Application of SCAP as target in treatment or prevention of aortic dissection
By studying the relationship between SCAP and vascular smooth muscle cells, SCAP is determined as a potential target for aortic dissection, solving the problem of lack of effective targets in the existing technology, providing new methods for treating and preventing aortic dissection, which significantly affects the The occurrence and development of diseases.
Patent Information
- Application Number
- CN202411413232.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-11
- Publication Date
- 2024-12-27
AI Technical Summary
The prior art has not yet explored the application of SCAP as a target in the treatment or prevention of aortic dissection, resulting in the lack of effective drug targets to prevent and treat this serious cardiovascular disease.
By studying the expression and function of SCAP in aortic dissection, SCAP was determined to be related to transposition and rearrangement of vascular smooth muscle cells, and in animal experiments, it was proved that the VSMC-specific SCAP knockout mouse model promoted the occurrence of aortic dissection, and then Identify SCAP as a potential target for the treatment and prevention of aortic dissection.
The downregulation of SCAP is confirmed in clinical samples and animal models to be associated with the occurrence and development of aortic dissection, providing a new target to intervene in the occurrence of aortic dissection with potential therapeutic and preventive value.
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Abstract
Description
Technical Field
[0001] The present invention relates to application of SCAP as a target in treating or preventing aortic dissection, and belongs to the technical field of biomedicine. Background Art
[0002] Aortic dissection is a serious cardiovascular disease caused by media separation and adventitia rupture caused by rupture of the intima of the vascular wall. The blood in the aortic lumen enters the aortic media from the tear of the aortic intima, causing the media to separate and expand along the long axis of the aorta to form a state of separation between the true and false cavities of the aortic wall. Aortic dissection is characterized by degradation of the extracellular matrix (ECM) and progressive phenotypic conversion of vascular smooth muscle cells (VSMC), leading to aortic aneurysm, dissection and rupture. The ECM of collagen and elastin plays an important role in vascular elasticity (compliance) and integrity. In aortic dissection, the aortic wall has increased hematoma, degradation of elastin, structural incompleteness and reduced collagen fibers. VSMCs show a series of phenotypic conversions, from a static (contractile) state responsible for regulating blood flow and blood pressure, to a proliferative (synthetic) phenotype capable of synthesizing matrix metalloproteinase-2 and matrix metalloproteinase-9. The radial rearrangement of VSMCs fails, and the loss of VSMCs in the vascular wall leads to structural incompleteness. Therefore, targeting the radial rearrangement of smooth muscle cells may be an effective strategy for the treatment of aortic dissection.
[0003] Sterol regulatory element binding protein (SREBP) cleavage activation protein (SCAP) is an endoplasmic reticulum protein that can sense changes in intracellular cholesterol levels. It participates in regulating intracellular cholesterol levels and is essential for maintaining normal physiological functions of cells. It is crucial for the survival and growth of mammals. Cholesterol can bind to a variety of transmembrane proteins to help maintain and change their conformations. Intracellular cholesterol homeostasis is a complex metabolic regulatory network, which is jointly determined by the dynamic relationship between biosynthesis, exogenous uptake, efflux and storage. Studies have shown that vascular smooth muscle cell proliferation is abnormal in mice with specific knockout of SCAP, and the skeleton proteins in SCAP knockout smooth muscle cells are clustered and abnormally distributed, and the secretion of extracellular matrix is reduced, resulting in abnormal smooth muscle cell translocation and reduced angiogenesis. All of these can indicate that SCAP can affect the expression of angiogenic factors. However, the role of SCAP in vascular diseases has not been well studied.
[0004] At present, there are no reports on the application of SCAP as a target in the treatment or prevention of aortic dissection at home and abroad. Summary of the invention
[0005] The technical problem to be solved by the present invention is to provide a new drug action target for clinical prevention and treatment of aortic dissection.
[0006] In order to achieve the above object, the present invention provides the use of SCAP as a target in the treatment and prevention of aortic dissection.
[0007] The present invention first determined the relationship between SCAP and VSMC transposition rearrangement in aortic dissection, proved that SCAP expression was downregulated in aortic dissection samples in clinical samples, and proved in animal experiments that VSMC-specific SCAP knockout mice promoted the occurrence and development of aortic dissection. The present invention determined that SCAP caused the occurrence of aortic dissection by affecting VSMC radial rearrangement. BRIEF DESCRIPTION OF THE DRAWINGS
[0008] Figure 1 In the figure, A shows the expression of SCAP in clinical aortic dissection specimens and normal control specimens in the GEO database. B shows the HE staining, MASSON staining, and EVG staining detection of aortic tissue in the normal control group and aortic dissection group; C shows the mRNA expression of SCAP in the normal control group and aortic dissection group; D shows the protein expression of SCAP in the normal control group and aortic dissection group.
[0009] Figure 2 In the figure, A is a schematic diagram of the modeling of aortic dissection in two different genotype mice (WT and SCAP KD); B is a survival curve of two different genotype mice (WT and SCAP KD); C is a general cardiovascular diagram of two different genotype mice (WT and SCAP KD). D is a HE staining and EVG staining detection diagram of two different genotype mice (WT and SCAP KD).
[0010] Figure 3 A is the elastin immunofluorescence staining of aortic tissue in the aortic dissection group and the normal control group; B is the fibronectin immunofluorescence staining of aortic tissue in the aortic dissection group and the normal control group; C is the vimentin immunofluorescence staining of aortic tissue in the aortic dissection group and the normal control group; D is the P-MLC immunofluorescence staining of aortic tissue in the aortic dissection group and the normal control group.
[0011] Figure 4 In the figure, A is the elastin immunofluorescence staining of two mice with different genotypes (WT and SCAP KD); B is the fibronectin immunofluorescence staining of two mice with different genotypes (WT and SCAP KD); C is the vimentin immunofluorescence staining of two mice with different genotypes (WT and SCAP KD); D is the P-MLC immunofluorescence staining of two mice with different genotypes (WT and SCAP KD). DETAILED DESCRIPTION
[0012] The following is a further detailed description in conjunction with the drawings in the specific examples of the present invention to ensure that relevant researchers can understand the present invention more comprehensively. The experimental methods used in the present invention are all conventional methods in the art, and the reagents used are all commercially available. For animal experiments, all were approved by the Animal Ethics Committee of Chongqing Medical University and followed the "Guidelines for the Care and Use of Laboratory Animals".
[0013] Clinical samples: Clinical samples were donated by the Department of Cardiovascular Surgery, Xinqiao Hospital, Army Medical University.
[0014] Animal model and treatment: We used the Cre-Loxp system to establish a mouse model and transformed SCAP - / - The mice were crossed with SM22a-Cre transgenic mice to obtain offspring. Since VSMC-specific knockout of SCAP mice resulted in embryonic lethality, SM22-SCAP was used in subsequent animal experiments. + / - The construction process of the aortic dissection model is as follows: We selected 4-week-old male mice and fed them with BAPN solution for 4 weeks. Then, the mice were killed 8 weeks later. The hearts and the entire aorta of the mice were taken for photographic analysis to observe their pathological changes.
[0015] Hematoxylin and eosin (HE) staining: Clinical samples and mouse aorta tissue were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5um slices. The slices were placed in different gradients of xylene and concentration gradients of ethanol for dewaxing and rehydration. Stain with hematoxylin staining solution for 5-10 minutes, and rinse off excess staining solution in tap water for about 10 minutes. Then stain with eosin staining solution for 30 seconds. Then dehydrate and make transparent with different gradients of ethanol and xylene, and seal with neutral gum: xylene = 1:1 sealing agent. Use a tissue slice scanner for observation and subsequent records.
[0016] MASSON staining: Fix the tissue in 4% paraformaldehyde, perform conventional dehydration and embedding, slice 5um continuously, and dewax to water in a conventional manner. Slice into mordant solution (cover), mordant at room temperature overnight, and then rinse with running water for 10 minutes. Drip with lapis lazuli blue staining solution for 2-3 minutes, wash with water twice, 15 seconds each time. Drip with Mayer hematoxylin staining solution for 2-3 minutes, wash with distilled water twice, 15 seconds each time. Differentiate with acidic ethanol differentiation solution for a few seconds until the tissue turns completely red, wash with water to terminate differentiation, and rinse with distilled water for 10 minutes. Drip with Ponceau fuchsin staining solution for 10 minutes, wash with distilled water twice, 15 seconds each time. Treat with phosphomolybdic acid solution for about 10 minutes. Pour off the upper liquid and directly add aniline blue staining solution for 5 minutes. After washing away the aniline blue solution with weak acid solution, continue to drip weak acid working solution to cover the slices for 2 minutes. Dehydrate with 95% ethanol for 30 seconds, dehydrate with anhydrous ethanol twice, clear with xylene twice, and seal with sealing medium.
[0017] EVG staining: The tissue was fixed in 4% paraformaldehyde solution and dewaxed to distilled water. The slices were immersed in Victoria Blue B staining solution for 15 minutes. Differentiated in 95% ethanol for a few seconds, then washed twice with distilled water; stained with Ponceau staining solution for 5 minutes, directly differentiated and dehydrated with anhydrous ethanol, transparentized with xylene, and finally sealed with neutral gum, observed and recorded under a microscope.
[0018] Immunofluorescence staining: clinical samples and mouse aorta samples were dewaxed and fixed with 4% paraformaldehyde, washed and blocked, and then the primary antibodies of collagen, fibronectin, vimentin or P-MLC were added and incubated overnight at 4°C. After washing, the secondary antibodies were incubated, and DAPI was counterstained. After washing, the slides were sealed with anti-fluorescence quencher and then photographed and observed using a confocal microscope.
[0019] Western blots: clinical samples were placed in RIPA lysis buffer and homogenized with a 4°C tissue disruptor to extract proteins. Appropriate gel was selected according to the molecular weight for gel preparation and sample loading electrophoresis experiments. Constant current electrophoresis was then performed. The membrane was sealed and incubated with the primary antibody at 4°C overnight. After washing, the secondary antibody was incubated and then exposed to the light and the grayscale value was calculated and quantified using Image J.
[0020] Example 1 Downregulation of SCAP in Clinical Samples of Aortic Dissection
[0021] In the GEO database, SCAP expression was downregulated in clinical aortic dissection specimens compared with the control group ( Figure 1 A); HE and MASSON staining can be used to observe the increase of intramural hematoma, severe degradation of elastic fibers, large amounts of collagen fibers leaking out, and varying degrees of structural integrity damage in clinical aortic dissection samples ( Figure 1 B); Real-time fluorescence quantitative PCR and protein immunoblotting (Western blots) were used to detect SCAP. The results showed that compared with the control group, the expression of SCAP in patients with aortic dissection was downregulated ( Figure 1 CD).
[0022] Example 2 Relationship between SCAP and aortic dissection in mice
[0023] Two different genotype mice (WT and SCAP KD) were treated in the model group ( Figure 2 A), VSMC-specific SCAP knockout significantly reduced the survival rate of mice compared with the WT group ( Figure 2 B). After 8 weeks of age, the mice were killed and the aorta and heart were stripped. The perivascular fat tissue and muscle tissue were removed with ophthalmic forceps. Obvious aortic dissection bulge was observed in the VSMC-specific SCAP knockout group ( Figure 2 C), then the aorta was cut open at the location where the aorta root was connected to the heart, the aortic arch and abdominal aorta were cut and embedded with OCT, the tissue blocks were cut into 5um slices, and HE and MASSON staining were performed. The results showed that the vascular structure in the VSMC-specific SCAP knockout group was obviously disordered, with elastin degradation, incomplete structure and a large amount of collagen fibers exposed ( Figure 2 D).
[0024] Example 3 Relationship between SCAP and VSMC translocation and rearrangement in aortic tissue of patients with aortic dissection in clinical samples
[0025] Immunofluorescence staining was used to detect elastin, fibronectin, vimentin, and P-MLC in the aortic dissection group and the normal control group. The results showed that the expression of elastin and fibronectin was reduced in the aortic dissection group, and vimentin and P-MLC were aggregated in an eccentric distribution ( Figure 3 AD).
[0026] Example 4 SCAP affects VSMC translocation rearrangement in vivo
[0027] Immunofluorescence staining was used to detect elastin, fibronectin, vimentin, and P-MLC in two different genotypes of mice (WT and SCAP KD). The results showed that VSMC-specific SCAP knockout led to a decrease in the expression of elastin and fibronectin, and the eccentric distribution of vimentin and P-MLC was more obvious ( Figure 4 AD).
Claims
1. Application of SCAP as a target in the treatment or prevention of aortic dissection.
2. The use according to claim 1, characterized in that The SCAP leads to the occurrence and development of aortic dissection by regulating the arrangement of VSMCs.
3. The use according to claim 2, characterized in that The SCAP regulates the arrangement of VSMCs by participating in the translocation and redirection of VSMCs, thereby affecting the structural integrity of the vascular wall and causing aortic dissection.
4. The use as claimed in claim 1, comprising an in vivo use, wherein an aortic dissection model is constructed using VSMC-specific SCAP knockout mice, wherein the degree of aortic dissection is significantly higher than that of the control group.