Application of ANGPTL2 protein in the treatment and evaluation of predicting occlusion after coronary artery bypass grafting

Abstract

This invention relates to the cardiovascular field. Specifically, it relates to the application of ANGPTL2 protein in the treatment and assessment of predicting occlusion after coronary artery bypass grafting (CABG). The biomarker of this invention can accurately target treatment and predict the risk of long-term occlusion of the venous graft after CABG.

Description

Technical Field

[0001] This invention relates to the field of coronary artery disease prognosis technology, specifically to the application of ANGPTL2 in predicting the probability of venous graft occlusion after coronary artery bypass grafting. Background Technology

[0002] Coronary artery bypass grafting (CABG) is one of the main methods for revascularization in patients with coronary artery disease. However, approximately 40% of venous grafts and 10% of arterial grafts become occluded within the first 10 years post-procedure, severely impacting patient prognosis. Unlike early graft occlusion, which is usually attributed to acute thrombosis within the graft due to technical, catheter-related, or external factors, mid- to long-term graft occlusion is a naturally occurring process. Intimal hyperplasia of the graft, caused by the proliferation and phenotypic transformation of vascular smooth muscle cells (VSMCs), is one of the main pathological mechanisms. Summary of the Invention

[0003] Based on the inventors' research findings, this invention provides a gene encoding the ANGPTL2 protein and the application of the ANGPTL2 protein as a target in the preparation of drugs for treating occlusion after coronary artery bypass grafting.

[0004] The present invention also provides the gene encoding ANGPTL2 protein and the application of ANGPTL2 protein as a target in the preparation of reagents, kits, test strips or chips for postoperative evaluation, efficacy assessment and occlusion recurrence monitoring of coronary artery bypass grafting.

[0005] Furthermore, the ANGPTL2 protein belongs to the epicardial fat secretion protein family. Elevated levels of the gene encoding ANGPTL2 or the expression level of the ANGPTL2 protein relative to patients with a low epicardial fat attenuation index (Low-pFAI) may indicate a risk of occlusion after coronary artery bypass grafting.

[0006] Optionally, the reagent is a mass spectrometry identification reagent, an antibody, or an antigen-binding fragment thereof. The test sample for the reagent, kit, test strip, or chip is selected from the pericardial fat and its secretions of the human heart.

[0007] Furthermore, the reference values ​​for the reagents, kits, test strips, or chips represent the levels of the protein biomarkers in individuals with a low-pFAI (low-epardymal fat decay index) of epicardial fat.

[0008] This invention can target and treat long-term venous graft occlusion after CABG surgery, and can predict the risk of long-term venous graft occlusion after CABG surgery with high sensitivity and accuracy. Attached Figure Description

[0009] Figure 1 shows the statistical graph of the changes in FAI value of mouse visceral fat after 8 weeks of high-fat feeding and CT three-dimensional reconstruction of mouse visceral fat tissue; Figure 1 (A) shows the three color images from left to right after 0, 4 and 8 weeks of high-fat feeding, which are the images of mouse visceral fat tissue three-dimensional reconstruction based on CT; Figure 1 (B) shows the statistics of mouse visceral fat volume (left) and mouse visceral fat FAI value (right).

[0010] Figure 2 shows the results of protein sequencing of epigastric fat (EAT) secretions and their corresponding validation, as well as the expression of ANGPTL2 in mouse adipose tissue; Figure 2 (A) Heatmap of EAT secretion proteomics sequencing results; (B) Bubble chart, (C) Volcano chart (n = 15); (D) CCK-8 assay to assess VSMC cell viability (n = 6); (E) Relative mRNA levels of ANGPTL2 in low pFAI and high pFAI EAT (n = 15); (F and G) Western Blots: ANGPTL2 protein expression levels in low pFAI and high pFAI EAT (left panel) and statistical graph (right panel, n = 6); (H) ELISA determination of ANGPTL2 content in low pFAI and high pFAI EAT (n = 15); (I) Immunofluorescence staining images of ANGPTL2 (red) and PLIN1 (green) in EAT and quantitative statistical graph of the percentage of ANGPTL2-positive cells (n = 6); (J) Immunohistochemical staining of ANGPTL2 in low-pFAI and high-pFAI EAT; (K) ELISA determination of ANGPTL2 concentration in secretions of low-pFAI and high-pFAI EAT (n = 15); (L) ELISA determination of serum ANGPTL2 concentration in patients with low-pFAI and high-pFAI (n = 15); (M) Western blot analysis of relative mRNA levels of ANGPTL2 in VAT of mice with low-FAI (normal diet) and high-FAI (high-fat diet); (N) Protein expression levels of ANGPTL2 in VAT of mice with low-FAI and high-FAI (left panel) and statistical plot (right panel, N = 6); (O) Immunohistochemical staining of ANGPTL2 in VAT of mice with low-FAI and high-FAI.

[0011] Figure 3 illustrates whether the effect of High-FAI adipose tissue in promoting intimal hyperplasia is mainly mediated by local secretion of ANGPTL2; Figure 3 (A) Schematic diagram of the procedure for knocking down ANGPTL2 in VAT of high-FAI mice and performing fat transplantation in a carotid artery ligation model; (B) Relative mRNA levels of Angptl2 in high-FAI VAT after AAV-shNC or AAV-shAngptl2 treatment (n = 6); (C) Western Blots: Protein expression levels of ANGPTL2 in high-FAI VAT after AAV-shNC or AAV-shAngptl2 treatment (left panel) and statistical graph (right panel, n = 6); (D) HE staining and PCNA immunohistochemical staining of the ligated carotid artery after perivascular fat transplantation following AAV-shNC or AAV-shAngptl2 treatment of high-FAI VAT, and statistical graphs of neointimal area, neointimal / mediaal area, and percentage of PCNA-positive cells in the neointimal region (n = 6); (E) Schematic diagram of fat grafting in a carotid artery ligation model with ANGPTL2 overexpression in VAT of low FAI mice; (F) Relative mRNA levels of Angptl2 in low FAI VAT after AAV-NC or AAV-Angptl2-OE treatment (n = 6); (G) Western Blots: Protein expression levels of ANGPTL2 in low FAI VAT after AAV-NC or AAV-Angptl2-OE treatment (left panel) and statistical plot (right panel, n = 6); (H) Western Blots: Flag protein expression levels in low FAI VAT after transplantation with AAV-N and AAV-Angptl2-OE treatment (left panel) and statistical plot (right panel, n = 3); (I) Western Blots: Flag protein expression levels in the left carotid artery of mice without fat grafting, and in the right carotid artery (unoperated side) and left carotid artery (operated side) of mice receiving AAV-Angptl2-OE fat grafting (left panel) and statistical plot (right panel, n = 3). 3) (J) After treatment with AAV-NC or AAV-Angptl2-OE and low FAI VAT, perivascular fat grafting was performed. The ligated carotid artery was stained with HE and PCNA immunohistochemical staining, and the new intima area, new intima / media area and percentage of PCNA positive cells in the new intima were statistically analyzed (n = 6).

[0012] Figure 4 shows the results of promoting neointimal proliferation in the mouse carotid artery and transplanted vein after local overexpression of AAV-mediated ANGPTL2 in the carotid artery ligation model and the vein transplantation model; Figure 4 (A) HE staining of mouse carotid artery after local AAV-NC or AAV-Angptl2 treatment and ligation at the proximal 200-600 μm (left), and statistical graphs of neointimal area and neointimal / media area (right, n = 6); (B) RT-qPCR analysis of the relative mRNA levels of Pcna, Ccnd1, Acta2, and Myh11 in the sham-operated group and the carotid artery ligation group after AAV-NC or AAV-Angptl2 treatment (n = 4); (C) Immunofluorescence staining images of α-SMA (green) and PCNA (red) in the carotid artery of ligated mice after AAV-NC or AAV-Angptl2 treatment (top) and statistical graphs of the percentage of PCNA-positive cells in the neointimal area (bottom, n = 6); (D) HE staining and immunohistochemical staining images of PCNA in mouse intravenous grafts after AAV-NC or AAV-Angptl2 treatment (left), and a statistical graph of neointimal area and percentage of PCNA-positive cells in neointimal (right, n = 6). Detailed Implementation

[0013] Unless otherwise specified, the scientific and technical terms used in this article are intended for understanding by those skilled in the art.

[0014] Epicardial adipose tissue (EAT) is located between the myocardium and the visceral pericardium, primarily distributed in the atrioventricular groove and interventricular groove, thus effectively covering the coronary arteries. In coronary artery bypass grafting (CABG), the removal of EAT around the coronary arteries is a routine surgical procedure that rarely attracts the attention of cardiac surgeons. However, although EAT plays a minor role in CABG, the role of EAT and its secretions in venous graft occlusion warrants further consideration. First, EAT is directly adjacent to the venous graft, and for some time post-surgery, certain segments of the graft may even be surrounded by EAT. Second, EAT secretes abundant adipokines (such as adiponectin and leptin), which act directly on surrounding blood vessels and tissues via paracrine signals.

[0015] This invention, through research on epicardial adipose tissue and its conditioned culture medium from patients with High-pFAI and Low-pFAI, found that patients with a high fat decay index (High-pFAI) were more prone to venous graft occlusion in the long term (≥1 year) after coronary artery bypass grafting. Furthermore, the level of ANGPTL2 protein (sequence number ENSG00000136859) in the epicardial adipose tissue and its conditioned culture medium was higher in the High-pFAI group than in the Low-pFAI group. Simultaneously, ELISA, immunohistochemical staining, RT-PCR, and Western blot experiments demonstrated that the High-FAI group highly expressed ANGPTL2 in the visceral adipose tissue of mice.

[0016] Further validation in a mouse model confirmed the high expression of ANGPTL2 in High-FAI adipose tissue, and demonstrated that the role of High-FAI adipose tissue in promoting vascular intimal proliferation is mainly mediated through paracrine ANGPTL2. Specifically, High-FAI adipose tissue highly expresses ANGPTL2, and ANGPTL2 promotes angiogenesis and intimal proliferation through paracrine means. Furthermore, using hydrogels to deliver AAV overexpressing ANGPTL2 around the ligated carotid artery and transplanted vein, it was verified that AAV-mediated smooth muscle-specific ANGPTL2 overexpression can promote angiogenesis and intimal proliferation.

[0017] The present invention will be further explained and illustrated below with reference to the embodiments. All materials used in the following embodiments are commercially available products.

[0018] Example 1:

[0019] This example demonstrates that patients with a high fat attenuation index (High-pFAI) in epicardial adipose tissue and those with a low fat attenuation index (Low-pFAI) are more prone to venous graft occlusion (in this study, "High-pFAI" is defined as a pFAI value greater than the first quartile (-66.76 HU), and "Low-pFAI" is defined as a pFAI value less than the third quartile (-76.09 HU)). The specific research details are as follows: Research subjects: The study included CT imaging data of patients who underwent CABG surgery at the Department of Cardiac Surgery, Xijing Hospital, and who underwent coronary artery enhancement CT examination at the Department of Radiology, Xijing Hospital, 1-3 years post-surgery.

[0020] Research methods and related results: (1) Based on CT imaging data, the patency of the bypass grafts in patients was graded according to the Fitzgibbon classification criteria (A, B, O); (2) Based on CT imaging data, the volume of epicardial fat and pFAI value (an independent risk factor for restenosis of bypass grafts after coronary artery bypass grafting) were measured using image analysis software; A: patency of the graft is greater than 50%; B: patency of the graft is less than 50%; O: complete occlusion of the graft. In this study, grades A and B were defined as the non-occlusion group after coronary artery bypass grafting; A total of 97 patients were included in this study, of which 35 were in the bypass graft occlusion group and 62 were in the non-occlusion group; there was no significant difference in baseline data between the two groups (see Table 1 for comparison of clinical baseline data between the non-occlusion group and the occlusion group). (3) The differences in imaging parameters of epicardial fat between the non-occlusion group and the occlusion group after surgery were compared, and the differences (the parameters listed in the left column of Table 2) were further analyzed by logistic regression with the patency of the bypass graft to obtain the OR value. The results are shown in Table 2. Univariate and multivariate logistic regression analysis showed that high pFAI value was a risk factor for bypass graft occlusion (OR according to the number of patients: 1.292 (1.149-1.453, P<0.001); OR according to the number of venous grafts: 1.396 (1.268-1.537, P<0.001), which indicates that patients with high fat attenuation index (High-pFAI) are more likely to have venous graft occlusion than patients with low fat attenuation index (Low-pFAI).

[0021] Table 1

[0022] Table 2

[0023] Example 2: This example demonstrates that ANGPTL2 can serve as a protein biomarker for predicting the risk of venous graft occlusion. The specific methods and results are as follows: Research subjects: The samples used in this study included epicardial fat and its conditioned medium from 15 patients with High-pFAI and 15 patients with Low-pFAI, all obtained from epicardial fat tissue samples collected by the Department of Cardiovascular Surgery, First Affiliated Hospital of Air Force Medical University, from patients undergoing venous grafting. The specimen acquisition was performed by qualified clinicians without interfering with the original surgical and treatment protocols. The acquisition of clinical epicardial fat tissue samples and related research have been approved by the Ethics Committee of Xijing Hospital, Air Force Medical University (Ethics Committee Approval No.: KY-20222232-F-1).

[0024] The C57BL / 6J mice (8 weeks old, male) used were purchased from the Experimental Animal Center of Air Force Medical University. The mice were housed under standard conditions, with the temperature controlled between 22 and 24°C, and a circadian rhythm of 12 hours of light and 12 hours of darkness. All mouse housing and experimental procedures complied with the "Regulations on the Management of Experimental Animals" and were approved by the Ethics Committee of Air Force Medical University.

[0025] Research Methods: (1) Proteomics sequencing The proteomics testing was commissioned to Standard Biotech (Qingdao) Co., Ltd. The simplified steps are as follows: (1.1) Sample grouping and preparation: A pFAI value greater than the first quartile (-66.76 HU) was defined as "High-pFAI," and a pFAI value less than the third quartile (-76.09 HU) was defined as "Low-pFAI." Epicardial lipid secretions from 15 patients were included for proteomics analysis. (1.2) Selection of proteomics quantification technology: Data Independent Acquisition (DIA) technology was selected for proteomics quantification in this study; (1.3) Protein extraction: Proteins were extracted from the samples and the protein concentration in each sample was determined using the Bradford method; (1.4) Protein quantification: After enzymatic desalting, liquid chromatography-mass spectrometry was used for analysis. Data was acquired using the DIA method, and the acquired data were combined with a database for quantitative analysis.

[0026] (2) Epicardial fat and its conditioned medium and culture medium The obtained epicardial fat clinical specimens were placed on a cell manipulation table and rinsed three times with PBS. The adipose tissue was trimmed using sterilized surgical instruments (scissors and forceps), removing vascular tissue and areas burned by electrocautery. The adipose tissue was then cut into pieces approximately 1 mm in size. 3 The tissue was cut into small fragments; the adipose tissue was rinsed again with PBS; the adipose tissue was cultured in DMEM / F12 1:1 medium containing 10% FBS and 1% penicillin and streptomycin for 24 h; the adipose tissue culture medium was replaced with DMEM / F12 1:1 medium without FBS and containing 1% penicillin and streptomycin, and cultured for 24 h; after 24 h, the conditioned medium was collected, frozen at -80℃, and the previous step was repeated, collecting the conditioned medium twice for subsequent concentration steps; Concentration of epicardial fat conditioned culture medium: Thaw the conditioned culture medium derived from epicardial fat tissue of the same patient and transfer it to the same centrifuge tube, mixing thoroughly. Transfer the conditioned culture medium to the inner tube of an Amicon Ultra-15-3K ultrafiltration centrifuge tube and tighten the cap. Then, centrifuge at 14000g for approximately 30 minutes in a centrifuge pre-cooled to 4°C. Observe the liquid level in the inner tube periodically, controlling the concentration ratio to approximately 25-fold. Use a pipette tip to aspirate the concentrated liquid from the inner tube and collect it in the centrifuge tube. Quantify the protein concentration in the concentrated liquid using the BCA method. Label the concentrated conditioned culture medium and freeze it at -80°C.

[0027] The main antibodies used in the method are shown in Table 3.

[0028] Table 3

[0029] The main reagents and consumables used are shown in Table 4.

[0030] Table 4

[0031] (3) CT measurement and three-dimensional reconstruction of abdominal fat in mice High-FAI group: Mice were fed high-fat (60FDC) purified rat food for 8 weeks; Low-FAI group (normal feeding): Mice were fed normal sterilized rat food for 8 weeks.

[0032] Mice were fed a high-fat diet, and their lower abdomens were scanned by CT scans after 4 and 8 weeks of feeding. The lower abdomen was defined as the area from the fourth lumbar vertebra (L4) to the caudal vertebra 44. The X-ray source was set to 90 kV, 80 μA, and the field of view was 72 mm (voxel size 144 μm, scan time 4 min).

[0033] Three-dimensional CT reconstruction of the ventral abdominal fat (VAT) of mice was performed using Materialise Mimics Medical 21.0 software (Technologielaan 15, Leuven, Belgium), and the VAT volume and radioactivity were calculated. The abdominal fat of mice fed a high-fat diet was designated as the High-FAI group, while the abdominal fat of normally fed mice was designated as the Low-FAI group.

[0034] (4) Immunohistochemical staining of mouse peritoneal fat Peritoneal adipose tissue was collected from mice fed a high-fat diet for 8 weeks and a normal diet for 8 weeks, respectively. After fixation with 4% PFA for 48 hours, the tissue was embedded in paraffin. Sections were 5 µm thick, and immunohistochemical staining was performed using an immunohistochemical detection kit. Specifically, the paraffin sections were routinely dewaxed and rehydrated, then subjected to heat antigen retrieval with 1× sodium citrate antigen retrieval solution at 100°C for 20 minutes, followed by cooling in deionized water. Once cooled to room temperature, the sections were blocked with QuickBlock™ blocking solution for 20 minutes. Afterward, the sections were incubated with primary antibody overnight at 4°C, and then incubated with HRP-labeled anti-rabbit / mouse polymer as secondary antibody for 30 minutes at room temperature. The results were observed under an inverted microscope using DAB as the chromogenic reagent. Hematoxylin staining was performed for 2 minutes, the sections were dehydrated, and mounted. The sections were scanned using a Pannoramic 250 FLASH III digital scanner (3DHISTECH, Hungary).

[0035] (5) Immunofluorescence staining of mouse peritoneal fat The obtained mouse peritoneal fat paraffin sections were dewaxed and rehydrated with a series of ethanol solutions (100%, 95%, 85%, 75%) for 3 min each; then soaked in distilled water for 2 min; the paraffin sections were placed in sodium citrate antigen retrieval solution, boiled in a water bath for 20 min, and cooled to room temperature; rapid blocking solution was added to the tissue samples and incubated for about 15 min; the tissue samples were incubated overnight at 4°C with primary antibody, followed by TBST washing three times for 5 min each time; the tissue samples were incubated at room temperature in the dark with fluorescent secondary antibody for 1 h, followed by TBST washing three times for 5 min each time; the tissue samples were incubated at 37°C for 10 min with DAPI staining solution, followed by TBST washing three times for 5 min each time; the sections were mounted with anti-fluorescence quenching mounting medium and temporarily stored at 4°C; the sections were scanned using a laser confocal microscope.

[0036] (6) CCK-8 detection Cell viability was assessed using the CCK-8 assay according to the CCK-8 kit instructions. Simply put, primary rat smooth muscle cells were seeded in 96-well plates at 2 × 10⁶ cells per well. 3 Cells were starved of serum for 24 h, and then treated with drugs (CysC, SOX1, NID2, ANGPTL2, and ECM1) or PBS for 48 h. Afterwards, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37 °C for 2 h. Absorbance was measured at 450 nm.

[0037] (7) Determination of EAT and ANGPTL2 levels in human and mouse serum The ANGPTL2 ELISA kit was used for the assay. After processing the samples according to the kit instructions, the assay was performed following the manufacturer's instructions. For serum: whole blood samples collected in serum separation tubes were incubated at room temperature for 2 hours or overnight at 2-8°C, then centrifuged at 1000×g for 20 minutes. The supernatant was collected and stored at -20°C or -80°C for later use. For tissue homogenization: tissue was rinsed with pre-cooled PBS to remove residual blood, weighed, and then minced. The minced tissue was added to a glass homogenizer with an appropriate volume of PBS (generally at a weight / volume ratio of 1:9), and homogenized thoroughly on ice or using a homogenizer. Finally, the homogenate was centrifuged at 5000×g for 5-10 minutes, and the supernatant was collected for assay.

[0038] (8) Extraction of total protein from human peritoneal fat and mouse peritoneal fat Add one 2mm, two 1mm, and several 0.5mm zirconia beads to each of the 1.5 mL centrifuge tubes containing adipose tissue, add 80 μL of prepared lysis buffer, and place the tubes in a high-throughput tissue homogenizer. Run the homogenizer at 70 Hz for 120 s four times to completely lyse the adipose tissue. Place the centrifuge tubes on ice and lyse for 5 min. Place the centrifuge tubes in a centrifuge pre-cooled to 4°C and centrifuge at 12000 r / min for 15 min. Transfer the supernatant to a new centrifuge tube. Take 4 μL of the supernatant for BCA protein quantification and measure the protein concentration. Adjust the protein concentration of all samples to be consistent with PBS, add loading buffer, vortex to mix, boil for 10 min, and freeze at -20°C for later use.

[0039] (9) BCA protein quantification Prepare standard protein samples: Dilute the standard protein samples in the BCA kit to prepare a series of gradient standard protein samples; Prepare BCA working solution: Mix the two working solutions at a ratio of 50:1 and mix thoroughly; Load samples: Add 20 μL of protein standard or protein sample to be tested to each well in the 96 plate; Add BCA working solution: Add 200 μL of BCA working solution to each well in the 96 plate and incubate at 37℃ in the dark for 30 min; Measure absorbance: Measure the absorbance value at 562 nm using an ELISA reader; Data processing: Calculate the protein standard curve based on the standard protein samples and calculate the protein concentration in the protein sample to be tested. (10) Western Blot Gel preparation: Prepare the gel according to the instructions of the SDS-PAGE gel preparation kit; Electrophoresis buffer and transfer buffer: Prepare the electrophoresis buffer and transfer buffer according to the formula in the table below; Table 5 Electrophoresis Solution Formulation

[0040] Table 6 Transfer Buffer Formulation

[0041] Assemble the gel and electrophoresis tank, and add an appropriate amount of electrophoresis buffer. Add equal amounts of protein sample to each well of the gel sequentially. Set the power supply voltage to 80V and perform electrophoresis until the markers of each molecular weight are fully separated. Transfer: Cut the PVDF membrane to a suitable size and activate it by soaking it in methanol for 15 seconds. Pry open the glass plate and remove the gel, then cut it to a suitable size. Prepare a "transfer sandwich" in the following order: sponge, thick filter paper, gel, PVDF membrane, thick filter paper, sponge. Place this sandwich in the transfer clamp and insert it into the transfer tank. Add transfer buffer pre-cooled to 4°C and an ice pack to the transfer tank. Set the power supply to 90V and transfer for 60-100 minutes. Blocking: After transfer, remove the PVDF membrane and incubate it with 1% BSA solution at room temperature for 1 hour. The membrane was then washed once with TBST for 5 min; membrane cutting: the PVDF membrane was cut at an appropriate position according to the molecular weight of the target protein and the marker position; primary antibody incubation: the PVDF membrane was incubated overnight at 4°C with the corresponding protein antibody; membrane washing: the membrane was washed three times with TBST for 10 min each time; secondary antibody incubation: the PVDF membrane was incubated at room temperature for 2 h with the corresponding secondary antibody; membrane washing: the membrane was washed three times with TBST for 10 min each time; luminescence: the PVDF bands were developed using a chemiluminescence imaging system.

[0042] (10) Statistical analysis The data in this study were statistically analyzed using GraphPad Prism 9.0, and all values ​​are expressed as mean ± standard deviation. Independent samples were used to compare the means between two groups. t For comparisons among multiple groups, analysis of variance (ANOVA) was used. (Two-tailed) P A value <0.05 is considered statistically significant. P <0.05, P <0.01, P <0.001.

[0043] result: Figure 1 is a statistical graph showing the changes in FAI value of visceral fat in mice after 8 weeks of high-fat feeding, based on CT three-dimensional reconstruction. The results show that the visceral fat volume and FAI value of mice were significantly increased after 8 weeks of high-fat feeding.

[0044] Figure 2 shows the results and validation of protein sequencing of EAT secretions from the epididymal adipose tissue of the heart. Example 1: This study demonstrated that patients with a high fat attenuation index (High-pFAI) are more prone to venous graft occlusion than those with a low fat attenuation index (Low-pFAI). "High-pFAI" was defined as a pFAI value greater than the first quartile (-66.76 HU), and "Low-pFAI" was defined as a pFAI value less than the third quartile (-76.09 HU).

[0045] Figure 2A -C represents proteomics sequencing, and the results demonstrated that the differences between the two groups of secretions were statistically significant. Table 7 lists the top 10 differentially expressed proteins. Figure 2D After excluding extracellular matrix components, the CCK-8 assay was used to screen for molecules with the most significant effect on promoting VSMC proliferation among CysC, SOX1, NID2, ANGPTL2, and ECM1. The results showed that ANGPTL2 significantly increased the proliferation level of VSMCs more than other secreted proteins. Figure 2E Based on the results of RT-qPCR, the mRNA expression of ANGPTL2 in the EAT of High-pFAI was significantly higher than that in the EAT of Low-pFAI. Figure 2F Western blotting and ELISA were used to demonstrate that the High-pFAI group had a higher ANGPTL2 protein level.

[0046] Table 7

[0047] Figure 2I Immunofluorescence and immunohistochemistry experiments were performed on EAT by J and J respectively to verify the above conclusions. The results were the same as those of the above conclusions. Figure 2K The concentrations of EAT secreted products and serum ANGPTL2 in the High-pFAI group and Low-pFAI group were measured by ELISA. The results showed that the concentration of ANGPTL2 in the EAT secreted products of the High-pFAI group was significantly higher than that in the Low-pFAI group, while there was no significant difference in the concentration of ANGPTL2 in serum.

[0048] Figure 2M -O represents the expression of ANGPTL2 in mouse High-FAI adipose tissue (from high-fat diet mice) and Low-FAI adipose tissue (from normal diet mice), as well as RT-qPCR, Western Blots, and IHC staining. The results all showed that the transcriptional and expression levels of ANGPTL2 in mouse High-FAI adipose tissue were higher than those in the Low-FAI group, consistent with the results of human EAT samples.

[0049] The above results demonstrate that there are significant differences between the secretory products produced by High-pFAI EAT and Low-pFAI EAT. In particular, the concentration of ANGPTL2 in the EAT secretory products of High-pFAI is significantly higher than that of the Low-pFAI group. ANGPTL2 can serve as a protein biomarker for predicting the risk of venous graft occlusion.

[0050] Example 3: This embodiment is a study on an animal model related to coronary artery bypass grafting. The specific methods and results of this embodiment are as follows: Animal models and experimental materials: This section uses commonly used animal models for studying coronary artery bypass grafting: Pressure-induced endarterial neoplasia model: Mice were anesthetized with 2.5% isoflurane, with 1% used for maintenance. After exposing the left common carotid artery, the distal end of the common carotid artery was ligated with 6-0 silk suture. Seven days after ligation, mice were sacrificed, and the carotid arteries were harvested for RT-qPCR detection. Twenty-eight days after ligation, the ligated left and right carotid arteries were cut and fixed in 4% paraformaldehyde for subsequent studies.

[0051] Mouse vein transplantation model: The mouse vein transplantation model was established using the "cuff" technique. Specifically, mice were anesthetized with 2.5% isoflurane, with 1% used for maintenance. After exposing the right common carotid artery of the recipient mouse, the proximal and distal ends of the vessel were clamped with two vascular clamps, and the vessel between the two clamps was cut open. Simultaneously, an 8-10 mm section of the inferior vena cava was isolated and removed from another donor mouse. The inferior vena cava from the donor mouse was then anastomosed to the right carotid artery of the recipient mouse using the "cuff" technique, with an 8-0 suture. Successful vein transplantation was considered successful if blood flow was observed through the vessel after releasing the clamps. Vein transplantation samples were collected 28 days after modeling for subsequent research.

[0052] Perivascular fat grafting (ATT) model in mice: After successfully establishing a pressure-induced intimal neoplasia model or a vein transplantation model, the target vessel in the recipient mouse was exposed. Simultaneously, approximately 50 mg of adipose tissue was harvested from the epididymis of the donor mouse. The adipose tissue was then implanted around the target vessel. Four weeks after transplantation, the adipose tissue graft and the target vessel were removed and fixed in 4% paraformaldehyde for further analysis.

[0053] The main antibodies used in this section are shown in Table 8.

[0054] Table 8

[0055] The main reagents and consumables used in this section are shown in Table 9.

[0056] Table 9

[0057] Research Methods: Recombinant adeno-associated virus (AAV)-mediated ANGPTL2 knockdown or overexpression: ANGPTL2 knockdown AAV and AAV overexpression were designed and manufactured by Shanghai Hanheng Biotechnology Co., Ltd. To knock down or overexpress ANGPTL2 in mouse adipose tissue, adipose-specific AAV-shAngptl2 or AAV-Angptl2-OE was injected using a microsyringe (50 μL / mouse, concentration 1×10⁻⁶). 12 (Vg / mL) injected at multiple points into the epididymal adipose tissue. ANGPTL2 is overexpressed perivascularly; 1×10 8 PFU-specific smooth muscle-specific AAV-Angptl2 was dissolved in 40 μL of 30% Pluronic gel (Beyotime, Shanghai, China, ST501-10 g). After successfully establishing carotid artery ligation or vein transplantation models, the gel containing AAV-Angptl2 was delivered perivascularly to the exposed target vessel according to the above method.

[0058] The remaining experimental methods are the same as in Example 1.

[0059] result: Figure 3 shows a two-part experiment to verify whether the effect of High-FAI adipose tissue in promoting endometrial hyperplasia is mainly mediated by local secretion of ANGPTL2. Figure 3A (and 3E). Recombinant AAV virus was constructed to knock down or overexpress mouse ANGPTL2. AAV-shAngptl2 was locally injected into the paraepididymal fat (VAT) of mice on a high-fat diet. RT-qPCR and Western blots showed that AAV-shAngptl2 successfully knocked out ANGPTL2 in VAT. Figure 3B And 3C). Then, VAT from AAV-shAngptl2-infected high-fat-fed mice was transplanted around the carotid arteries of ligated recipient mice; 28 days later, HE and IHC staining were used to observe the intimal hyperplasia of the ligated carotid arteries. The results showed that the transplanted adipose tissue remained viable; in addition, the neointimal area, neointimal / media ratio, and percentage of PCNA-positive cells in the neointimal tissue of the ligated carotid arteries in the AAV-shAngptl2 group were all lower than those in the AAV-NC group ( Figure 3D ).

[0060] Subsequently, the VAT of normal-diet mice was locally infected with AAV overexpressing ANGPTL2 (AAV-Angptl2-OE), and the successful overexpression of ANGPTL2 was verified by RT-qPCR and Western Blots. Figure 3FAnd 3G). Then, AAV-infected VAT was transplanted around the ligated carotid artery using the method described above. Next, Flag markers were detected by Western blotting to assess the expression and secretion of ANGPTL2 in adipose tissue and blood vessels. The results showed that Flag expression in VAT was significantly increased after AAV-Angptl2-OE injection and remained at the same level for 4 weeks post-transplantation. Figure 3H Furthermore, 4 weeks post-transplantation, the Flag level detected in the left carotid artery (operated side) was higher than that in the right carotid artery (unoperated side). Figure 3I ).

[0061] The above results indicate that transplanted AAV-Angptl2-OE-VAT can effectively express ANGPTL2 and secrete it into peripheral blood vessels; 28 days after ATT treatment, the AAV-Angptl2-OE-ATT group had a higher neointimal area and neointimal / media ratio (HE staining) in the ligated arteries. Figure 3J Following AAV-Angptl2-OE-ATT, the percentage of PCNA-positive cells detected by IHC increased ( Figure 3J It is evident that High-FAI adipose tissue highly expresses ANGPTL2, and ANGPTL2 promotes angiogenesis and intimal hyperplasia through paracrine mechanisms.

[0062] Figure 4 illustrates the effect of ANGPTL2-overexpressing AAV delivered via hydrogel around the ligated carotid artery and transplanted vein, verifying its role in promoting angiogenesis and intimal hyperplasia. Carotid artery HE staining showed that, compared to the control group, the AAV-Angptl2 group exhibited significantly increased angiogenesis intimal area and neointima / media ratio. Figure 4A RT-qPCR was used to detect mouse carotid artery mRNA expression. The results showed that perivascular overexpression of Angptl2 significantly increased the transcriptional levels of vascular smooth muscle cell proliferation markers and decreased the transcriptional levels of their contractile phenotype markers. Figure 4B Immunofluorescence staining showed that after ANGPTL2 overexpression, the number of PCNA-positive cells in the carotid neointima significantly increased. Figure 4C Similarly, the above treatment was repeated in a mouse vein transplantation model. Results showed that HE staining in the AAV-Angptl2 group indicated a higher intimal area of ​​the transplanted blood vessel compared to the AAV-NC group, and a higher proportion of PCNA-positive cells detected by IHC staining. Figure 4D ).

[0063] In summary, AAV-mediated smooth muscle-specific ANGPTL2 overexpression can promote angiogenesis and intimal proliferation.

Claims

1. The gene encoding ANGPTL2 protein and its application as a target in the preparation of drugs for treating occlusion after coronary artery bypass grafting.

2. The application of the gene encoding ANGPTL2 protein and ANGPTL2 protein as a target in the preparation of reagents, kits, test strips or chips for postoperative evaluation, efficacy assessment and occlusion recurrence monitoring of coronary artery bypass grafting.

3. The application according to claim 2, characterized in that, The ANGPTL2 protein is an epicardial lipid secretion protein.

4. The application according to claim 2, characterized in that, Elevated levels of the gene encoding the ANGPTL2 protein or the expression level of the ANGPTL2 protein relative to the low fat decay index of epicardial fat in patients may indicate a risk of occlusion after coronary artery bypass grafting.

5. The application according to claim 2, characterized in that, The reagents are mass spectrometry identification reagents, antibodies, or their antigen-binding fragments.

6. The application as described in claim 2, characterized in that, The reagents, kits, test strips, or chips used to detect samples are selected from the epigastric fat and its secretions of the human heart.

7. The application as described in claim 2, characterized in that, The reference values ​​for the reagents, kits, test strips, or chips represent the levels of the protein biomarkers in individuals with a low epicardial fat attenuation index.

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