Use of tnfaip6 in the treatment of aortic aneurysm and aortic dissection

Abstract

The application discloses application of TNFAIP6 in treatment of aortic aneurysm and aortic dissection, and discloses, for the first time, a key role of TNFAIP6 in occurrence and development of aortic aneurysm and aortic dissection, and provides a brand-new strategy of using TNFAIP6 and a modulator thereof for diagnosis, prevention and treatment of aortic aneurysm and aortic dissection, and solves a clinical problem that there is no effective drug intervention means for AAD at present.

Description

Technical Field

[0001] This invention belongs to the field of cardiovascular disease prevention and treatment technology, specifically involving the application of tumor necrosis factor-induced gene 6 protein (TNFAIP6) in the diagnosis, prevention and treatment of aortic aneurysm and aortic dissection. Background Technology

[0002] Aortic aneurysms and aortic dissections are highly fatal large vessel diseases. Aortic aneurysms manifest as irreversible localized dilation of the aorta, while aortic dissections result from intimal tears leading to stratification of the vessel wall, creating a "true lumen" and a "false lumen." Both share similar pathological bases, involving severe degradation and remodeling imbalance of the extracellular matrix in the vessel wall, ultimately leading to aortic rupture and massive hemorrhage, severely endangering life. Currently, fundamental treatments for aortic aneurysms / dissections are limited to surgical or endovascular repair, which are highly invasive, risky, and expensive. Clinically, there is a severe lack of effective drugs that can slow or halt the progression of aortic aneurysms / dissections. Therefore, elucidating the key molecular mechanisms of aortic aneurysm / dissection development and identifying new intervention targets is of significant clinical and social importance.

[0003] The extracellular matrix is ​​crucial for maintaining the structural integrity and functional homeostasis of the aortic wall. In the progression of aortic aneurysms / dissections, disruption of collagen and elastin networks, abnormal proteoglycan structure, and metabolic disorders of hyaluronic acid are common characteristics. Hyaluronic acid (HA), as the framework of the extracellular matrix, directly affects the signal transduction of proteoglycans and various growth factors (such as VEGF and TGF-β) attached to it, thereby regulating the function of vascular smooth muscle cells, endothelial cells, and immune cells.

[0004] Tumor necrosis factor-induced gene 6 (TNFAIP6) is a secreted glycoprotein with a unique linker protein domain that catalyzes the covalent binding of serum interstitial α-inhibitor heavy chains (HC) to hyaluronic acid, forming a stable HC-HA complex. This process is crucial for enhancing the stability of the hyaluronic acid network, resisting degradation, and regulating hyaluronic acid-dependent cellular signaling. While TNFAIP6 has been extensively studied in inflammation and tissue remodeling in diseases such as arthritis and tumors, its role in aortic aneurysm / dissection, a core disease of vascular remodeling, remains unreported. Whether and how it participates in and regulates the progression of aortic aneurysm / dissection is completely unknown.

[0005] Aortic aneurysms and aortic dissections are highly fatal large vessel diseases. Currently, fundamental treatments for aortic aneurysms / dissections are limited to surgical or endovascular repair, which are highly invasive, risky, and expensive. Clinically, there is a severe lack of effective drugs to slow or halt the progression of aortic aneurysms / dissections. Our previous research utilized vascular tissue from aortic dissection patients and mouse models of aortic aneurysms / dissections, combining proteomics and transcriptomics technologies with analysis of extracellular matrix-related gene databases. This screening revealed that TNFAIP6 showed the greatest fold change in extracellular matrix-related genes in both mouse and human aortic aneurysm / dissection samples. During the research process, we noted that the core pathological mechanism of aortic aneurysms / dissections is the severe imbalance between extracellular matrix degradation and remodeling, but its regulatory mechanism remains unclear. Tumor necrosis factor-induced gene 6 (TNFAIP6) is an important regulator of extracellular matrix remodeling. It can catalyze the covalent binding of hyaluronic acid (HA) to heavy chains (HC) and enhance HA stability. However, the role of TNFAIP6 in aortic aneurysm / dissection, a vascular remodeling disease, has not been reported to date. Therefore, investigating the expression changes and functions of TNFAIP6 in aortic aneurysm / dissection may reveal new disease mechanisms and intervention targets.

[0006] Currently, clinical interventions for aortic aneurysms / dissections primarily involve surgery and endovascular repair. These methods are all physical repairs, highly invasive and risky, and are only suitable for patients with large aneurysms or those where dissection has already occurred; they cannot be used for early intervention or to slow disease progression. Regarding drug development, there are currently no approved drugs that can fundamentally prevent aortic dilation or dissection progression. Some studies have explored the use of statins and doxycycline, but their clinical efficacy is unclear or controversial, and they have not been translated into effective treatment options. Related patents also fail to elucidate the crucial role of TNFAIP6 in aortic aneurysms / dissections. Summary of the Invention

[0007] The purpose of this invention is to provide the application of tumor necrosis factor-induced gene 6 protein (TNFAIP6) in the diagnosis, prevention, and treatment of aortic aneurysms and aortic dissections. This invention reveals for the first time the crucial role of TNFAIP6 in the development and progression of aortic aneurysms and aortic dissections, and provides a novel strategy for the diagnosis, prevention, and treatment of aortic aneurysms and aortic dissections using TNFAIP6 and its modulators, thereby addressing the current clinical challenge of lacking effective drug interventions for AAD.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] In a first aspect, the present invention seeks protection for the use of a reagent for detecting TNFAIP6 levels in biological samples in either (1) or (2) below:

[0010] (1) Use in the preparation of products for the diagnosis or auxiliary diagnosis of aortic aneurysm and / or aortic dissection;

[0011] (2) Application in screening drugs for the treatment of aortic aneurysm and / or aortic dissection.

[0012] Furthermore, the biological sample is selected from serum, plasma, or aortic tissue; the product is a kit or a biochip.

[0013] Secondly, the present invention seeks protection for the use of TNFAIP6 in the preparation of medicaments for the prevention and / or treatment of aortic aneurysms and / or aortic dissections.

[0014] Furthermore, the drug comprises TNFAIP6 and a pharmaceutically acceptable carrier.

[0015] Furthermore, the drug also contains matrix metalloproteinase inhibitors and / or anti-inflammatory drugs.

[0016] Thirdly, the present invention claims protection for a method for screening candidate drugs for the prevention and / or treatment of aortic aneurysm and / or aortic dissection, comprising the following steps:

[0017] (1) Construct animal models of aortic aneurysm and / or aortic dissection;

[0018] (2) Provide the test compound;

[0019] (3) Detect the expression level or activity of TNFAIP6 in the animal model. If the expression of TNFAIP6 is upregulated or its activity is enhanced, the compound is determined to be a candidate drug.

[0020] The core improvement of this invention lies in the first-ever linking TNFAIP6 with the diagnosis and treatment of aortic aneurysm / dissection (AAD). Prior to this invention, the role of TNFAIP6 in AAD was completely unknown. This invention not only reveals the potential of TNFAIP6 as a diagnostic biomarker for AAD, but also demonstrates that recombinant TNFAIP6 protein can be used to treat AAD.

[0021] In our previous studies, we used vascular tissues from patients with aortic dissection and mouse models of aortic aneurysm / dissection, combined with proteomics and transcriptomics techniques, and further analyzed them in conjunction with an extracellular matrix-related gene database. We found that the extracellular matrix-related TNFAIP6 protein was significantly increased in the lesion tissues of aortic aneurysm / dissection. Tumor necrosis factor-induced gene 6 (TNFAIP6), a known secreted protein that catalyzes the formation of a stable complex between hyaluronic acid and heavy chains, had a completely unexplored role in aortic aneurysm / dissection, a core disease of vascular remodeling. This prompted us to conduct in-depth functional research on it.

[0022] Clinical sample testing:

[0023] We collected serum samples from patients with aortic aneurysm / dissection and healthy controls. ELISA analysis revealed that TNFAIP6 levels in the serum of patients were significantly higher than those in the healthy controls. Further logistic regression analysis suggested that serum TNFAIP6 levels could serve as a potential diagnostic biomarker to distinguish AAD patients from healthy individuals.

[0024] Animal model validation:

[0025] Expression level: In two animal models of aortic aneurysm / dissection (BAPN drinking water induction model and PPE incubation induction model), we found by Western blotting and immunofluorescence that the expression level of TNFAIP6 protein in the diseased aortic tissue was significantly upregulated, consistent with clinical findings.

[0026] Treatment efficacy: In a PPE-induced aortic aneurysm / dissection model, mice were intraperitoneally injected with recombinant TNFAIP6 protein (15 μg / kg, once daily for 2 weeks). Results showed that, compared to the untreated group, the aortic dilation was significantly reduced in the treated group (confirmed by gross morphological observation and quantitative measurement of the maximum diameter of the isolated aorta). Histological staining (HE, EVG) and transmission electron microscopy further confirmed the improved extracellular matrix structure of the aortic wall in the treated group.

[0027] The mechanism of action of this invention lies in the fact that the occurrence and development of aortic aneurysms / dissections are closely related to the degradation and stability of hyaluronic acid (HA) in the extracellular matrix. TNFAIP6, as a key catalytic protein, can mediate the covalent binding of serum-derived heavy chains (HC) to the HA backbone, forming a stable HC-HA complex. This complex can effectively resist degradation by hyaluronidase, enhance the structural and functional stability of the extracellular matrix, and thus protect the integrity of the vascular wall.

[0028] This invention demonstrates that intraperitoneal administration of recombinant TNFAIP6 protein in a PPE-induced aortic aneurysm / dissection model effectively reduces vasodilation and improves extracellular matrix structure, indicating that TNFAIP6 has a clear protective effect in aortic aneurysm / dissection.

[0029] TNFAIP6 can be used in combination with matrix metalloproteinase inhibitors (such as doxycycline) or other anti-inflammatory drugs to achieve better therapeutic effects.

[0030] The TNFAIP6 protein (also known as TSG-6, tumor necrosis factor alpha induced protein 6) described in this invention is tumor necrosis factor alpha induced protein 6, including human and mouse homologous proteins. The NCBI Gene ID of the TNFAIP6 protein from house mice is 21930, the RefSeq accession number for the reference mRNA sequence is NM_009398.2, and the RefSeq accession number for the corresponding reference protein sequence is NP_033424.1. The corresponding accession numbers for the human TNFAIP6 protein are 7130, NM_007115.4, and NP_009046.2.

[0031] The recombinant TNFAIP6 protein used in this invention is a commercially available recombinant human TSG-6 protein, NM_007115.4, NP_009046.2 (catalog number BA68888, concentration ≥50 μg / mL), which can be obtained through conventional commercial channels. Those skilled in the art can also prepare functionally equivalent recombinant TNFAIP6 proteins based on the disclosed coding sequence using conventional genetic engineering methods. For example, the coding sequence shown in NM_007115.4 can be cloned into a prokaryotic or eukaryotic expression vector, transformed into suitable host cells, and then induced to express and purified.

[0032] The beneficial effects of this invention are:

[0033] This invention reveals for the first time the crucial role of TNFAIP6 in the development and progression of aortic aneurysm / dissection (AAD) and provides a novel strategy for diagnosis, prevention, and treatment based on TNFAIP6 and its modulators. Specific beneficial effects include: (1) Correlation analysis and serum testing confirm that TNFAIP6 levels can effectively predict the occurrence of aortic dissection, providing a new biomarker for early warning and non-invasive diagnosis of AAD; (2) It confirms that exogenous administration of recombinant TNFAIP6 protein can significantly improve aortic aneurysm / dissection lesions in mice, filling the current clinical gap in effective intervention methods for AAD. This invention also provides a novel pathway with translational value for the diagnosis, prognosis, and targeted therapy of AAD. Attached Figure Description

[0034] Figure 1 This diagram illustrates the serum TNFAIP6 levels in control patients and patients with aortic aneurysm / dissection.

[0035] Figure 2The receiver operating characteristic (ROC) curves of the predictive model, constructed based on demographic data of control group patients and aortic aneurysm / dissection patients, demonstrate the predictive performance of the logistic regression model for aortic aneurysm / dissection. The horizontal axis represents specificity, and the vertical axis represents sensitivity.

[0036] Figure 3 This diagram illustrates the expression of TNFAIP6 protein in normal mice and mice with aortic aneurysm / dissection.

[0037] Figure 4 This diagram illustrates the phenotypic changes after intraperitoneal administration of recombinant TNFAIP6 protein in a normal control mouse model and a porcine pancreatic elastase (PPE)-induced aortic aneurysm / dissection model. Detailed Implementation

[0038] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0039] Example 1:

[0040] Of the 40 subjects included, 20 were in the control group and 20 were in the aortic aneurysm / dissection group. 5 ml of peripheral venous blood was collected from each of the 20 patients using anticoagulant-free vacuum blood collection tubes. After collection, the samples were allowed to stand at room temperature for 30–60 minutes until complete coagulation. They were then centrifuged at 3000 rpm for 15 minutes. After centrifugation, the supernatant serum was collected, and TNFAIP6 levels were detected using an ELISA kit. (N=20, ***p < 0.001). ELISA testing of serum TNFAIP6 levels in the subjects revealed that serum TNFAIP6 levels in aortic aneurysm / dissection patients were significantly higher than those in the control group (e.g., ...). Figure 1 (As shown).

[0041] A binary logistic regression model was constructed, with aortic dissection diagnosis (yes / no) as the dependent variable and TNFAIP6 level, sex, age, and history of hypertension (yes / no) as independent variables. The logistic regression model coefficients and odds ratios (OR) were calculated. Logistic regression analysis showed that TNFAIP6 level was a significant predictor of AAD diagnosis (OR=1.058, 95% CI: 1.016–1.102; p=0.006). Specifically, for every 1 unit increase in TNFAIP6, the risk of AD increased by approximately 5.8%. Sex (male vs. female: OR=1.718, p=0.578), age (OR=0.944, p=0.198), and history of hypertension (yes / no: OR=1.928, p=0.421) did not reach statistical significance in the univariate model (as shown in Table 1).

[0042] Table 1. Logistic Regression Model Coefficients and OR Value

[0043] TNFAIP6 0.056 0.020 2.753 0.006 1.058 Gender (Male) 0.541 0.973 0.556 0.578 1.718 age -0.058 0.045 -1.286 0.198 0.944 History of hypertension (yes) 0.656 0.816 0.805 0.421 1.928

[0044] The predictive performance of the constructed binary logistic regression model for aortic aneurysm / dissection was evaluated using receiver operating characteristic (ROC) curves. The overall goodness of fit of the model was acceptable, with a residual bias of 40.541 (degrees of freedom = 35). The model exhibited good discriminative ability, with an area under the ROC curve (AUC) of 0.81 (95% CI: 0.69–0.93), indicating good discriminative power (e.g.,...). Figure 2 (As shown in Table 2). In terms of classification prediction, the overall accuracy of the model was 75.0% (30 / 40). Sensitivity and specificity were both 75.0%, and the positive and negative predictive values ​​were both 75.0% (as shown in Table 2).

[0045] Table 2 Model Prediction Performance Indicators

[0046] accuracy 75.0%（58.8%-87.3%） Sensitivity 75.0% Specificity 75.0% Positive predictive value 75.0% Negative predictive value 75.0% AUC 0.81（0.69-0.93） AIC 50.541

[0047] Example 2: Validation of TNFAIP6 expression under aortic aneurysm / dissection pathological conditions

[0048] This invention uses two aortic aneurysm / dissection (AAD) mouse models to verify the upregulation of TNFAIP6 expression in diseased aortic tissue at the protein level.

[0049] 1. Validation of the BAPN-induced AAD model

[0050] Three-week-old male C57BL / 6 mice were selected and treated with 0.25% β-aminopropionitrile (BAPN) in their drinking water for 4 weeks to establish an AAD model.

[0051] Western blot analysis was used to quantitatively detect the expression of TNFAIP6 in the aorta of mice. The results showed that, compared with the normal control group, the protein expression level of TNFAIP6 in the aortic tissue of the model group mice was significantly upregulated (N=6, **p < 0.01, ***p < 0.001), confirming the overall increased expression of TNFAIP6 in the development and progression of AAD. Figure 3 (A in the middle).

[0052] Immunofluorescence was used to detect the expression of TNFAIP6 in mouse aortic tissue. Aortic tissue sections were stained with immunofluorescence, with TNFAIP6 marked in red, α-SMA (a smooth muscle cell marker) marked in green, and DAPI used to stain the cell nuclei in blue. The results showed that TNFAIP6 was strongly expressed in the aortic wall of the model group mice. (N=3, scale bar: 50 μm) Figure 3 (B in the middle).

[0053] 2. Validation of the PPE perfusion-induced AAD model

[0054] Eight-week-old male C57BL / 6 mice were selected, and an AAD model was constructed by local incubation of 10 mg / ml porcine pancreatic elastase (PPE) in the subrenal abdominal aorta for 10 minutes. The mice were harvested 2 weeks after the operation.

[0055] Immunofluorescence staining was used to detect the expression of TNFAIP6 in mouse aortic tissue, employing the same TNFAIP6 (red) / α-SMA (green) / DAPI (blue) immunofluorescence staining method. Results showed that the expression level of TNFAIP6 in PPE-induced AAD lesions was significantly higher than that in the control group, and consistent with the expression pattern in the BAPN model (N=3, scale bar: 50 μm). Figure 3 (C in the middle).

[0056] In summary, this embodiment first confirmed the increased expression of TNFAIP6 in aortic aneurysm / dissection (AAD) using an animal model. In the BAPN water-drinking-induced AAD model, Western blotting ( Figure 3 Both A) and immunofluorescence staining (TNFAIP6 red fluorescence, α-SMA green fluorescence) showed significantly increased TNFAIP6 expression. Figure 3 (B in the text). In the PPE-induced AAD model, immunofluorescence staining also confirmed the specific high expression of TNFAIP6 in the diseased vascular tissue. Figure 3 (C in the middle).

[0057] Conclusion: The results of the two independent animal models consistently show that TNFAIP6 is specifically highly expressed in the pathological state of aortic aneurysm / dissection, suggesting that it may be involved in the pathological process of AAD.

[0058] Example 3: Intervention effect of recombinant TNFAIP6 protein on PPE-induced aortic aneurysm / dissection

[0059] To investigate the specific biological function of TNFAIP6 in aortic aneurysm / dissection (AAD), this invention utilizes a mouse AAD model induced by porcine pancreatic elastase (PPE) perfusion, intervenes by intraperitoneal injection of recombinant human TNFAIP6 protein, and systematically evaluates its effect on improving the disease phenotype.

[0060] 1. Model construction and dosing regimen

[0061] Eight-week-old male C57BL / 6 mice were used to establish an aortic aortic model by incubating the aorta with 10 mg / mL porcine pancreatic elastase (PPE) for 10 minutes. Immediately after modeling, the experimental group was given an intraperitoneal injection of recombinant human TNFAIP6 protein (15 μg / kg), while the control group was treated with an equal volume of PBS. The intervention was continued for 2 weeks before samples were collected for analysis.

[0062] 2. TNFAIP6 intervention improves aortic morphological phenotype.

[0063] Gross morphological observation: After dissection, it was observed that the aorta of mice in the PPE model group was significantly dilated and the vessel wall was thinned. However, after treatment with TNFAIP6 recombinant protein, the degree of aortic dilation was significantly reduced, and the appearance of the blood vessels was closer to that of the normal control group. Figure 4 In the figure, A, N = 6-14, and scale bar = 5 mm.

[0064] Quantitative measurement verification: The maximum outer diameter of the isolated aorta was precisely measured and statistically analyzed. The results showed that the maximum diameter of the TNFAIP6 treatment group was significantly smaller than that of the PPE model group (***p < 0.001, N=6-14), which quantitatively confirmed the therapeutic effect of TNFAIP6 in inhibiting aortic dilation. Figure 4 (B in the middle).

[0065] 3. TNFAIP6 intervention alleviates histopathological damage.

[0066] Tissue structure repair: Hematoxylin-eosin (HE) staining showed that the smooth muscle arrangement of the aortic media in the PPE model group was disordered and inflammatory cell infiltration was significant; elastic fiber (EVG) staining showed that elastic fibers were broken and severely degraded. In contrast, the integrity of the tunica media structure in the TNFAIP6 treatment group was maintained, inflammatory infiltration was reduced, and the continuity and density of elastic fibers were significantly improved. Figure 4In the figure, C, N = 7-9, scale bar = 100 μm, 500 μm).

[0067] Ultrastructural protection: Transmission electron microscopy (TEM) further confirmed that PPE-induced ultrastructural damage and elastic fiber breakage in smooth muscle cells were significantly reversed after TNFAIP6 treatment, and collagen fibers were reconnected. Figure 4 In the figure, D, N=3, scale bar=1 μm).

[0068] TNFAIP6 plays a protective role in PPE-induced aortic aneurysm / dissection. Through the establishment of a PPE-induced aortic aneurysm / dissection model, intraperitoneal administration of recombinant TNFAIP6 protein, morphological observation and quantitative analysis, we confirmed that TNFAIP6 participates in regulating the occurrence and development of aortic dissection.

[0069] In the PPE-induced model, intraperitoneal injection of recombinant human TNFAIP6 protein significantly reduced the degree of aortic dilation in mice. Figure 4 The results of the maximum outer diameter measurement (A in the figure) confirmed this therapeutic effect. Figure 4 B), HE and EVG staining showed significant improvement in histopathological damage ( Figure 4 (C) Transmission electron microscopy showed that the degree of elastic fiber breakage had improved. Figure 4 (D in the middle).

Claims

1. The reagents for detecting TNFAIP6 levels in biological samples are used in the following (1) or (2): (1) Use in the preparation of products for the diagnosis or auxiliary diagnosis of aortic aneurysm and / or aortic dissection; (2) Application in screening drugs for the treatment of aortic aneurysm and / or aortic dissection.

2. The application according to claim 1, characterized in that, The biological samples are selected from serum, plasma, or aortic tissue; the product is a kit or a biochip.

3. Application of TNFAIP6 in the preparation of drugs for the prevention and / or treatment of aortic aneurysm and / or aortic dissection.

4. The application according to claim 3, characterized in that, The drug contains TNFAIP6 and a pharmaceutically acceptable carrier.

5. The application according to claim 3 or 4, characterized in that, The drug also contains matrix metalloproteinase inhibitors and / or anti-inflammatory drugs.

6. A method for screening candidate drugs for the prevention and / or treatment of aortic aneurysm and / or aortic dissection, characterized in that, Includes the following steps: (1) Construct animal models of aortic aneurysm and / or aortic dissection; (2) Provide the test compound; (3) Detect the expression level or activity of TNFAIP6 in the animal model. If the expression of TNFAIP6 is upregulated or its activity is enhanced, the compound is determined to be a candidate drug.

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