Application of LRP1-beta chain oligopeptide as target in treatment of aortic dissection

Abstract

The invention discloses an application of a gene for coding LRP1-beta chain oligopeptide as a target in screening / preparing a medicine for treating aortic dissection, an application of a reagent for inducing overexpression of the LRP1-beta chain oligopeptide in preparing the medicine for treating the aortic dissection, and an application of the LRP1-beta chain oligopeptide as a biomarker in preparing an aortic dissection diagnostic reagent. Experiments prove for the first time that the LRP1-beta chain oligopeptide can inhibit phenotypic transformation of vascular smooth muscle cells and inflammatory infiltration of macrophages by down-regulating expression of OPN, alleviate inflammatory response and relieve or inhibit occurrence and development of aortic dissection, provides a new direction and thought for treatment of aortic dissection, and has broad application prospects in treatment of aortic dissection. The compound is expected to be applied to research and development of new drugs for treating aortic dissection

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of LRP1-β chain short peptide as a target in the treatment of aortic dissection. Background Technology

[0002] Aortic dissection (AD) is an acute aortic disease that occurs under conditions such as hypertension, where a tear in the vascular intima allows blood to flow into the arterial wall, causing separation of the intima and media and the formation of a false lumen. It can affect the thoracic and abdominal aorta. Clinically, AD is classified into type A (affecting the ascending aorta), type B (affecting the descending aorta), and non-A / non-B (affecting the aortic arch) depending on the location. In some AD patients, the affected aorta gradually dilates to form an aneurysm. This disease is characterized by its rapid onset, high mortality, and poor prognosis. In recent years, the detection and prevalence of this disease have been increasing, and the age of onset is trending younger. While surgical treatments for AD are relatively mature, the procedures are complex, difficult, and prone to various postoperative complications.

[0003] The specific pathogenesis of Alzheimer's disease (AD) remains incompletely understood. Extensive literature indicates that AD is closely related to factors such as vascular wall inflammation, phenotypic transformation of vascular smooth muscle cells (VSMCs), endothelial cell dysfunction, extracellular matrix (ECM) destruction, inflammatory responses, oxidative stress, hemodynamic disturbances, and mitochondrial dysfunction. Inflammation plays a crucial role in the pathogenesis of AD. When Alzheimer's disease (AD) occurs, lymphocytes, macrophages, and neutrophils infiltrate the aortic wall. Macrophages are the most significantly proliferating immune cell group within the diseased tissue, infiltrating the vessel wall early in the disease course and rapidly increasing infiltration in later stages. Pro-inflammatory macrophages predominate, exacerbating the local inflammatory cascade and promoting elastic fiber degradation by secreting inflammatory cytokines (osteopontin (OPN), interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), etc.) and matrix metalloproteinases (MMPs). VSMCs are the main components of the aortic wall media and play a crucial role in maintaining aortic function and homeostasis. Under physiological conditions, VSMCs regulate vascular tone through contractile function and maintain aortic structural integrity by secreting ECM proteins. Under pathological conditions, factors such as immune cell infiltration and ECM degradation jointly drive phenotypic transformation of VSMCs, inducing them to change from contractile to proliferative, migratory, inflammatory, and synthetic forms. Furthermore, the progressive loss of VSMCs is a key characteristic of aortic degeneration, and VSMCs participate in the progression of vascular wall lesions through various cell death mechanisms, including necrosis, apoptosis, and ferroptosis.

[0004] Osteopontin (OPN), a crucial inflammatory regulator, is closely associated with macrophage-mediated inflammatory responses in the pathological progression of aortic aneurysms and aortic dissections. Studies have shown that OPN can regulate macrophage differentiation towards a pro-inflammatory phenotype, enhance their infiltrative capacity, and induce apoptosis and phenotypic transformation of vascular smooth muscle cells (VSMCs), further exacerbating vascular wall damage. This forms a core pathological axis of "macrophage infiltration—OPN upregulation—VSMC phenotypic transformation / death—matrix degradation," ultimately leading to aortic structural destruction.

[0005] Low-density lipoprotein receptor-related protein 1 (LRP1), also known as CD91 or α2 macroglobulin receptor, is a type I transmembrane glycoprotein belonging to the low-density lipoprotein receptor superfamily. It is widely expressed in tissues and cells throughout the body. It is formed by the cleavage of a 600 kDa precursor protein into a 515 kDa α chain and an 85 kDa β chain, which are linked through non-covalent interactions. LRP1 is also an endocytic receptor, participating in the regulation of various important physiological processes in different cells by binding to and internalizing a variety of ligands with different structures and functions, including intracellular signal transduction, lipid homeostasis, and clearance of apoptotic cells. Reports indicate that LRP1 plays a crucial role in maintaining vascular integrity.

[0006] There are no reports on the application of LRP1-β chain peptides to aortic dissection in current technology. The commonly used drugs or treatments for aortic dissection have limitations in applicability, poor efficacy and side effects. Further research on the pathogenesis of aortic dissection and the search for new drug targets are of great significance for guiding the diagnosis, treatment and prognosis of aortic dissection in clinical practice. Summary of the Invention

[0007] This invention, through experimental research, has confirmed the correlation between the expression level of the aortic LRP1-β chain peptide and aortic dissection. It clarifies that the LRP1-β chain peptide can improve aortic dissection by downregulating OPN expression, inhibiting vascular smooth muscle cell phenotypic transformation and macrophage inflammatory infiltration, thus providing important theoretical basis for the diagnosis and treatment of aortic dissection. Based on this, this invention provides the following technical solution:

[0008] This invention provides the application of the gene encoding the aortic LRP1-β chain short peptide as a target in the screening / preparation of drugs for the treatment of aortic dissection, inducing high expression of the aortic LRP1-β chain short peptide to alleviate or inhibit the occurrence and development of aortic dissection.

[0009] In the above-mentioned application technology solution, the gene encoding the LRP1-β chain short peptide is the LRP1 gene.

[0010] In the above-mentioned application technology, by upregulating the protein expression of the aortic LRP1-β chain short peptide and upregulating the expression of OPN, the phenotypic transformation of VSMCs and the inflammatory infiltration of macrophages are inhibited, thereby achieving the purpose of alleviating or inhibiting the occurrence and development of aortic dissection.

[0011] This invention also provides the application of reagents that induce high expression of the aortic LRP1-β chain short peptide in the preparation of drugs for treating aortic dissection.

[0012] In the above application technical solution, the reagent for inducing the expression of LRP1-β chain short peptide is a small molecule or low molecular weight compound, an LRP1-β chain short peptide protein-specific binder, or an overexpression molecule. The LRP1-β chain short peptide protein-specific binder includes antibodies, peptide antibodies, lectins, and binders targeting LRP1-β chain short peptides.

[0013] Preferably, the overexpression molecule is plasmid DNA.

[0014] This invention also provides the application of LRP1-β short peptide as a biomarker in the preparation of diagnostic reagents for aortic dissection.

[0015] In the above-mentioned application technology, low expression of LRP1-β chain short peptide indicates that the patient has aortic dissection, or indicates a poor prognosis for patients with aortic dissection.

[0016] The beneficial effects of this invention are: For the first time, experiments have demonstrated that the LRP1-β chain peptide can inhibit VSMC phenotypic transformation and macrophage inflammatory infiltration by downregulating OPN expression, thereby alleviating or inhibiting the development of aortic dissection. Developing drugs targeting the LRP1-β chain peptide can reverse the downregulation of aortic LRP1-β chain peptide expression caused by aortic dissection, downregulate OPN protein expression, inhibit VSMC phenotypic transformation and macrophage inflammatory infiltration, and reduce the inflammatory response, thereby achieving the goal of alleviating or treating aortic dissection. This provides a new direction and approach for the treatment of aortic dissection and holds promise for application in the development of new drugs for the treatment of aortic dissection. Attached Figure Description

[0017] Figure 1 This study established an animal model of aortic dissection induced by β-aminopropionitrile (BAPN) in normal control C57BL / 6 mice.

[0018] Figure 2 This is a comparison of the rate of change in body weight between normal control mice and BAPN-induced mice; *, P < 0.05, **, P < 0.01, ***, P < 0.001, and ****, P < 0.0001.

[0019] Figure 3 The survival curves of mice in the normal control group and mice in the BAPN-induced group are compared; *, P<0.05, **, P<0.01, ***, P<0.001, and ****, P<0.0001.

[0020] Figure 4 This is a comparison of gross aortic specimens from normal control mice and BAPN-induced mice after modeling.

[0021] Figure 5 This study compared the histopathological changes (HE staining) of the aorta between normal control mice and BAPN-induced mice, and quantitatively assessed the ratio of true lumen circumference to maximum media thickness; *, P<0.05, **, P<0.01, ***, P<0.001, and ****, P<0.0001.

[0022] Figure 6 This is a comparison of the degree of elastic fiber rupture in the aortic tissue of normal control mice and BAPN-induced mice (EVG staining).

[0023] Figure 7 This is a comparison of the degree of collagen fiber degradation in the aortic tissue of normal control mice and BAPN-induced mice (Masson staining).

[0024] Figure 8 This is a comparison of LRP1 protein expression levels in the aortic tissue of normal control mice and BAPN-induced mice.

[0025] Figure 9 This is a comparison of the spatial localization and expression distribution of the LRP1-β chain in the aortic tissue of normal control mice and BAPN-induced mice (immunofluorescence).

[0026] Figure 10 It is BAPN-induced LRP1-β chain 2AAAA wild type (LRP1-β chain) 2AAAA-wiletype Mice and LRP1-β chain 2AAAA mutant (LRP1-β chain 2AAAA Establishment of a mouse model of aortic dissection disease.

[0027] Figure 11 It is the LRP1-β chain. 2AAAA-wiletype Mouse and LRP1-β chain 2AAAA LRP1 genotyping in mice.

[0028] Figure 12 It is BAPN-induced LRP1-β chain 2AAAA-wiletype Mouse and LRP1-β chain 2AAAA Comparison of weight change rates in mice; *, P < 0.05, **, P < 0.01, ***, P < 0.001, and ****, P < 0.0001.

[0029] Figure 13 It is BAPN-induced LRP1-β chain 2AAAA-wiletype Mouse and LRP1-β chain 2AAAA Survival curves of mice were compared; *, P<0.05, **, P<0.01, ***, P<0.001, and ****, P<0.0001.

[0030] Figure 14 It is BAPN-induced LRP1-β chain 2AAAA-wiletype Mouse and LRP1-β chain 2AAAA Gross specimens of the aorta in mice after modeling were established.

[0031] Figure 15 It is BAPN-induced LRP1-β chain 2AAAA-wiletype Mouse and LRP1-β chain 2AAAA Comparison of histopathological changes in mouse aorta (HE staining) and quantitative assessment of the ratio of true lumen circumference to maximum media thickness; *, P<0.05, **, P<0.01, ***, P<0.001, and ****, P<0.0001.

[0032] Figure 16 It is BAPN-induced LRP1-β chain 2AAAA-wiletype Mouse and LRP1-β chain 2AAAA Comparison of the degree of elastic fiber rupture in mouse aortic tissue (EVG staining).

[0033] Figure 17 It is BAPN-induced LRP1-β chain 2AAAA-wiletype Mouse and LRP1-β chain 2AAAA Comparison of the degree of collagen fiber degradation in mouse aortic tissue (Masson staining).

[0034] Figure 18 It is BAPN-induced LRP1-β chain 2AAAA-wiletype Mouse and LRP1-β chain 2AAAA Comparison of OPN protein expression levels in mouse aortic tissue.

[0035] Figure 19 It is BAPN-induced LRP1-β chain 2AAAA-wiletype Mouse and LRP1-β chain 2AAAA Comparison of expression levels of NF-κBp65, IL-6, OPN and α-SMAmRNA in mouse aortic tissue.

[0036] Figure 20 It is α-SMA ( Figure 20 A), OPN Figure 20 B) and F4 / 80 Figure 20 C) BAPN-induced LRP1-β chain 2AAAA-wiletype Mouse and LRP1-β chain 2AAAA Comparison of spatial localization and expression distribution in mouse aortic tissue (immunofluorescence). Detailed Implementation

[0037] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the chemical and biological reagents used are conventional reagents in the art and are commercially available.

[0039] Main reagent sources:

[0040] LRP1-β chain 2AAAA-wiletype Mouse and LRP1-β chain 2AAAA The mice were donated by the U.S. Southwestern Medical Center.

[0041]

[0042] The antibody source information used in the examples is as follows:

[0043] Antibody name Company Name Product batch number Rabbit anti-OPN antibody Proteintech 22952-1-AP Rabbit anti-LRP1 antibody Selleck A5229 Mouse anti-F4 / 80 antibody CellSignaling Technology 71299T Mouse anti-β-actin antibody Proteintech 66009-1-Ig Rabbit anti-α-SMA antibody Proteintech 14395-1-AP

[0044] Example 1

[0045] I. Materials and Methods

[0046] 1. Obtaining mice with specific gene mutations and animal experiments

[0047] LRP1-β chain 2AAAA-wiletype Mouse and LRP1-β chain 2AAAA The mice were donated by the US Southwestern Medical Center. Mating yielded 3-4 week old male LRP1-β chain mice. 2AAAA-wiletype Mouse and LRP1-β chain 2AAAA Five mice per group were given BAPN in their drinking water for four weeks, with the water changed every three days. Mice were housed in ventilated cages with constant temperature and humidity, and subjected to light-dark cycles every 12 hours. After modeling, the mice were fasted overnight, weighed, and euthanized by cervical dislocation. The thoracic and abdominal aortas were harvested, and the aortic tissue was divided into two parts: 1) The most severely diseased portion was directly embedded in an OCT scanner for frozen sectioning and subsequent pathological staining. 2) The remaining portions were flash-frozen in liquid nitrogen and then temporarily stored at -80°C for the extraction of total protein and total RNA for subsequent Western blot (WB) and real-time quantitative PCR (RT-qPCR) experiments.

[0048] 2. Immunoblotting

[0049] Tissue samples were lysed using RIPA lysis buffer (containing 1% phosphatase inhibitor and 1% protease inhibitor). Protein samples were separated by electrophoresis on SDS-PAGE gels, followed by routine membrane transfer, blocking, primary antibody incubation, secondary antibody incubation, and finally development using chemiluminescence. The grayscale values ​​of the target proteins were analyzed using ImageJ software.

[0050] 3. Real-time quantitative PCR

[0051] Total RNA was extracted from aortic tissue using RNAiso PLUS (Takara, Japan). The RNA was then reverse transcribed into cDNA using a reverse transcription reagent (Takara, Japan). The expression of genes such as OPN, NF-κB p65, and IL-6 was detected by RT-qPCR. The RT-qPCR amplification program was: 94℃ for 20 seconds; 94℃ for 10 seconds, 60℃ for 20 seconds, for 40 cycles.

[0052] 4. Immunofluorescence

[0053] After thawing frozen sections to room temperature, fix them with 4% paraformaldehyde for 30 minutes, wash them three times with PBST (5 minutes each time), permeabilize them with 0.3%-0.5% Triton X-100, block them with 3% BSA for 30-60 minutes, add primary antibody and incubate overnight at 4°C, wash them three times with PBS, add fluorescent secondary antibody and incubate at room temperature for 60 minutes under light-protected conditions, wash them three times with PBS, stain the nuclei with DAPI for 5 minutes, wash them with PBS, add anti-quenching mounting medium and mount them, and observe them under a fluorescence microscope.

[0054] 5. HE staining

[0055] After thawing frozen sections to room temperature, fix them with 4% paraformaldehyde for 30 minutes, rinse with running water; stain with hematoxylin for 5-10 minutes (time controlled for microscopic examination), rinse with running water, differentiate with 1% hydrochloric acid ethanol for 5-10 seconds, rinse with running water for 15-20 minutes to return to blue; counterstain with eosin for 30-60 seconds, rinse with running water for 5-10 minutes, dehydrate with a gradient of ethanol (3 seconds each for 85%, 95%, and anhydrous ethanol), clear with xylene for 3 seconds, and mount immediately with neutral resin.

[0056] 6. Masson staining

[0057] After thawing frozen sections to room temperature, fix them with 4% paraformaldehyde for 30 minutes and rinse with running tap water; stain with hematoxylin for 5-30 seconds, differentiate with 1% hydrochloric acid ethanol for 5-10 seconds, rinse with running water for 10-15 minutes to regain blue color; stain with ponceau red and hematoxylin for 5 minutes and rinse with running water; treat with phosphomolybdic acid for 1-5 minutes (differentiation), stain with aniline blue for 10-60 seconds (adjust according to sample), wash with weak acid working solution, dehydrate with a gradient of ethanol (85%, 95%, and anhydrous ethanol, 3 seconds each), clear with xylene for 3 seconds, and mount immediately with neutral resin.

[0058] 7. EVG staining

[0059] After thawing frozen sections to room temperature, fix them with 4% paraformaldehyde for 30 minutes and rinse with running water. Add the prepared Weigert oxidant to the sections for 3 minutes and rinse with running water for 1 minute. Add Weigert bleach to the sections for 3 minutes and rinse with running water for 1 minute. Stain with Weigert fuchsin solution at room temperature, cover and immerse for 1-3 hours. Differentiate with acidic differentiation solution until no staining solution remains, then rinse with running water to stop differentiation. Stain with modified VG staining solution for 5-10 minutes and rinse with running water for 30 seconds. Dehydrate with a gradient of ethanol (85%, 95%, and anhydrous ethanol, 3 seconds each), clear with xylene for 3 seconds, and immediately mount with neutral resin.

[0060] 8. Statistical Analysis

[0061] All data in the examples are expressed as mean ± standard error (mean ± SEM). Graphpad Prism 8.0 software was used for plotting and statistical analysis. The differences between two groups were tested using independent samples t-tests, and the differences between multiple groups were tested using two-way ANOVA. A p-value < 0.05 was considered statistically significant.

[0062] II. Results

[0063] 1. The expression level of the aortic LRP1-β chain short peptide is negatively correlated with aortic dissection (AD).

[0064] LRP1 is widely expressed in various tissues and cells throughout the body and can participate in the regulation of various important physiological processes in different cells by binding to and internalizing a variety of ligands with different structures and functions. Although it has been reported that LRP1 plays a key role in maintaining vascular integrity, its expression and function in AD have not been reported. Therefore, in order to explore the expression changes of aortic LRP1 in the development and progression of AD, we established a β-aminopropionitrile (BAPN)-induced AD mouse model (…). Figure 1 A) After treatment, mouse aortic tissue was taken for pathological staining and Western blot (WB) analysis. Figure 1 B).

[0065] The results showed that, compared with normal control mice, AD model mice exhibited significantly increased mortality and weight loss. Figure 2 ,3). Based on gross morphological observation of the aortic tissue ( Figure 4 ) and various pathological staining results ( Figure 5-7 In the AD model group, the aorta of mice showed more significant pathological damage characteristics, specifically: a significant reduction in the true lumen circumference. Figure 5 A) The maximum thickness of the median membrane increased significantly. Figure 5 B) Elastic fiber breakage is more severe. Figure 6 And a significant increase in collagen fiber deposition ( ) Figure 7 This indicates that the aortic dissection disease model has been successfully established. Western blot and immunofluorescence staining results consistently showed that the expression level of LRP1 in the aortic tissue of AD model mice was significantly reduced. Figure 8 (9). In summary, the results indicate that the expression of aortic LRP1 (especially its β-chain short peptide form) is downregulated in the BAPN-induced AD mouse model.

[0066] 2. LRP1-β chain peptide improves aortic dissection by inhibiting VSMC phenotypic transformation and macrophage inflammatory infiltration.

[0067] To further explore the association between the LRP1-β chain short peptide and aortic dissection, we induced the LRP1-β chain using BAPN. 2AAAA-Wiletype Mouse and LRP1-β chain 2AAAA Aortic dissection in mice Figure 10 LRP1-β chain 2AAAA Mutant short peptides are novel short peptides constructed by mutating all four amino acids (N-asparagine; P-proline; V-valine; Y-tyrosine) of the distal NPxY motif of the LRP1 β-chain short peptide to A-alanine (AAAA). After genotyping and confirmation, they are used for subsequent animal model construction. Figure 11 ).

[0068] like Figure 12 and 13 As shown, the LRP1-β chain 2AAAA-Wiletype Mouse and LRP1-β chain 2AAAA No statistically significant differences were observed in mortality or weight change in mice; however, it is noteworthy that the LRP1-β chain... 2AAAA Mice exhibited a high mortality trend. This was based on gross morphological observations of the aortic tissue (…). Figure 14 ) and various pathological staining results ( Figure 15-17 LRP1-β chain 2AAAA The mouse aorta exhibited more pronounced pathological damage characteristics, specifically a significant reduction in the true lumen circumference. Figure 15 A) The maximum thickness of the median membrane increased significantly. Figure 15 B) Elastic fiber breakage is more severe. Figure 16 And a significant increase in collagen fiber deposition ( ) Figure 17 ).

[0069] via WB ( Figure 18 ), RT-qPCR Figure 19 ) and immunofluorescence ( Figure 20 A and 20B) A series of experiments confirmed that: compared to the LRP1-β chain 2AAAA-Wiletype Mouse, LRP1-β chain 2AAAAIn mice, the systolic marker α-SMA was significantly upregulated at both the mRNA and protein levels, while the synthetic marker OPN showed the opposite trend. LRP1-β chain 2AAAA In the mouse aorta, the inflammatory cytokine IL-6 and the key transcription factor p65 of the classical inflammatory signaling pathway NF-κB are upregulated at the mRNA level. Figure 19 This suggests the LRP1-β chain. 2AAAA The inflammatory response in the aorta of mice was exacerbated. Immunofluorescence staining further confirmed that more severe macrophage infiltration occurred in the aorta of this mouse model. Figure 20 C). In summary, the results indicate that the aortic LRP1-β chain peptide improves aortic dissection by downregulating OPN expression, inhibiting VSMC phenotypic transformation and macrophage inflammatory infiltration.

Claims

1. The application of a gene encoding the LRP1-β chain short peptide as a target in the screening / preparation of drugs for the treatment of aortic dissection, characterized in that: The gene encoding the LRP1-β chain peptide is regulated, and high expression of the LRP1-β chain peptide helps alleviate or inhibit the occurrence and development of aortic dissection.

2. The application according to claim 1, characterized in that: The gene encoding the LRP1-β chain peptide is the LRP1 gene.

3. The application according to claim 1, characterized in that: By upregulating the expression of the aortic LRP1-β chain short peptide and downregulating the expression of OPN, the inflammatory infiltration of macrophages and the phenotypic transformation of vascular smooth muscle cells are inhibited, thereby alleviating or inhibiting the development of aortic dissection.

4. The application according to claim 3, characterized in that: LRP1-β short peptide attenuates or inhibits aortic dissection by regulating OPN expression.

5. Application of reagents that induce LRP1-β chain overexpression in the preparation of drugs for the treatment of aortic dissection.

6. The application according to claim 5, characterized in that: It regulates the expression of LRP1-β chain peptide in the aorta. Increased expression of LRP1-β chain peptide inhibits phenotypic transformation of vascular smooth muscle cells and inflammatory infiltration of macrophages.

7. The application according to claim 5, characterized in that: The reagents for inducing LRP1-β chain peptide overexpression are small molecule or low molecular weight compounds, LRP1-β chain peptide protein-specific binders, or overexpression molecules. The LRP1-β chain peptide protein-specific binders include antibodies, peptide antibodies, lectins, and binders targeting LRP1-β chain peptides.

8. The application according to claim 7, characterized in that: The overexpressed molecule is plasmid DNA.

9. Application of LRP1-β chain short peptide as a biomarker in the preparation of diagnostic reagents for aortic dissection.

10. The application according to claim 9, characterized in that: Low expression of LRP1-β short peptides may indicate aortic dissection or a poor prognosis in patients with aortic dissection.

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