Application of WWP1 in preparation of medicine for preventing and / or treating aortic dissection

Abstract

The invention relates to the technical field of molecular biology and medicine, and particularly discloses application of WWP1 in preparation of a medicine for preventing and / or treating aortic dissection, and relates to application of WWP1 in prevention and treatment of aortic dissection. Molecular biology experiment detection of the expression level of the WWP1 in aortic dissection aortic tissue and the expression level of smooth muscle cell contraction and secretion phenotype marker genes proves that the expression level of the WWP1 in an aortic dissection aortic tissue sample is obviously higher than that of a control aortic tissue; and overexpression of the WWP1 in an animal model significantly reduces the morbidity of the aortic dissection and improves the contraction phenotype of the aortic smooth muscle, i.e., the protectiveness of the WWP1 in the morbidity process of the aortic dissection is improved. Based on the research result, the WWP1 can be used as a drug target for screening, preventing and treating the aortic dissection.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and medical technology, specifically to the use of WWP1 in the preparation of drugs for the prevention and / or treatment of aortic dissection. Background Technology

[0002] Aortic dissection (AD) is a highly fatal cardiovascular emergency. It is most common in people aged 65-75, with large-scale epidemiological studies estimating an incidence of approximately 2.4-3.5 cases per 100,000 people annually. Besides age, key risk factors include hypertension, dyslipidemia (especially associated with atherosclerosis), and hereditary connective tissue diseases (such as Marfan syndrome). The core pathological basis of AD lies in the interaction between abnormal aortic media structure and hemodynamic stress. Media degeneration (such as elastic fiber rupture, smooth muscle cell apoptosis, and myxoid matrix accumulation) creates the conditions for dissection, while hypertension, atherosclerosis, and hereditary connective tissue diseases (such as Marfan syndrome) can accelerate media thinning. At the hemodynamic level, shear stress on the aortic wall and intravascular pressure fluctuations (especially high-speed blood flow impact) work together on the intima, ultimately leading to intimal tearing. When blood rushes into the tunica media to form a false lumen, the true lumen becomes compressed and narrowed, leading to ischemia in important branch vessels. This further activates systemic inflammatory responses and disrupts the coagulation system, creating a vicious cycle.

[0003] The structural integrity of the aortic wall depends on the synergistic action of elastic fibers, collagen fibers, and vascular smooth muscle cells in the aortic media. Among these, abnormalities in the TGF-β signaling pathway, increased activity of matrix metalloproteinases (MMPs), and an imbalance of their tissue inhibitors (TIMPs) are key molecular mechanisms leading to medial degeneration. In genetically related dissections, such as Marfan syndrome and Loeys-Dietz syndrome, mutations in the FBN1 gene or abnormal TGF-β receptor function can directly cause impaired synthesis and dysregulation of medial structural proteins. Furthermore, inflammatory cell infiltration, oxidative stress, and phenotypic transformation of vascular smooth muscle cells also participate in the occurrence and development of dissections, forming a complex pathological network.

[0004] E3 ubiquitin ligase 1 (WWP1), containing the WW domain, belongs to the HECT family of E3 ubiquitin ligases. As a key regulator of protein post-translational modification, WWP1 participates extensively in cellular life activities, including signal transduction, transcriptional regulation, cell proliferation, and apoptosis, by specifically recognizing substrates and catalyzing their ubiquitination. In pathological conditions, abnormal expression or altered activity of WWP1 is a key factor driving disease progression. However, in the field of aortic dissection, the function and mechanism of action of WWP1 remain unclear. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing the application of WWP1 in the preparation of drugs for the prevention and / or treatment of aortic dissection.

[0006] To solve the above-mentioned technical problems, the present invention discloses the following technical solution:

[0007] In a first aspect, the present invention discloses the use of WWP1 in the preparation of medicaments for the prevention and / or treatment of aortic dissection.

[0008] In some embodiments, the nucleotide sequence encoding WWP1 is as shown in NCBI's Gene ID: 107568, specifically nucleotides 19,608,296 - 19,709,004 in column NC_000070.7.

[0009] In some embodiments, the prevention and / or treatment of aortic dissection includes: reducing the incidence of aortic dissection, and / or alleviating smooth muscle cell phenotypic transformation, and / or antagonizing the increase in WWP1 protein levels caused by aortic dissection, and / or increasing WWP1 protein levels, i.e., increasing the level of WWP1 that is protectively increased during the pathogenesis of aortic dissection.

[0010] In some embodiments, the relief of smooth muscle cell phenotypic transformation includes antagonizing the decrease in the level of α-SMA protein, a smooth muscle contractile phenotypic marker induced by aortic dissection.

[0011] Secondly, this invention discloses the use of recombinant vectors overexpressing WWP1 in the preparation of medicaments for the prevention and / or treatment of aortic dissection.

[0012] In some embodiments, the nucleotide sequence encoding WWP1 is as shown in NCBI's Gene ID: 107568, specifically nucleotides 19,608,296 - 19,709,004 in column NC_000070.7.

[0013] In some embodiments, the recombinant vector is an adeno-associated virus vector.

[0014] In some embodiments, the recombinant vector contains the nucleotide sequence shown in SEQ ID NO.1.

[0015] In some embodiments, the prevention and / or treatment of aortic dissection includes: reducing the incidence of aortic dissection, and / or alleviating smooth muscle cell phenotypic transformation, and / or antagonizing the increase in WWP1 protein levels caused by aortic dissection, and / or increasing WWP1 protein levels, i.e., increasing the level of WWP1 that is protectively increased during the pathogenesis of aortic dissection.

[0016] In some embodiments, the relief of smooth muscle cell phenotypic transformation includes antagonizing the decrease in the level of α-SMA protein, a smooth muscle contractile phenotypic marker induced by aortic dissection.

[0017] Thirdly, the present invention discloses a product for diagnosing aortic dissection.

[0018] In some embodiments, the product includes reagents for detecting aortic dissection markers, including WWP1.

[0019] In some embodiments, the nucleotide sequence encoding WWP1 is as shown in NCBI's Gene ID: 107568, specifically nucleotides 19,608,296 - 19,709,004 in column NC_000070.7.

[0020] The present invention provides the application of WWP1 as a drug target for the prevention and / or treatment of aortic dissection.

[0021] This invention, through molecular biological experiments comparing the expression levels of WWP1 in aortic dissection tissue with the expression levels of smooth muscle cell contraction and secretion phenotypic marker genes, confirmed that the expression level of WWP1 in aortic dissection tissue samples was significantly higher than that in control aortic tissue. Based on this, WWP1 can be used to screen drug targets for the prevention and treatment of aortic dissection, as well as products for diagnosing aortic dissection. Furthermore, this invention, by packaging a WWP1 overexpression plasmid with adeno-associated virus and injecting it into mice via the tail vein, alleviated the incidence of BAPN-induced aortic dissection and increased the expression levels of aortic smooth muscle cell contraction phenotypic genes, demonstrating that in vivo administration of the WWP1 overexpression plasmid can alleviate BAPN-induced smooth muscle cell phenotypic transformation and prevent the occurrence of aortic dissection.

[0022] Beneficial effects:

[0023] This invention reveals that overexpression of WWP1 can effectively alleviate smooth muscle cell phenotypic transformation and aortic dilation lesions caused by aortic dissection, thereby preventing and treating aortic dissection and improving survival rates. This invention provides a novel method for preventing and / or treating aortic dissection, which has the potential to become an effective clinical prevention strategy for aortic dissection and rupture. This invention also shows promising clinical application prospects for the prevention of Alzheimer's disease (AD). Attached Figure Description

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0025] Figure 1 The left side shows the bands of WWP1, the contraction phenotype marker α-SMA, and the internal reference protein GAPDH in the aortic tissue of the control mouse aortic dissection model mouse as detected by Western Blot. The right side shows the relative expression levels of WWP1 and α-SMA. * indicates that the expression level of WWP1 protein in the BAPN group is significantly different from that in the Sham group, with P < 0.05.

[0026] Figure 2 Survival curves (left) and incidence of various aortic events (right) of mice injected via tail vein with WWP1 overexpression plasmid or NC control plasmid packaged with adeno-associated virus vector after being fed with ordinary drinking water and water containing BAPN, respectively.

[0027] Figure 3 Representative aortic ultrasound images of mice from four groups.

[0028] Figure 4 The maximum average diameter of the aorta is shown in the four groups of mice; **** indicates that the maximum average diameter of the aorta in the BAPN+WWP1 group is significantly different from that in the BAPN+NC group, with P < 0.0001.

[0029] Figure 5 The images show a representative gross aortic image (left) and a representative HE-stained scan image (right) of the mouse described in section 2.

[0030] Figure 6 The relative protein expression levels of WWP1 and α-SMA in mouse aortic tissue were detected by Western blotting; *, * indicate that the difference in WWP1 protein expression level between the BAPN+WWP1 group and the BAPN+NC group was P<0.05, and the difference in α-SMA protein expression level was P<0.01. Detailed Implementation

[0031] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0032] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0033] Example 1

[0034] 1. Constructing a mouse aortic dissection model

[0035] Control group (Sham): C57BL / 6 mice of a specific age (usually 3-4 weeks old) were given free access to autoclaved water.

[0036] Model group (0.5% BAPN): C57BL / 6 mice of a specific age (usually 3-4 weeks old) were given free access to water containing the LOX inhibitor β-aminopropionitrile (BAPN) at a concentration of 5 g / L for 4 weeks to inhibit the cross-linking of collagen and elastin in the aortic wall, thereby weakening its structure and inducing aortic dissection.

[0037] like Figure 1 As shown, the level of WWP1 protein in the aortic dissection tissue of the model group was increased compared with that of the control, while the level of α-SMA was relatively decreased. This indicates that the mouse aortic dissection model was successfully established.

[0038] 2. Experimental Grouping

[0039] The experimental mice were divided into four groups based on whether they drank water containing BAPN or were injected with a negative control adeno-associated virus / WWP1 overexpressing adeno-associated virus. The four groups were: the ordinary drinking water injection negative control adeno-associated virus group (abbreviated as Sham+NC (negative control, nonsense sequence) group), the ordinary drinking water and WWP1 overexpressing group (abbreviated as Sham+ WWP1 group), the BAPN-containing drinking water injection negative control adeno-associated virus group (abbreviated as BAPN+NC group), and the BAPN-containing drinking water and WWP1 overexpressing group (abbreviated as BAPN+ WWP1 group). There were 20 mice in each group.

[0040] The adeno-associated virus (AAV)-packaged overexpression plasmid AAV9-SM22α-gfp-WWP1 mus and control AAV9-SM22α-gfp-NC were constructed and synthesized by Suzhou Jima Gene Co., Ltd. The sequence encoding WWP1 is shown in NCBI Gene ID: 107568. The linearized plasmid was transfected into 293A cells to construct AAV9-SM22α-gfp-WWP1 mus (sequence shown in SEQ ID NO.1) or AAV9-SM22α-gfp-NC, which drives WWP1 expression by a smooth muscle cell-specific promoter (SM22α). The AAV9-SM22α-gfp-WWP1 mus or AAV9-SM22α-gfp-NC was injected via tail vein at a dose of 5 x 11^vg / mouse to achieve overexpression of the WWP1 gene, resulting in mice with high WWP1 expression in smooth muscle cells (WWP1 group) and control mice (NC group).

[0041] 3. Experimental Methods

[0042] When mice die during the modeling process, the deaths are recorded in a timely manner to plot survival curves, and autopsies are performed to confirm the cause of death. The morphology and diameter of the aorta are monitored by small animal ultrasound every two weeks. Finally, gross dissection and histopathological staining (such as H&E and EVG staining) are performed, and the diameter is combined to analyze aortic dilation, the incidence of aortic dissection, and typical pathological changes.

[0043] (1) Aortic ultrasound and aortic dissection in mice

[0044] Mice were anesthetized by inhalation (using isoflurane) and fixed in a supine position on a constant-temperature heating plate. Hair removal cream was applied to the chest and heart areas for thorough hair removal, followed by the application of ultrasound coupling gel to ensure effective sound wave transmission. A high-frequency small animal ultrasound imaging system was used for scanning: the ascending aorta and aortic arch were clearly displayed in the parasternal long-axis section, and the aortic diameter was accurately measured in the transverse section. The thoracic and abdominal aortas were systematically scanned, and the integrity of the aortic wall structure, the presence of intimal tears, intramural hematomas, or false lumens—characteristic lesions of dissection—were carefully observed. Static and dynamic images were saved, and the aortic diameter, location and extent of dissection, and blood flow signals were recorded for subsequent quantitative analysis and model validation.

[0045] Mice under deep anesthesia were perfused with saline via the left ventricle to flush out blood from the vessels, followed by perfusion fixation with pre-cooled 4% paraformaldehyde phosphate buffer to ensure the vascular morphology was well preserved in vivo. The skin and body wall were incised along the midline of the sternum and abdomen to fully expose the thoracic and abdominal cavities. Surrounding tissues and organs were carefully dissected to fully expose the entire aorta, from the ascending aorta to the bifurcation of the common iliac artery. Using fine forceps and scissors, the aorta was carefully freed from the surrounding connective tissue, and the aortic arch, thoracic aorta, and abdominal aorta were completely removed. The removed aorta was immediately macroscopically observed and photographed to record any obvious lesions such as vascular dilation, aneurysm formation, intramural hematoma, or adventitia rupture and hemorrhage.

[0046] (2) HE staining

[0047] Mouse aortic tissue blocks that had been fixed, dehydrated, and embedded in paraffin were cut into continuous sections with a thickness of 4-6 μm using a microtome. These sections were then mounted on glass slides and baked in a 60-65°C oven to melt the paraffin and ensure its firm adhesion. The sections were then immersed twice in xylene to completely remove the paraffin from the tissue. The dewaxed sections were then treated sequentially with a gradient of ethanol (100%, 95%, 85%, 70%), and finally rinsed with distilled water to replace the ethanol in the tissue, preparing for staining with water-soluble dyes. The hydrated sections were then immersed in hematoxylin staining solution for several minutes, followed by brief differentiation with acidic ethanol solution and a blue recovery with a weakly alkaline solution (such as ammonia or PBS). This step specifically stains the cell nuclei blue-purple. After a brief rinse with running water, the sections were immersed in eosin staining solution for several seconds to several minutes. This step non-specifically stains extracellular components such as cytoplasm, collagen fibers, and muscle fibers pink to red. The stained sections were then dehydrated again by sequentially passing them through a series of ethanol solutions (70%, 85%, 95%, 100%). The dehydrated sections were then immersed in xylene to clear them, increasing the tissue's translucency. After removing the sections from the xylene, a suitable amount of neutral resin was applied to the tissue area, and the sections were sealed with coverslips for long-term preservation and microscopic observation.

[0048] (3) Tissue protein extraction and Western Blot

[0049] Frozen mouse aortic tissue was washed in pre-chilled PBS, finely shredded with fine scissors, and placed in pre-chilled centrifuge tubes. A potent lysis buffer containing protease and phosphatase inhibitors (RIPA) was added to the tissue fragments, and the tissue was sonicated on ice to ensure complete lysis. The lysis mixture was centrifuged at 4°C and 12,000 rpm for 15 minutes, and the supernatant (total protein solution) was carefully aspirated and transferred to new pre-chilled centrifuge tubes. The protein concentration of each sample was determined using a BCA protein quantification kit, and all samples were adjusted to a uniform concentration using lysis buffer or loading buffer based on the results.

[0050] Mix the adjusted protein sample with 5X SDS-PAGE loading buffer and heat in a 95-100°C metal bath for 5-10 minutes to denature the protein. Then, add an equal volume of protein (e.g., 20-40 μg) to the wells of an SDS-polyacrylamide gel. First, compress the sample into a single line in the stacking gel at a constant voltage of 80V, then switch to a constant voltage of 120V for separating gel electrophoresis until the pre-stained protein marker band of the target protein is separated to the appropriate position. Using a wet transfer method, transfer the protein band from the gel to a PVDF membrane at a constant current (200-300 mA) for 60-120 minutes at low temperature. Immerse the transferred membrane in 5% skim milk or BSA blocking buffer and block on a shaker at room temperature for 1-2 hours to block non-specific binding sites. Incubate the membrane overnight at 4°C with specific primary antibodies against the target protein and internal control protein. After washing the membrane with TBST, the secondary antibody labeled with horseradish peroxidase (HRP) corresponding to the species of the primary antibody was incubated at room temperature for 1-2 hours. The target area on the membrane was uniformly covered with the working solution of the ECL chemiluminescence kit, and the signal was acquired in a dark room using a chemiluminescence imaging system to obtain protein band images. Gray-scale quantitative analysis was performed using image analysis software.

[0051] 4. Experimental Results

[0052] (1) Effects of overexpressing WWP1

[0053] like Figure 2 As shown, WWP1 overexpression significantly inhibited BAPN-induced disease progression, manifested as a reduction in aortic dilation and dissection, as well as a decrease in the incidence of aortic rupture as the endpoint event. Ultimately, it resulted in a significant prolongation of survival and an overall improvement in survival prognosis. At week 4, the survival rate of the BAPN+WWP1 group increased to 80% (16 / 20), which was much higher than the 60% (12 / 20) of the BAPN+NC group.

[0054] like Figure 3As shown, in the representative aortic ultrasound images, the maximum diameter of the ascending aorta (Asc Ao: L) in the BAPN+WWP1 group was 1.351 mm, and the maximum diameter of the aortic arch (Trans Arch: L) was 1.690 mm, significantly lower than the 2.024 mm and 2.787 mm in the BAPN+NC group, and comparable to the 1.284 mm and 1.449 mm in the Sham+NC group. Figure 4 As shown, the maximum average aortic diameter in the BAPN+WWP1 group was 1.56 mm, which was significantly lower than the 1.88 mm in the BAPN+NC group (P < 0.0001). This study indicates that overexpression of WWP1 significantly alleviates BAPN-induced aortic dilatation.

[0055] like Figure 5 As shown, overexpression of WWP1 significantly improved pathological changes such as increased aortic neutrophil infiltration induced by BAPN.

[0056] like Figure 6 As shown, overexpression of WWP1 effectively antagonizes the decrease in α-SMA protein levels induced by BAPN; at the same time, overexpression of WWP1 can also effectively further increase the WWP1 protein level induced by BAPN, thereby enhancing the protective increase of WWP1 in the pathogenesis of aortic dissection.

[0057] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Application of WWP1 in the preparation of medicaments for the prevention and / or treatment of aortic dissection.

2. The application according to claim 1, characterized in that, The nucleotide sequence encoding WWP1 is shown in NCBI's GeneID: 107568.

3. The application according to claim 1, characterized in that, The prevention and / or treatment of aortic dissection includes any one or more of the following: To reduce the incidence of aortic dissection, Relieve smooth muscle cell phenotypic transformation Increase the level of WWP1 in the protective increase during the pathogenesis of aortic dissection.

4. The application according to claim 3, characterized in that, The relief of smooth muscle cell phenotypic transformation includes antagonizing the decrease in the level of α-SMA protein, a phenotypic marker of smooth muscle contraction induced by aortic dissection.

5. Application of recombinant vectors overexpressing WWP1 in the preparation of drugs for the prevention and / or treatment of aortic dissection.

6. The application according to claim 5, characterized in that, The nucleotide sequence encoding WWP1 is shown in NCBI's GeneID: 107568.

7. The application according to claim 5, characterized in that, The recombinant vector is an adeno-associated virus vector.

8. The application according to claim 5, characterized in that, The recombinant vector contains the nucleotide sequence shown in SEQ ID NO.

1.

9. The application according to claim 5, characterized in that, The prevention and / or treatment of aortic dissection includes any one or more of the following: To reduce the incidence of aortic dissection, Relieve smooth muscle cell phenotypic transformation Increase the level of WWP1 in the protective increase during the pathogenesis of aortic dissection; Preferably, the relief of smooth muscle cell phenotypic transformation includes antagonizing the decrease in the level of α-SMA protein, a smooth muscle contraction phenotypic marker induced by aortic dissection.

10. A product for diagnosing aortic dissection, characterized in that, It includes reagents for detecting aortic dissection markers, including WWP1.

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