Application of NETs in treatment of postoperative adverse events in aortic dissection cavity and prognosis evaluation

Abstract

The invention discloses application of NETs in treatment of postoperative adverse events and prognosis evaluation in an aortic dissection cavity, and belongs to the technical field of biological medicine. The invention finds that the composition protein CitH3 of NETs is closely related to poor aortic remodeling of the aortic dissection, is an independent risk factor of AAEs after intracavitary treatment of a patient with the aortic dissection, and can be used as a key marker for prognosis evaluation of the aortic dissection. Therefore, the invention provides an application of a biomarker such as CitH3 and the like in risk assessment of adverse events occurring after treatment in the aortic dissection cavity. The peripheral circulation NETs marker CitH3 provided by the invention provides an accurate, effective, simple, convenient and easy-to-popularize quantitative index and risk stratification method for the assessment of the overall prognosis of interlayer intracavity treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an application of NETs in adverse events and prognosis evaluation after intracavitary treatment of aortic dissection. Background Art

[0002] Aortic dissection (AD) is caused by proximal tearing of the aortic intima or ruptured bleeding of the vasa vasorum in the aortic wall. The torn intima extends forward or backward, promoting and continuing the separation of the aortic wall, forming a true and false lumen. Once the false lumen ruptures, the patient may die in a very short time, and there is almost no possibility of rescue. It is a critical cardiovascular disease. The incidence of acute dissection can reach 3-5 / 100,000, and the incidence in the middle-aged population rises to 15-20 / 100,000. The average age of onset is 63 years old, and most of them are men (65%). Acute dissection patients usually have no signs before the onset of the disease, and lack typical symptoms when they develop. Common clinical manifestations include sudden severe chest pain, back pain and high blood pressure, as well as dizziness, abdominal pain and limb ischemia caused by tearing of the intima to the branch artery. It is easy to be misdiagnosed or missed, delaying the best time for treatment. After the onset of acute dissection, the mortality rate increases by 1% to 3% per hour, the 24-hour mortality rate is as high as 21%, and the 7-day mortality rate is as high as 74%.

[0003] Thoracic endovascular aortic repair (TEVAR) is currently the preferred treatment for type B aortic dissection, with the advantages of less trauma, high success rate, rapid postoperative recovery, and definite efficacy. The first TEVAR was successfully performed in 1996. Over the decades of development and iteration, new surgical instruments and technologies have emerged in an endless stream, and treatment concepts have also changed accordingly. Evidence-based medicine based on large-scale clinical trials shows that compared with the best medical treatment, patients with complex dissection who receive TEVAR have better short-term and long-term prognosis and significant clinical benefits; compared with open surgery, TEVAR has better short-term prognosis and similar long-term prognosis. TEVAR mainly seals the proximal intimal rupture of the aorta, restores positive blood flow in the true lumen, promotes thrombosis of the false lumen and aortic remodeling; however, simply sealing the proximal rupture leaves an imbalanced distribution of blood flow in the distal true and false lumens. In some patients, the distal aortic blood flow continues to flow backward into the false lumen, and the weak wall of the false lumen that is not full-thickness continues to expand and form a tumor, that is, the aortic remodeling is poor or negative, increasing the risk of aortic rupture, leading to complications such as endoleak, new distal rupture, proximal retrograde type A dissection, and organ dysfunction. Improving aortic remodeling has become the key to further improve the overall prognosis of dissection. In-depth research on aortic remodeling after endovascular treatment of dissection, exploring indicators that effectively predict the effect of aortic remodeling, accurately identifying high-risk patients with aortic-related adverse events (AAEs) after surgery, and providing close follow-up and active intervention, so as to optimize the effect of endovascular treatment, improve the prognosis of patients with dissection, and further reduce the risk of aortic-related death and adverse events are the key and difficulty in the current diagnosis and treatment of dissection.

[0004] At present, the research on aortic remodeling in dissected aorta mainly focuses on the changes in aortic morphology and structure based on imaging, as well as the research on biomarkers based on multi-omics such as chips and sequencing technology. Factors such as the degree of thrombosis of the false lumen of the dissection and the angle of the aortic arch are closely related to aortic remodeling. However, there is no recognized imaging-based morphological measurement method and evaluation standard for the results and characteristics of aortic remodeling. In the process of disease occurrence and development, biomarkers can accurately reflect the pathophysiological changes of the body. In recent years, some progress has been made in the study of the prognosis of endovascular treatment of dissected aorta, including D-dimer, inflammation and lipid metabolism-related markers, smooth muscle and extracellular matrix markers, and microRNA (miRNA). These biomarkers may work synergistically with existing clinically known risk factors to identify high-risk groups for poor aortic remodeling. Due to the limitations of limited sensitivity and specificity, inconsistent results between different studies, difficulty in replication, and the fact that the case-control studies of acute dissections are mainly based on cases of subacute and chronic dissections, most of the existing candidate biomarkers are still in the preclinical research stage. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a method for predicting the overall prognosis of endovascular treatment of aortic dissection based on the level of extracellular trap formation of peripheral neutrophils. Based on a multi-omics strategy, a molecular marker that can be detected in peripheral venous blood and can effectively predict the aortic remodeling effect and aortic-related adverse events after endovascular treatment is determined, and high-risk patients with aortic-related adverse events after surgery are identified. At the same time, potential auxiliary treatment targets are revealed to optimize the effect of endovascular treatment, improve the prognosis of patients with dissection, and achieve survival benefits for patients.

[0006] The neutrophil extracellular trap (NETs) described in the present invention is a mesh structure composed of DNA, histones and antimicrobial proteins. More specifically, NETs is based on DNA as the skeleton, in which are embedded histones (mainly citrullinated histone 3, CitH3), neutrophil elastase (NE), myeloperoxidase (MPO), antimicrobial peptides (LL-37), serine proteases, etc. Therefore, the detection of NETs is also based on these main components.

[0007] The present invention unexpectedly found that CitH3, a constituent protein of NETs, ​​is closely related to adverse aortic remodeling in aortic dissection. CitH3 is an independent risk factor for the occurrence of AAEs after endovascular treatment of patients with aortic dissection and can be used as a key marker for the prognosis assessment of aortic dissection.

[0008] One object of the present invention is to provide a biomarker, such as CitH3, for use in the risk assessment of adverse events after endovascular treatment of aortic dissection. A second object of the present invention is to provide a kit for assessing the risk of aorta-related adverse events after endovascular treatment of aortic dissection. A third object of the present invention is to provide a method for assessing the risk of aorta-related adverse events after endovascular treatment of aortic dissection.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] In the first aspect of the present invention, the present invention provides a biomarker for use in the preparation and / or screening of products for assessing the risk of aortic-related adverse events after endovascular treatment of aortic dissection, wherein the marker is selected from one or a combination of two or more of CitH3, cf-DNA, MPO, NE, IL-1β, and IL-6.

[0011] Preferably, the biomarker is CitH3 and / or MPO.

[0012] More preferably, the biomarker is CitH3.

[0013] The endovascular treatment of aortic dissection described in the present invention is thoracic endovascular aortic repair (TEVAR).

[0014] The aorta-related adverse events after endovascular treatment of aortic dissection described in the present invention include, but are not limited to, endoleak, new distal stent rupture (dSINE), retrograde type A dissection (RTAD), distal aortic aneurysm, branch artery occlusion or stenosis, aortic rupture and death.

[0015] In the second aspect of the present invention, the present invention provides an application of a substance for detecting a biomarker in the preparation and / or screening of a product for assessing the risk of aortic-related adverse events after endovascular treatment of aortic dissection, wherein the marker is selected from one or a combination of two or more of CitH3, cf-DNA, MPO, NE, IL-1β, and IL-6.

[0016] Preferably, the biomarker is CitH3 and / or MPO.

[0017] More preferably, the biomarker is CitH3.

[0018] The biomarker detection material includes any reagent required for detecting the protein content or gene expression level of the biomarker in a sample by RTPCR, RT qPCR, biochip detection, Southern blotting, in situ hybridization, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA).

[0019] In a specific embodiment of the present invention, the substance for detecting biomarkers is a reagent for detecting the content of the biomarker protein in a sample to be tested by enzyme-linked immunosorbent assay.

[0020] The sample of the present invention is selected from the blood, plasma, serum or aortic dissection tissue of patients with aortic dissection.

[0021] In a third aspect of the present invention, the present invention provides a kit for assessing the risk of aorta-related adverse events after endovascular treatment of aortic dissection, the kit comprising an antibody that specifically binds to the biomarker described in the second aspect of the present invention.

[0022] Furthermore, the kit also includes other conventional reagents required for enzyme-linked immunosorbent assay (ELISA), including but not limited to enzyme-labeled secondary antibodies and color development solutions conventionally used in the art.

[0023] In a fourth aspect of the present invention, the present invention provides a method for detecting the level of a biomarker in a sample not for the purpose of treatment or diagnosis, the method is used to assess the risk of aorta-related adverse events after endovascular treatment of aortic dissection, the method comprising the following steps:

[0024] (1) Obtaining samples to be tested;

[0025] (3) adding antibodies that specifically bind to the biomarker, incubating, and washing;

[0026] (4) Add enzyme-labeled secondary antibody for incubation and wash;

[0027] (5) Signal detection based on enzyme-linked immunosorbent assay.

[0028] The sample to be tested is selected from blood, plasma, serum or aortic dissection tissue of patients with aortic dissection.

[0029] The marker is selected from one or a combination of two or more of CitH3, cf-DNA, MPO, NE, IL-1β, and IL-6.

[0030] Preferably, the biomarker is CitH3 and / or MPO.

[0031] More preferably, the biomarker is CitH3.

[0032] In the specific operation of the present invention, the method for detecting CitH3 levels comprises the following steps:

[0033] i. Collect about 2 ml of peripheral blood using an anticoagulant blood collection tube (purple head), centrifuge at 4°C for 20 min, and collect the supernatant;

[0034] ii. Set up sample wells, standard wells and blank wells, and set up duplicate wells. Add 100 μl of sample or standard to each well, mix thoroughly, and incubate in a 37°C incubator for 40 minutes.

[0035] iii. Add 350 μl of 1× washing solution to each microwell, let it stand for 15 seconds, then discard and spin dry, washing 5 times;

[0036] iv. Except for the blank wells, add 50 μl of CitH3 primary antibody working solution and deionized water to each microwell, react at 37°C for 20 minutes, and wash the plate as before;

[0037] v. Add 100 μl of enzyme-labeled secondary antibody working solution to each microwell, react at 37°C for 10 minutes, and wash the plate as before;

[0038] vi. Add 100 μl of substrate working solution to each microwell and incubate in the dark at 37°C for 15 minutes. Add 100 μl of stop solution to each microwell and mix well. After 1 minute, place the microplate in an ELISA reader, read the OD value, and calculate the CitH3 concentration.

[0039] When the CitH3 concentration in the sample to be tested is ≥6.91ng / ml, it indicates that the patient has a high risk of aorta-related adverse events after endovascular treatment. When the CitH3 concentration is <6.91ng / ml, it indicates that the patient has a low risk of aorta-related adverse events after endovascular treatment.

[0040] The present invention detects and analyzes the number and proportion of various inflammatory cells in the patient's peripheral circulation according to the above method, and statistically compares the occurrence of aortic adverse events in the patients according to the patients' clinical follow-up information. The results show that by measuring CitH3 in the patient's peripheral circulation to reflect the level of NETs formation, the patients with dissection are divided into a high CitH3 group and a low CitH3 group. The high CitH3 group has more significant inflammatory cell infiltration, lower aortic media thickness, and a higher incidence of aorta-related adverse events. Therefore, it can be used as a molecular marker for effectively predicting the aortic remodeling effect and aorta-related adverse events after endovascular treatment.

[0041] The peripheral circulating NETs marker CitH3 provided by the present invention provides an accurate, effective, simple, convenient and easy-to-promote quantitative index and risk stratification method for evaluating the overall prognosis of intracavitary treatment of dissection. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Violin plots were drawn based on the levels of CitH3, cf-DNA, MPO, NE, IL-1B, and IL-6 according to whether AAEs occurred.

[0043] Figure 2 ROC curve of CitH3 relative to AAEs.

[0044] Figure 3 Kaplan-Meier curves were drawn based on the CitH3 cutoff value. DETAILED DESCRIPTION

[0045] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] 1. Research subjects

[0047] This study selected patients with type B aortic dissection who underwent TEVAR treatment at Zhongshan Hospital Affiliated to Fudan University between November 2016 and November 2020. All patients provided written informed consent. Inclusion and exclusion criteria were:

[0048] Inclusion criteria:

[0049] i. Preoperative CT angiography (CTA) and intraoperative digital subtraction angiography (DSA) confirmed type B aortic dissection;

[0050] ii. The patient is diagnosed with type B aortic dissection for the first time and has not undergone previous cardiac or great vascular surgery (open surgery for Marfan syndrome or bicuspid aortic valve malformation, etc.);

[0051] iii. Preoperative clinical data (current medical history, personal history, family history, laboratory tests, imaging examinations, etc.), surgical records and postoperative course of disease are complete.

[0052] Exclusion criteria: Patients who met any of the following criteria were excluded:

[0053] i. Patients diagnosed with aortic intramural hematoma or aortic ulcer by CTA or DSA;

[0054] ii. Suffering from complications that may affect the inflammatory cell count, such as blood system diseases, autoimmune diseases, active malignant tumors, receiving radiotherapy and chemotherapy, acute / chronic infectious diseases, taking anti-inflammatory drugs in the past 3 months, etc.;

[0055] iii. Abnormal body temperature before surgery, fever of unknown cause, or obvious local or systemic infection symptoms;

[0056] iv. Patients who were lost to follow-up or not followed up for 1 year after surgery.

[0057] 2. Research Methods

[0058] i. The endpoint of this study was aortic adverse events (AAEs) after TEVAR during the follow-up period. AAEs were diagnosed by CTA and / or DSA, including endoleak, new distal stent rupture (dSINE), retrograde type A dissection (RTAD), distal aortic aneurysm, branch artery occlusion or stenosis, aortic rupture and death. The patient's age, gender, current medical history, personal history, family history, laboratory tests, imaging examinations and surgical records were collected, including classification (complex / uncomplicated), course of disease (acute / subacute / chronic), primary rupture location, proximal anchoring zone length (distance from dissection rupture to the posterior edge of the left subclavian artery), subclavian artery (LSA) coverage, stent type / size, magnification, whether branch stent technology was used, etc. Potential confounders considered in this study included demographic characteristics, comorbidities (hypertension, history of smoking and drinking, diabetes, angina pectoris, myocardial infarction, ischemic or hemorrhagic stroke or transient ischemic attack, chronic kidney disease, heart failure, peripheral arterial disease), medication use (antihypertensive drugs, antiplatelet drugs, anticoagulants, and statins), and aortic dissection-related variables (intervention timing - acute, subacute, and chronic), location of primary rupture (Z3 or Z4), length of proximal landing zone, and use of branch stents.

[0059] ii. Statistical methods: The sample of 201 patients (75 in the AAEs group and 126 in the non-AAEs group) included in this study had a power of 95.2% to detect differences in the proportion of patients with the primary endpoint at a two-sided significance level of 0.05. Continuous variables were expressed as means with standard deviations (SDs) or medians with interquartile ranges (IQRs). Student's t test or Mann-Whitney U test was used for continuous variables, and Pearson's chi-square test or Fisher's exact test was used for categorical variables. Kolmogorov-Smirnov test was used to test for normality. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated by logistic regression. Receiver operating characteristic (ROC) curves were used to determine the optimal cutoff values ​​of the continuous variables NLR, MLR, PLR, SII, and SIRI for predicting the occurrence of AAEs. Kaplan-Meier curves and log-rank tests were used for survival analysis. Univariate Cox regression analysis was used to preliminarily analyze the risk factors for AAEs. Variables with statistical significance (p<0.2) in the univariate Cox analysis were included in the multivariate Cox regression to determine independent risk factors. Multiple imputation was used to handle missing data. P<0.05 was considered statistically significant.

[0060] 3. Measurement of NET levels in peripheral circulation

[0061] Peripheral venous blood was collected from the included patients before surgery. About 2 ml of peripheral blood was collected using an anticoagulant blood collection tube (purple head), centrifuged at 4°C for 20 min, the supernatant was taken, incubated at 37°C for 40 min, 1× washing solution 350 μl was added, and the solution was discarded and dried after standing for 15 seconds, and washed 5 times. 50 μl of CitH3 primary antibody working solution and deionized water were added, reacted at 37°C for 20 min, and the plate was washed as before.

[0062] Add 100 μl of enzyme-labeled antibody working solution, react at 37°C for 10 minutes, and wash the plate as before. Add 100 μl of substrate working solution, incubate at 37°C in the dark for 15 minutes, then add 100 μl of stop solution and mix well. After 1 minute, place in a microplate reader, read the OD value, and calculate the concentration.

[0063] 4. Correlation analysis between NETs level and prognosis

[0064] The number and proportion of various inflammatory cells in the peripheral circulation of the two groups of patients were detected and analyzed, and the occurrence of aortic adverse events in the two groups of patients was statistically compared based on the clinical follow-up information of the patients. Univariate and multivariate regression analysis was performed to avoid bias caused by confounding factors. The Youden index of CitH3 concentration was used as the threshold for the level of NETs formation in the peripheral circulation. CitH3 concentrations greater than or equal to 7ng / ml were considered high peripheral circulation high NETs formation levels, and CitH3 concentrations less than 7ng / ml were considered low NETs formation levels.

[0065] The levels of CitH3, cf-DNA, MPO, NE, IL-1B, and IL-6 in the peripheral blood of all enrolled patients were tested, and violin plots were made based on whether AAEs occurred. The results are as follows Figure 1 As shown in the results, patients with dissection who developed AAEs after endovascular treatment had higher plasma CitH3 and MPO levels, and the difference was statistically significant ( Figure 1 ).

[0066] When the critical threshold of CitH3 was 6.91 ng / ml, the sensitivity and specificity of predicting the occurrence of AAEs after endovascular treatment in patients with dissection were 0.6 and 0.9, respectively, and the area under the curve was 0.75 ( Figure 2 ).

[0067] CitH3 was divided into high level and low level with a critical value of 6.91ng / ml. The Kaplan-Meier curve showed that patients with high level of CitH3 had a higher incidence of aortic-related adverse events than those with low level of CitH3 ( Figure 3 ).

[0068] Univariate Cox regression analysis was used to preliminarily analyze the risk factors for AAEs; variables with statistical significance (p<0.2) in the univariate Cox analysis were included in the multivariate Cox regression to determine independent risk factors. Multiple imputation was used to handle missing data values. P<0.05 was considered to be statistically significant. The results are shown in Table 1:

[0069] Table 1 Cox regression analysis results

[0070]

[0071] Note: D-dimer refers to D-dimer, Neutrophil refers to neutrophil, NLR refers to neutrophil to lymphocyte ratio, MLR refers to monocyte to lymphocyte ratio, SIRI refers to systemic inflammatory response index.

[0072] The results of the multivariate Cox regression model in the above table showed that CitH3 was an independent risk factor for poor prognosis of dissection.

[0073] Based on the above results, NETs play a key role in the onset, progression and outcome of aortic dissection. CitH3, a NETs marker in the peripheral circulation, can effectively predict the occurrence of aortic-related adverse events before surgery and timely and accurately identify high-risk patients with poor prognosis. For this reason, it is necessary to consider strengthening perioperative management and adopting more rigorous follow-up strategies for these patients, which will help promote benign aortic remodeling, improve the effect of endovascular treatment, and improve the overall prognosis of patients with dissection, providing a more scientific, objective and detailed basis for personalized and precise treatment of dissection.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of a biomarker in the preparation and / or screening of a product for assessing the risk of aortic-related adverse events after endovascular treatment of aortic dissection, wherein the biomarker is selected from one or a combination of two or more of CitH3, cf-DNA, MPO, NE, IL-1β, and IL-6.

2. Use of a substance for detecting biomarkers in the preparation and / or screening of products for assessing the risk of aortic-related adverse events after endovascular treatment of aortic dissection, wherein the marker is selected from one or a combination of two or more of CitH3, cf-DNA, MPO, NE, IL-1β, and IL-6.

3. The use according to claim 2, characterized in that: The biomarkers are CitH3 and / or MPO.

4. The use according to claim 3, characterized in that: The biomarker is CitH3.

5. The use according to claim 2, characterized in that: The biomarker detection material includes any reagent required for detecting the protein content or gene expression level of the biomarker in a sample by RTPCR, RT qPCR, biochip detection, Southern blotting, in situ hybridization, immunohistochemistry, and enzyme-linked immunosorbent assay.

6. A kit for evaluating the risk of aorta-related adverse events after endovascular treatment of aortic dissection, the kit comprising an antibody that specifically binds to the biomarker described in claim 2.

7. The kit according to claim 6, characterized in that The kit also includes other conventional reagents required for enzyme-linked immunosorbent assay, including enzyme-labeled secondary antibody and color development solution.

8. A method for detecting the level of a biomarker in a sample not for the purpose of treatment or diagnosis, the method being used to assess the risk of aorta-related adverse events after endovascular treatment of aortic dissection, the method comprising the following steps: (1) Obtaining samples to be tested; (3) adding antibodies that specifically bind to the biomarker, incubating, and washing; (4) Add enzyme-labeled secondary antibody for incubation and wash; (5) Signal detection based on enzyme-linked immunosorbent assay; The sample to be tested is selected from the blood, plasma, serum or aortic dissection tissue of patients with aortic dissection; the marker is selected from one or a combination of two or more of CitH3, cf-DNA, MPO, NE, IL-1β, and IL-6.

9. The method according to claim 8, characterized in that The biomarker is CitH3 and / or MPO, preferably, the biomarker is CitH3.

10. The method according to claim 9, characterized in that The method for detecting CitH3 levels comprises the following steps: i. Collect about 2 ml of peripheral blood using an anticoagulant blood collection tube (purple head), centrifuge at 4°C for 20 min, and collect the supernatant; ii. Set up sample wells, standard wells and blank wells, and set up duplicate wells. Add 100 μl of sample or standard to each well, mix thoroughly, and incubate in a 37°C incubator for 40 minutes. iii. Add 350 μl of 1× washing solution to each microwell, let it stand for 15 seconds, then discard and spin dry, washing 5 times; iv. Except for the blank wells, add 50 μl of CitH3 primary antibody working solution and deionized water to each microwell, react at 37°C for 20 minutes, and wash the plate as before; v. Add 100 μl of enzyme-labeled secondary antibody working solution to each microwell, react at 37°C for 10 minutes, and wash the plate as before; vi. Add 100 μl of substrate working solution to each microwell and incubate in the dark at 37°C for 15 minutes. Add 100 μl of stop solution to each microwell and mix well. After 1 minute, place the microplate in an ELISA reader, read the OD value, and calculate the CitH3 concentration.

Images