differences in gut microbiota between patients with acute coronary syndrome (ACS) and those with stable angina (SAP), and found that Proteus mirabilis was positively correlated with clinical indicators of myocardial injury.

[0032] Example 2: Proteus mirabilis promotes plaque instability in animal models To verify the pathogenicity of Proteus mirabilis, ApoE was used. - / - Mice were fed a high-fat diet for 12 weeks to establish an atherosclerosis model. The mice were divided into three groups: a control group (administered PBS by gavage), a heat-inactivated *Proteus mirabilis* group (*P. mirabilis past), and a live *P. mirabilis* group, administered by gavage every 3 days. Figure 2 (A). After 12 weeks, compared with the control group and the heat-inactivated bacteria group, the mice in the live bacteria group showed significantly slower weight gain and significantly higher serum total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) levels. Figure 2 Medium CG).

[0033] En Face Oil Red O staining of the mouse aorta showed that the plaque coverage area was significantly increased in the live bacteria group. Figure 2Middle H, I). Histological analysis of the aortic root further confirmed that the plaques in the viable bacterial group were larger and had more prominent lipid cores (Oil Red O and H&E staining, Figure 2 (J, K, M, N), and the fiber cap is significantly thinner (Masson dyeing, Figure 2 J, O). Immunofluorescence staining showed decreased expression of the VSMC marker αSMA and increased expression of the macrophage marker Mac2 in the viable bacterial plaques. Figure 2 P, Q).

[0034] These results demonstrate that live Proteus mirabilis can exacerbate atherosclerosis in mice and induce plaques to develop unstable characteristics (whether this result is representative of the disease), while heat-inactivated bacteria do not have this effect, indicating that the biological activity of the bacteria is key to its pathogenicity.

[0035] Figure 2 Proteus mirabilis promotes plaque progression and instability in atherosclerotic mice. A: Experimental design. ApoE - / - Mice were fed a high-fat diet for 12 weeks. Red bars represent administration of heat-inactivated Proteus mirabilis (Pm past), live Proteus mirabilis (Pm) (4 × 10^6 CFU), or PBS via oral tube feeding every 3 days. Mice were sacrificed after 12 weeks. n=5 per group. B: Gram staining of Proteus mirabilis, scale bar = 50 μm. C: Changes in mouse body weight gain during 12 weeks. DG: Representative bar graphs showing different serum lipid levels among the three groups, including total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c). HI: ApoE in the three groups. - / - Representative Oil Red O staining of mouse aorta and quantification of lesion coverage area. JQ: 3 groups of ApoE - / - Representative Oil Red O, H&E, and Masson staining of atherosclerotic plaques in the aortic root of mice, as well as co-staining of αSMA (a contraction phenotype marker) and Mac2 (an inflammation phenotype marker), scale bar = 200 μm; DAPI was used to label DNA (blue), αSMA was blue, and Mac2 was red. Three groups of ApoE... - / -Quantitative analysis of plaque size, necrotic area, fibrous cap thickness (yellow arrows), and αSMA and Mac2 positive areas in mouse atherosclerotic plaques. Results are expressed as mean ± standard error (Figures CG, I, KO, Q). Data were analyzed by one-way ANOVA. **** P<0.0001; *** P<0.001; ** P<0.01; * P<0.05. TC, total cholesterol; TG, total triglycerides; LDL-c, low-density lipoprotein cholesterol; HDL-c, high-density lipoprotein cholesterol.

[0036] Example 3: Isolation, characterization, and in vivo tracing of Proteus mirabilis extracellular vesicles (Pm-EVs) Extracellular vesicles of *Proteus mirabilis* (Pm-EVs): *Proteus mirabilis* was cultured in 500 mL of TSB medium under microaerophilic conditions at 37 °C. After culture, to remove bacterial cells, the cells were removed by repeated differential centrifugation at 4 °C at 300 g for 15 min, 3000 g for 20 min, and 12,000 g for 30 min. The collected sterile supernatant was filtered through a 0.22 μm sterile filter and then ultracentrifuged at 135,000 g for 70 min at 4 °C using an SW32Ti rotor to concentrate and precipitate the vesicles. The precipitate was resuspended in sterile PBS and then ultracentrifuged at 120,000 g for 35 min at 4 °C using a 70Ti rotor for secondary precipitation. Finally, the supernatant was removed, the purified vesicles were resuspended in PBS, and their protein concentration was determined by the BCA method. The vesicles were then frozen at -80 °C for subsequent experiments. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) showed that Pm-EVs exhibited a typical spherical bilayer film structure with an average particle size of approximately 150.2 nm. Figure 3 A, B). Western blot analysis confirmed that Pm-EVs carried the membrane protein OmpA and lipopolysaccharide LPS from their mother bacteria. Figure 3 C).

[0037] To investigate whether Pm-EVs can be taken up by vascular cells and cross the intestinal barrier, Pm-EVs were labeled with CellMask fluorescent dye. In the in vitro experiment (the procedure was as follows: first, the cell membrane of Pm-EVs was labeled with CellMask dye and incubated in the dark for 15 minutes; then, the labeled vesicles were precipitated and purified by ultracentrifugation at 135,000 g, 4°C for 70 minutes, and finally resuspended in sterile PBS; next, labeled Pm-EVs at a concentration of 10 μg / ml were co-incubated with mouse aortic smooth muscle cell line (MOVAS) at 37°C, 5% CO2 for 6 hours; after incubation, the cells were washed with PBS, and the nuclei were stained with Hoechst; finally, imaging was performed using an Olympus confocal microscope), the labeled Pm-EVs were successfully internalized by mouse aortic smooth muscle cells (MOVAS). Figure 3 D). In in vivo experiments (the specific procedure for in vivo experiments was to administer 20 μg of labeled Pm-EVs orally via gavage to ApoE cells that had been fed a high-fat diet for 8 weeks). - / - Mice. Twenty-four hours after gavage, mice were sacrificed and samples were collected. Serum fluorescence signals were analyzed using a multi-well microplate reader. Simultaneously, the aorta was completely dissected for in vitro fluorescence signal analysis, and frozen sections were prepared for direct microscopic observation of the specific distribution of fluorescence signals within the vessel wall. - / - Mice were orally administered labeled Pm-EVs via gavage. Significant fluorescence signals were detected in mouse serum 24 hours later, with fluorescence enrichment observed in the aorta (especially in plaque-prone areas such as the aortic arch). Figure 3 F, G). Frozen sections of the aorta showed that the fluorescence signal was mainly concentrated in the vascular media near the intima (F, G). Figure 3 H).

[0038] These results indicate that Pm-EVs can cross the intestinal barrier into the systemic circulation and accumulate in targeted areas of atherosclerotic plaques.

[0039] Extracellular vesicles (Pm-EVs) derived from Proteus mirabilis promoted plaque instability in animal models, as shown in the test results. Figure 3 I~T.

[0040] Figure 3This study demonstrates the systemic translocation of extracellular vesicles (EVs) from *Proteus mirabilis* and their disruption of atherosclerotic plaque stability. A: Morphology of *Proteus mirabilis*-derived EVs (Pm-EVs) under transmission electron microscopy, scale bar = 50 μm. B: Mean diameter of Pm-EVs measured using nanoparticle tracking analysis (NTA). C: Detection of OmpA and LPS in *Proteus mirabilis* and Pm-EVs by Western blotting; total protein was visualized using Coomassie Brilliant Blue staining as a loading control, with a loading amount of 30 μg of protein. D: Uptake of CellMask-labeled Pm-EVs by MOVAS cells; Hoechst was used to label DNA (blue), and Pm-EVs are shown in red, scale bar = 10 μm. E: [The text abruptly ends here, likely due to an incomplete translation or a missing section.] - / - Six hours after oral administration of CellMask and CellMask-labeled Pm-EVs to mice via tube, the distribution of Pm-EVs was detected in vitro by fluorescence imaging, with n=3 per group. F: Bar graph showing the content of CellMask-Pm-EVs in mouse serum, excitation wavelength = 645 nm, emission wavelength = 670 nm. G: Twenty-four hours after oral administration of CellMask and CellMask-labeled Pm-EVs to mice via tube, the distribution of Pm-EVs in the aorta was detected in vitro by fluorescence imaging, excitation wavelength = 630 nm, emission wavelength = 700 nm. H: Representative fluorescence image of a frozen section of a transverse section of the mouse aorta; the lower right corner shows the vascular lumen; DAPI was used to label DNA (blue); autofluorescence of the blood vessel is green; Pm-EVs are red; scale bar = 50 μm. I: Experimental design; ApoE - / - Mice were fed a high-fat diet for 12 weeks. Red bars indicate intraperitoneal injections of Pm-EVs (10 μg / 100 μL) or PBS every 3 days. Mice were sacrificed after 12 weeks, with n=5 per group. J: Changes in mouse body weight gain during 12 weeks. KL: ApoE levels in the two groups. - / - Representative Oil Red O staining of mouse aorta and quantification of lesion coverage area. MT: ApoE in two groups. - / - Representative Oil Red O, H&E, and Masson staining of atherosclerotic plaques in the aortic root of mice, as well as co-staining of αSMA (a contraction phenotype marker) and Mac2 (an inflammation phenotype marker), scale bar = 200 μm; DAPI was used to label DNA (blue), αSMA was blue, and Mac2 was red. Two groups of ApoE... - / -Quantitative analysis of plaque size, necrosis area, fibrous cap thickness, and αSMA and Mac2 positive areas in mouse atherosclerotic plaques. Results are expressed as mean ± standard error (Figures F, J, L, NR, T). Data were analyzed using t-tests. ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05.

[0041] Pm-EVs induce phenotypic transformation and lipid accumulation in vascular smooth muscle cells (VSMCs), and the test results are as follows: Figure 4 As shown in the figure, Pm-EVs downregulate VSMC contractile markers (αSMA, SM22α), upregulate inflammatory markers (CD68, Mac2), and promote intracellular lipid accumulation in vitro.

[0042] Figure 4Pm-EVs induce phenotypic transformation and lipid accumulation in vascular smooth muscle cells. AC: Representative Western blot and quantitative data show the expression of αSMA, SM22α, and GAPDH after 48 hours of treatment with Pm-EVs (10 μg / mL) or PBS. GAPDH was used as a loading control. n=3 per group. DE: qRT-PCR analysis of the mRNA levels of Acta2 and Tagln in the two groups of cells. Gapdh was used as an internal control. n=5 per group. FG: Representative immunofluorescence microscopy images and quantitative data show the expression of CD68 in MOVAS cells treated with PBS, and in MOVAS cells loaded with ox-LDL (50 μg / mL) and co-treated with Pm-EVs (10 μg / mL) or PBS for 48 hours. DAPI was used to label DNA (blue), and CD68 was shown in green. n=5 per group. Scale bar = 10 μm. HJ: Representative Western blot (F) and quantitative data show the expression of αSMA, SM22α, Mac2, and β-tubulin among the three groups; β-tubulin was used as a loading control, n=3 per group. KP: qRT-PCR analysis of mRNA levels of Acta2, Tagln, Cd68, Tnf-α, Mcp1, and IL-6 in the three groups of cells; Gapdh was used as an internal control, n=3 per group. QR: Representative Oil Red O staining and quantification of Oil Red O staining area in the three groups of cells, scale bar = 100 μm, n=5 per group. S: Stacked bar plots show the cholesterol ester (CE), free cholesterol (FC), and total cholesterol content among the three groups, n=5 per group. Results are expressed as mean ± standard error (Figure BE, GP, RS). Data were analyzed by t-test (Figure BE) or one-way ANOVA (Figure GP, RS). ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05.

[0043] Transcriptome analysis revealed that inhibition of the FAK / SRC pathway is a key mechanism by which Pm-EVs induce phenotypic transformation in vascular smooth muscle cells (VSMCs), with results as follows: Figure 5 As shown in the figure, Pm-EVs treatment significantly suppressed cell adhesion-related genes (especially the FAK / SRC pathway) in VSMCs. Figure 5Transcriptomic analysis revealed that FAK / SRC pathway inhibition is a key mechanism by which Pm-EVs induce phenotype transformation in VSMCs. A: Principal component analysis (PCA) was performed on the transcriptomic profiles of MOVAS cells treated with PBS, and MOVAS cells treated with ox-LDL (50 μg / mL) and Pm-EVs (10 μg / mL) or PBS for 48 hours, with n=3 for each group. B: A representative transcriptomic analysis heatmap shows the changes in genes related to MOVAS phenotype transformation among the three groups; this heatmap is generated based on fold change and FPKM values, with numbers shown in the heatmap indicating |fold change| ≥ 2; negative values ​​indicate downregulation of differentially expressed genes (DEGs), and positive values ​​indicate upregulation. C: Analysis of the top 10 enriched KEGG pathways among the upregulated DEGs; comparison of the ox-LDL + Pm-EVs group with the ox-LDL group; the numbers shown are P < 0.05. D: GO enrichment analysis of key functional modules associated with VSMC phenotypic transition; comparison of the ox-LDL + Pm-EVs group with the ox-LDL group, showing P < 0.05. E: Representative immunofluorescence micrographs showing p-Src expression in PBS-treated MOVAS cells, and in MOVAS cells treated with ox-LDL (50 μg / mL) and Pm-EVs (10 μg / mL) or PBS for 48 hours; DAPI was used to label DNA (blue), BODIPY in green, and p-Src in red; n=5 per group, scale bar = 10 μm. FG: Representative Western blot (E) and quantitative data showing the expression of FAK, p-FAK, Src, and p-Src across the three groups; GAPDH was used as a loading control; n=3 per group.

[0044] Example 4: HpmA is a key effector protein in Pm-EVs that induces VSMC phenotypic transformation. Pm-EVs were pretreated with DNase, RNase, or protease before being co-cultured with MOVAS cells. The specific steps were as follows: First, 100 μL of Pm-EVs solution at a concentration of 1 mg / ml was lysed through five freeze-thaw cycles, followed by 30 seconds of sonication and 30 seconds of incubation on ice to expose the internal DNA, RNA, or protein. Then, the lysed Pm-EVs were divided into three groups for enzyme treatment: one group was treated with RNase A at a final concentration of 10 µg / ml and incubated at 37°C for 15 minutes to degrade RNA; another group was treated with DNase I at 1 U / ml and incubated at 37°C for 15 minutes to degrade DNA; and the third group was treated with proteinase K at 20 µg / ml and incubated at 55°C for 15 minutes to degrade protein. Samples treated with proteinase K were further inactivated by heating at 95°C for 5 minutes after incubation. These Pm-EVs treated with different enzymes were then used in subsequent cell co-culture experiments. The results showed that only Pm-EVs treated with protease lost their ability to induce VSMC phenotypic transformation, indicating that the effect was mediated by proteins.

[0045] Subsequently, mass spectrometry proteomics was used to analyze the protein components of Pm-EVs, and it was found that compared with whole bacterial lysates, the virulence factors HPMA (protein ID P16466, NCBI) and FlgB were significantly enriched in Pm-EVs. Figure 6 (A, B). Functional validation was performed by synthesizing recombinant HPMA and FlgB proteins. In vitro experiments: All cells (except the blank control group) were co-incubated with 50 μg / mL oxidized low-density lipoprotein (ox-LDL) to simulate the cellular environment of atherosclerosis. Based on this, the experimental groups were further treated with 10 μg / mL Pm-EVs, purified and endotoxin-free recombinant HPMA protein, or recombinant FlgB protein for 48 hours, respectively. After incubation, the phosphorylation levels of FAK and SRC proteins, as well as the expression levels of phenotype-related proteins such as αSMA, SM22α, and Mac2, were detected by Western blot. Simultaneously, CD68 expression was observed by immunofluorescence staining, and the degree of intracellular lipid accumulation was assessed using Oil Red O staining. In vitro experiments confirmed that recombinant HPMA protein can fully mimic the biological effects of Pm-EVs, including inhibiting FAK / SRC phosphorylation, downregulating VSMC contractile markers (αSMA, SM22α), upregulating inflammatory markers (CD68, Mac2), and promoting lipid accumulation. Figure 6 CK), while FlgB protein has no such effect.

[0046] Molecular docking simulations predict that HPMA can bind directly to the FAK protein. Figure 6L). Co-immunoprecipitation assays further confirmed that, intracellularly, the His-tagged HPMA protein does indeed interact directly with the endogenous FAK protein. Figure 6 N).

[0047] These results clearly indicate that HPMA is a key functional protein in Pm-EVs that mediates VSMC phenotypic transformation, and its mechanism is achieved by directly binding to and inhibiting FAK.

[0048] Figure 6 The virulence factor HPMA mediates the VSMC phenotypic transformation induced by Pm-EVs through inhibition of FAK. This figure uses proteomics to identify HPMA as a key effector protein in Pm-EVs and confirms that recombinant HPMA can mimic the function of Pm-EVs and can directly bind to FAK.

[0049] Figure 6The virulence factor HPMA mediates Pm-EVs-induced VSMC phenotypic shift by inhibiting FAK. A: Venn diagram shows proteins expressed individually or co-expressed by *Proteus mirabilis* (Pm) and its extracellular vesicles (Pm-EVs). B: Volcano diagram shows proteins upregulated in the Pm-EVs group compared to the Pm group (-log10(P)>1, |log2(fold change)| ≥ 2); HPMA and FlgB are virulence factors specific to *Proteus mirabilis*. CH: Representative Western blot and quantitative data show the expression of FAK, p-FAK, Src, p-Src, αSMA, SM22α, and Mac2 in MOVAS cells treated with PBS, and in MOVAS cells treated with ox-LDL (50 μg / mL) and Pm-EVs (10 μg / mL), HPMA (10 μg / mL), FlugB (10 μg / mL), or PBS for 48 hours; GAPDH was used as a loading control; n=3 per group. IK: Representative immunofluorescence micrographs and quantitative data show the expression of CD68 among the 5 groups; DAPI was used to label DNA (blue), CD68 was green, n=5 per group, scale bar=10μm; representative Oil Red O staining and quantification of Oil Red O staining area for the 5 groups, scale bar=100μm, n=5 per group. L: Molecular docking of key proteins FlugB (P96972) and HPMA (P16466-4W8Q, NCBI) in Pm-EVs with the core target FAK (Q05397-6YOJ); FAK is yellow, FlugB is blue, and HPMA is red. M: Representative immunofluorescence micrographs showing the expression of αSMA and His-tagged HPMA in MOVAS cells co-treated with ox-LDL (50 μg / mL) and HPMA (10 μg / mL) for 48 hours; DAPI was used to label DNA (blue), αSMA is red, and HPMA-His tag is green, n=3 per group, scale bar=10 μm. N: Co-immunoprecipitation of FAK and HPMA-His tag. Results are expressed as mean ± standard error (Figure DH, JK). Data were analyzed by one-way ANOVA. **** P<0.0001; * P<0.05.

[0050] Example 5: EVs as carriers are crucial for HPMA to exert its pathogenic effect in vivo. To compare the pathogenic efficacy of HPMA protein itself and its carrier Pm-EVs in vivo, ApoE was administered via oral gavage or tail vein injection. - / - Recombinant HpmA protein or an equivalent amount of Pm-EVs in mice Figure 7A). Specific procedures: Every 3 days, mice in different groups were administered 4 mg / kg of Pm-EVs, 4 mg / kg of recombinant HPMA protein, or PBS solution as a control group via two different routes of administration (oral gavage or tail vein injection). After 12 weeks of the experiment, all mice were sacrificed. Subsequently, Oil Red O staining of the entire aorta and Oil Red O and H&E histological staining of the plaques at the aortic root were performed to quantitatively analyze the plaque coverage area, plaque size, and necrotic core area of ​​each group of mice, thereby assessing the impact of different treatments on atherosclerotic lesions.

[0051] The results showed that intravenous injection of HPMA or Pm-EVs, as well as oral administration of Pm-EVs, significantly exacerbated atherosclerotic plaque formation in mice. Figure 7 B, C). However, oral administration of recombinant HPMA protein had a much weaker effect, with plaque area and necrotic core size significantly smaller than the other three groups (B, C). Figure 7 DH).

[0052] This comparative experiment strongly demonstrates that although HPMA is the final effector molecule, it cannot effectively enter the circulatory system and reach target tissues to exert its effects when administered via the digestive tract without the protection and delivery of EVs. Therefore, Pm-EVs play an indispensable and crucial role in mediating the stability, systemic transport, and vascular targeting of HPMA.

[0053] Figure 7 EV-mediated delivery enhances the pro-atherosclerotic effect of HPMA in vivo. This figure compares the effects of recombinant HPMA and Pm-EVs on plaque formation in mice under different administration routes, demonstrating that EVs, as carriers, are crucial for HPMA to exert its pathogenic effect.

[0054] Figure 7 EV-mediated delivery enhanced the pro-atherosclerotic effect of HPMA in vivo. A: Experimental design; ApoE - / - Mice were fed a normal diet for 12 weeks. Red bars indicate administration of Pm-EVs (4 mg / kg), HPMA (4 mg / kg), or PBS every 3 days via oral tube feeding or intravenous injection. Mice were sacrificed after 12 weeks, with n=6 per group. BC: 6 groups of ApoE - / - Representative Oil Red O staining of mouse aorta and quantification of lesion coverage area. DH: 6 groups of ApoE - / - Representative Oil Red O and H&E staining of atherosclerotic plaques in the aortic root of mice; scale bar = 200 μm; 6 groups of ApoE staining. - / -Quantitative analysis of plaque size and necrosis area in atherosclerotic plaques in mice. Results are expressed as mean ± standard error (Figure C, EH). Data were analyzed by one-way ANOVA. **** P<0.0001; *** P<0.001; ** P<0.01.

[0055] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of a reagent for detecting the abundance of Proteus mirabilis in the preparation of products for diagnosing, screening, or assessing atherosclerotic plaque instability and / or acute coronary syndrome, characterized in that: The biological sample used for testing the product is feces.

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